Mol Cell Biochem DOI 10.1007/s11010-015-2401-7
Effect of microRNA-203 on tumor growth in human hypopharyngeal squamous cell carcinoma Ru Wang1 • Jugao Fang1,2 • Hongzhi Ma1 • Lin Feng1 • Meng Lian1 Fan Yang1 • Haizhou Wang1 • Qi Wang1,2 • Xiaohong Chen1,2
•
Received: 29 November 2014 / Accepted: 27 March 2015 Ó Springer Science+Business Media New York 2015
Abstract MicroRNAs (MiRNAs) have been recognized to regulate cancer initiation and progression in carcinogenesis as either oncogenes or tumor suppressor genes, but their role in hypopharyngeal cancer development is not clearly defined. To determine whether miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma, we conducted experiments on the functional study of miRNA-203 and identification of miRNA-203 regulated target genes in hypopharyngeal cancer cells. We found that cell proliferation and cell colony-forming increased more in the miRNA-203 upregulated cancer cells than in the negative control cancer cells. Up-regulation of miRNA-203 accelerated cell cycle progression in hypopharyngeal cancer cells. TP63 and B3GNT5 mRNAs were identified and validated as targets of miRNA-203. However, transwell assay and wound scratch assay showed that miRNA-203 did not involve in invasion and metastasis in hypopharyngeal cancer cells. According to the results, we conclude that miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma. These results provide the convincing evidence for the first time that up-regulation of miRNA203 contributes to the malignancy of hypopharyngeal squamous cell carcinoma, possibly through down-regulating TP63 and B3GNT5.
& Jugao Fang [email protected] 1
Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China
2
Key Laboratory of Otorhinolaryngology Head and Neck Surgery, Ministry of Education, Beijing Institute of Otorhinolaryngology, Beijing 100005, China
Keywords Hypopharyngeal neoplasm Hsa-mir-203 Cell proliferation Cell cycle
Introduction Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck aggressive malignancies, with an incidence of about 10 cases per million people every year [1]. More than 75 % patients with HSCC are at an advanced stage at the time of diagnosis [2]. So HSCC has a very poor prognosis with five-year survival rates ranging from 30 to 35 % [3, 4]. Although the locoregional control of the cancer has been significantly improved in the last decades because of the advent of various new treatments including surgery, radiotherapy, and chemotherapy, survival rates of HSCC patients have not yet markedly improved. Therefore, understanding the molecular oncogenic pathways underlying HSCC can bring insight into the search for improved therapy and prevention of the disease. It is known that microRNAs (miRNAs) are endogenous small non-coding RNA molecules (19-22 bases in length), which regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner [5]. A growing number of evidence suggest that miRNAs are aberrantly expressed in many kinds of human cancers and that miRNAs play significant roles in initiation, development, and metastasis of these cancers [6, 7]. However, the function of miRNAs in hypopharyngeal cancer development has not been determined. To determine whether microRNA-203 (miR-203) promotes tumor growth in human HSCC, we conducted experiments on the functional study of miR-203 and identification of miR-203 regulated target genes in the HSCC cell line FaDu. MTT assay and colony formation
123
Mol Cell Biochem
assay were performed to evaluate the effect of miR-203 on cell proliferation in FaDu cells. To elucidate how miR-203 affects cell proliferation, we tested cell cycle by PI-FACS in FaDu cells. The expression levels of miR-203 relating target genes were detected by quantitative real-time PCR (qRT- PCR).
Materials and methods Reagents Antibiotic solution (104 U penicillin, 10 mg streptomycin, 25 ug amphotericin B), dimethyl sulfoxide (DMSO), and propidium iodide (PI) were purchased from Sigma Chemicals (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco (Cambrex, MD, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). Plasmid Extraction Kit was obtained from Promega (Madison, WI, USA). MiScript Reverse Transcription Kit was purchased from Qiagen (Hilden, Germany), and SYBR Green Master Mix Kit was purchased from TaKaRa (Dalian, China). Cell culture and transfection HSCC cell line FaDu was conserved in our own laboratory. Cells were cultured in DMEM supplemented with 10 % FBS and 100 units/ml penicillin at 37 °C in a humidified 5 % carbon dioxide incubator. According to the manufacturer’s instructions,miR-203 was transfected into cells by lentivirus vectors. MiR-203 over-expression lentivirus vector (miR203-up-LV) and negative control lentivirus vector (NC-LV) harboring green fluorescent protein (GFP) were constructed by Genechem (Shanghai, China). FaDu cells were plated in six-well plates (cell density 20 %) for 24 h, and when the cells reached 30 % confluency, the lentiviruses were added into FaDu cells for transfection. After 24 h transfection, the culture media was replaced with fresh DMEM, and cells were returned to the incubator for additional 48 h transfection. After 72 h transfection, the mean percentage of FaDu cells expressing GFP was calculated under a fluorescence microscope (IX70, Olympus, Japan). RNA isolation and qRT- PCR According to manufacturer’s instructions, total RNA was extracted using Trizol reagent (Invitrogen). For miR-203 qRT-PCR, total RNA was reverse-transcribed with a miRNAspecific primer using the MiScript Reverse Transcription Kit and then qRT-PCR was performed using a miRNA-specific primer on the ABI 9700 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA); U6 snRNA served as an
123
endogenous control for normalization. qRT- PCR analyses for target mRNAs were performed using SYBR Green Master Mix Kit. The primer sequences for PCR amplification of B3GNT5 gene were 5-GGGCCTCGCTACCAATACTTG-3 and 5-CGGAACGTCGATCATAGTTTTCA-3; TP63 gene were 5-GTCATTTGATTCGAGTAGAGGGG-3 and 5-CTG GGGTGGCTCATAAGGT-3; and VSNL1 gene were 5-TGC CTTCCGAACCTTCGAC-3 and 5-CATCTCCACTCGGG TGATCT-3. GAPDH was applied as an internal control. The primer sequences of GAPDH were 5-TGACTTCAACAG CGACACCCA-3 and 5-CACCCTGTTGCTGTAGCCAA A-3. For relative quantification, 2-DDCt was calculated and used as an indication of the relative expression levels [8]. MTT cell proliferation and colony formation assay The MTT cell proliferation and colony formation assays were performed as previously described [9]. After 0, 24, 48, 72, and 96 h of transfection, cell proliferation was evaluated by using the MTT according to manufacturer’s instructions. Briefly, 10 ll of MTT solution was added to the culture medium, and incubated for additional 4 h. The absorbance was determined at 490 nm wavelength. For cell colony formation assay, a total of 800 cells were plated in complete growth media and allowed to grow until visible colonies formed (14 days). Cell colonies were fixed with paraformaldehyde, stained with GIEMSA, washed and air dried. PI-FACS cell cycle assay The cell cycle distribution was analyzed by flow cytometry (FACSCalibur, BD, USA) as described [10]. In brief, cells were suspended in 0.5 ml solution containing 20 lg/ml propidium iodide and 50 lg/ml Rnase, and then analyzed using flow cytometry. Histograms were used to represent the percentage of cells in each phase of the cell cycle (G0/ G1, S, and G2/M). Transwell migration assay Cell invasion assay was performed as described from the BD BiocoatTM MatrigelTM Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA). Cells were harvested and resuspended in serum-free medium. 5 9 105 cells of the prepared cell suspension were added into the chamber. The cells were allowed to migrate for 24 h at 37 °C in a humidified incubator with 5 % CO2. Non-invading cells were gently removed from the interior of the inserts with a cotton-tipped swab. Invasive cells were stained with GIEMSA staining. Once the filters had dried, cells were counted in five randomly selected non-overlapping fields.
