Med Oncol (2014) 31:129 DOI 10.1007/s12032-014-0129-y
ORIGINAL PAPER
Prognostic potential of microRNA-138 and its target mRNA PDK1 in sera for patients with non-small cell lung cancer Lihong Han • Guoxiu Zhang • Nali Zhang Haiyan Li • Yanyan Liu • Aiguo Fu • Youguang Zheng
•
Received: 23 June 2014 / Accepted: 7 July 2014 / Published online: 27 July 2014 Ó Springer Science+Business Media New York 2014
Abstract microRNA (miR)-138 has been recognized as a potential tumor suppressor via regulating 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in nonsmall cell lung cancer (NSCLC) cells. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients’ prognosis. miR-138 and PDK1 mRNA expressions in 100 NSCLCs and 100 healthy control sera were detected by quantitative real-time PCR. miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (P \ 0.001), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (P \ 0.001). In addition, miR-138 downregulation and PDK1 upregulation were both significantly associated with advanced tumor-node-metastasis (TNM) stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. Moreover, the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P \ 0.001). Notably, NSCLC patients with combined miR-138 downregulation and PDK1 upregulation (miR-138-low/PDK1-high) had shortest overall survival (P \ 0.001). Furthermore, multivariate analysis showed that miR-138 expression L. Han N. Zhang H. Li Y. Liu A. Fu Y. Zheng (&) Department of Respiratory Medicine, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, Henan Province, China e-mail: [email protected] G. Zhang First Affiliated Hospital to Henan University of Science and Technology, Luoyang 471009, Henan Province, China
(P = 0.01), PDK1 expression (P = 0.01), and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001) were all independent prognostic factors for overall survival in NSCLC patients. Deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. More importantly, miR-138 and PDK1 may synergistically predict patients’ prognosis and their combination may represent a promising prognostic biomarker of human NSCLC. Keywords microRNA-138 3-Phosphoinositidedependent protein kinase-1 Non-small cell lung cancer Quantitative real-time PCR Prognosis
Introduction Lung cancer, as one of the most lethal cancers worldwide, causes millions of deaths annually [1]. Many patients with lung cancer are in the advanced stages of the disease at the time of diagnosis, which lead to poor survival. The 5-year survival rate for patients with lung cancer was 10 to 20 % before the year 2000 [2]. Non-small cell lung cancer (NSCLC), the most frequently occurring category of lung cancer, accounts for approximately 80 % of all cases [3]. Recent studies have reported that the incidence of NSCLC may be closely related to multiple factors, including tobacco use, radon exposure, sex and ethnicity [4]. Although prolongation of life span and improvements in quality of life have been brought about by the development of supportive care and molecular-targeting drugs, the prognosis of NSCLC patients is still poor. The overall 5-year survival rate for NSCLC is only 17.1 % in USA [5]. Thus, it is extremely necessary to explore early predictive biomarkers for NSCLC in order to establish the early
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diagnosis, which in turn further contributes to the improvement of clinical outcome. However, the molecular mechanisms of NSCLC carcinogenesis have not been fully elucidated, resulting in the lack of the biomarkers with satisfactory sensitivity and specificity available for this malignancy. Because of its noninvasiveness and easy detection, serum biomarkers are considered as good tools for identifying malignancy and are more helpful in clinical practice [6]. Hence, accumulating investigations are performed to identify reliable serum biomarkers for human NSCLC. MicroRNAs (miRNAs), a species of abundant small noncoding, 18 to 25 nucleotide-long, naturally occurring RNA molecules interfere with the degradation of their target mRNAs and then inhibit protein translation by binding to the 30 untranslated region of mRNA to yield an RNA-induced silencing complex [7]. To date, there have been at least 1,000 loci encoding miRNAs in humans. Some miRNAs may have as many as a few thousand targets and bioinformatics data indicate that miRNAs have the potential to regulate at least 20–30 % of human genes [8]. Growing evidence show that miRNAs play crucial roles in various human biological processes such as differentiation, cell proliferation, apoptosis, epithelial-mesenchymal-transition (EMT) and cell metabolism [9]. More recently, expression profiling of many miRNAs in various normal and disease tissues demonstrated unique spatial and temporal expression patterns [10]. The dysregulation of miRNAs has been found to be implicated into cancer initiation and progression. An increasing number of miRNAs have been functionally characterized as oncogenes or tumor suppressor genes in various cancer types [11]. Some onco-miRNAs can inhibit tumor suppressor genes or other genes that modulate cell differentiation and apoptosis, stimulate cell proliferation, vasculogenesis and tumorigenesis. By contrast, tumor suppressive miRNAs can negatively modulate oncogenes or genes that inhibit cell differentiation and apoptosis. Accumulating studies also identified several miRNAs in serum and plasma in a remarkably stable form that is protected from endogenous RNase activity, suggesting that serum miRNAs may be used as molecular biomarkers for diagnosis of cancer and prediction of prognosis [12]. miR-138 is a family of microRNA precursors found in animals, including humans [13]. Two miR-138 genes were initially predicted in the mouse genome termed ‘‘miR-138-1 and miR-138-2,’’ and their human homologs were mapped to chromosome 3p21.33 and 16q13, respectively [14]. miR-138 has been used as an example of the post-transcriptional regulation of miRNA, due to the finding that the precursor is expressed ubiquitously, but the mature product is found only in specific cell types [15]. Recent studies have demonstrated that miR-138 is involved in several
