Mol Cell Biochem (2014) 388:39–49 DOI 10.1007/s11010-013-1897-y
Prognostic significance of microRNA-141 expression and its tumor suppressor function in human pancreatic ductal adenocarcinoma Zi-Man Zhu • Yue-Fang Xu • Qin-Jun Su • Jun-Dong Du • Xiang-Long Tan • Yu-Liang Tu Jing-Wang Tan • Hua-Bao Jiao
•
Received: 6 September 2013 / Accepted: 5 November 2013 / Published online: 17 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Increasing evidence shows that dysregulation of microRNAs is correlated with tumor development. This study was performed to determine the expression of miR141 and investigate its clinical significance in pancreatic ductal adenocarcinoma (PDAC). Taqman quantitative RTPCR was used to detect miR-141 expressions in 94 PDAC tissues and 16 nontumorous pancreatic tissues. Correlations between miR-141 expression and clinicopathologic features and prognosis of patients were statistically analyzed. The effects of miR-141 expression on growth and apoptosis of PDAC cell line (PANC-1) were determined by MTT, colony formation, and flow cytometry assays. Potential target genes were identified by luciferase reporter and Western blot assays. The expression level of miR-141 in PDAC tissues was significantly lower than that in corresponding nontumorous tissues. Downregulation of miR141 correlated with poorer pT and pN status, advanced clinical stage, and lymphatic invasion. Also, low miR-141 expression in PDAC tissues was significantly correlated Zi-Man Zhu, Yue-Fang Xu and Qin-Jun Su should be regarded as joint first authors for their equal contributions. Z.-M. Zhu J.-D. Du X.-L. Tan Y.-L. Tu J.-W. Tan H.-B. Jiao (&) Department of Hepatobiliary Surgery, First Hospital Affiliated to the Chinese PLA General Hospital, Fucheng Road 51, Haidian District, Beijing 100048, China e-mail: [email protected] Y.-F. Xu Department of Pharmacy, First Hospital Affiliated to the Chinese PLA General Hospital, Fucheng Road 51, Haidian District, Beijing 100048, China Q.-J. Su Department of Pathology, Lanzhou Military Area General Hospital, Lanzhou 730050, China
with shorter overall survival, and multivariate analysis showed that miR-141 was an independent prognostic factor for PDAC patients. Further, functional researches suggested that miR-141 inhibits growth and colony formation, and enhances caspase-3-dependent apoptosis in PANC-1 cells by targeting Yes-associated protein-1 (YAP1). Therefore, miR-141 is an independent prognostic factor for PDAC patients, and functions as a tumor suppressor gene by targeting YAP1. Keywords MicroRNA-141 Pancreatic ductal adenocarcinoma YAP1 Prognosis
Introduction Pancreatic cancer is one of the most aggressive human malignant tumors and a leading cause of cancer-related mortality around the world [1]. Of these, pancreatic ductal adenocarcinoma (PDAC) accounts for *90 % of all the cases. Despite advances in surgery, chemotherapy, and other therapies, the overall survival of patients with PDAC is quite low [2]. Therefore, a better understanding of the biology of PDAC will be helpful for the identification of novel molecular markers to predict the prognosis of the patients. Extensive researches have characterized the complex genetic networks and transcriptomics alterations underlying the progression of PDAC [3]. Recently, the discovery of a new class of microRNAs (miRNAs) has provided additional insights, potentially explaining the gap that exists between tumor genotype and phenotype [4, 5]. MiRNAs can downregulate gene expression by inducing the degradation or impairing the translation of target mRNAs. Increasing evidences have shown that miRNAs
123
40
play critical roles in many human key biologic processes including tissue differentiation, cell proliferation, and embryonic development [6–8]. As such, the dysregulation of miRNAs and their targets are correlated with tumor development [9]. In this context, miRNAs have been identified as promising alternative biomarkers for early detection of cancer, informing prognosis, and monitoring treatment response [10]. Recent studies have found that dysregulation of miRNAs is involved in the development of PADC [11, 12]. Previously, miR-141 has been reported to function as a tumor suppressor in many tumors including renal cell carcinoma, colorectal cancer, and gastric cancer [13–15]. However, the status of miR-141 and its clinical value in human PDAC are unclear and remain to be elucidated. In this study, our purpose is to quantify expression levels of miR-141 in the clinical samples of PDAC, and to evaluate their correlation with clinicopathological features of PDAC patients. Also, the correlation of miR-141 expression with prognosis of patients is evaluated. In addition, the effects of miR-141 expression on malignant phenotypes of PDAC cells and the possible molecular mechanisms will be further investigated.
Materials and methods Patients and tissue samples Primary tumor specimens were obtained from 94 patients diagnosed with PDAC, who underwent complete resection at our Hospital between 2006 and 2010. The original histopathologic slide sets and reports were obtained from each case, and these were reviewed to confirm the diagnosis of PDAC. None of the patients had received chemotherapy or radiotherapy before surgery. Experienced pathologists provided detailed pathologic diagnosis according to UICC TNM classification standards (2002). Informed consent was obtained from all patients in writing. The ethics committee of Nanjing Medical University approved the study protocol. Also, 16 paired of PDAC and corresponding nontumorous pancreatic tissues were also collected. Samples were snap-frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Cell culture Human PDAC cell line (PANC-1) was purchased from Shanghai Institute Chinese academy of science, and cultured in RPMI 1640 media (Invitrogen, CA, USA) supplemented with 10 % fetal bovine serum (FBS) and 100 lM each of penicillin and streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C.
123
Mol Cell Biochem (2014) 388:39–49
Transfection of plasmid vectors, miRNA mimics or inhibitor, and siRNA A DNA fragment containing the hsa-miR-141 or YAP1 coding region was amplified from human pancreatic tissue genomic DNA and cloned into pEGFP-N1 or pcDNA3.0 (?) vector which was named pEGFP/miR-141 or pcDNA/ YAP1, respectively. The siRNA/YAP1 (sc-3863) and control siRNA (siRNA/control) were purchased through Santa Cruz Biotechnology. The transient or stable transfection was performed using LipofectamineTM 2000 (Invitrogen, CA, USA) following the manufacturer’s instructions. The G418-resistant cells were named PANC1/miR-141 or PANC-1/miR-NC, respectively. Taqman quantitative RT-PCR assay Total RNA isolated was used, TRIzol Reagent (Invitrogen, CA, USA). TaqMan qRT-PCR assay was used to assess the expression of miR-141 with kits from Applied Biosystems (Foster City, CA, USA) as described previously [16]. We calculated 4Ct (target-reference) which is equal to the difference between threshold cycles for miR-141 (i.e., target) and the threshold cycle for U6 RNA (i.e., reference) (i.e., DCt (target-reference) = Ct target-Ct reference). The foldchange between patient or cell sample and a normal control for miR-141 was calculated with the 2-DDCt method. qRTPCR was repeated in triplicate for each sample. Western blotting assay The level of YAP1, cleaved or total caspase-3, cleaved or total PARP, and GAPDH protein were analyzed using standard Western blot procedures as described previously with the rabbit antihuman YAP1, cleaved or total caspase3, cleaved or total PARP, and GAPDH primary antibody (Santa Cruz Biotechnology) [17]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The transfected PANC-1 cells (6.0 9 103 cells/well) were plated in 96-well plates. Then, for 6 days, a batch of cells were stained with 20 lL sterile MTT dye (5 mg/mL) at 37 °C for 4 h every day, and then, the culture medium was removed, and 150 lL of DMSO was added and thoroughly mixed in for 10 min. Spectrometric absorbance at 490 nm was measured using a microplate reader. Colony formation assay The stably transfected cells were plated in 10-cm culture dishes. After 14 days, cells were fixed with methanol, and
Mol Cell Biochem (2014) 388:39–49
41
stained with 0.1 % crystal violet. Visible colonies were manually counted. Flow cytometry detection of apoptosis The cells were recovered by centrifugation for evaluation of apoptotic cells with the use of double staining with annexin V-fluoresein isothiocyanate (Annexin V-FITC) and propidium iodide, (PI) (BioVision, St Pete Beach, FL) according to the manufacturer’s instructions. Luciferase reporter assay The 30 -UTR sequence of YAP1, predicted to interact with miR-141, was amplified using primers (sense, 50 -GCGAATTCCCAAAGCACTATTTGT-30 ; reverse, 50 -GGCTCGAGA-AGCACTCATTTCTC-30 ) and cloned into the EcoRI and XhoI sites of pGL3-Basic vector (Promega, Madison, WI, USA) that was named pGL3-YAP1/30 -UTR-wt. Putative binding sites of miR-141 in YAP1 30 -UTR were mutated using the QuikChange site-directed mutagenesis kit (Stratagene, Germany) to obtain pGL3-YAP1/30 -UTRmt. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instruction. Statistical analyses Analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). The Kruskal–Wallis H or Mann– Whitney U test was used to investigate clinicopathological significance of miR-141 expression. Overall survival curves were plotted using the Kaplan–Meier method and were evaluated for the statistical significance using a logrank test. The significance of different variables with respect to survival was analyzed using the multivariate Cox proportional hazards model. The correlation was evaluated by Spearman’s rank correlation coefficients. P \ 0.05 was considered to indicate a statistically significant difference.