Mol Cell Biochem
Wound scratch assay Wound scratch assay was performed according to the methods previously described [11]. Cells from each group that was seeded on a six-well plate were scraped with a sterile 100 ul pipette tip to create two linear regions devoid of cells. After scratching, the cells were washed with phosphate-buffered saline (PBS) medium three times and incubated at 37 °C. Cells migrated into the wound surface and average distance of migrating cells was determined under an inverted microscopy at specified time points. The experiments were performed in triplicate and repeated at least three times. Statistical analysis All data were expressed as mean ± SD of three independent experiments, in which each assay was performed in triplicate. The Student t test was used to evaluate the differences between the miR-203-up-LV group and NC-LV group using SPSS 20.0 software. P \ 0.05 was considered as statistically significant.
Results MiR-203 expression was up-regulated by recombinant lentivirus in FaDu cells The lentiviral vector system (miR-203-up-LV and NC-LV) up-regulated the expression of miR-203 in FaDu cells. As expected, a high percentage ([80 %) of FaDu cells expressed GFP after lentivirus transfection (Fig. 1a), indicating the high efficiency and stability of the transfection. Furthermore, the miR-203 level increased in the miR-203up-LV group more than that in the NC-LV group (Fig. 1b). It suggested that the miR-203-up-LV was effective to upregulate miR-203 in FaDu cells. Over-expression of miR-203 promoted cell proliferation in FaDu cells MTT assay and colony formation assay showed that Ectopic expression of miR-203 significantly promoted the growth of FaDu cells. Cell proliferation increased more in the miR203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells (Fig. 2a). Colony formation assay was performed to evaluate the long-term impact of miR-203 overexpression on the proliferation of FaDu cells. The colony size and numbers (Fig. 2b) increased more in the miR-203up-LV infected FaDu cells than in the NC-LV infected FaDu cells. Taken together, these data suggested that miR-
203 may function as an oncogenic regulator of human hypopharyngeal cancer. Over-expression of miR-203 accelerated cell cycle progression in FaDu cells PI-FACS analysis elucidated that miR-203 accelerated cell cycle progression in FaDu cells. The cells in G1 phase decreased more in the miR-203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells. The cells in G2/M phase increased in the miR-203-up-LV infected FaDu cells more than that in the NC-LV infected FaDu cells (Fig. 2c). It suggested that the over-expression of miR-203 decreased the percentage of G1 phase cells and revealed a higher proportion of cells at G2/M phase. In conclusion, miR-203 may regulate cell proliferation by accelerating cell cycle progression in human hypopharyngeal cancer cells. Dysregulation of TP63 and B3GNT5 in miR-203 over-expressed FaDu cells To understand how miR-203 affects the proliferation of FaDu cells further, bioinformatic miRNA target prediction tools (TargetScan, PicTar, and MiRanda) searched three potential miR-203 target genes [12]. TP-63, B3GNT5, and VSNL1 were identified as target genes of miR-203 in HSCC. The expression of TP63 and B3GNT5 mRNA was decreased in the miR-203-up-LV infected FaDu cells more than that in the NC-GFP-LV infected FaDu cells. However, no statistically significant difference was observed in the expression of VSNL1 (Fig. 3). These findings suggested that miR-203 may promote cell proliferation by regulating the expression of TP63 and B3GNT5 negatively in FaDu cells. The effect of miR-203 on migration and invasion ability of FaDu cells Transwell migration assay and wound scratch assay showed that miR-203 did not affect cell migration and invasion ability in FaDu cells. The migration and invasion ability of FaDu cells enhanced more in the miR-203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells (Fig. 4). However, the difference between the two groups was not statistically significance (P [ 0.05). The results illustrated that miR-203 may not be invovled in cell migration and invasion of FaDu cells.