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biological processes, including the development of mammary glands, regulating of dendritic spine morphogenesis, modulation of cardiac patterning during embryonic development and thermotolerance acquisition [16]. Under the pathological conditions, miR-138 has been reported to function as a tumor suppressor in a variety of human cancers, including glioblastoma, neuroblastoma, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, hepatocellular carcinoma, cholangiocarcinoma, colorectal cancer and ovarian cancer [17–22]. In particular, in lung cancer, Wang et al. [20] in 2011 reported that the upregulation of miR-138 could increase the sensitivity of NSCLC cells to cisplatin in vitro drug sensitivity assay and could increase apoptosis assessed by flow cytometry, suggesting that this miRNA could play an important role in the development of cisplatin resistance in NSCLC; Zhang et al. [23] in 2013 defined a tumor suppressor function for miR-138 in NSCLC and confirmed that miR-138 could inhibit tumor growth through repression of EZH2 in this cancer; Yang et al. [24] in 2014 found that miR-138/ SENP1 cascade may be relative to radiosensitization in lung cancer cells and may be a potential radiotherapy target; and meanwhile, Ye et al. [25] also indicated that miR138 could inhibit cell proliferation by targeting 3-phosphoinositide-dependent protein kinase-1 (PDK1) in NSCLC cells, which imply the crucial roles of miR-138/ PDK1 cascade in NSCLC. However, its clinical significance in NSCLC remains poorly understood. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients’ prognosis.
Materials and methods Patients and tissue samples This study was approved by the ethics committee of Zhengzhou University & First Affiliated Hospital to Henan University of Science and Technology, and all specimens were collected after obtaining the patients’ informed consents. Serum samples in the current study were obtained from 100 patients with NSCLC (70 males and 30 females, median age 60.18 years, range 30–88 years) and 100 healthy volunteers (65 males and 35 females, median age 59.22 years, range 28–80 years) serving as age-matched normal controls at the Department of Respiratory Medicine, Luoyang Central Hospital Affiliated to Zhengzhou University & First Affiliated Hospital to Henan University of Science and Technology between January 2006 and December 2010. None of the NSCLC patients had received
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Table 1 Associations of microRNA (miR)-138 or 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in sera of non-small cell lung cancer (NSCLC) patients with different clinicopathological characteristics Clinicopathological characteristics
No. of cases
miR-138 expression
P
High (n, %)
Low (n, %)
Gender
PDK1 expression
P
High (n, %)
Low (n, %)
18 (60.00)
12 (40.00)
42 (60.00)
28 (40.00)
20 (62.50)
12 (37.50)
40 (58.82)
28 (41.18)
26 (54.17)
22 (45.83)
34 (65.38)
18 (34.62)
NS
Female
30
10 (33.33)
20 (66.67)
Male Age (years)
70
28 (40.00)
42 (60.00)
NS
NS
\ 60
32
11 (34.38)
21 (65.62)
C 60
68
27 (39.71)
41 (60.29)
Smoke
NS
NS
Non-smokers
48
20 (41.67)
28 (58.33)
Smokers
52
18 (34.62)
34 (65.38)
Histological type
NS
NS
NS
Adenocarcinoma
48
16 (33.33)
32 (66.67)
32 (66.67)
16 (33.33)
Squamous cell carcinoma
26
11 (42.31)
15 (57.69)
13 (50.00)
13 (50.00)
Others
26
11 (42.31)
15 (57.69)
15 (57.69)
11 (42.31)
Differentiation
NS
NS
Well
15
8 (53.33)
7 (46.67)
7 (46.67)
8 (53.33)
Moderate
30
10 (33.33)
20 (66.67)
18 (60.00)
12 (40.00)
Poor
55
20 (36.36)
35 (63.64)
35 (63.64)
20 (36.36)
48
26 (54.17)
22 (45.83)
20 (41.67)
28 (58.33)
52
12 (23.07)
40 (76.93)
40 (76.93)
12 (23.07)
Negative
42
22 (52.38)
20 (47.62)
20 (47.62)
22 (52.38)
Positive
58
16 (27.59)
42 (72.41)
40 (68.97)
18 (31.03)
TNM stage I–II III
0.002
0.002
Lymph node metastasis
chemotherapy or radiotherapy before surgery. The clinical and laboratory data reported in the current study were obtained at the time of serum sampling. The histological grade of cancers was assessed according to the standard of the World Health Organization. Cancers were classified according to the 7th edition of the TNM staging system. Detailed clinicopathological characteristics were refined from the medical records and are summarized in Table 1. All NSCLC patients were performed a follow-up examination. The latest follow-up was updated in December 2013, and the median follow-up for overall survival was 50.62 months (range 2–66 months) for patients who remained alive at the time of this analysis. Serum samples were obtained from the patients at the time of diagnosis. After centrifugation at 3,000 rpm for 15 min at room temperature, clotted serum samples were separated and stored in aliquots at -80 °C until use. Quantitative real-time PCR (qRT-PCR) assay Expression levels of miR-138 and PDK1 mRNA in serum samples obtained from NSCLC patients and healthy
0.01
0.01
controls were detected by qRT-PCR assay according to the previous studies [26, 27]. In brief, total RNA was extracted by using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The relative expression levels of miR-138 and PDK1 mRNA were, respectively, determined using the mirVana qRT-PCR microRNA Detection kit (Ambion) and the standard SYBR-Green RT-PCR Kit (Takara, Otsu, Japan) following the manufacturer’s instructions. Specific primer sets for miR-138 and U6 (used as an internal reference) were obtained from Ambion. The sequences of the qRT-PCR primers for PDK1 and GAPDH used as an endogenous control were validated previously [28] and synthesized by Invitrogen. Real-time PCR was performed using the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). The comparative cycle threshold (Ct) method was used to calculate the relative expression levels of miR-138 to U6 and PDK1 mRNA to GAPDH mRNA. As there were no absolute criteria for the serum levels of miR-138 and PDK1 mRNA in NSCLC patients, we selected the median value of the relative expression levels for miR-138 or PDK1 mRNA as cutoff points, considering