Fig. 1 qRT-PCR assay was used to detect the expression of miR-141 in PDAC and corresponding non-tumorous pancreatic tissues. Downregulation (a) and downregulation (b) of miR-141 expression. qRT-PCR analysis of miR-141 expression was performed in PDAC tissues (n = 16) and corresponding non-tumorous pancreatic tissues (n = 16). U6 was used as an internal control. c The mean level of miR-141 expression in PDAC tissues (n = 16) was significantly lower than that in corresponding non-tumorous tissues (n = 16) (P \ 0.001). Each experiment was performed at least in triplicate. *P \ 0.05 versus control
tissues was significantly higher than that in nontumorous pancreatic tissues (P \ 0.001; Fig. 1c). Thus, downregulation of miR-141 might play a critical role during PDAC development. Clinicopathological significance of miR-141 expression in human PDAC
Results Expression of miR-141 was significantly downregulated in human PDAC tissues First, Taqman quantitative (q) RT-PCR assay was performed to detect miR-141 expression in 16 PDAC tissues and nontumorous pancreatic tissues. Compared with nontumorous pancreatic tissues, the level of miR-141 expression was lower in 12 PDAC tissues (Fig. 1a), and the expression of miR-141 was found to be upregulated in only four cases (Fig. 1b). The mean level of miR-141 in PDAC
Next, we investigated the correlation of miR-141 expression with clinicopathological features of PDAC patients. Expression of miR-141 was analyzed by relative quantity (RQ) using the equation RQ = 2-DDCT (CT = threshold cycle to detectfluorescence). According to the mean RQ of miR-141 in 94 PDAC tissues, (1.54 ± 2.65), 53 (56.4 %) patients were classified as low-miR-141 group (B1.54) and 41 (33.6 %) as high-miR-141 group ([1.54). By statistical analysis (Table 1), it was observed that low miR-141 expression was closely correlated with pT status, pN status, advanced clinical stage, and higher incidence of lymphatic
123
42
Mol Cell Biochem (2014) 388:39–49
0.014*
18.73 % in those with low expression. Moreover, patients with high miR-141 expression had significantly longer 5-year median overall survival (OS) than those with low miR-141 expression (log-rank test; P = 0.017; Fig. 2b): 48.7 months in patients with high expression versus 26.4 months in those with low expression. To further determine the prognostic value of miR-141 for PDAC patients, univariate and multivariate data analyses were performed using the Cox proportional hazards regression model (Table 2). Univariate analyses showed that pT status, clinical stage, residual tumor category, and the status of miR-141 expression were significantly correlated with over survival of PDAC patients (P = 0.024, 0.013, 0.005 and 0.034, respectively). Multivariate analyses showed that that the status of miR-141 expression and other factors (clinical stage, status of lymphatic invasion, and residual tumor category) were independent prognostic factors for PDAC patients (HR 2.55, 95 % CI 2.08–4.71, P 0.019).
0.001*
Effects of miR-141 on growth, colony formation, and apoptosis in PDAC cells
Table 1 Relationship of miR-141 expression with various clinicopathological factors of PDAC patients Factors
MiR-141 expression Low (n = 53)
High (n = 41)
32
31
21
10
B55
19
11
[55
34
30
Head
17
20
Body or tail
37
21
pT1/pT2
23
29
pT3/pT4
30
12
pN0
20
26
pN1
33
15
Gender Male Female Age (year)
0.119
0.352
Tumor location
0.087
pT category
0.008*
pN category
Clinical stage I/II
18
28
35
13
Negative
22
27
Positive
31
14
Negative
32
24
Positive
21
17
Negative
41
30
Positive
12
11
R0
38
34
R1
15
7
III/IV Lymphatic invasion
P value
0.019*
Vessel invasion
0.857
Neural invasion
0.640
Residual tumor category
0.202
* P \ 0.05 was considered statistically significant
invasion (P = 0.008, 0.014, 0.001, and 0.019, respectively). However, there were no significant differences in gender, age, tumor location, vessel invasion, neural invasion, and residual tumor status between the low miR-141 group and high miR-141 group (P = 0.119, 0.352, 0.087, 0.857, 0.640, and 0.202, respectively). Prognostic significance of miR-141 expression in human PDAC By survival analysis, low miR-144 expression was observed to be in correlation with a significantly lower 5-year recurrence free survival (RFS) rate of PDAC patients (P = 0.031; Fig. 2a): 24.56 % in patients with high expression versus
123
To determine the effect of miR-141 on malignant phenotypes of PDAC cells, pEGFP/miR-141- and pEGFP/miR-NCexpressing vectors were stably transfected into PANC-1 cells. Then, qRT-PCR assay was performed to detect miR141 expression in PANC-1/miR-141 or PANC-1/miR-NC cells. Compared with PANC-1/miR-NC cells, the expression of miR-141 in PANC-1/miR-141 cells was significantly upregulated by about 321.5 % (P \ 0.01; Fig. 3a). The results of the proliferation assays performed using MTT and colony formation assays are shown in the figure. As shown in Fig. 3b, PANC-1/miR-141 cells grew significantly slowly compared with PANC-1/miR-NC, and the inhibitory rate reached about 65.8 % at day 6 (P \ 0.01). It was revealed that the colony growth ability of PANC-1/miR-141 cells was significantly reduced compared with PANC-1/miR-NC cells (P \ 0.05; Fig. 3c). FACS analysis of apoptosis showed that PANC-1/miR-141 cells represented significant apoptosis enhancement (P \ 0.05; Fig. 3d). Further, upregulation of miR-141 could lead to the increased expression of cleaved caspase-3 or PARP protein in PANC-1 cells, but the expression of total caspase-3 or PARP protein showed no difference between PANC-1/miR-141 and PANC-1/miRNC cells (Fig. 3e). Thus, these data indicated that upregulation of miR-141 inhibited growth of PDAC cells by enhancing caspase-3-dependent apoptosis. YAP1 was a functional target of miR-141 in PDAC cells To further investigate the underlying molecular mechanism of miR-141 in the regulation of growth, we interrogated the
Mol Cell Biochem (2014) 388:39–49
43
Fig. 2 Kaplan–Meier survival curve of PDAC patients. a MiR141 expression was correlated with recurrence-free survival in PDAC patients (P = 0.031). b MiR-141 expression was correlated with overall survival in PDAC patients (P = 0.017). Corresponding P values analyzed by log-rank tests were indicated
Table 2 Univariate and multivariate Cox regression analyses of prognostic factors for PDAC Factors
Univariate analysis
Multivariate analysis
HR (95 % CI)
P value
HR (95 % CI)
P value
Gender
2.12 (0.76–3.12)
0.188
Age
1.65 (0.92–1.97)
0.214
Tumor location
2.04 (0.86–2.66)
0.321
0.98 (0.42–1.05)
0.321
pT category pN category
1.65 (1.21–2.52)
0.024*
2.86 (0.68–3.55)
0.108
1.12 (0.72–1.76)
0.193
1.71 (0.88–2.33)
0.086
Clinical stage
3.28 (2.11–4.54)
0.013*
3.09 (2.32–4.68)
0.008*
Lymphatic invasion Vessel invasion
1.05 (0.53–1.93)
0.087
2.44 (1.73–3.03)
0.003*
0.77 (0.58–1.32)
0.128
2.05 (1.53–3.69)
0.021*
Neural invasion
2.65 (0.77–2.81)
0.211
1.50 (0.83–1.74)
0.286
Residual tumor category
3.08 (2.12–3.49)
0.005*
1.92 (1.22–2.84)
0.032*
MiR-141
expression
1.78
2.55
(2.08–4.71)
0.019*
(1.18–2.91)
0.034*
HR hazard ratio, 95 % CI 95 % confidence interval * P \ 0.05
TargetScan and PicTar databases to search for the target genes of miR-141, and Yes-associated protein-1 (YAP1), the nuclear effector of the Hippo pathway, was identified as a putative target of miR-141. Previously, it was reported that miR-141 could confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma [18]. However, whether miR-141
functions as a tumor suppressor by targeting YAP1 in PDAC is still unclear. First, we analyzed the effect of miR141 expression on YAP1 protein expression. As shown in Fig. 4a, miR-141 mimics could lead to the decreased YAP1 protein (P \ 0.05) and miR-141 inhibitor lead to the increased YAP1 protein (P \ 0.01) in PANC-1 cells. To further confirm direct targeting of YAP1 by miR-141, we performed luciferase reporter assay integrating sequences of the YAP1 30 -UTR containing the binding sites for miR141 or corresponding sequences whose binding sites were mutated into a luciferase reporter vector (Fig. 4b). Results indicated that miR-141 mimics could decrease the luciferase activity of the pGL3-YAP1/30 -UTR-wt vector (P \ 0.01) but did not affect the luciferase activity of pGL3-YAP1/30 -UTR-mut in PANC-1 cells (P [ 0.05; Fig. 4c). However, miR-141 inhibitor could increase the luciferase activity of the pGL3-YAP1/30 -UTR-wt vector (P \ 0.05) but did not affect the luciferase activity of pGL3-YAP1/30 -UTR-mut in PANC-1 cells (P [ 0.05; Fig. 4c). In addition, we detected the expression of YAP1 protein in above 16 PDAC tissues and nontumorous pancreatic tissues by using Western blotting assay, and found that the relative level of YAP1 protein in PDAC tissues was significantly higher than that in nontumorous tissues (P = 0.008; Fig. 4d). Furthermore, by using linear regression analysis, there was an inverse correlation between miR-141 expression and YAP1 protein in PDAC tissues (Spearman rank test r = -0.213; P = 0.023; Fig. 4e). SiRNA targeting YAP1 significantly inhibits growth and induces apoptosis in PDAC cells To investigate the biologic functions of YAP1 in PDAC cells, siRNA targeting YAP1 (siRNA/YAP1) was transfected into PANC-1 cells, and Western blotting assay
123
44
Mol Cell Biochem (2014) 388:39–49
Fig. 3 Upregulation of miR-141 could significantly inhibit growth and enhance apoptosis in PANC-1 cells. a qRT-PCR assay was used to determine the expression of miR-141 in PANC-1/miR-141 or PANC-1/miR-NC cells. U6 was used as an internal control. b. The growth of PANC-1/miR-141 or PANC-1/miR-NC cells was monitored by MTT assay every day for next 6 days. Bars represent mean ± SD. c The stably transfected PANC-1 cells were trypsinized and seeded in six-well plate for colony formation assay. After 2 weeks, colonies were stained with 0.1 % crystal violet,
photographed and counted using imaging system. Bars represent the mean of total number of colonies ± SD. d Flow cytomeric analysis of apoptosis in PANC-1/miR-141 or PANC-1/miR-NC cells. e Western blot analysis of cleaved caspase-3 or PARP, total caspase-3 or PARP protein expression. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
confirmed that siRNA/YAP1 could significantly downregulate the expression of YAP1 protein in PANC-1 cells (P \ 0.05; Fig. 5a). Results from MTT assay showed that siRNA-mediated YAP1 inhibition could result in the decreased growth of PANC-1 cells (Fig. 5b). Moreover, siRNA-mediated YAP1 inhibition could significantly reduce the activity of colony formation in PANC-1 cells (P \ 0.05; Fig. 5c). Flow cytomeric analysis indicated that the apoptotic rate of siRNA/YAP1-transfected PANC-1 cells was significantly increased by about 16.5 % compared with siRNA/control-transfected cells (P \ 0.01; Fig. 5d). Also, we determined the effect of siRNA/YAP1 on the expression of cleaved or total caspase-3 or PARP protein in PANC-1 cells. Results showed that the expression of cleaved caspase-3 or PARP protein in siRNA/ YAP1-transfected PANC-1 cells was significantly
upregulated in comparison with that in siRNA/controltransfected cells. However, the expression of total caspase3 or PARP protein showed no difference between two cells (Fig. 5e). These data suggested that siRNA targeting YAP1 could mimic the tumor suppressor function of miR-141 in PDAC.