Discussion Dysregulation of oncogene and tumor suppressor gene expression has been identified as one of the critical causes of tumorigenesis. The discovery of miRNAs provides
123
Mol Cell Biochem Fig. 1 The expression of miR203 was dramatically overexpressed by miR-203-up-LV transfection. a Microscope pictures of GFP-expressing cells 96 h after miR-203-up-LV or NC-LV transfection, including fluorescence microscope images and light microscope images (magnification, 1009). b Quantitative analysis of miR203 by qRT-PCR. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
A
micro up (×100)
NC (×100)
Relative miR-203 level
B
200
*
180 160 140 120 100 80 60 40 20 0
additional new insights into the molecular mechanism of gene expression regulation. It has been reported that miRNAs regulate gene expression by inducing target mRNAs cleavage or translational repression through binding to its 30 -UTR. Ectopic expression of miRNAs was recently found in cancer and recognized as a biomarker of cancer [13, 14]. Studies on miRNAs expression profiling and their functions in cancer have indeed provided
123
NC
micro up
evidence of tumor development and progression. However, up to date, almost none of identified miRNAs have been investigated to elucidate their precise biological roles in HSCC. Therefore, it is essential to fully assess the biological function and molecular mechanism of cancerrelated miRNAs in HSCC. MiRNA expression patterns are highly specific to different cell-type and cellular differentiation status. A
Mol Cell Biochem
Fig. 2 The effect of miR-203 over-expression on the growth of FaDu cells. a MTT assay of cell proliferation in FaDu cells infected with miR-203-up-LV or NC-LV, values are mean ± SD of six wells. The asterisk under the line denotes statistically significant differences from the control group, P \ 0.05. b MiR-203 over-expression increased colony number of FaDu cells. MiR-203-up-LV and NCLV infected FaDu cells were cultured for 2 weeks and stained with
GIEMSA. Quantitative analysis of colony formation was shown. c Up-regulation of miR-203 accelerated cell cycle progression. PIFACS analysis of cell cycle in FaDu cells infected with miR-203-upLV or NC-LV and the cell percentage at different phases were indicated. Bars represent mean ± SD from three independent experiments run in duplicate. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
123
Mol Cell Biochem
number of miRNAs have been reported to be up-regulated in one type of cancer but down-regulated in other cancers. For some miRNA, both up-regulation and down-regulation have been reported in the same cancer type by different studies. MiR-7 has been reported as up and down-regulated in HNSCC [15]. MiR-34b is widely reported to be upregulated in HNSCC, but this is contrary as miR-34b has been shown to act as a tumor suppressor in a feedback loop with the proto-oncogene Met [16]. MiR-203 is a small, single-stranded, nonprotein-coding RNAs of 22 nucleotides, located at chromosome 14q 32-33. It acts as a tumor suppressor or oncogene by regulating cell differentiation, proliferation, invasion, metastasis, and apoptosis of tumor cells via actions on its target genes [17, 18]. MiR203 exerts a tumor-suppressive role in many types of cancer. For example, miR-203 has been validated as a tumor suppressor in lung, pancreatic, breast, esophageal, hepatocellular, laryngeal cancer as a result of its ability to directly target genes such as Src, Caveolin-1, SNAI2, Ran, and Survivin [19–24]. However, it was reported that miR203 was over-expressed in pancreatic adenocarcinoma samples compared with chronic pancreatitis and normal pancreas samples. The up-regulation of miR-203 was a novel biomarker of poor prognosis in pancreatic adenocarcinoma patients [25]. In epithelial ovarian cancer (EOC), the expression of miR-203 was significantly higher in EOC tissues and the up-regulation of miR-203 may serve as molecular marker to predict the aggressive tumor progression and unfavorable prognosis of EOC patients [26]. In HSCC, miR-203 was reported to be downregulated in few clinical samples of HSCC [27]. However, there is no report on the functional study of miR-203 in FaDu cells. In our study, we provide convincing evidence that miR-203 can promote tumor growth in HSCC. Our results demonstrated that over-expression of miR-203 significantly promoted cell proliferation and accelerated cell cycle progression in FaDu cells. Studies on molecular mechanism of miR-203 in tumorigenesis revealed that TP63 and B3GNT5 may be target genes of miR-203. TP53 family members are shown acting as candidate drivers of miRNAs over-expression [28]. Expression of TP63 is significantly correlated with expression of miRNAs whose promoters contain TP53 family binding sites in head and neck carcinomas [29, 30]. Differential expression of TP63 in verrucous carcinoma of the head and neck correlates inversely with the expression of miR-203 [31]. In our study, the expression of TP63 is remarkable downregulated in miR-203-up-LV-infected FaDu cells, suggesting that TP63 is a target gene of miR-203 in HSCC. B3GNT5 encodes a member of the beta-1, 3-N-acetylglucosaminyltransferase family. This enzyme is a type II
123
Fig. 3 QRT-PCR analysis of miR-203-related target genes in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Bars represent mean ± SD from three independent experiments run in duplicate. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
membrane protein and it is identified as the most likely candidate for lactotriaosylceramide synthase [32, 33]. In our study, the expression of B3GNT5 is obviously downregulated when miR-203 is over-expressed in FaDu cells, suggesting that B3GNT5 is a target gene of miR-203 in HSCC. However, the function of TP63 and B3GNT5 in HSCC is still unclear. There is no report on it by far. Further studies are needed to elucidate the molecular mechanism and signaling pathways controlling occurrence and progression of HSCC. In conclusion, the data presented here suggest for the first time that miR-203 plays carcinogenic role and B3GNT5 and TP63 are identified as target genes of miR-203 in hypopharyngeal tumor. The identification and characterization of the carcinogenic effects of miR-203 may provide a novel therapeutic approach for HSCC treatment.
Mol Cell Biochem
A invasion and metastasis rate
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
B
NC
micro up
Fig. 4 The effect of miR-203 on cell migration in FaDu cells. a Transwell assay in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Migrated cells on the lower surface of the transwell filter were stained by GIEMSA, photographs were taken at 24 h postmigration (magnification, 2009). The number of migrated cells in the miR-203-up-LV group and NC-GFP-LV group were shown.
b Wound scratch assay in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Wound margin distance was measured in the scratch assay. Movement of FaDu cells into the wound was shown for miR-203-up-LV and NC-LV transfected cells and quantification of the wound healing assay. Bars show mean ± SD from three independent experiments run in duplicate, P [ 0.05
Acknowledgments The authors are thankful for the financial support from Beijing Natural Science Foundation of China (7112029).
3. Hoffman HT, Karnell LH, Shah JP, Ariyan S, Brown GS, Fee WE, Glass AG, Goepfert H, Ossoff RH, Fremgen AM (1997) Hypopharyngeal cancer patient care evaluation. Laryngoscope 107:1005–1017 4. Bova R, Goh R, Poulson M, Coman WB (2005) Total pharyngolaryngectomy for squamous cell carcinoma of hypopharynx: a review. Laryngoscope 115:864–869 5. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114. doi:10.1038/nrg2290 6. Nelson KM, Weiss GJ (2008) MicroRNAs and cancer: past, present, and potential future. Mol Cancer Ther 7:3655–3660. doi:10.1158/1535-7163 7. Esquela-Kerscher A, Slack FJ (2006) Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6:259–269
Conflicts of interest
All authors declare no conflicts of interest.