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different expression on healthy controls and NSCLC patients. This method considered the following values as criteria for the high expression group: miR-138 C 2.44 and PDK1 C 3.60. Statistical analysis All computations were carried out using the software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. The correlation between miR-138 and PDK1 mRNA expression was calculated using Spearman’s test. The survival probabilities were determined using the Kaplan–Meier analysis, and the significance of differences was analyzed by the log-rank test. Multivariate analysis of the prognostic factors was performed with Cox regression model. P \ 0.05 was considered statistically significant.
Results miR-138 downregulation and PDK1 upregulation in sera of NSCLC patients versus healthy controls As shown in Fig. 1, miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (NSCLC vs. control 2.47 ± 1.23 vs. 4.46 ± 1.32, P \ 0.001, Fig. 1a), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (NSCLC vs. control 3.90 ± 1.38 vs. 1.13 ± 0.34, P \ 0.001, Fig. 1b). More interestingly, the relative expression levels of miR-138 in sera of NSCLC patients were negatively correlated with those of PDK1 mRNA significantly (Spearman correlation coefficient r = -0.53, P \ 0.001, Fig. 1c). Based on the scoring system of the current study as described in Materials and methods section, of 100 patients with PCa, 12 (12.00 %) were both high expression of miR-138 and PDK1, 14 (14.00 %) were both low expression of miR-138 and PDK1, 26 (26.00 %) were miR-138-high and PDK1-low expression, and 48 (48.00 %) were miR-138-low and PDK1-high expression. miR-138 downregulation and PDK1 upregulation associated with aggressive progression of NSCLC patients Table 1 summarized the associations of miR-138 downregulation or PDK1 upregulation with clinicopathological characteristics of human NSCLC. We found that miR-138
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Fig. 1 Relative expression levels of miR-138 (a) and PDK1 mRNA (b) in sera of non-small cell lung cancer (NSCLC) patients and healthy controls. c Scatter diagram of the spearman correlation analysis between miR-138 and PDK1 mRNA expression levels in sera of NSCLC patients
downregulation and PDK1 upregulation were both significantly associated with advanced TNM stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. No statistically significant associations of miR-138 or PDK1 with age at diagnosis,
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Fig. 2 Kaplan–Meier survival curves of non-small cell lung cancer (NSCLC) patients. a The 5-year overall survival of NSCLC patients with high MiR-138 protein expression was significantly lower than that of NSCLC patients with low MiR-138 protein expression (P \ 0.001). b The 5-year overall survival of NSCLC patients with
high PDK1 protein expression was significantly lower than that of NSCLC patients with low PDK1 protein expression (P \ 0.001). c The 5-year overall survival of NSCLC patients with Bmi-high/ PDK1-high expression was significantly lowest of all three groups (P \ 0.001)
gender of patients, smoke status, histological type and differentiation were found (both P [ 0.05, Table 1).
carcinoma antigens, carcinoembryonic antigens and cytokeratin 19, none has been proved to be sensitive or specific enough in initial screening, staging, prognosis, prediction of relapse or therapeutic monitoring [12]. The identification of effective biomarkers involved in the initiation and development of NSCLC is a fundamental step to improve the prognosis of this disease. The current study focused on the clinical significance of preoperative serum level of miR-138 and its target gene PDK1 in NSCLC patients. Our data showed that the aberrant expressions of miR-138 and PDK1 mRNA in patients’ sera appeared to be correlated with TNM stage and positive lymph node metastasis, as well as patients’ survival. These strong associations suggest that the deregulation of miR-138/PDK1 cascade may promote tumor progression and that miR-138 and/or PDK1 could possibly be used as a biomarker for a more aggressive phenotype of human NSCLC. To the best of our knowledge, this is the first study to demonstrate the prognostic value of miR-138/PDK1 cascade in this malignancy. MiR-138 is broadly conserved among vertebrates and is highly expressed in brain tissues [13]. It regulates a number of essential biological processes, including the development of mammary glands, regulating dendritic spine morphogenesis, modulating cardiac patterning during embryonic development and thermotolerance acquisition [14]. MiR-138 has been observed to be downregulated and act as a tumor suppressive miRNA in a variety of cancer types. For example, ectopic expression of miRNA-138 could effectively inhibit glioblastoma cell proliferation in vitro and tumorigenicity in vivo via inhibition of EZH2CDK4/6-pRb-E2F1 signal loop [17]; miR-138 could induce cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma; miR-138 could suppress epithelialmesenchymal transition in squamous cell carcinoma cell
Association of miR-138 and PDK1 mRNA expression with overall survival in NSCLC patients The Kaplan–Meier plot showed that the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P \ 0.001, Fig. 2a, b). More importantly, there was a trend toward the poorest overall survival in the patient group with combined low miR-138 expression and high PDK1 expression (P \ 0.001, Fig. 2c). Notably, the overall survival curve for patients with miR-138-high/ PDK1-high or miR-138-low/PDK1-low was relatively close to those with miR-138-high/PDK1-low, but was dramatically discrepant from those of the miR-138-low/ PDK1-high group (Fig. 2c). In multivariate analysis, Cox proportional hazards model showed that miR-138 expression (P = 0.01, Table 2), PDK1 expression (P = 0.01, Table 2) and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001, Table 2) were all independent prognostic factors for overall survival in NSCLC patients.