123
Restoration of YAP1 partially rescues the functions of miR-141 upregulation in PDAC cells The above results suggested that silencing of YAP1 could mimic the upregulation of miR-141 and that miR141 could post-transcriptionally regulate the expression of YAP1 by directly binding to its 30 -UTR. We hypothesized that overexpression of YAP1 directly
Mol Cell Biochem (2014) 388:39–49
45
Fig. 4 YAP1 was a direct target of miR-141 in human PDAC cells. a 48 h after PANC-1 cells was transfected with miR-141 mimics (or miR-NC mimics) or miR-141 inhibitor (miR-NC inhibitor), Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b The binding sites and the corresponding mutated sequences within the YAP1/30 -UTR for miR141 were presented. c Analysis of luciferase activity. PANC-1 cells were co-transfected with miR-141 (or miR-NC) mimics or miR-141 (or miR-NC) inhibitor and pGL3-Basic vector with YAP1/30 -UTR-wt or YAP1/30 -UTR-mut. After 48 h, the luciferase activity was determined. Values are presented as relative luciferase activity after
normalization to Renilla luciferase activity. d Western blotting assay was used to detect the expression of YAP1 protein in PDAC tissues and corresponding non-tumorous pancreatic tissues (n = 16; P = 0.008). GAPDH was used as an internal control. e There was a statistically significant inverse correlation between miR-141 and YAP1 protein expression levels in 16 PDAC tissues (Spearman’s correlation analysis, r = -0.213; P = 0.023). The data are expressed as the mean value ± SEM of the results obtained from three independent experiments. *P \ 0.05, **P \ 0.01 versus control. NS no significance versus control
mediated miR-141-mediated growth inhibition in PDAC cells. To further confirm that PANC-1 cells were cotransfected with miR-141 mimics and pcDNA/YAP1 or pcDNA/control, respectively. Western blotting assay showed that pcDNA/YAP1 could rescue miR-141-mediated YAP1 downregulation in PANC-1 cells (Fig. 6a). By functional analyses, it was observed that overexpression of YAP1 could partially restore the inhibition of growth and colony formation in PANC-1 cells induced by upregulation of miR-141 (Fig. 6b, c). Moreover, overexpression of YAP1 could restore miR-141-induced apoptosis enhancement and the increased expression of cleaved caspase-3 or PARP protein in PANC-1 cells (Fig. 6e, f). Taken together, our data showed that miR141 exerted its functions in PDAC cells by way of regulating of YAP1 expression.
Discussion In this study, we investigated the clinicopathologic or prognostic significance of miR-141 expression in human PDAC and its effects on malignant phenotypes of PDAC cells. Low miR-141 expression was found to be significantly correlated with status of pT or pN, clinical stage, and lymphatic invasion, and the status of miR-141 expression was an independent prognostic factor for PDAC patients. Also, miR-141 could significantly inhibit growth, reduce colony formation, and enhance apoptosis in PDAC cells via downregulating YAP1. MiRNAs regulate the expression of target genes by interfering with transcription or inhibiting translation [19]. Increasing evidence shows that miRNAs play a crucial role in almost every cellular biologic process, such as
123
46
Mol Cell Biochem (2014) 388:39–49
Fig. 5 SiRNA-mediated YAP1 inhibition could significantly inhibit growth and enhance apoptosis in PANC-1 cells. a 48 h after PANC-1 cells were transfected with siRNA/YAP1 or siRNA/control, Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b The growth of siRNA/ YAP1 or siRNA/control-transfected PANC-1 cells was monitored by MTT assay every day for next 5 days. Bars represent mean ± SD. c The siRNA/YAP1 or siRNA/control-transfected PANC-1 cells were trypsinized and seeded in six-well plate for colony formation assay.
After 2 weeks, colonies were stained with 0.1 % crystal violet, photographed and counted using imaging system. Bars represent the mean of total number of colonies ± SD. d Flow cytomeric analysis of apoptosis in siRNA/YAP1 or siRNA/control-transfected PANC-1 cells. e Western blotting detection of cleaved caspase-3, total caspase3, cleaved PARP and total PARP proteins in siRNA/YAP1 or siRNA/ control-transfected PANC-1 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
metabolism, differentiation, and apoptosis [20, 21]. Currently, it was widely accepted that dysregulation of miRNAs could contribute to tumor development by acting as oncogenes or tumor suppressor genes [22, 23]. Some researchers have reported the diagnostic or prognostic values of miR-141 in human tumors. Chen et al. [24] showed that circulating plasma miR-141 might be a novel biomarker for metastatic colon cancer and predicts poor prognosis. Leskela¨ et al. [25] reported that low miR-200 family expression had a trend toward poor PFS of ovarian cancer patients (HR [ 2.0, P values 0.051, 0.054, and 0.079 for miR-200c, miR-141, and miR-429, respectively, multivariate analysis). By comparing circulating microRNA-141 with circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining response to treatment in patients with metastatic prostate cancer, Gonzales et al. [26] showed that miR-141 demonstrated a similar ability to predict clinical progression when
compared with other clinically validated biomarkers and demonstrated high correlation with changes of the other biomarkers. These data suggested that miR-141 might be an interesting candidate diagnostic or prognostic molecular marker for tumor patients. Here, we first detect the expression of miR-141 in PDAC tissues, and found that the level of miR-141 expression in PDAC tissues was significantly lower than that in nontumorous tissues. By statistical analyses, low miR-141 expression was found to correlate with pT and pN status, clinical stage, and lymphatic invasion, suggesting that downregulation of miR-141 might play critical roles in PDAC development. Importantly, multivariate analyses showed that low miR-141 expression was a poor prognostic factor for PDAC patients. To the best of our knowledge, this is the first report about prognostic value of miR-141 in human PDAC. As a tumor suppressor gene, miR-141 has been found to be downregulated in many human malignancies. Yu et al.