References 1. Davies L, Welch HG (2006) Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg 135:451–457 2. Smith RB, Apostolakis LW, Karnell LH, Koch BB, Robinson RA, Zhen W, Menck HR, Hoffman HT (2003) National cancer data base report on osteosarcoma of the head and neck. Cancer 98:1670–1680
123
Mol Cell Biochem 8. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-DDCt) method. Methods 25(4):402–408 9. Zhang R, Ma L, Zheng M, Ren J, Wang T, Meng Y, Zhao J, Jia L, Yao L, Han H, Li K, Yang A (2010) Survivin knockdown by short hairpin RNA abrogates the growth of human hepatocellular carcinoma xenografts in nude mice. Cancer Gene Ther 17:275–288 10. Fang J, Yu Z, Lian M, Ma H, Tai J, Zhang L, Han D (2012) Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma. Mol Cell Biochem 360:301–307. doi:10.1007/ s11010-011-1069-x 11. Xie J, Tan ZH, Tang X, Mo MS, Liu YP, Gan RL, Li Y, Zhang L, Li GQ (2014) MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis. World J Gastroenterol 20(46):17439–17447. doi:10.3748/wjg.v20.i46. 17439 12. Thomas M, Lieberman J, Lal A (2010) Desperately seeking microRNA targets. Nat Struct Mol Biol 17:1169–1174 13. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 14. Cheng Q, Yi B, Wang A, Jiang X (2013) Exploring and exploiting the fundamental role of microRNAs in tumor pathogenesis. Onco Targets Ther 6:1675–1684. doi:10.2147/OTT. S52730 15. Sethi N, Wright A, Wood H, Rabbitts P (2014) MicroRNAs and head and neck cancer: reviewing the first decade of research. Eur J Cancer 50(15):2619–2635. doi:10.1016/j.ejca.2014.07.012 16. Wang LG, Ni Y, Su BH, Mu XR, Shen HC, Du JJ (2013) MicroRNA-34b functions as a tumor suppressor and acts as a nodal point in the feedback loop with Met. Int J Oncol 42(3):957–962. doi:10.3892/ijo.2013.1767 17. Yi R, Poy MN, Stoffel M, Fuchs E (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452:225–229 18. He JH, Li YM, Li YG, Xie XY, Wang L, Chun SY, Cheng WJ (2013) hsa-miR-203 enhances the sensitivity of leukemia cells to arsenic trioxide. Exp Ther Med 5(5):1315–1321 19. Wang N, Liang H, Zhou Y, Wang C, Zhang S, Pan Y, Wang Y, Yan X, Zhang J, Zhang CY, Zen K, Li D, Chen X (2014) miR203 suppresses the proliferation and migration and promotes the apoptosis of lung cancer cells by targeting SRC. PLoS One 9:e105570 20. Miao L, Xiong X, Lin Y, Cheng Y, Lu J, Zhang J, Cheng N (2014) miR-203 inhibits tumor cell migration and invasion via caveolin-1 in pancreatic cancer cells. Oncol Lett 7(3):658–662 21. Zhang Z, Zhang B, Li W, Fu L, Fu L, Zhu Z, Dong JT (2011) Epigenetic silencing of miR-203 upregulates SNAI2 and contributes to the invasiveness of malignant breast cancer cells. Genes Cancer 2:782–791. doi:10.1177/1947601911429743 22. Zhang F, Yang Z, Cao M, Xu Y, Li J, Chen X, Gao Z, Xin J, Zhou S, Zhou Z, Yang Y, Sheng W, Zeng Y (2014) MiR-203 suppresses tumor growth and invasion and down-regulates MiR21 expression through repressing Ran in esophageal cancer. Cancer Lett 342:121–129. doi:10.1016/j
123
23. Wei W, Wanjun L, Hui S, Dongyue C, Xinjun Y, Jisheng Z (2013) miR-203 inhibits proliferation of HCC cells by targeting survivin. Cell Biochem Funct 31:82–85. doi:10.1002/cbf 24. Bian K, Fan J, Zhang X, Yang XW, Zhu HY, Wang L, Sun JY, Meng YL, Cui PC, Cheng SY, Zhang J, Zhao J, Yang AG, Zhang R (2012) MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin. FEBS Lett 586:804–809 25. Ikenaga N, Ohuchida K, Mizumoto K, Yu J, Kayashima T, Sakai H, Fujita H, Nakata K, Tanaka M (2010) MicroRNA-203 expression as a new prognostic marker of pancreatic adenocarcinoma. Ann Surg Oncol 17:3120–3128 26. Wang S, Zhao X, Wang J, Wen Y, Zhang L, Wang D, Chen H, Chen Q, Xiang W (2013) Upregulation of microRNA-203 is associated with advanced tumor progression and poor prognosis in epithelial ovarian cancer. Med Oncol 30:681. doi:10.1007/ s12032-013-0681-x 27. Kikkawa N, Hanazawa T, Fujimura L, Nohata N, Suzuki H, Chazono H, Sakurai D, Horiguchi S, Okamoto Y, Seki N (2010) miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC). Br J Cancer 103(6):877–884. doi:10.1038/sj.bjc.6605811 28. Knouf EC, Garg K, Arroyo JD, Correa Y, Sarkar D, Parkin RK, Wurz K, O’Briant KC, Godwin AK, Urban ND, Ruzzo WL, Gentleman R, Drescher CW, Swisher EM, Tewari M (2012) An integrative genomic approach identifies p73 and p63 as activators of miR-200 microRNA family transcription. Nucleic Acids Res 40:499–510. doi:10.1093/nar/gkr731 29. Bailey SG, Sanchez-Elsner T, Stephanou A, Cragg MS, Townsend PA (2010) Regulating the genome surveillance system: miRNAs and the p53 super family. Apoptosis 15:541–552. doi:10.1007/s10495-010-0456-1 30. Ory B, Ellisen LW (2011) A microRNA-dependent circuit controlling p63/p73 homeostasis: p53 family cross-talk meets therapeutic opportunity. Oncotarget 2(3):259–264 31. Odar K, Bostjancic E, Gale N, Glavac D, Zidar N (2012) Differential expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins PTEN and p63 in verrucous carcinoma of the head and neck. Histopathology 61(2):257–265. doi:10.1111/j.1365-2559.2012.04242.x 32. Wang Z, Wen L, Ma X, Chen Z, Yu Y, Zhu J, Wang Y, Liu Z, Liu H, Wu D, Zhou D, Li Y (2012) High expression of lactotriaosylceramide, a differentiation-associated glycosphingolipid, in the bone marrow of acute myeloid leukemia patients. Glycobiology 22:930–938. doi:10.1093/glycob/cws061 33. Togayachi A, Akashima T, Ookubo R, Kudo T, Nishihara S, Iwasaki H, Natsume A, Mio H, Inokuchi J, Irimura T, Sasaki K, Narimatsu H (2001) Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids. J Biol Chem 276:22032–22040
Effect of microRNA-203 on tumor growth in human hypopharyngeal squamous cell carcinoma Ru Wang1 • Jugao Fang1,2 • Hongzhi Ma1 • Lin Feng1 • Meng Lian1 Fan Yang1 • Haizhou Wang1 • Qi Wang1,2 • Xiaohong Chen1,2
•
Received: 29 November 2014 / Accepted: 27 March 2015 Ó Springer Science+Business Media New York 2015
Abstract MicroRNAs (MiRNAs) have been recognized to regulate cancer initiation and progression in carcinogenesis as either oncogenes or tumor suppressor genes, but their role in hypopharyngeal cancer development is not clearly defined. To determine whether miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma, we conducted experiments on the functional study of miRNA-203 and identification of miRNA-203 regulated target genes in hypopharyngeal cancer cells. We found that cell proliferation and cell colony-forming increased more in the miRNA-203 upregulated cancer cells than in the negative control cancer cells. Up-regulation of miRNA-203 accelerated cell cycle progression in hypopharyngeal cancer cells. TP63 and B3GNT5 mRNAs were identified and validated as targets of miRNA-203. However, transwell assay and wound scratch assay showed that miRNA-203 did not involve in invasion and metastasis in hypopharyngeal cancer cells. According to the results, we conclude that miRNA-203 can promote tumor growth in human hypopharyngeal squamous cell carcinoma. These results provide the convincing evidence for the first time that up-regulation of miRNA203 contributes to the malignancy of hypopharyngeal squamous cell carcinoma, possibly through down-regulating TP63 and B3GNT5.
