MiR-214Suppresses The Growth And Invasiveness Of Cervical Cancer Cells By Targeting GALNT7

Objective:Cervical cancer is a complex disease involving the abnormal expression of many oncogenes and tumor suppressor genes. Previous studies have revealed several genes associated with human cervical cancer. microRNA(miRNA) is a class of single-stranded non-coding regulatory small molecule RNA, can bind to the3’untranslated regions (3’UTRs) of target mRNAs through base pairing, either preventing their translation or causing target degradation (the post-transcriptional regulation). The regulation of target gene expression levels are regulated by miRNAs, and it plays a very important role in tumorigenesis and development. GALNT7is one member of the UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (GalNAc-T or GALNT) family. GALNTs initiate mucin-type O-linked glycosylation in the Golgi apparatus by catalyzing the transfer of GalNAc to serine and threonine residues on target proteins. Because O-linked glycosylation proceeds step-wise, addition of GalNAc to serine or threonine represents the first committed step in mucin biosynthesis. Despite this apparent simplicity, multiple GALNT family members appear to be necessary to fully glycosylate their protein substrates. Aberrant glycosylation is a well described hallmark of many human cancers and is associated with cell growth, differentiation, transformation, adhesion, metastasis, and tumor immune surveillance.Methods:Firstly, we used bioinformation and identified that GALNT7is a direct target gene of miR-214, and the reliability was confirmed by EGFP reporter experiment. The mRNA levels and protein levels of GALNT7in miR-214over-expressed cervical cancer cells or miR-214low-expresssed cervical cancer cells were detected with qRT-PCR and Western blot. In order to confirm the regulating role of miR-214in target gene GALNT7expression, miR-214and GALNT7’s expression were tested in human cervical cancer tissues and adjacent normal tissues with qRT-PCR and Immunohistochemistry. Then the cell phenotype of miR-214(overexpression and lowexpression) was detected in human cervical cancer cells using MTT assay, colony formation assay, and transwell assays. The knockdown of GALNT7markedly inhibits cervical cancer cell proliferation, migration and invasion; whereas ectopic expression of GALNT7significantly enhances these properties, indicating that GALNT7might function as an oncogene in cervical cancer. The restoration of GALNT7expression can counteract the effect of miR-214on cell proliferation, migration and invasiveness of cervical cancer cells.Results:We identified GALNT7as one of the candidate target genes for miR-214. The GALNT7mRNA3’UTR contains the potential binding site of miR-214. The EGFP reporter experiment also confirmed that miR-214can directly bind to the GALNT7mRNA3’UTR and negatively regulate the gene expression. After overexpression or lowexpression of miR-214in cervical cancer cell lines, mRNA level and protein level of GALNT7were changed by using qRT-PCR and Western blot. Campared to adjacent normal tissues, the expression of miR-214in cervical cancer tissues was lower and the GALNT7mRNA and protein’s expression were higher. Finally, we found that after knockdown of GALNT7, the cell proliferation, migration and invasiveness activity were all inhibited. In cervical cancer cell lines, overexpression of miR-214can not only effect the cells viability but also decrease the colony formation, migration and invasiveness, and vice versa. Knockdown of GALNT7inhibits the proliferation, migration and invasiveness of HeLa and C33A cells. Restoration of GALNT7counteracts the effects of miR-214expression.Conclusion:miR-214is expressed at a low level in cervical cancer compared with normal cervical tissues, and overexpression of miR-214inhibits cell growth and invasion. A new target gene of miR-214, GALNT7, was found to be up-regulated in cervical cancer tissues. These findings indicate that inhibition of miR-214in cervical cancer may contribute to the malignant phenotype by maintaining a high level of GALNT7. Thus, the identification of miR-214and its target gene, GALNT7, in cervical cancer may help us to understand potential molecular mechanisms of tumorigenesis and may provide new prognostic markers for the management of cervical cancer in the future.