Discussion Reliable prognostic biomarkers are very helpful to make accurate decisions regarding effective treatment strategies for NSCLC patients. Although several serologic biomarkers, which are preferable to monitor using a noninvasive method, have been screened, such as squamous cell
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Table 2 Cox multivariate analysis Parameter
Risk ratio
95 % confidence interval
P
Differentiation
1.26
0.83–2.18
NS
TNM stage
7.97
2.26–16.98
\0.001
Lymph node metastasis
4.82
1.92–10.18
0.01
miR-138 expression
4.20
2.02–9.08
0.01
PDK1 expression
3.80
1.92–7.69
0.02
Combined miR-138/PDK1 expression
6.88
2.33–15.32
\0.001
lines [19]; and miR-138 could suppress ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1a [22]. These findings suggest that downregulation of miR-138, which can lead to increased cell proliferation and invasion, may be advantageous for cancer cell survival and growth. PDK1, encoded by the gene PDPK1, belongs to the family of AGC kinases (serine and threonine kinases) that show a sequence homology in their catalytic domain to cAMPdependent protein kinase 1, cyclic guanosine monophosphate-dependent protein kinase and protein kinase C (PKC) [29]. It is ubiquitously expressed in human tissues and plays a crucial role in signaling pathways activated by several growth factors and hormones [30]. PDK1 functions as a molecular kinase belonging to the phosphoinositide-3kinase (PI3K) signaling pathway and is involved in the regulation of various physiological processes relevant to metabolism, growth, proliferation and survival [31]. In 2005, Bayascas and colleagues initially discovered that PDK1 might be a viable target in cancer [32]. After that, subsequent studies have demonstrated the role of PDK1 in a variety of human cancers. For example, silencing of PDK1 gene expression by RNA interference suppresses growth of esophageal cancer [33]; downregulation of PDK1 levels could inhibits migration and experimental metastasis of human breast cancer cells [34]. These findings suggest that elevated activation of PDK1 may induce tumorigenesis by enhancing cell proliferation and inhibiting apoptosis. In particular, the previous study of Ye and colleagues identified PDK1 as a direct target of miR-138 in NSCLC cell lines and also found that the proliferation of NSCLC cells could be suppressed by miR-138 in a concentrationdependent manner, which was similar with the effect of knockdown of PDK1 by siRNA [25]. On this basis, we hypothesized that the miR-138/PDK1 cascade might be associated with aggressive progression of human NSCLC, and the prognostic value of the combination of miR-138 and PDK1 (miR-138/PDK1) might be better than miR-138 or PDK1 alone. To validate this hypothesis, we analyzed the correlations of miR-138/PDK1 combined expression,
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miR-138 expression and PDK1 expression with various clinicopathological characteristics and overall survival of NSCLC patients. Our data did not only showed significant associations between miR-138 downregulation or PDK1 upregulation and advanced TNM stage and positive lymph node metastasis, but also demonstrated that miR-138/ PDK1 combined expression, miR-138 expression and PDK1 expression were all independent prognostic factors for overall survival of NSCLC patients. More interestingly, miR-138/PDK1 combined expression could more efficiently predict the prognosis in NSCLC patients, implying that the combined detection of miR-138 and PDK1 expression could be used to design optimal and individualized treatment. Additional research would be required to confirm our findings using a large and independent cohort. In conclusion, the deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. Aberrant expression of miR-138 and PDK1 may be closely correlated with the biological behavior of this malignancy. More importantly, miR-138 and PDK1 may synergistically predict patients’ prognosis and their combination may represent a promising prognostic biomarker of human NSCLC. Conflict of interest
None.