& Jugao Fang [email protected] 1
Department of Otorhinolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, China
2
Key Laboratory of Otorhinolaryngology Head and Neck Surgery, Ministry of Education, Beijing Institute of Otorhinolaryngology, Beijing 100005, China
Keywords Hypopharyngeal neoplasm Hsa-mir-203 Cell proliferation Cell cycle
Introduction Hypopharyngeal squamous cell carcinoma (HSCC) is one of the most common head and neck aggressive malignancies, with an incidence of about 10 cases per million people every year [1]. More than 75 % patients with HSCC are at an advanced stage at the time of diagnosis [2]. So HSCC has a very poor prognosis with five-year survival rates ranging from 30 to 35 % [3, 4]. Although the locoregional control of the cancer has been significantly improved in the last decades because of the advent of various new treatments including surgery, radiotherapy, and chemotherapy, survival rates of HSCC patients have not yet markedly improved. Therefore, understanding the molecular oncogenic pathways underlying HSCC can bring insight into the search for improved therapy and prevention of the disease. It is known that microRNAs (miRNAs) are endogenous small non-coding RNA molecules (19-22 bases in length), which regulate protein-coding gene expression by repressing translation or cleaving RNA transcripts in a sequence-specific manner [5]. A growing number of evidence suggest that miRNAs are aberrantly expressed in many kinds of human cancers and that miRNAs play significant roles in initiation, development, and metastasis of these cancers [6, 7]. However, the function of miRNAs in hypopharyngeal cancer development has not been determined. To determine whether microRNA-203 (miR-203) promotes tumor growth in human HSCC, we conducted experiments on the functional study of miR-203 and identification of miR-203 regulated target genes in the HSCC cell line FaDu. MTT assay and colony formation
123
Mol Cell Biochem
assay were performed to evaluate the effect of miR-203 on cell proliferation in FaDu cells. To elucidate how miR-203 affects cell proliferation, we tested cell cycle by PI-FACS in FaDu cells. The expression levels of miR-203 relating target genes were detected by quantitative real-time PCR (qRT- PCR).
Materials and methods Reagents Antibiotic solution (104 U penicillin, 10 mg streptomycin, 25 ug amphotericin B), dimethyl sulfoxide (DMSO), and propidium iodide (PI) were purchased from Sigma Chemicals (St. Louis, MO, USA). Fetal bovine serum (FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Gibco (Cambrex, MD, USA). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). Plasmid Extraction Kit was obtained from Promega (Madison, WI, USA). MiScript Reverse Transcription Kit was purchased from Qiagen (Hilden, Germany), and SYBR Green Master Mix Kit was purchased from TaKaRa (Dalian, China). Cell culture and transfection HSCC cell line FaDu was conserved in our own laboratory. Cells were cultured in DMEM supplemented with 10 % FBS and 100 units/ml penicillin at 37 °C in a humidified 5 % carbon dioxide incubator. According to the manufacturer’s instructions,miR-203 was transfected into cells by lentivirus vectors. MiR-203 over-expression lentivirus vector (miR203-up-LV) and negative control lentivirus vector (NC-LV) harboring green fluorescent protein (GFP) were constructed by Genechem (Shanghai, China). FaDu cells were plated in six-well plates (cell density 20 %) for 24 h, and when the cells reached 30 % confluency, the lentiviruses were added into FaDu cells for transfection. After 24 h transfection, the culture media was replaced with fresh DMEM, and cells were returned to the incubator for additional 48 h transfection. After 72 h transfection, the mean percentage of FaDu cells expressing GFP was calculated under a fluorescence microscope (IX70, Olympus, Japan). RNA isolation and qRT- PCR According to manufacturer’s instructions, total RNA was extracted using Trizol reagent (Invitrogen). For miR-203 qRT-PCR, total RNA was reverse-transcribed with a miRNAspecific primer using the MiScript Reverse Transcription Kit and then qRT-PCR was performed using a miRNA-specific primer on the ABI 9700 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA); U6 snRNA served as an
123
endogenous control for normalization. qRT- PCR analyses for target mRNAs were performed using SYBR Green Master Mix Kit. The primer sequences for PCR amplification of B3GNT5 gene were 5-GGGCCTCGCTACCAATACTTG-3 and 5-CGGAACGTCGATCATAGTTTTCA-3; TP63 gene were 5-GTCATTTGATTCGAGTAGAGGGG-3 and 5-CTG GGGTGGCTCATAAGGT-3; and VSNL1 gene were 5-TGC CTTCCGAACCTTCGAC-3 and 5-CATCTCCACTCGGG TGATCT-3. GAPDH was applied as an internal control. The primer sequences of GAPDH were 5-TGACTTCAACAG CGACACCCA-3 and 5-CACCCTGTTGCTGTAGCCAA A-3. For relative quantification, 2-DDCt was calculated and used as an indication of the relative expression levels [8]. MTT cell proliferation and colony formation assay The MTT cell proliferation and colony formation assays were performed as previously described [9]. After 0, 24, 48, 72, and 96 h of transfection, cell proliferation was evaluated by using the MTT according to manufacturer’s instructions. Briefly, 10 ll of MTT solution was added to the culture medium, and incubated for additional 4 h. The absorbance was determined at 490 nm wavelength. For cell colony formation assay, a total of 800 cells were plated in complete growth media and allowed to grow until visible colonies formed (14 days). Cell colonies were fixed with paraformaldehyde, stained with GIEMSA, washed and air dried. PI-FACS cell cycle assay The cell cycle distribution was analyzed by flow cytometry (FACSCalibur, BD, USA) as described [10]. In brief, cells were suspended in 0.5 ml solution containing 20 lg/ml propidium iodide and 50 lg/ml Rnase, and then analyzed using flow cytometry. Histograms were used to represent the percentage of cells in each phase of the cell cycle (G0/ G1, S, and G2/M). Transwell migration assay Cell invasion assay was performed as described from the BD BiocoatTM MatrigelTM Invasion Chamber (BD Biosciences, Franklin Lakes, NJ, USA). Cells were harvested and resuspended in serum-free medium. 5 9 105 cells of the prepared cell suspension were added into the chamber. The cells were allowed to migrate for 24 h at 37 °C in a humidified incubator with 5 % CO2. Non-invading cells were gently removed from the interior of the inserts with a cotton-tipped swab. Invasive cells were stained with GIEMSA staining. Once the filters had dried, cells were counted in five randomly selected non-overlapping fields.
