Dysregulated Mir-27A-3P Promotes Nasopharyngeal Carcinoma Cell Proliferation and Migration by Targeting Mapk10

ONCOLOGY REPORTS 37: 2679-2687, 2017 Dysregulated miR-27a-3p promotes nasopharyngeal carcinoma cell proliferation and migration by targeting Mapk10 LIHUA LI1 and ZHAOHUI LUO2 1Department of Radiotherapy, Hunan Cancer Hospital, Changsha, Hunan 410013; 2Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, P.R. China Received November 6, 2016; Accepted January 16, 2017 DOI: 10.3892/or.2017.5544 Abstract. miRNA-27a-3p is an important regulator of carci- it plays an important role in the occurrence and progression nogenesis and other pathological processes. However, its of cancer. Zhou et al revealed that miR-27a-3p functions as an role in laryngeal carcinoma is still unknown. In our previous oncogene in gastric cancer by targeting BTG2 (8). miR-27 may research, we found that miR-27a-3p expression was upregu- also play a key role in osteoblast proliferation, apoptosis and lated in nasopharyngeal carcinoma (NPC) using a microarray differentiation through the post-transcriptional regulation of chip. In the present study, we identified miR-27a-3p as an sFRP1 (9). Additionally, miR-27a/b promoted angiogenesis by endogenous promoter of metastatic invasion. The expression targeting the angiogenesis inhibitor SEMA6A, which controls levels of miR-27a-3p were correlated with human metastatic the repulsion of neighboring endothelial cells (10). progression outcomes and Kaplan-Meier survival. In silico Research on nasopharyngeal carcinoma (NPC) has database analyses revealed that Mapk10 is a potential target of mirrored other cancer research programs. Studies have largely miR-27a-3p, and luciferase reporter assay results revealed that focused on molecular defects, including the dysregulation miR-27a-3p directly inhibits the Mapk10 3' untranslated region of miRNAs. To date, several miRNAs have been shown to (3'UTR). Real-time PCR and western blotting results ascer- target specific mRNAs to regulate the progression of NPC. tained that Mapk10 expression was regulated by miR-27a-3p. miR-216b (11), miR-18a (12), miR-218 (13), miR-26a/b (14,15), In addition, miR-27a-3p gain-of-function promoted cell prolif- miR-10b (16), let-7 (17), miR-141 (18) and miR-200a (19) eration, migration and invasion in 5-8 F NPC cells. These have been shown to possess tumor-suppressive functions in effects partially depended on Mapk10, and loss of miR-27a-3p NPC. Not surprisingly, Epstein-Barr virus-encoded miRNAs function had the opposite effects. have oncogenic properties (20-22). Our previous research described the roles of miR-18a in the progression of NPC Introduction and the mechanism by which miR-18a promotes malignant tumor progression. Effort has also been made to associate Aberrant expression of microRNAs (miRNAs) and of factors miR-18a and Dicer with NPC (12). miR-27a-3p was revealed in their biogenesis have been frequently observed in different to be upregulated in miRNA expression profile studies from types of cancer (1). Disruption of miRNA expression patterns two independent laboratories. Using deep sequencing screen is now recognized as a hallmark of human cancer (2-4). Each laser-microdissected biopsies from 12 NPC and 8 chronic miRNA is estimated to regulate an average of several hundred nasopharyngitis patients, Zhang et al identified miR-27a-5p protein-coding genes, and ~60% of proteins in cells are as differentially expressed (23). In a previous study, we also thought to be regulated by miRNAs (5,6). Changes in miRNA demonstrated that miR-27a-3p was significantly upregulated expression patterns have been linked to profound effects on during NPC progression (24), suggesting an oncogenic role for cell phenotypes, and miRNAs have an emerging role in diverse miR-27a-3p in NPC. physiological and pathological processes (7). Notably, the TargetScan, PicTar, miRNAome and miRanda predicted dysregulation of miR-27a in various types of cancer suggests thousands of target genes for miR-27a-3p. The method used to select putative target genes of miR-27a-3p is another high- light of the present study. Since miRNAs downregulate gene expression by targeting the 3' untranslated region (3'UTR) of Correspondence to: Dr Zhaohui Luo, Department of Neurology, mRNAs (24,25), NPC cDNA expression data (GSE12452) Xiangya Hospital, Central South University, 87 Xiangya Road, were downloaded from the publicly accessible National Kaifu, Changsha, Hunan 410008, P.R. China Center for Biotechnology Information-Gene Expression E-mail: [email protected] Omnibus (GEO). We identified Mapk10 as a target gene of miR-27a-3p, and the cDNA data revealed that Mapk10 expres- Key words: miR-27a-3p, Mapk10, nasopharyngeal carcinoma, sion was downregulated in NPC. metastatic invasion, proliferation In the present study, we demonstrated that miR-27a-3p, which was predicted to target Mapk10 in silico, was upregulated in tumors of various origins and in the 5-8 F cell line. 2680 LI and LUO: miR-27a-3p targets Mapk10 in nasopharyngeal carcinomas