123
Mol Cell Biochem (2014) 388:39–49
47
Fig. 6 Overexpression of YAP1 could rescue the functions of miR141 upregulation in PANC-1 cells. 48 h after PANC-1 cells were cotransfected with miR-141 mimics and pcDNA/YAP1 or pcDNA/ control, those cells were collected for further assays. a Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b MTT analysis of growth in above PANC-1 cells. c Analysis of colony formation capacity of
above PANC-1 cells using above methods. d Flow cytomeric analysis of apoptosis in above PANC-1 cells. e Western blotting detection of cleaved caspase-3, total caspase-3, cleaved PARP and total PARP proteins in above PANC-1 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
[13] show that microRNA-141 is downregulated in human renal cell carcinoma and regulates cell survival by targeting CDC25B. In esophageal cancer, inhibitions of SOX17 by microRNA-141 and methylation activate the WNT-signaling pathway, which promotes esophageal tumorigenesis [27]. Also, Hu et al. [14] reported that microRNA-141 could regulate Smad interacting protein 1 (SIP1) and inhibit migration and invasion of colorectal cancer cells. More importantly, the status of miR-141 expression is found to be correlated with the responses of tumor patients to chemotherapy or molecular-targeted therapy. Imanaka et al. [18] showed that microRNA-141 could confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. A study by Berkers et al. [28] reported that microRNA-141’s downregulation-driven epithelial-to-mesenchymal transition in clear cell renal cell carcinoma was linked to an unfavorable response to sunitinib therapy, and reintroduction of microRNA-141 in vitro led to epithelial-to-mesenchymal transition reversal and increased sensibility to a hypoxic environment. From these data, it could be found that
downregulation of miR-141 might play critical roles in tumor development. Here, we performed functional analyses, and found that upregulation of miR-141 could inhibit growth, reduce colony formation, and increase apoptosis in PANC-1 cells. Sequentially, further study is needed to characterize the molecular mechanism. Analysis basedon the TargetScan and PicTar databases revealed that the YesAssociated Protein-1 (YAP1) was a potential target of miR141. YAP1, also known as YAP or YAP65, was first identified by virtue of its ability to associate with the SH3 domain of Yes-and Src-protein-tyrosine kinases [29]. As a nuclear effector of the Hippo pathway, YAP1 is found to play an important role in tumorigenesis and progression of multiple cancers. Recent studies have demonstrated that YAP is aberrantly expressed in multiple types of human cancers, including hepatocellular carcinoma, breast cancer, oral squamous cell carcinomas, and esophageal squamous cell carcinomas [30–33]. Recently, YAP1 is found to overexpress in pancreatic cancer tissues and potentially play an important role in the clonogenicity and growth of pancreatic cancer cells [34]. However, the molecular
123
48
mechanisms involved in YAP1 overexpression in pancreatic cancer are unclear. Although miR-141 has been reported to confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma, whether the expression of YAP1 could be regulated by miR-141 and the roles of YAP1 in miR-141mediated functions in human PDAC were not reported. Our reporter assays demonstrated that YAP1 was a target of miR-141 in PDAC, and this finding was further confirmed by the results of Western blotting analysis. In addition, it was found that the expression of miR-141 was inversely correlated with YAP1 protein in PDAC tissues. Consequently, we hypothesized that miR-141 might function as a tumor suppressor via downregulation of YAP1. To further testify this hypothesis, siRNA technology was employed to knockdown the endogenous expression of YAP1 in PANC1 cells, and results indicated that siRNA/YAP1 could mimic the effects of miR-141 mimics on malignant phenotypes of PANC-1 cells. More importantly, overexpression of YAP1 could rescue not only the downregulation of YAP1 but also the growth inhibition and caspase-3dependent apoptosis enhancement in PANC-1 cells induced by miR-141 upregulation. These findings support the notion that miR-141 inhibits the growth of PDAC cells by targeting YAP1. Of course, this study has several limits. First, as the number of patients in this study is smaller, further study involving a larger case population is necessary to confirm the prognostic value of miR-141 expression in PDAC. Second, we only used a PDAC cell line, and more PDAC cell lines should be used to confirm the functions of miR-141. In conclusion, our findings provide the first evidence that miR-141 was downregulated in PDAC tissues, and low miR-141 expression might be a poor prognostic factor for patients. Also, functional analyses indicate that miR-141 functions as a tumor suppressor gene in PDAC by partially targeting YAP1. Expanding insights into the key role of dysregulated miRNAs involved in PDAC progression will be helpful for the investigation of the complicated molecular pathogenesis of PDAC and the development of novel therapeutic regimens for PADC therapy. Acknowledgments The authors wish to thank Prof. Rui Wang and Dr. Ming Sun for their technical support and sincere help.
References 1. Jemal A, Siegel R, Xu J et al (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300 2. Giovinazzo F, Turri G, Zanini S et al (2012) Clinical implications of biological markers in pancreatic ductal adenocarcinoma. Surg Oncol 21:e171–e182
123
Mol Cell Biochem (2014) 388:39–49 3. Krautz C, Ru¨ckert F, Saeger HD et al (2011) An update on molecular research of pancreatic adenocarcinoma. Anticancer Agents Med Chem 11:411–417 4. Pasquinelli AE, Ruvkun G (2002) Control of developmental timing by micrornas and their targets. Annu Rev Cell Dev Biol 18:495–513 5. Ke XS, Liu CM, Liu DP et al (2003) MicroRNAs: key participants in gene regulatory networks. Curr Opin Chem Biol 7:516–523 6. Takaya T, Nishi H, Horie T et al (2012) Roles of microRNAs and myocardial cell differentiation. Prog Mol Biol Transl Sci 111:139–152 7. Sharma A, Wu JC (2013) MicroRNA expression profiling of human-induced pluripotent and embryonic stem cells. Methods Mol Biol 936:247–256 8. Jovanovic M, Hengartner MO (2006) MiRNAs and apoptosis: RNAs to die for. Oncogene 25:6176–6187 9. Dalmay T, Edwards DR (2006) MicroRNAs and the hallmarks of cancer. Oncogene 25:6170–6175 10. Shen J, Stass SA, Jiang F (2013) MicroRNAs as potential biomarkers in human solid tumors. Cancer Lett 329:125–136 11. Zhang L, Jamaluddin MS, Weakley SM et al (2011) Roles and mechanisms of microRNAs in pancreatic cancer. World J Surg 35:1725–1731 12. Wang J, Sen S (2011) MicroRNA functional network in pancreatic cancer: from biology to biomarkers of disease. J Biosci 36:481–491 13. Yu XY, Zhang Z, Liu J et al (2013) MicroRNA-141 is downregulated in human renal cell carcinoma and regulates cell survival by targeting CDC25B. Onco Targets Ther 6:349–354 14. Hu M, Xia M, Chen X et al (2010) MicroRNA-141 regulates Smad interacting protein 1 (SIP1) and inhibits migration and invasion of colorectal cancer cells. Dig Dis Sci 55:2365–2372 15. Du Y, Xu Y, Ding L et al (2009) Down-regulation of miR-141 in gastric cancer and its involvement in cell growth. J Gastroenterol 44:556–561 16. Zhang Z, Li Z, Gao C et al (2008) MiR-21 plays a pivotal role in gastric cancer pathogenesis and progression. Lab Invest 88:1358–1366 17. Wang R, Huang J, Feng B et al (2012) Identification of ING4 (inhibitor of growth 4) as a modulator of docetaxel sensitivity in human lung adenocarcinoma. Mol Med 18:874–886 18. Imanaka Y, Tsuchiya S, Sato F et al (2011) MicroRNA-141 confers resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. J Hum Genet 56:270–276 19. Nolan T, Cogoni C (2004) The long hand of the small RNAs reaches into several levels of gene regulation. Biochem Cell Biol 82:472–481 20. Ferna´ndez-Hernando C, Ramı´rez CM, Goedeke L et al (2013) MicroRNAs in metabolic disease. Arterioscler Thromb Vasc Biol 33:178–185 21. Crippa S, Cassano M, Sampaolesi M (2012) Role of miRNAs in muscle stem cell biology: proliferation, differentiation and death. Curr Pharm Des 18:1718–1729 22. Zhang B, Pan X, Cobb GP et al (2007) MicroRNAs as oncogenes and tumor suppressors. Dev Biol 302:1–12 23. Lee YS, Dutta A (2006) MicroRNAs: small but potent oncogenes or tumor suppressors. Curr Opin Investig Drugs 7:560–564 24. Cheng H, Zhang L, Cogdell DE et al (2011) Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS ONE 6:e17745 25. Leskela¨ S, Leandro-Garcı´a LJ, Mendiola M et al (2010) The miR200 family controls beta-tubulin III expression and is associated with paclitaxel-based treatment response and progression-free
Mol Cell Biochem (2014) 388:39–49
26.
27.
28.
29.