Mol Cell Biochem
Wound scratch assay Wound scratch assay was performed according to the methods previously described [11]. Cells from each group that was seeded on a six-well plate were scraped with a sterile 100 ul pipette tip to create two linear regions devoid of cells. After scratching, the cells were washed with phosphate-buffered saline (PBS) medium three times and incubated at 37 °C. Cells migrated into the wound surface and average distance of migrating cells was determined under an inverted microscopy at specified time points. The experiments were performed in triplicate and repeated at least three times. Statistical analysis All data were expressed as mean ± SD of three independent experiments, in which each assay was performed in triplicate. The Student t test was used to evaluate the differences between the miR-203-up-LV group and NC-LV group using SPSS 20.0 software. P \ 0.05 was considered as statistically significant.
Results MiR-203 expression was up-regulated by recombinant lentivirus in FaDu cells The lentiviral vector system (miR-203-up-LV and NC-LV) up-regulated the expression of miR-203 in FaDu cells. As expected, a high percentage ([80 %) of FaDu cells expressed GFP after lentivirus transfection (Fig. 1a), indicating the high efficiency and stability of the transfection. Furthermore, the miR-203 level increased in the miR-203up-LV group more than that in the NC-LV group (Fig. 1b). It suggested that the miR-203-up-LV was effective to upregulate miR-203 in FaDu cells. Over-expression of miR-203 promoted cell proliferation in FaDu cells MTT assay and colony formation assay showed that Ectopic expression of miR-203 significantly promoted the growth of FaDu cells. Cell proliferation increased more in the miR203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells (Fig. 2a). Colony formation assay was performed to evaluate the long-term impact of miR-203 overexpression on the proliferation of FaDu cells. The colony size and numbers (Fig. 2b) increased more in the miR-203up-LV infected FaDu cells than in the NC-LV infected FaDu cells. Taken together, these data suggested that miR-
203 may function as an oncogenic regulator of human hypopharyngeal cancer. Over-expression of miR-203 accelerated cell cycle progression in FaDu cells PI-FACS analysis elucidated that miR-203 accelerated cell cycle progression in FaDu cells. The cells in G1 phase decreased more in the miR-203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells. The cells in G2/M phase increased in the miR-203-up-LV infected FaDu cells more than that in the NC-LV infected FaDu cells (Fig. 2c). It suggested that the over-expression of miR-203 decreased the percentage of G1 phase cells and revealed a higher proportion of cells at G2/M phase. In conclusion, miR-203 may regulate cell proliferation by accelerating cell cycle progression in human hypopharyngeal cancer cells. Dysregulation of TP63 and B3GNT5 in miR-203 over-expressed FaDu cells To understand how miR-203 affects the proliferation of FaDu cells further, bioinformatic miRNA target prediction tools (TargetScan, PicTar, and MiRanda) searched three potential miR-203 target genes [12]. TP-63, B3GNT5, and VSNL1 were identified as target genes of miR-203 in HSCC. The expression of TP63 and B3GNT5 mRNA was decreased in the miR-203-up-LV infected FaDu cells more than that in the NC-GFP-LV infected FaDu cells. However, no statistically significant difference was observed in the expression of VSNL1 (Fig. 3). These findings suggested that miR-203 may promote cell proliferation by regulating the expression of TP63 and B3GNT5 negatively in FaDu cells. The effect of miR-203 on migration and invasion ability of FaDu cells Transwell migration assay and wound scratch assay showed that miR-203 did not affect cell migration and invasion ability in FaDu cells. The migration and invasion ability of FaDu cells enhanced more in the miR-203-up-LV infected FaDu cells than in the NC-LV infected FaDu cells (Fig. 4). However, the difference between the two groups was not statistically significance (P [ 0.05). The results illustrated that miR-203 may not be invovled in cell migration and invasion of FaDu cells.
Discussion Dysregulation of oncogene and tumor suppressor gene expression has been identified as one of the critical causes of tumorigenesis. The discovery of miRNAs provides
123
Mol Cell Biochem Fig. 1 The expression of miR203 was dramatically overexpressed by miR-203-up-LV transfection. a Microscope pictures of GFP-expressing cells 96 h after miR-203-up-LV or NC-LV transfection, including fluorescence microscope images and light microscope images (magnification, 1009). b Quantitative analysis of miR203 by qRT-PCR. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
A
micro up (×100)
NC (×100)
Relative miR-203 level
B
200
*
180 160 140 120 100 80 60 40 20 0
additional new insights into the molecular mechanism of gene expression regulation. It has been reported that miRNAs regulate gene expression by inducing target mRNAs cleavage or translational repression through binding to its 30 -UTR. Ectopic expression of miRNAs was recently found in cancer and recognized as a biomarker of cancer [13, 14]. Studies on miRNAs expression profiling and their functions in cancer have indeed provided
123
NC
micro up
evidence of tumor development and progression. However, up to date, almost none of identified miRNAs have been investigated to elucidate their precise biological roles in HSCC. Therefore, it is essential to fully assess the biological function and molecular mechanism of cancerrelated miRNAs in HSCC. MiRNA expression patterns are highly specific to different cell-type and cellular differentiation status. A
Mol Cell Biochem
Fig. 2 The effect of miR-203 over-expression on the growth of FaDu cells. a MTT assay of cell proliferation in FaDu cells infected with miR-203-up-LV or NC-LV, values are mean ± SD of six wells. The asterisk under the line denotes statistically significant differences from the control group, P \ 0.05. b MiR-203 over-expression increased colony number of FaDu cells. MiR-203-up-LV and NCLV infected FaDu cells were cultured for 2 weeks and stained with