Furthermore, we determined that Mapk10 expression was MTT assay. Cells (5-8 F) were seeded at a density of inversely correlated with miR-27a-3p expression, suggesting 2x103 cells/well in 96-well plates 24 h before transfection. The that miR-27a-3p may regulate Mapk10. Our results indicated cells were incubated in growth medium for 24 h. A volume of that miR-27a-3p directly binds to and downregulates Mapk10 20 µl of MTT solution (5 mg/ml) was added to each well, and the levels in vitro. Importantly, the miR-27a-3p-mediated increase plates were incubated at 37̊C for an additional 4 h. The media in NPC cell proliferation, migration and invasion partially was removed, and 150 µl dimethyl sulfoxide (DMSO) was added depended on Mapk10. Altogether, our results demonstrated to each well. The plates were shaken for 10 min to dissolve the that miR-27a-3p regulates Mapk10 and provide evidence for MTT formazan crystals. The optical density (OD) of each well the development of novel cancer therapies. was determined with a scanning multi-well spectrophotometer at a wavelength of 490 nm. The experiment was repeated 3 times, Materials and methods and 6 parallel samples were assessed each time. Cell lines and human subjects. The NPC cell lines 5-8 F, Wound closure assay. The cells were transfected with 6-10 B and HK-1 (Cancer Research Institute of Central synthetic miRNA mimics or the 2'-O-methyl-modified inhibi- South University, Changsha, China) were cultured in RPMI- tors in a 6-well dish until they reached 90% confluency. A 1640 medium supplemented with penicillin G (100 U/ml), wound was created using a sterile 10-µl pipette tip, and then streptomycin (100 mg/ml) and 10% fetal calf serum (FCS). the dish was washed with 1X phosphate-buffered saline (PBS) Immortalized normal nasopharynx epithelial NP69 cells were to remove detached cells. Subsequently, the cells were cultured cultured in RPMI-1640 medium supplemented with peni- in medium with 2% serum and treated with 5 mM hydroxy- cillin G (100 U/ml), streptomycin (100 mg/ml), 10% FCS and urea (26). Migration at the wound site was documented using growth factors. The cells were grown at 37̊C in a humidified a microscope (Nikon Corporation, Tokyo, Japan) at different atmosphere of 5% CO2 and were routinely sub-cultured using time-points (0, 24 and 48 h). 0.25% (w/v) trypsin-EDTA solution. Transwell migration assay. Before the cells were seeded, Ethical standards. All procedures were in accordance with Corning Costar Transwell 24-well plates (8-µm pores) Corning, the standards of the relevant Ethics Committees for human Corning, NY, USA) were coated with Matrigel (BD Biosciences, experimentation (institutional and national) and with the 1964 Franklin Lakes, NJ, USA) and placed in the cell culture hood and later versions of the Declaration of Helsinki. for 1 h at 37̊C. A total of 1x105 cells were seeded in the wells after they were transfected, and were cultured in medium with Vectors, oligonucleotides, antibodies and siRNA. Synthetic 2% serum. Normal growth medium was placed in the bottom miRNA mimics and 2'-O-methyl-modified inhibitors, wells. The cells were then allowed to migrate for 24 or 48 h. The ‘antagomiRs’, were purchased from the GenePharma Company migrated cells were fixed with 100% methanol for 1 min and (Shanghai, China). An miR-insensitive Mapk10 construct was allowed to air dry. The cells that invaded the lower surface of purchased from GeneCopoeia Company (Guangzhou, China). the membrane were stained by dipping the inserts in a staining The rabbit anti-human Mapk10 antibody was purchased from solution for 20 min. The stained cells were then counted. Cell Signaling Technology, Inc. (Beverly, MA, USA). The Mapk10 siRNA (S11163) was purchased from Life Technologies Western blotting assay. The protein used for western blot- Company (Shanghai, China). The Mapk10-3 siRNA produced ting was extracted using mRIPA buffer containing protease the best results and was used in subsequent experiments. inhibitors (50 mM Tris, pH 7.4; 100 mM NaCl; 1% Nonidet P-40; 0.5% deoxycholic acid; 0.1% SDS; 10 µg/ml aprotinin; Quantitative real-time PCR. Expression of the miRNAs was 10 µg/ml leupeptin; and 1 mM PMSF). The proteins were evaluated using SYBR-Green quantitative real-time RT-PCR quantified using the BCA™ protein assay kit (Pierce, Rockford, (Takara, Dalian, China). Total RNA was extracted from cells IL, USA). The western blotting system was established using and samples using TRIzol® reagent (Invitrogen, Carlsbad, CA, a Bio-Rad Bis-Tris gel system according to the manufacturer's USA). The individual miRNA qPCR Quantitation kits were instructions (Bio-Rad, Hercules, CA, USA). The primary purchased from GenePharma Company. The quantitative PCR antibodies were prepared in 5% blocking buffer. The primary was performed according to the manufacturer's instructions. antibody (against Mapk10 or GAPDH) was incubated with the The qPCR cycle was 98̊C for 2 min, 40 cycles of 95̊C for membrane overnight at 4̊C.

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