survival in ovarian cancer patients. Endocr Relat Cancer 18: 85–95 Gonzales JC, Fink LM, Goodman OB Jr et al (2011) Comparison of circulating MicroRNA 141 to circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining treatment response in patients with metastatic prostate cancer. Clin Genitourin Cancer 9:39–45 Jia Y, Yang Y, Zhan Q et al (2012) Inhibition of SOX17 by microRNA 141 and methylation activates the WNT signaling pathway in esophageal cancer. J Mol Diagn 14:577–585 Berkers J, Govaere O, Wolter P et al (2013) A possible role for microRNA-141 down-regulation in sunitinib resistant metastatic clear cell renal cell carcinoma through induction of epithelial-tomesenchymal transition and hypoxia resistance. J Urol 189: 1930–1938 Sudol M, Shields DC, Farooq A (2012) Structures of YAP protein domains reveal promising targets for development of new cancer drugs. Semin Cell Dev Biol 23:827–833
49 30. Tschaharganeh DF, Chen X, Latzko P et al (2013) Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma. Gastroenterology 144(1530–42): e12 31. Yuan M, Tomlinson V, Lara R et al (2008) Yes-associated protein (YAP) functions as a tumor suppressor in breast. Cell Death Differ 15:1752–1759 32. Li SY, Hu JA, Wang HM (2013) Expression of Yes-associated protein 1 gene and protein in oral squamous cell carcinoma. Chin Med J (Engl) 126:655–658 33. Yeo MK, Kim SH, Kim JM et al (2012) Correlation of expression of phosphorylated and non-phosphorylated Yes-associated protein with clinicopathological parameters in esophageal squamous cell carcinoma in a Korean population. Anticancer Res 32:3835–3840 34. Diep CH, Zucker KM, Hostetter G et al (2012) Down-regulation of Yes associated protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS ONE 7: e32783
123
Prognostic significance of microRNA-141 expression and its tumor suppressor function in human pancreatic ductal adenocarcinoma Zi-Man Zhu • Yue-Fang Xu • Qin-Jun Su • Jun-Dong Du • Xiang-Long Tan • Yu-Liang Tu Jing-Wang Tan • Hua-Bao Jiao
•
Received: 6 September 2013 / Accepted: 5 November 2013 / Published online: 17 November 2013 Ó Springer Science+Business Media New York 2013
Abstract Increasing evidence shows that dysregulation of microRNAs is correlated with tumor development. This study was performed to determine the expression of miR141 and investigate its clinical significance in pancreatic ductal adenocarcinoma (PDAC). Taqman quantitative RTPCR was used to detect miR-141 expressions in 94 PDAC tissues and 16 nontumorous pancreatic tissues. Correlations between miR-141 expression and clinicopathologic features and prognosis of patients were statistically analyzed. The effects of miR-141 expression on growth and apoptosis of PDAC cell line (PANC-1) were determined by MTT, colony formation, and flow cytometry assays. Potential target genes were identified by luciferase reporter and Western blot assays. The expression level of miR-141 in PDAC tissues was significantly lower than that in corresponding nontumorous tissues. Downregulation of miR141 correlated with poorer pT and pN status, advanced clinical stage, and lymphatic invasion. Also, low miR-141 expression in PDAC tissues was significantly correlated Zi-Man Zhu, Yue-Fang Xu and Qin-Jun Su should be regarded as joint first authors for their equal contributions. Z.-M. Zhu J.-D. Du X.-L. Tan Y.-L. Tu J.-W. Tan H.-B. Jiao (&) Department of Hepatobiliary Surgery, First Hospital Affiliated to the Chinese PLA General Hospital, Fucheng Road 51, Haidian District, Beijing 100048, China e-mail: [email protected] Y.-F. Xu Department of Pharmacy, First Hospital Affiliated to the Chinese PLA General Hospital, Fucheng Road 51, Haidian District, Beijing 100048, China Q.-J. Su Department of Pathology, Lanzhou Military Area General Hospital, Lanzhou 730050, China
with shorter overall survival, and multivariate analysis showed that miR-141 was an independent prognostic factor for PDAC patients. Further, functional researches suggested that miR-141 inhibits growth and colony formation, and enhances caspase-3-dependent apoptosis in PANC-1 cells by targeting Yes-associated protein-1 (YAP1). Therefore, miR-141 is an independent prognostic factor for PDAC patients, and functions as a tumor suppressor gene by targeting YAP1. Keywords MicroRNA-141 Pancreatic ductal adenocarcinoma YAP1 Prognosis
Introduction Pancreatic cancer is one of the most aggressive human malignant tumors and a leading cause of cancer-related mortality around the world [1]. Of these, pancreatic ductal adenocarcinoma (PDAC) accounts for *90 % of all the cases. Despite advances in surgery, chemotherapy, and other therapies, the overall survival of patients with PDAC is quite low [2]. Therefore, a better understanding of the biology of PDAC will be helpful for the identification of novel molecular markers to predict the prognosis of the patients. Extensive researches have characterized the complex genetic networks and transcriptomics alterations underlying the progression of PDAC [3]. Recently, the discovery of a new class of microRNAs (miRNAs) has provided additional insights, potentially explaining the gap that exists between tumor genotype and phenotype [4, 5]. MiRNAs can downregulate gene expression by inducing the degradation or impairing the translation of target mRNAs. Increasing evidences have shown that miRNAs
123
40
play critical roles in many human key biologic processes including tissue differentiation, cell proliferation, and embryonic development [6–8]. As such, the dysregulation of miRNAs and their targets are correlated with tumor development [9]. In this context, miRNAs have been identified as promising alternative biomarkers for early detection of cancer, informing prognosis, and monitoring treatment response [10]. Recent studies have found that dysregulation of miRNAs is involved in the development of PADC [11, 12]. Previously, miR-141 has been reported to function as a tumor suppressor in many tumors including renal cell carcinoma, colorectal cancer, and gastric cancer [13–15]. However, the status of miR-141 and its clinical value in human PDAC are unclear and remain to be elucidated. In this study, our purpose is to quantify expression levels of miR-141 in the clinical samples of PDAC, and to evaluate their correlation with clinicopathological features of PDAC patients. Also, the correlation of miR-141 expression with prognosis of patients is evaluated. In addition, the effects of miR-141 expression on malignant phenotypes of PDAC cells and the possible molecular mechanisms will be further investigated.
Materials and methods Patients and tissue samples Primary tumor specimens were obtained from 94 patients diagnosed with PDAC, who underwent complete resection at our Hospital between 2006 and 2010. The original histopathologic slide sets and reports were obtained from each case, and these were reviewed to confirm the diagnosis of PDAC. None of the patients had received chemotherapy or radiotherapy before surgery. Experienced pathologists provided detailed pathologic diagnosis according to UICC TNM classification standards (2002). Informed consent was obtained from all patients in writing. The ethics committee of Nanjing Medical University approved the study protocol. Also, 16 paired of PDAC and corresponding nontumorous pancreatic tissues were also collected. Samples were snap-frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Cell culture Human PDAC cell line (PANC-1) was purchased from Shanghai Institute Chinese academy of science, and cultured in RPMI 1640 media (Invitrogen, CA, USA) supplemented with 10 % fetal bovine serum (FBS) and 100 lM each of penicillin and streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C.
123
Mol Cell Biochem (2014) 388:39–49
Transfection of plasmid vectors, miRNA mimics or inhibitor, and siRNA A DNA fragment containing the hsa-miR-141 or YAP1 coding region was amplified from human pancreatic tissue genomic DNA and cloned into pEGFP-N1 or pcDNA3.0 (?) vector which was named pEGFP/miR-141 or pcDNA/ YAP1, respectively. The siRNA/YAP1 (sc-3863) and control siRNA (siRNA/control) were purchased through Santa Cruz Biotechnology. The transient or stable transfection was performed using LipofectamineTM 2000 (Invitrogen, CA, USA) following the manufacturer’s instructions. The G418-resistant cells were named PANC1/miR-141 or PANC-1/miR-NC, respectively. Taqman quantitative RT-PCR assay Total RNA isolated was used, TRIzol Reagent (Invitrogen, CA, USA). TaqMan qRT-PCR assay was used to assess the expression of miR-141 with kits from Applied Biosystems (Foster City, CA, USA) as described previously [16]. We calculated 4Ct (target-reference) which is equal to the difference between threshold cycles for miR-141 (i.e., target) and the threshold cycle for U6 RNA (i.e., reference) (i.e., DCt (target-reference) = Ct target-Ct reference). The foldchange between patient or cell sample and a normal control for miR-141 was calculated with the 2-DDCt method. qRTPCR was repeated in triplicate for each sample. Western blotting assay The level of YAP1, cleaved or total caspase-3, cleaved or total PARP, and GAPDH protein were analyzed using standard Western blot procedures as described previously with the rabbit antihuman YAP1, cleaved or total caspase3, cleaved or total PARP, and GAPDH primary antibody (Santa Cruz Biotechnology) [17]. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay The transfected PANC-1 cells (6.0 9 103 cells/well) were plated in 96-well plates. Then, for 6 days, a batch of cells were stained with 20 lL sterile MTT dye (5 mg/mL) at 37 °C for 4 h every day, and then, the culture medium was removed, and 150 lL of DMSO was added and thoroughly mixed in for 10 min. Spectrometric absorbance at 490 nm was measured using a microplate reader. Colony formation assay The stably transfected cells were plated in 10-cm culture dishes. After 14 days, cells were fixed with methanol, and
Mol Cell Biochem (2014) 388:39–49
41
stained with 0.1 % crystal violet. Visible colonies were manually counted. Flow cytometry detection of apoptosis The cells were recovered by centrifugation for evaluation of apoptotic cells with the use of double staining with annexin V-fluoresein isothiocyanate (Annexin V-FITC) and propidium iodide, (PI) (BioVision, St Pete Beach, FL) according to the manufacturer’s instructions. Luciferase reporter assay The 30 -UTR sequence of YAP1, predicted to interact with miR-141, was amplified using primers (sense, 50 -GCGAATTCCCAAAGCACTATTTGT-30 ; reverse, 50 -GGCTCGAGA-AGCACTCATTTCTC-30 ) and cloned into the EcoRI and XhoI sites of pGL3-Basic vector (Promega, Madison, WI, USA) that was named pGL3-YAP1/30 -UTR-wt. Putative binding sites of miR-141 in YAP1 30 -UTR were mutated using the QuikChange site-directed mutagenesis kit (Stratagene, Germany) to obtain pGL3-YAP1/30 -UTRmt. Luciferase reporter assay was performed using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI) as per the manufacturer’s instruction. Statistical analyses Analyses were performed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). The Kruskal–Wallis H or Mann– Whitney U test was used to investigate clinicopathological significance of miR-141 expression. Overall survival curves were plotted using the Kaplan–Meier method and were evaluated for the statistical significance using a logrank test. The significance of different variables with respect to survival was analyzed using the multivariate Cox proportional hazards model. The correlation was evaluated by Spearman’s rank correlation coefficients. P \ 0.05 was considered to indicate a statistically significant difference.