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ORIGINAL PAPER
Prognostic potential of microRNA-138 and its target mRNA PDK1 in sera for patients with non-small cell lung cancer Lihong Han • Guoxiu Zhang • Nali Zhang Haiyan Li • Yanyan Liu • Aiguo Fu • Youguang Zheng
•
Received: 23 June 2014 / Accepted: 7 July 2014 / Published online: 27 July 2014 Ó Springer Science+Business Media New York 2014
Abstract microRNA (miR)-138 has been recognized as a potential tumor suppressor via regulating 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in nonsmall cell lung cancer (NSCLC) cells. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients’ prognosis. miR-138 and PDK1 mRNA expressions in 100 NSCLCs and 100 healthy control sera were detected by quantitative real-time PCR. miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (P \ 0.001), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (P \ 0.001). In addition, miR-138 downregulation and PDK1 upregulation were both significantly associated with advanced tumor-node-metastasis (TNM) stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. Moreover, the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P \ 0.001). Notably, NSCLC patients with combined miR-138 downregulation and PDK1 upregulation (miR-138-low/PDK1-high) had shortest overall survival (P \ 0.001). Furthermore, multivariate analysis showed that miR-138 expression L. Han N. Zhang H. Li Y. Liu A. Fu Y. Zheng (&) Department of Respiratory Medicine, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang 471009, Henan Province, China e-mail: [email protected] G. Zhang First Affiliated Hospital to Henan University of Science and Technology, Luoyang 471009, Henan Province, China
(P = 0.01), PDK1 expression (P = 0.01), and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001) were all independent prognostic factors for overall survival in NSCLC patients. Deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. More importantly, miR-138 and PDK1 may synergistically predict patients’ prognosis and their combination may represent a promising prognostic biomarker of human NSCLC. Keywords microRNA-138 3-Phosphoinositidedependent protein kinase-1 Non-small cell lung cancer Quantitative real-time PCR Prognosis
Introduction Lung cancer, as one of the most lethal cancers worldwide, causes millions of deaths annually [1]. Many patients with lung cancer are in the advanced stages of the disease at the time of diagnosis, which lead to poor survival. The 5-year survival rate for patients with lung cancer was 10 to 20 % before the year 2000 [2]. Non-small cell lung cancer (NSCLC), the most frequently occurring category of lung cancer, accounts for approximately 80 % of all cases [3]. Recent studies have reported that the incidence of NSCLC may be closely related to multiple factors, including tobacco use, radon exposure, sex and ethnicity [4]. Although prolongation of life span and improvements in quality of life have been brought about by the development of supportive care and molecular-targeting drugs, the prognosis of NSCLC patients is still poor. The overall 5-year survival rate for NSCLC is only 17.1 % in USA [5]. Thus, it is extremely necessary to explore early predictive biomarkers for NSCLC in order to establish the early
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diagnosis, which in turn further contributes to the improvement of clinical outcome. However, the molecular mechanisms of NSCLC carcinogenesis have not been fully elucidated, resulting in the lack of the biomarkers with satisfactory sensitivity and specificity available for this malignancy. Because of its noninvasiveness and easy detection, serum biomarkers are considered as good tools for identifying malignancy and are more helpful in clinical practice [6]. Hence, accumulating investigations are performed to identify reliable serum biomarkers for human NSCLC. MicroRNAs (miRNAs), a species of abundant small noncoding, 18 to 25 nucleotide-long, naturally occurring RNA molecules interfere with the degradation of their target mRNAs and then inhibit protein translation by binding to the 30 untranslated region of mRNA to yield an RNA-induced silencing complex [7]. To date, there have been at least 1,000 loci encoding miRNAs in humans. Some miRNAs may have as many as a few thousand targets and bioinformatics data indicate that miRNAs have the potential to regulate at least 20–30 % of human genes [8]. Growing evidence show that miRNAs play crucial roles in various human biological processes such as differentiation, cell proliferation, apoptosis, epithelial-mesenchymal-transition (EMT) and cell metabolism [9]. More recently, expression profiling of many miRNAs in various normal and disease tissues demonstrated unique spatial and temporal expression patterns [10]. The dysregulation of miRNAs has been found to be implicated into cancer initiation and progression. An increasing number of miRNAs have been functionally characterized as oncogenes or tumor suppressor genes in various cancer types [11]. Some onco-miRNAs can inhibit tumor suppressor genes or other genes that modulate cell differentiation and apoptosis, stimulate cell proliferation, vasculogenesis and tumorigenesis. By contrast, tumor suppressive miRNAs can negatively modulate oncogenes or genes that inhibit cell differentiation and apoptosis. Accumulating studies also identified several miRNAs in serum and plasma in a remarkably stable form that is protected from endogenous RNase activity, suggesting that serum miRNAs may be used as molecular biomarkers for diagnosis of cancer and prediction of prognosis [12]. miR-138 is a family of microRNA precursors found in animals, including humans [13]. Two miR-138 genes were initially predicted in the mouse genome termed ‘‘miR-138-1 and miR-138-2,’’ and their human homologs were mapped to chromosome 3p21.33 and 16q13, respectively [14]. miR-138 has been used as an example of the post-transcriptional regulation of miRNA, due to the finding that the precursor is expressed ubiquitously, but the mature product is found only in specific cell types [15]. Recent studies have demonstrated that miR-138 is involved in several
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biological processes, including the development of mammary glands, regulating of dendritic spine morphogenesis, modulation of cardiac patterning during embryonic development and thermotolerance acquisition [16]. Under the pathological conditions, miR-138 has been reported to function as a tumor suppressor in a variety of human cancers, including glioblastoma, neuroblastoma, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, hepatocellular carcinoma, cholangiocarcinoma, colorectal cancer and ovarian cancer [17–22]. In particular, in lung cancer, Wang et al. [20] in 2011 reported that the upregulation of miR-138 could increase the sensitivity of NSCLC cells to cisplatin in vitro drug sensitivity assay and could increase apoptosis assessed by flow cytometry, suggesting that this miRNA could play an important role in the development of cisplatin resistance in NSCLC; Zhang et al. [23] in 2013 defined a tumor suppressor function for miR-138 in NSCLC and confirmed that miR-138 could inhibit tumor growth through repression of EZH2 in this cancer; Yang et al. [24] in 2014 found that miR-138/ SENP1 cascade may be relative to radiosensitization in lung cancer cells and may be a potential radiotherapy target; and meanwhile, Ye et al. [25] also indicated that miR138 could inhibit cell proliferation by targeting 3-phosphoinositide-dependent protein kinase-1 (PDK1) in NSCLC cells, which imply the crucial roles of miR-138/ PDK1 cascade in NSCLC. However, its clinical significance in NSCLC remains poorly understood. The aim of this study was to investigate miR-138 and PDK1 mRNA expression in serum of NSCLC and their associations with patients’ prognosis.