GIEMSA. Quantitative analysis of colony formation was shown. c Up-regulation of miR-203 accelerated cell cycle progression. PIFACS analysis of cell cycle in FaDu cells infected with miR-203-upLV or NC-LV and the cell percentage at different phases were indicated. Bars represent mean ± SD from three independent experiments run in duplicate. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
123
Mol Cell Biochem
number of miRNAs have been reported to be up-regulated in one type of cancer but down-regulated in other cancers. For some miRNA, both up-regulation and down-regulation have been reported in the same cancer type by different studies. MiR-7 has been reported as up and down-regulated in HNSCC [15]. MiR-34b is widely reported to be upregulated in HNSCC, but this is contrary as miR-34b has been shown to act as a tumor suppressor in a feedback loop with the proto-oncogene Met [16]. MiR-203 is a small, single-stranded, nonprotein-coding RNAs of 22 nucleotides, located at chromosome 14q 32-33. It acts as a tumor suppressor or oncogene by regulating cell differentiation, proliferation, invasion, metastasis, and apoptosis of tumor cells via actions on its target genes [17, 18]. MiR203 exerts a tumor-suppressive role in many types of cancer. For example, miR-203 has been validated as a tumor suppressor in lung, pancreatic, breast, esophageal, hepatocellular, laryngeal cancer as a result of its ability to directly target genes such as Src, Caveolin-1, SNAI2, Ran, and Survivin [19–24]. However, it was reported that miR203 was over-expressed in pancreatic adenocarcinoma samples compared with chronic pancreatitis and normal pancreas samples. The up-regulation of miR-203 was a novel biomarker of poor prognosis in pancreatic adenocarcinoma patients [25]. In epithelial ovarian cancer (EOC), the expression of miR-203 was significantly higher in EOC tissues and the up-regulation of miR-203 may serve as molecular marker to predict the aggressive tumor progression and unfavorable prognosis of EOC patients [26]. In HSCC, miR-203 was reported to be downregulated in few clinical samples of HSCC [27]. However, there is no report on the functional study of miR-203 in FaDu cells. In our study, we provide convincing evidence that miR-203 can promote tumor growth in HSCC. Our results demonstrated that over-expression of miR-203 significantly promoted cell proliferation and accelerated cell cycle progression in FaDu cells. Studies on molecular mechanism of miR-203 in tumorigenesis revealed that TP63 and B3GNT5 may be target genes of miR-203. TP53 family members are shown acting as candidate drivers of miRNAs over-expression [28]. Expression of TP63 is significantly correlated with expression of miRNAs whose promoters contain TP53 family binding sites in head and neck carcinomas [29, 30]. Differential expression of TP63 in verrucous carcinoma of the head and neck correlates inversely with the expression of miR-203 [31]. In our study, the expression of TP63 is remarkable downregulated in miR-203-up-LV-infected FaDu cells, suggesting that TP63 is a target gene of miR-203 in HSCC. B3GNT5 encodes a member of the beta-1, 3-N-acetylglucosaminyltransferase family. This enzyme is a type II
123
Fig. 3 QRT-PCR analysis of miR-203-related target genes in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Bars represent mean ± SD from three independent experiments run in duplicate. The asterisk above the bar denotes statistically significant differences from the control group, P \ 0.05
membrane protein and it is identified as the most likely candidate for lactotriaosylceramide synthase [32, 33]. In our study, the expression of B3GNT5 is obviously downregulated when miR-203 is over-expressed in FaDu cells, suggesting that B3GNT5 is a target gene of miR-203 in HSCC. However, the function of TP63 and B3GNT5 in HSCC is still unclear. There is no report on it by far. Further studies are needed to elucidate the molecular mechanism and signaling pathways controlling occurrence and progression of HSCC. In conclusion, the data presented here suggest for the first time that miR-203 plays carcinogenic role and B3GNT5 and TP63 are identified as target genes of miR-203 in hypopharyngeal tumor. The identification and characterization of the carcinogenic effects of miR-203 may provide a novel therapeutic approach for HSCC treatment.
Mol Cell Biochem
A invasion and metastasis rate
1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0
B
NC
micro up
Fig. 4 The effect of miR-203 on cell migration in FaDu cells. a Transwell assay in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Migrated cells on the lower surface of the transwell filter were stained by GIEMSA, photographs were taken at 24 h postmigration (magnification, 2009). The number of migrated cells in the miR-203-up-LV group and NC-GFP-LV group were shown.
b Wound scratch assay in FaDu cells infected with miR-203-up-LV and NC-GFP-LV. Wound margin distance was measured in the scratch assay. Movement of FaDu cells into the wound was shown for miR-203-up-LV and NC-LV transfected cells and quantification of the wound healing assay. Bars show mean ± SD from three independent experiments run in duplicate, P [ 0.05
Acknowledgments The authors are thankful for the financial support from Beijing Natural Science Foundation of China (7112029).
3. Hoffman HT, Karnell LH, Shah JP, Ariyan S, Brown GS, Fee WE, Glass AG, Goepfert H, Ossoff RH, Fremgen AM (1997) Hypopharyngeal cancer patient care evaluation. Laryngoscope 107:1005–1017 4. Bova R, Goh R, Poulson M, Coman WB (2005) Total pharyngolaryngectomy for squamous cell carcinoma of hypopharynx: a review. Laryngoscope 115:864–869 5. Filipowicz W, Bhattacharyya SN, Sonenberg N (2008) Mechanisms of post-transcriptional regulation by microRNAs: are the answers in sight? Nat Rev Genet 9:102–114. doi:10.1038/nrg2290 6. Nelson KM, Weiss GJ (2008) MicroRNAs and cancer: past, present, and potential future. Mol Cancer Ther 7:3655–3660. doi:10.1158/1535-7163 7. Esquela-Kerscher A, Slack FJ (2006) Oncomirs—microRNAs with a role in cancer. Nat Rev Cancer 6:259–269
Conflicts of interest
All authors declare no conflicts of interest.