Fig. 1 qRT-PCR assay was used to detect the expression of miR-141 in PDAC and corresponding non-tumorous pancreatic tissues. Downregulation (a) and downregulation (b) of miR-141 expression. qRT-PCR analysis of miR-141 expression was performed in PDAC tissues (n = 16) and corresponding non-tumorous pancreatic tissues (n = 16). U6 was used as an internal control. c The mean level of miR-141 expression in PDAC tissues (n = 16) was significantly lower than that in corresponding non-tumorous tissues (n = 16) (P \ 0.001). Each experiment was performed at least in triplicate. *P \ 0.05 versus control
tissues was significantly higher than that in nontumorous pancreatic tissues (P \ 0.001; Fig. 1c). Thus, downregulation of miR-141 might play a critical role during PDAC development. Clinicopathological significance of miR-141 expression in human PDAC
Results Expression of miR-141 was significantly downregulated in human PDAC tissues First, Taqman quantitative (q) RT-PCR assay was performed to detect miR-141 expression in 16 PDAC tissues and nontumorous pancreatic tissues. Compared with nontumorous pancreatic tissues, the level of miR-141 expression was lower in 12 PDAC tissues (Fig. 1a), and the expression of miR-141 was found to be upregulated in only four cases (Fig. 1b). The mean level of miR-141 in PDAC
Next, we investigated the correlation of miR-141 expression with clinicopathological features of PDAC patients. Expression of miR-141 was analyzed by relative quantity (RQ) using the equation RQ = 2-DDCT (CT = threshold cycle to detectfluorescence). According to the mean RQ of miR-141 in 94 PDAC tissues, (1.54 ± 2.65), 53 (56.4 %) patients were classified as low-miR-141 group (B1.54) and 41 (33.6 %) as high-miR-141 group ([1.54). By statistical analysis (Table 1), it was observed that low miR-141 expression was closely correlated with pT status, pN status, advanced clinical stage, and higher incidence of lymphatic
123
42
Mol Cell Biochem (2014) 388:39–49
0.014*
18.73 % in those with low expression. Moreover, patients with high miR-141 expression had significantly longer 5-year median overall survival (OS) than those with low miR-141 expression (log-rank test; P = 0.017; Fig. 2b): 48.7 months in patients with high expression versus 26.4 months in those with low expression. To further determine the prognostic value of miR-141 for PDAC patients, univariate and multivariate data analyses were performed using the Cox proportional hazards regression model (Table 2). Univariate analyses showed that pT status, clinical stage, residual tumor category, and the status of miR-141 expression were significantly correlated with over survival of PDAC patients (P = 0.024, 0.013, 0.005 and 0.034, respectively). Multivariate analyses showed that that the status of miR-141 expression and other factors (clinical stage, status of lymphatic invasion, and residual tumor category) were independent prognostic factors for PDAC patients (HR 2.55, 95 % CI 2.08–4.71, P 0.019).
0.001*
Effects of miR-141 on growth, colony formation, and apoptosis in PDAC cells
Table 1 Relationship of miR-141 expression with various clinicopathological factors of PDAC patients Factors
MiR-141 expression Low (n = 53)
High (n = 41)
32
31
21
10
B55
19
11
[55
34
30
Head
17
20
Body or tail
37
21
pT1/pT2
23
29
pT3/pT4
30
12
pN0
20
26
pN1
33
15
Gender Male Female Age (year)
0.119
0.352
Tumor location
0.087
pT category
0.008*
pN category
Clinical stage I/II
18
28
35
13
Negative
22
27
Positive
31
14
Negative
32
24
Positive
21
17
Negative
41
30
Positive
12
11
R0
38
34
R1
15
7
III/IV Lymphatic invasion
P value
0.019*
Vessel invasion
0.857
Neural invasion
0.640
Residual tumor category
0.202
* P \ 0.05 was considered statistically significant
invasion (P = 0.008, 0.014, 0.001, and 0.019, respectively). However, there were no significant differences in gender, age, tumor location, vessel invasion, neural invasion, and residual tumor status between the low miR-141 group and high miR-141 group (P = 0.119, 0.352, 0.087, 0.857, 0.640, and 0.202, respectively). Prognostic significance of miR-141 expression in human PDAC By survival analysis, low miR-144 expression was observed to be in correlation with a significantly lower 5-year recurrence free survival (RFS) rate of PDAC patients (P = 0.031; Fig. 2a): 24.56 % in patients with high expression versus
123
To determine the effect of miR-141 on malignant phenotypes of PDAC cells, pEGFP/miR-141- and pEGFP/miR-NCexpressing vectors were stably transfected into PANC-1 cells. Then, qRT-PCR assay was performed to detect miR141 expression in PANC-1/miR-141 or PANC-1/miR-NC cells. Compared with PANC-1/miR-NC cells, the expression of miR-141 in PANC-1/miR-141 cells was significantly upregulated by about 321.5 % (P \ 0.01; Fig. 3a). The results of the proliferation assays performed using MTT and colony formation assays are shown in the figure. As shown in Fig. 3b, PANC-1/miR-141 cells grew significantly slowly compared with PANC-1/miR-NC, and the inhibitory rate reached about 65.8 % at day 6 (P \ 0.01). It was revealed that the colony growth ability of PANC-1/miR-141 cells was significantly reduced compared with PANC-1/miR-NC cells (P \ 0.05; Fig. 3c). FACS analysis of apoptosis showed that PANC-1/miR-141 cells represented significant apoptosis enhancement (P \ 0.05; Fig. 3d). Further, upregulation of miR-141 could lead to the increased expression of cleaved caspase-3 or PARP protein in PANC-1 cells, but the expression of total caspase-3 or PARP protein showed no difference between PANC-1/miR-141 and PANC-1/miRNC cells (Fig. 3e). Thus, these data indicated that upregulation of miR-141 inhibited growth of PDAC cells by enhancing caspase-3-dependent apoptosis. YAP1 was a functional target of miR-141 in PDAC cells To further investigate the underlying molecular mechanism of miR-141 in the regulation of growth, we interrogated the
Mol Cell Biochem (2014) 388:39–49
43
Fig. 2 Kaplan–Meier survival curve of PDAC patients. a MiR141 expression was correlated with recurrence-free survival in PDAC patients (P = 0.031). b MiR-141 expression was correlated with overall survival in PDAC patients (P = 0.017). Corresponding P values analyzed by log-rank tests were indicated
Table 2 Univariate and multivariate Cox regression analyses of prognostic factors for PDAC Factors
Univariate analysis
Multivariate analysis
HR (95 % CI)
P value
HR (95 % CI)
P value
Gender
2.12 (0.76–3.12)
0.188
Age
1.65 (0.92–1.97)
0.214
Tumor location
2.04 (0.86–2.66)
0.321
0.98 (0.42–1.05)
0.321
pT category pN category
1.65 (1.21–2.52)
0.024*
2.86 (0.68–3.55)
0.108
1.12 (0.72–1.76)
0.193
1.71 (0.88–2.33)
0.086
Clinical stage
3.28 (2.11–4.54)
0.013*
3.09 (2.32–4.68)
0.008*
Lymphatic invasion Vessel invasion
1.05 (0.53–1.93)
0.087
2.44 (1.73–3.03)
0.003*
0.77 (0.58–1.32)
0.128
2.05 (1.53–3.69)
0.021*
Neural invasion
2.65 (0.77–2.81)
0.211
1.50 (0.83–1.74)
0.286
Residual tumor category
3.08 (2.12–3.49)
0.005*
1.92 (1.22–2.84)
0.032*
MiR-141
expression
1.78
2.55
(2.08–4.71)
0.019*
(1.18–2.91)
0.034*
HR hazard ratio, 95 % CI 95 % confidence interval * P \ 0.05
TargetScan and PicTar databases to search for the target genes of miR-141, and Yes-associated protein-1 (YAP1), the nuclear effector of the Hippo pathway, was identified as a putative target of miR-141. Previously, it was reported that miR-141 could confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma [18]. However, whether miR-141
functions as a tumor suppressor by targeting YAP1 in PDAC is still unclear. First, we analyzed the effect of miR141 expression on YAP1 protein expression. As shown in Fig. 4a, miR-141 mimics could lead to the decreased YAP1 protein (P \ 0.05) and miR-141 inhibitor lead to the increased YAP1 protein (P \ 0.01) in PANC-1 cells. To further confirm direct targeting of YAP1 by miR-141, we performed luciferase reporter assay integrating sequences of the YAP1 30 -UTR containing the binding sites for miR141 or corresponding sequences whose binding sites were mutated into a luciferase reporter vector (Fig. 4b). Results indicated that miR-141 mimics could decrease the luciferase activity of the pGL3-YAP1/30 -UTR-wt vector (P \ 0.01) but did not affect the luciferase activity of pGL3-YAP1/30 -UTR-mut in PANC-1 cells (P [ 0.05; Fig. 4c). However, miR-141 inhibitor could increase the luciferase activity of the pGL3-YAP1/30 -UTR-wt vector (P \ 0.05) but did not affect the luciferase activity of pGL3-YAP1/30 -UTR-mut in PANC-1 cells (P [ 0.05; Fig. 4c). In addition, we detected the expression of YAP1 protein in above 16 PDAC tissues and nontumorous pancreatic tissues by using Western blotting assay, and found that the relative level of YAP1 protein in PDAC tissues was significantly higher than that in nontumorous tissues (P = 0.008; Fig. 4d). Furthermore, by using linear regression analysis, there was an inverse correlation between miR-141 expression and YAP1 protein in PDAC tissues (Spearman rank test r = -0.213; P = 0.023; Fig. 4e). SiRNA targeting YAP1 significantly inhibits growth and induces apoptosis in PDAC cells To investigate the biologic functions of YAP1 in PDAC cells, siRNA targeting YAP1 (siRNA/YAP1) was transfected into PANC-1 cells, and Western blotting assay
123
44
Mol Cell Biochem (2014) 388:39–49
Fig. 3 Upregulation of miR-141 could significantly inhibit growth and enhance apoptosis in PANC-1 cells. a qRT-PCR assay was used to determine the expression of miR-141 in PANC-1/miR-141 or PANC-1/miR-NC cells. U6 was used as an internal control. b. The growth of PANC-1/miR-141 or PANC-1/miR-NC cells was monitored by MTT assay every day for next 6 days. Bars represent mean ± SD. c The stably transfected PANC-1 cells were trypsinized and seeded in six-well plate for colony formation assay. After 2 weeks, colonies were stained with 0.1 % crystal violet,
photographed and counted using imaging system. Bars represent the mean of total number of colonies ± SD. d Flow cytomeric analysis of apoptosis in PANC-1/miR-141 or PANC-1/miR-NC cells. e Western blot analysis of cleaved caspase-3 or PARP, total caspase-3 or PARP protein expression. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
confirmed that siRNA/YAP1 could significantly downregulate the expression of YAP1 protein in PANC-1 cells (P \ 0.05; Fig. 5a). Results from MTT assay showed that siRNA-mediated YAP1 inhibition could result in the decreased growth of PANC-1 cells (Fig. 5b). Moreover, siRNA-mediated YAP1 inhibition could significantly reduce the activity of colony formation in PANC-1 cells (P \ 0.05; Fig. 5c). Flow cytomeric analysis indicated that the apoptotic rate of siRNA/YAP1-transfected PANC-1 cells was significantly increased by about 16.5 % compared with siRNA/control-transfected cells (P \ 0.01; Fig. 5d). Also, we determined the effect of siRNA/YAP1 on the expression of cleaved or total caspase-3 or PARP protein in PANC-1 cells. Results showed that the expression of cleaved caspase-3 or PARP protein in siRNA/ YAP1-transfected PANC-1 cells was significantly
upregulated in comparison with that in siRNA/controltransfected cells. However, the expression of total caspase3 or PARP protein showed no difference between two cells (Fig. 5e). These data suggested that siRNA targeting YAP1 could mimic the tumor suppressor function of miR-141 in PDAC.