Materials and methods Patients and tissue samples This study was approved by the ethics committee of Zhengzhou University & First Affiliated Hospital to Henan University of Science and Technology, and all specimens were collected after obtaining the patients’ informed consents. Serum samples in the current study were obtained from 100 patients with NSCLC (70 males and 30 females, median age 60.18 years, range 30–88 years) and 100 healthy volunteers (65 males and 35 females, median age 59.22 years, range 28–80 years) serving as age-matched normal controls at the Department of Respiratory Medicine, Luoyang Central Hospital Affiliated to Zhengzhou University & First Affiliated Hospital to Henan University of Science and Technology between January 2006 and December 2010. None of the NSCLC patients had received
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Table 1 Associations of microRNA (miR)-138 or 3-phosphoinositide-dependent protein kinase-1 (PDK1) expression in sera of non-small cell lung cancer (NSCLC) patients with different clinicopathological characteristics Clinicopathological characteristics
No. of cases
miR-138 expression
P
High (n, %)
Low (n, %)
Gender
PDK1 expression
P
High (n, %)
Low (n, %)
18 (60.00)
12 (40.00)
42 (60.00)
28 (40.00)
20 (62.50)
12 (37.50)
40 (58.82)
28 (41.18)
26 (54.17)
22 (45.83)
34 (65.38)
18 (34.62)
NS
Female
30
10 (33.33)
20 (66.67)
Male Age (years)
70
28 (40.00)
42 (60.00)
NS
NS
\ 60
32
11 (34.38)
21 (65.62)
C 60
68
27 (39.71)
41 (60.29)
Smoke
NS
NS
Non-smokers
48
20 (41.67)
28 (58.33)
Smokers
52
18 (34.62)
34 (65.38)
Histological type
NS
NS
NS
Adenocarcinoma
48
16 (33.33)
32 (66.67)
32 (66.67)
16 (33.33)
Squamous cell carcinoma
26
11 (42.31)
15 (57.69)
13 (50.00)
13 (50.00)
Others
26
11 (42.31)
15 (57.69)
15 (57.69)
11 (42.31)
Differentiation
NS
NS
Well
15
8 (53.33)
7 (46.67)
7 (46.67)
8 (53.33)
Moderate
30
10 (33.33)
20 (66.67)
18 (60.00)
12 (40.00)
Poor
55
20 (36.36)
35 (63.64)
35 (63.64)
20 (36.36)
48
26 (54.17)
22 (45.83)
20 (41.67)
28 (58.33)
52
12 (23.07)
40 (76.93)
40 (76.93)
12 (23.07)
Negative
42
22 (52.38)
20 (47.62)
20 (47.62)
22 (52.38)
Positive
58
16 (27.59)
42 (72.41)
40 (68.97)
18 (31.03)
TNM stage I–II III
0.002
0.002
Lymph node metastasis
chemotherapy or radiotherapy before surgery. The clinical and laboratory data reported in the current study were obtained at the time of serum sampling. The histological grade of cancers was assessed according to the standard of the World Health Organization. Cancers were classified according to the 7th edition of the TNM staging system. Detailed clinicopathological characteristics were refined from the medical records and are summarized in Table 1. All NSCLC patients were performed a follow-up examination. The latest follow-up was updated in December 2013, and the median follow-up for overall survival was 50.62 months (range 2–66 months) for patients who remained alive at the time of this analysis. Serum samples were obtained from the patients at the time of diagnosis. After centrifugation at 3,000 rpm for 15 min at room temperature, clotted serum samples were separated and stored in aliquots at -80 °C until use. Quantitative real-time PCR (qRT-PCR) assay Expression levels of miR-138 and PDK1 mRNA in serum samples obtained from NSCLC patients and healthy
0.01
0.01
controls were detected by qRT-PCR assay according to the previous studies [26, 27]. In brief, total RNA was extracted by using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The relative expression levels of miR-138 and PDK1 mRNA were, respectively, determined using the mirVana qRT-PCR microRNA Detection kit (Ambion) and the standard SYBR-Green RT-PCR Kit (Takara, Otsu, Japan) following the manufacturer’s instructions. Specific primer sets for miR-138 and U6 (used as an internal reference) were obtained from Ambion. The sequences of the qRT-PCR primers for PDK1 and GAPDH used as an endogenous control were validated previously [28] and synthesized by Invitrogen. Real-time PCR was performed using the Applied Biosystems 7900 Fast Real-Time PCR system (Applied Biosystems, Foster City, California, USA). The comparative cycle threshold (Ct) method was used to calculate the relative expression levels of miR-138 to U6 and PDK1 mRNA to GAPDH mRNA. As there were no absolute criteria for the serum levels of miR-138 and PDK1 mRNA in NSCLC patients, we selected the median value of the relative expression levels for miR-138 or PDK1 mRNA as cutoff points, considering
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different expression on healthy controls and NSCLC patients. This method considered the following values as criteria for the high expression group: miR-138 C 2.44 and PDK1 C 3.60. Statistical analysis All computations were carried out using the software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. The correlation between miR-138 and PDK1 mRNA expression was calculated using Spearman’s test. The survival probabilities were determined using the Kaplan–Meier analysis, and the significance of differences was analyzed by the log-rank test. Multivariate analysis of the prognostic factors was performed with Cox regression model. P \ 0.05 was considered statistically significant.