References 1. Davies L, Welch HG (2006) Epidemiology of head and neck cancer in the United States. Otolaryngol Head Neck Surg 135:451–457 2. Smith RB, Apostolakis LW, Karnell LH, Koch BB, Robinson RA, Zhen W, Menck HR, Hoffman HT (2003) National cancer data base report on osteosarcoma of the head and neck. Cancer 98:1670–1680
123
Mol Cell Biochem 8. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-DDCt) method. Methods 25(4):402–408 9. Zhang R, Ma L, Zheng M, Ren J, Wang T, Meng Y, Zhao J, Jia L, Yao L, Han H, Li K, Yang A (2010) Survivin knockdown by short hairpin RNA abrogates the growth of human hepatocellular carcinoma xenografts in nude mice. Cancer Gene Ther 17:275–288 10. Fang J, Yu Z, Lian M, Ma H, Tai J, Zhang L, Han D (2012) Knockdown of zinc finger protein, X-linked (ZFX) inhibits cell proliferation and induces apoptosis in human laryngeal squamous cell carcinoma. Mol Cell Biochem 360:301–307. doi:10.1007/ s11010-011-1069-x 11. Xie J, Tan ZH, Tang X, Mo MS, Liu YP, Gan RL, Li Y, Zhang L, Li GQ (2014) MiR-374b-5p suppresses RECK expression and promotes gastric cancer cell invasion and metastasis. World J Gastroenterol 20(46):17439–17447. doi:10.3748/wjg.v20.i46. 17439 12. Thomas M, Lieberman J, Lal A (2010) Desperately seeking microRNA targets. Nat Struct Mol Biol 17:1169–1174 13. Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297 14. Cheng Q, Yi B, Wang A, Jiang X (2013) Exploring and exploiting the fundamental role of microRNAs in tumor pathogenesis. Onco Targets Ther 6:1675–1684. doi:10.2147/OTT. S52730 15. Sethi N, Wright A, Wood H, Rabbitts P (2014) MicroRNAs and head and neck cancer: reviewing the first decade of research. Eur J Cancer 50(15):2619–2635. doi:10.1016/j.ejca.2014.07.012 16. Wang LG, Ni Y, Su BH, Mu XR, Shen HC, Du JJ (2013) MicroRNA-34b functions as a tumor suppressor and acts as a nodal point in the feedback loop with Met. Int J Oncol 42(3):957–962. doi:10.3892/ijo.2013.1767 17. Yi R, Poy MN, Stoffel M, Fuchs E (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452:225–229 18. He JH, Li YM, Li YG, Xie XY, Wang L, Chun SY, Cheng WJ (2013) hsa-miR-203 enhances the sensitivity of leukemia cells to arsenic trioxide. Exp Ther Med 5(5):1315–1321 19. Wang N, Liang H, Zhou Y, Wang C, Zhang S, Pan Y, Wang Y, Yan X, Zhang J, Zhang CY, Zen K, Li D, Chen X (2014) miR203 suppresses the proliferation and migration and promotes the apoptosis of lung cancer cells by targeting SRC. PLoS One 9:e105570 20. Miao L, Xiong X, Lin Y, Cheng Y, Lu J, Zhang J, Cheng N (2014) miR-203 inhibits tumor cell migration and invasion via caveolin-1 in pancreatic cancer cells. Oncol Lett 7(3):658–662 21. Zhang Z, Zhang B, Li W, Fu L, Fu L, Zhu Z, Dong JT (2011) Epigenetic silencing of miR-203 upregulates SNAI2 and contributes to the invasiveness of malignant breast cancer cells. Genes Cancer 2:782–791. doi:10.1177/1947601911429743 22. Zhang F, Yang Z, Cao M, Xu Y, Li J, Chen X, Gao Z, Xin J, Zhou S, Zhou Z, Yang Y, Sheng W, Zeng Y (2014) MiR-203 suppresses tumor growth and invasion and down-regulates MiR21 expression through repressing Ran in esophageal cancer. Cancer Lett 342:121–129. doi:10.1016/j
123
23. Wei W, Wanjun L, Hui S, Dongyue C, Xinjun Y, Jisheng Z (2013) miR-203 inhibits proliferation of HCC cells by targeting survivin. Cell Biochem Funct 31:82–85. doi:10.1002/cbf 24. Bian K, Fan J, Zhang X, Yang XW, Zhu HY, Wang L, Sun JY, Meng YL, Cui PC, Cheng SY, Zhang J, Zhao J, Yang AG, Zhang R (2012) MicroRNA-203 leads to G1 phase cell cycle arrest in laryngeal carcinoma cells by directly targeting survivin. FEBS Lett 586:804–809 25. Ikenaga N, Ohuchida K, Mizumoto K, Yu J, Kayashima T, Sakai H, Fujita H, Nakata K, Tanaka M (2010) MicroRNA-203 expression as a new prognostic marker of pancreatic adenocarcinoma. Ann Surg Oncol 17:3120–3128 26. Wang S, Zhao X, Wang J, Wen Y, Zhang L, Wang D, Chen H, Chen Q, Xiang W (2013) Upregulation of microRNA-203 is associated with advanced tumor progression and poor prognosis in epithelial ovarian cancer. Med Oncol 30:681. doi:10.1007/ s12032-013-0681-x 27. Kikkawa N, Hanazawa T, Fujimura L, Nohata N, Suzuki H, Chazono H, Sakurai D, Horiguchi S, Okamoto Y, Seki N (2010) miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC). Br J Cancer 103(6):877–884. doi:10.1038/sj.bjc.6605811 28. Knouf EC, Garg K, Arroyo JD, Correa Y, Sarkar D, Parkin RK, Wurz K, O’Briant KC, Godwin AK, Urban ND, Ruzzo WL, Gentleman R, Drescher CW, Swisher EM, Tewari M (2012) An integrative genomic approach identifies p73 and p63 as activators of miR-200 microRNA family transcription. Nucleic Acids Res 40:499–510. doi:10.1093/nar/gkr731 29. Bailey SG, Sanchez-Elsner T, Stephanou A, Cragg MS, Townsend PA (2010) Regulating the genome surveillance system: miRNAs and the p53 super family. Apoptosis 15:541–552. doi:10.1007/s10495-010-0456-1 30. Ory B, Ellisen LW (2011) A microRNA-dependent circuit controlling p63/p73 homeostasis: p53 family cross-talk meets therapeutic opportunity. Oncotarget 2(3):259–264 31. Odar K, Bostjancic E, Gale N, Glavac D, Zidar N (2012) Differential expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins PTEN and p63 in verrucous carcinoma of the head and neck. Histopathology 61(2):257–265. doi:10.1111/j.1365-2559.2012.04242.x 32. Wang Z, Wen L, Ma X, Chen Z, Yu Y, Zhu J, Wang Y, Liu Z, Liu H, Wu D, Zhou D, Li Y (2012) High expression of lactotriaosylceramide, a differentiation-associated glycosphingolipid, in the bone marrow of acute myeloid leukemia patients. Glycobiology 22:930–938. doi:10.1093/glycob/cws061 33. Togayachi A, Akashima T, Ookubo R, Kudo T, Nishihara S, Iwasaki H, Natsume A, Mio H, Inokuchi J, Irimura T, Sasaki K, Narimatsu H (2001) Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids. J Biol Chem 276:22032–22040