123
Restoration of YAP1 partially rescues the functions of miR-141 upregulation in PDAC cells The above results suggested that silencing of YAP1 could mimic the upregulation of miR-141 and that miR141 could post-transcriptionally regulate the expression of YAP1 by directly binding to its 30 -UTR. We hypothesized that overexpression of YAP1 directly
Mol Cell Biochem (2014) 388:39–49
45
Fig. 4 YAP1 was a direct target of miR-141 in human PDAC cells. a 48 h after PANC-1 cells was transfected with miR-141 mimics (or miR-NC mimics) or miR-141 inhibitor (miR-NC inhibitor), Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b The binding sites and the corresponding mutated sequences within the YAP1/30 -UTR for miR141 were presented. c Analysis of luciferase activity. PANC-1 cells were co-transfected with miR-141 (or miR-NC) mimics or miR-141 (or miR-NC) inhibitor and pGL3-Basic vector with YAP1/30 -UTR-wt or YAP1/30 -UTR-mut. After 48 h, the luciferase activity was determined. Values are presented as relative luciferase activity after
normalization to Renilla luciferase activity. d Western blotting assay was used to detect the expression of YAP1 protein in PDAC tissues and corresponding non-tumorous pancreatic tissues (n = 16; P = 0.008). GAPDH was used as an internal control. e There was a statistically significant inverse correlation between miR-141 and YAP1 protein expression levels in 16 PDAC tissues (Spearman’s correlation analysis, r = -0.213; P = 0.023). The data are expressed as the mean value ± SEM of the results obtained from three independent experiments. *P \ 0.05, **P \ 0.01 versus control. NS no significance versus control
mediated miR-141-mediated growth inhibition in PDAC cells. To further confirm that PANC-1 cells were cotransfected with miR-141 mimics and pcDNA/YAP1 or pcDNA/control, respectively. Western blotting assay showed that pcDNA/YAP1 could rescue miR-141-mediated YAP1 downregulation in PANC-1 cells (Fig. 6a). By functional analyses, it was observed that overexpression of YAP1 could partially restore the inhibition of growth and colony formation in PANC-1 cells induced by upregulation of miR-141 (Fig. 6b, c). Moreover, overexpression of YAP1 could restore miR-141-induced apoptosis enhancement and the increased expression of cleaved caspase-3 or PARP protein in PANC-1 cells (Fig. 6e, f). Taken together, our data showed that miR141 exerted its functions in PDAC cells by way of regulating of YAP1 expression.
Discussion In this study, we investigated the clinicopathologic or prognostic significance of miR-141 expression in human PDAC and its effects on malignant phenotypes of PDAC cells. Low miR-141 expression was found to be significantly correlated with status of pT or pN, clinical stage, and lymphatic invasion, and the status of miR-141 expression was an independent prognostic factor for PDAC patients. Also, miR-141 could significantly inhibit growth, reduce colony formation, and enhance apoptosis in PDAC cells via downregulating YAP1. MiRNAs regulate the expression of target genes by interfering with transcription or inhibiting translation [19]. Increasing evidence shows that miRNAs play a crucial role in almost every cellular biologic process, such as
123
46
Mol Cell Biochem (2014) 388:39–49
Fig. 5 SiRNA-mediated YAP1 inhibition could significantly inhibit growth and enhance apoptosis in PANC-1 cells. a 48 h after PANC-1 cells were transfected with siRNA/YAP1 or siRNA/control, Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b The growth of siRNA/ YAP1 or siRNA/control-transfected PANC-1 cells was monitored by MTT assay every day for next 5 days. Bars represent mean ± SD. c The siRNA/YAP1 or siRNA/control-transfected PANC-1 cells were trypsinized and seeded in six-well plate for colony formation assay.
After 2 weeks, colonies were stained with 0.1 % crystal violet, photographed and counted using imaging system. Bars represent the mean of total number of colonies ± SD. d Flow cytomeric analysis of apoptosis in siRNA/YAP1 or siRNA/control-transfected PANC-1 cells. e Western blotting detection of cleaved caspase-3, total caspase3, cleaved PARP and total PARP proteins in siRNA/YAP1 or siRNA/ control-transfected PANC-1 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
metabolism, differentiation, and apoptosis [20, 21]. Currently, it was widely accepted that dysregulation of miRNAs could contribute to tumor development by acting as oncogenes or tumor suppressor genes [22, 23]. Some researchers have reported the diagnostic or prognostic values of miR-141 in human tumors. Chen et al. [24] showed that circulating plasma miR-141 might be a novel biomarker for metastatic colon cancer and predicts poor prognosis. Leskela¨ et al. [25] reported that low miR-200 family expression had a trend toward poor PFS of ovarian cancer patients (HR [ 2.0, P values 0.051, 0.054, and 0.079 for miR-200c, miR-141, and miR-429, respectively, multivariate analysis). By comparing circulating microRNA-141 with circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining response to treatment in patients with metastatic prostate cancer, Gonzales et al. [26] showed that miR-141 demonstrated a similar ability to predict clinical progression when
compared with other clinically validated biomarkers and demonstrated high correlation with changes of the other biomarkers. These data suggested that miR-141 might be an interesting candidate diagnostic or prognostic molecular marker for tumor patients. Here, we first detect the expression of miR-141 in PDAC tissues, and found that the level of miR-141 expression in PDAC tissues was significantly lower than that in nontumorous tissues. By statistical analyses, low miR-141 expression was found to correlate with pT and pN status, clinical stage, and lymphatic invasion, suggesting that downregulation of miR-141 might play critical roles in PDAC development. Importantly, multivariate analyses showed that low miR-141 expression was a poor prognostic factor for PDAC patients. To the best of our knowledge, this is the first report about prognostic value of miR-141 in human PDAC. As a tumor suppressor gene, miR-141 has been found to be downregulated in many human malignancies. Yu et al.