Results miR-138 downregulation and PDK1 upregulation in sera of NSCLC patients versus healthy controls As shown in Fig. 1, miR-138 expression level was significantly lower in NSCLC serum samples than in healthy control serum samples (NSCLC vs. control 2.47 ± 1.23 vs. 4.46 ± 1.32, P \ 0.001, Fig. 1a), while PDK1 mRNA expression level was significantly increased in NSCLC serum samples compared to healthy control serum samples (NSCLC vs. control 3.90 ± 1.38 vs. 1.13 ± 0.34, P \ 0.001, Fig. 1b). More interestingly, the relative expression levels of miR-138 in sera of NSCLC patients were negatively correlated with those of PDK1 mRNA significantly (Spearman correlation coefficient r = -0.53, P \ 0.001, Fig. 1c). Based on the scoring system of the current study as described in Materials and methods section, of 100 patients with PCa, 12 (12.00 %) were both high expression of miR-138 and PDK1, 14 (14.00 %) were both low expression of miR-138 and PDK1, 26 (26.00 %) were miR-138-high and PDK1-low expression, and 48 (48.00 %) were miR-138-low and PDK1-high expression. miR-138 downregulation and PDK1 upregulation associated with aggressive progression of NSCLC patients Table 1 summarized the associations of miR-138 downregulation or PDK1 upregulation with clinicopathological characteristics of human NSCLC. We found that miR-138
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Fig. 1 Relative expression levels of miR-138 (a) and PDK1 mRNA (b) in sera of non-small cell lung cancer (NSCLC) patients and healthy controls. c Scatter diagram of the spearman correlation analysis between miR-138 and PDK1 mRNA expression levels in sera of NSCLC patients
downregulation and PDK1 upregulation were both significantly associated with advanced TNM stage (both P = 0.002) and positive lymph node metastasis (both P = 0.01) of NSCLC patients. No statistically significant associations of miR-138 or PDK1 with age at diagnosis,
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Fig. 2 Kaplan–Meier survival curves of non-small cell lung cancer (NSCLC) patients. a The 5-year overall survival of NSCLC patients with high MiR-138 protein expression was significantly lower than that of NSCLC patients with low MiR-138 protein expression (P \ 0.001). b The 5-year overall survival of NSCLC patients with
high PDK1 protein expression was significantly lower than that of NSCLC patients with low PDK1 protein expression (P \ 0.001). c The 5-year overall survival of NSCLC patients with Bmi-high/ PDK1-high expression was significantly lowest of all three groups (P \ 0.001)
gender of patients, smoke status, histological type and differentiation were found (both P [ 0.05, Table 1).