123
Mol Cell Biochem (2014) 388:39–49
47
Fig. 6 Overexpression of YAP1 could rescue the functions of miR141 upregulation in PANC-1 cells. 48 h after PANC-1 cells were cotransfected with miR-141 mimics and pcDNA/YAP1 or pcDNA/ control, those cells were collected for further assays. a Western blotting assay was used to detect the expression of YAP1 protein. GAPDH was used as an internal control. b MTT analysis of growth in above PANC-1 cells. c Analysis of colony formation capacity of
above PANC-1 cells using above methods. d Flow cytomeric analysis of apoptosis in above PANC-1 cells. e Western blotting detection of cleaved caspase-3, total caspase-3, cleaved PARP and total PARP proteins in above PANC-1 cells. GAPDH was used as an internal control. Each experiment was performed at least in triplicate. NS no significance and *P \ 0.05, **P \ 0.01 versus control
[13] show that microRNA-141 is downregulated in human renal cell carcinoma and regulates cell survival by targeting CDC25B. In esophageal cancer, inhibitions of SOX17 by microRNA-141 and methylation activate the WNT-signaling pathway, which promotes esophageal tumorigenesis [27]. Also, Hu et al. [14] reported that microRNA-141 could regulate Smad interacting protein 1 (SIP1) and inhibit migration and invasion of colorectal cancer cells. More importantly, the status of miR-141 expression is found to be correlated with the responses of tumor patients to chemotherapy or molecular-targeted therapy. Imanaka et al. [18] showed that microRNA-141 could confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. A study by Berkers et al. [28] reported that microRNA-141’s downregulation-driven epithelial-to-mesenchymal transition in clear cell renal cell carcinoma was linked to an unfavorable response to sunitinib therapy, and reintroduction of microRNA-141 in vitro led to epithelial-to-mesenchymal transition reversal and increased sensibility to a hypoxic environment. From these data, it could be found that
downregulation of miR-141 might play critical roles in tumor development. Here, we performed functional analyses, and found that upregulation of miR-141 could inhibit growth, reduce colony formation, and increase apoptosis in PANC-1 cells. Sequentially, further study is needed to characterize the molecular mechanism. Analysis basedon the TargetScan and PicTar databases revealed that the YesAssociated Protein-1 (YAP1) was a potential target of miR141. YAP1, also known as YAP or YAP65, was first identified by virtue of its ability to associate with the SH3 domain of Yes-and Src-protein-tyrosine kinases [29]. As a nuclear effector of the Hippo pathway, YAP1 is found to play an important role in tumorigenesis and progression of multiple cancers. Recent studies have demonstrated that YAP is aberrantly expressed in multiple types of human cancers, including hepatocellular carcinoma, breast cancer, oral squamous cell carcinomas, and esophageal squamous cell carcinomas [30–33]. Recently, YAP1 is found to overexpress in pancreatic cancer tissues and potentially play an important role in the clonogenicity and growth of pancreatic cancer cells [34]. However, the molecular
123
48
mechanisms involved in YAP1 overexpression in pancreatic cancer are unclear. Although miR-141 has been reported to confer resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma, whether the expression of YAP1 could be regulated by miR-141 and the roles of YAP1 in miR-141mediated functions in human PDAC were not reported. Our reporter assays demonstrated that YAP1 was a target of miR-141 in PDAC, and this finding was further confirmed by the results of Western blotting analysis. In addition, it was found that the expression of miR-141 was inversely correlated with YAP1 protein in PDAC tissues. Consequently, we hypothesized that miR-141 might function as a tumor suppressor via downregulation of YAP1. To further testify this hypothesis, siRNA technology was employed to knockdown the endogenous expression of YAP1 in PANC1 cells, and results indicated that siRNA/YAP1 could mimic the effects of miR-141 mimics on malignant phenotypes of PANC-1 cells. More importantly, overexpression of YAP1 could rescue not only the downregulation of YAP1 but also the growth inhibition and caspase-3dependent apoptosis enhancement in PANC-1 cells induced by miR-141 upregulation. These findings support the notion that miR-141 inhibits the growth of PDAC cells by targeting YAP1. Of course, this study has several limits. First, as the number of patients in this study is smaller, further study involving a larger case population is necessary to confirm the prognostic value of miR-141 expression in PDAC. Second, we only used a PDAC cell line, and more PDAC cell lines should be used to confirm the functions of miR-141. In conclusion, our findings provide the first evidence that miR-141 was downregulated in PDAC tissues, and low miR-141 expression might be a poor prognostic factor for patients. Also, functional analyses indicate that miR-141 functions as a tumor suppressor gene in PDAC by partially targeting YAP1. Expanding insights into the key role of dysregulated miRNAs involved in PDAC progression will be helpful for the investigation of the complicated molecular pathogenesis of PDAC and the development of novel therapeutic regimens for PADC therapy. Acknowledgments The authors wish to thank Prof. Rui Wang and Dr. Ming Sun for their technical support and sincere help.
References 1. Jemal A, Siegel R, Xu J et al (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300 2. Giovinazzo F, Turri G, Zanini S et al (2012) Clinical implications of biological markers in pancreatic ductal adenocarcinoma. Surg Oncol 21:e171–e182
123
Mol Cell Biochem (2014) 388:39–49 3. Krautz C, Ru¨ckert F, Saeger HD et al (2011) An update on molecular research of pancreatic adenocarcinoma. Anticancer Agents Med Chem 11:411–417 4. Pasquinelli AE, Ruvkun G (2002) Control of developmental timing by micrornas and their targets. Annu Rev Cell Dev Biol 18:495–513 5. Ke XS, Liu CM, Liu DP et al (2003) MicroRNAs: key participants in gene regulatory networks. Curr Opin Chem Biol 7:516–523 6. Takaya T, Nishi H, Horie T et al (2012) Roles of microRNAs and myocardial cell differentiation. Prog Mol Biol Transl Sci 111:139–152 7. Sharma A, Wu JC (2013) MicroRNA expression profiling of human-induced pluripotent and embryonic stem cells. Methods Mol Biol 936:247–256 8. Jovanovic M, Hengartner MO (2006) MiRNAs and apoptosis: RNAs to die for. Oncogene 25:6176–6187 9. Dalmay T, Edwards DR (2006) MicroRNAs and the hallmarks of cancer. Oncogene 25:6170–6175 10. Shen J, Stass SA, Jiang F (2013) MicroRNAs as potential biomarkers in human solid tumors. Cancer Lett 329:125–136 11. Zhang L, Jamaluddin MS, Weakley SM et al (2011) Roles and mechanisms of microRNAs in pancreatic cancer. World J Surg 35:1725–1731 12. Wang J, Sen S (2011) MicroRNA functional network in pancreatic cancer: from biology to biomarkers of disease. J Biosci 36:481–491 13. Yu XY, Zhang Z, Liu J et al (2013) MicroRNA-141 is downregulated in human renal cell carcinoma and regulates cell survival by targeting CDC25B. Onco Targets Ther 6:349–354 14. Hu M, Xia M, Chen X et al (2010) MicroRNA-141 regulates Smad interacting protein 1 (SIP1) and inhibits migration and invasion of colorectal cancer cells. Dig Dis Sci 55:2365–2372 15. Du Y, Xu Y, Ding L et al (2009) Down-regulation of miR-141 in gastric cancer and its involvement in cell growth. J Gastroenterol 44:556–561 16. Zhang Z, Li Z, Gao C et al (2008) MiR-21 plays a pivotal role in gastric cancer pathogenesis and progression. Lab Invest 88:1358–1366 17. Wang R, Huang J, Feng B et al (2012) Identification of ING4 (inhibitor of growth 4) as a modulator of docetaxel sensitivity in human lung adenocarcinoma. Mol Med 18:874–886 18. Imanaka Y, Tsuchiya S, Sato F et al (2011) MicroRNA-141 confers resistance to cisplatin-induced apoptosis by targeting YAP1 in human esophageal squamous cell carcinoma. J Hum Genet 56:270–276 19. Nolan T, Cogoni C (2004) The long hand of the small RNAs reaches into several levels of gene regulation. Biochem Cell Biol 82:472–481 20. Ferna´ndez-Hernando C, Ramı´rez CM, Goedeke L et al (2013) MicroRNAs in metabolic disease. Arterioscler Thromb Vasc Biol 33:178–185 21. Crippa S, Cassano M, Sampaolesi M (2012) Role of miRNAs in muscle stem cell biology: proliferation, differentiation and death. Curr Pharm Des 18:1718–1729 22. Zhang B, Pan X, Cobb GP et al (2007) MicroRNAs as oncogenes and tumor suppressors. Dev Biol 302:1–12 23. Lee YS, Dutta A (2006) MicroRNAs: small but potent oncogenes or tumor suppressors. Curr Opin Investig Drugs 7:560–564 24. Cheng H, Zhang L, Cogdell DE et al (2011) Circulating plasma MiR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS ONE 6:e17745 25. Leskela¨ S, Leandro-Garcı´a LJ, Mendiola M et al (2010) The miR200 family controls beta-tubulin III expression and is associated with paclitaxel-based treatment response and progression-free
Mol Cell Biochem (2014) 388:39–49
26.
27.
28.
29.
survival in ovarian cancer patients. Endocr Relat Cancer 18: 85–95 Gonzales JC, Fink LM, Goodman OB Jr et al (2011) Comparison of circulating MicroRNA 141 to circulating tumor cells, lactate dehydrogenase, and prostate-specific antigen for determining treatment response in patients with metastatic prostate cancer. Clin Genitourin Cancer 9:39–45 Jia Y, Yang Y, Zhan Q et al (2012) Inhibition of SOX17 by microRNA 141 and methylation activates the WNT signaling pathway in esophageal cancer. J Mol Diagn 14:577–585 Berkers J, Govaere O, Wolter P et al (2013) A possible role for microRNA-141 down-regulation in sunitinib resistant metastatic clear cell renal cell carcinoma through induction of epithelial-tomesenchymal transition and hypoxia resistance. J Urol 189: 1930–1938 Sudol M, Shields DC, Farooq A (2012) Structures of YAP protein domains reveal promising targets for development of new cancer drugs. Semin Cell Dev Biol 23:827–833
49 30. Tschaharganeh DF, Chen X, Latzko P et al (2013) Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma. Gastroenterology 144(1530–42): e12 31. Yuan M, Tomlinson V, Lara R et al (2008) Yes-associated protein (YAP) functions as a tumor suppressor in breast. Cell Death Differ 15:1752–1759 32. Li SY, Hu JA, Wang HM (2013) Expression of Yes-associated protein 1 gene and protein in oral squamous cell carcinoma. Chin Med J (Engl) 126:655–658 33. Yeo MK, Kim SH, Kim JM et al (2012) Correlation of expression of phosphorylated and non-phosphorylated Yes-associated protein with clinicopathological parameters in esophageal squamous cell carcinoma in a Korean population. Anticancer Res 32:3835–3840 34. Diep CH, Zucker KM, Hostetter G et al (2012) Down-regulation of Yes associated protein 1 expression reduces cell proliferation and clonogenicity of pancreatic cancer cells. PLoS ONE 7: e32783
123