carcinoma antigens, carcinoembryonic antigens and cytokeratin 19, none has been proved to be sensitive or specific enough in initial screening, staging, prognosis, prediction of relapse or therapeutic monitoring [12]. The identification of effective biomarkers involved in the initiation and development of NSCLC is a fundamental step to improve the prognosis of this disease. The current study focused on the clinical significance of preoperative serum level of miR-138 and its target gene PDK1 in NSCLC patients. Our data showed that the aberrant expressions of miR-138 and PDK1 mRNA in patients’ sera appeared to be correlated with TNM stage and positive lymph node metastasis, as well as patients’ survival. These strong associations suggest that the deregulation of miR-138/PDK1 cascade may promote tumor progression and that miR-138 and/or PDK1 could possibly be used as a biomarker for a more aggressive phenotype of human NSCLC. To the best of our knowledge, this is the first study to demonstrate the prognostic value of miR-138/PDK1 cascade in this malignancy. MiR-138 is broadly conserved among vertebrates and is highly expressed in brain tissues [13]. It regulates a number of essential biological processes, including the development of mammary glands, regulating dendritic spine morphogenesis, modulating cardiac patterning during embryonic development and thermotolerance acquisition [14]. MiR-138 has been observed to be downregulated and act as a tumor suppressive miRNA in a variety of cancer types. For example, ectopic expression of miRNA-138 could effectively inhibit glioblastoma cell proliferation in vitro and tumorigenicity in vivo via inhibition of EZH2CDK4/6-pRb-E2F1 signal loop [17]; miR-138 could induce cell cycle arrest by targeting cyclin D3 in hepatocellular carcinoma; miR-138 could suppress epithelialmesenchymal transition in squamous cell carcinoma cell
Association of miR-138 and PDK1 mRNA expression with overall survival in NSCLC patients The Kaplan–Meier plot showed that the overall survival of NSCLC patients with low miR-138 expression or high PDK1 mRNA expression was obviously shorter than those with high miR-138 expression or low PDK1 mRNA expression (both P \ 0.001, Fig. 2a, b). More importantly, there was a trend toward the poorest overall survival in the patient group with combined low miR-138 expression and high PDK1 expression (P \ 0.001, Fig. 2c). Notably, the overall survival curve for patients with miR-138-high/ PDK1-high or miR-138-low/PDK1-low was relatively close to those with miR-138-high/PDK1-low, but was dramatically discrepant from those of the miR-138-low/ PDK1-high group (Fig. 2c). In multivariate analysis, Cox proportional hazards model showed that miR-138 expression (P = 0.01, Table 2), PDK1 expression (P = 0.01, Table 2) and combined expression of miR-138 and PDK1 (miR-138/PDK1, P = 0.001, Table 2) were all independent prognostic factors for overall survival in NSCLC patients.
Discussion Reliable prognostic biomarkers are very helpful to make accurate decisions regarding effective treatment strategies for NSCLC patients. Although several serologic biomarkers, which are preferable to monitor using a noninvasive method, have been screened, such as squamous cell
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Table 2 Cox multivariate analysis Parameter
Risk ratio
95 % confidence interval
P
Differentiation
1.26
0.83–2.18
NS
TNM stage
7.97
2.26–16.98
\0.001
Lymph node metastasis
4.82
1.92–10.18
0.01
miR-138 expression
4.20
2.02–9.08
0.01
PDK1 expression
3.80
1.92–7.69
0.02
Combined miR-138/PDK1 expression
6.88
2.33–15.32
\0.001
lines [19]; and miR-138 could suppress ovarian cancer cell invasion and metastasis by targeting SOX4 and HIF-1a [22]. These findings suggest that downregulation of miR-138, which can lead to increased cell proliferation and invasion, may be advantageous for cancer cell survival and growth. PDK1, encoded by the gene PDPK1, belongs to the family of AGC kinases (serine and threonine kinases) that show a sequence homology in their catalytic domain to cAMPdependent protein kinase 1, cyclic guanosine monophosphate-dependent protein kinase and protein kinase C (PKC) [29]. It is ubiquitously expressed in human tissues and plays a crucial role in signaling pathways activated by several growth factors and hormones [30]. PDK1 functions as a molecular kinase belonging to the phosphoinositide-3kinase (PI3K) signaling pathway and is involved in the regulation of various physiological processes relevant to metabolism, growth, proliferation and survival [31]. In 2005, Bayascas and colleagues initially discovered that PDK1 might be a viable target in cancer [32]. After that, subsequent studies have demonstrated the role of PDK1 in a variety of human cancers. For example, silencing of PDK1 gene expression by RNA interference suppresses growth of esophageal cancer [33]; downregulation of PDK1 levels could inhibits migration and experimental metastasis of human breast cancer cells [34]. These findings suggest that elevated activation of PDK1 may induce tumorigenesis by enhancing cell proliferation and inhibiting apoptosis. In particular, the previous study of Ye and colleagues identified PDK1 as a direct target of miR-138 in NSCLC cell lines and also found that the proliferation of NSCLC cells could be suppressed by miR-138 in a concentrationdependent manner, which was similar with the effect of knockdown of PDK1 by siRNA [25]. On this basis, we hypothesized that the miR-138/PDK1 cascade might be associated with aggressive progression of human NSCLC, and the prognostic value of the combination of miR-138 and PDK1 (miR-138/PDK1) might be better than miR-138 or PDK1 alone. To validate this hypothesis, we analyzed the correlations of miR-138/PDK1 combined expression,
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miR-138 expression and PDK1 expression with various clinicopathological characteristics and overall survival of NSCLC patients. Our data did not only showed significant associations between miR-138 downregulation or PDK1 upregulation and advanced TNM stage and positive lymph node metastasis, but also demonstrated that miR-138/ PDK1 combined expression, miR-138 expression and PDK1 expression were all independent prognostic factors for overall survival of NSCLC patients. More interestingly, miR-138/PDK1 combined expression could more efficiently predict the prognosis in NSCLC patients, implying that the combined detection of miR-138 and PDK1 expression could be used to design optimal and individualized treatment. Additional research would be required to confirm our findings using a large and independent cohort. In conclusion, the deregulation of miR-138/PDK1 cascade may be implicated in carcinogenesis and cancer progression of human NSCLC. Aberrant expression of miR-138 and PDK1 may be closely correlated with the biological behavior of this malignancy. More importantly, miR-138 and PDK1 may synergistically predict patients’ prognosis and their combination may represent a promising prognostic biomarker of human NSCLC. Conflict of interest
None.
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