The Role Of MiR-320a And Its Mechanism In Gastric Cancer Cells

Objective:MicroRNAs(miRNAs)are a class of small non-coding RNAs of 18-25 nucleotides in length,which repress protein translation through binding to target mRNAs.miRNAs involve in essential biological functions related to cell growth,migration,invasion and metastasis.Changes in miRNA expression profiles have been observed in a variety of human diseases,including cancer.Gastric cancer(GC)is the fourth most common cancer and the second leading cause of cancer death in the world.Recent evidence showed that changed expressions or activities of specific genes are associated with the pathogenesis of GC,including miRNA.Previous studies in our lab have showed that miR-320a promoted apoptosis in hepatocellular carcinoma(HCC).cells.The objective of this study is to explore the expression of miR-320a in GC and its influence on biological behavior of GC cells and to identify its direct target gene and investigate its role in GC cells.Furthermore,we aim to investigate the molecular mechanisms of miR-320a in GC.The study will provide theoretical basis and targets for cancer therapies.Methods:qRT-PCR was used to detect the different expression of miR-320a in human GC and adjacent normal tissues,and also in two GC cells lines and normal gastric epithelial cells.With the expression of miR-320a was enhanced or decreased in GC cell lines MGC-803 and BGC-823,the cell phenotypes were measured by MTT assay,Transwell migration and invasion assay.Subsequently,the candidate target gene(USP14)of miR-320a was predicted by the bioinformatics analysis and was validated by EGFP reporter experiment.Furthermore,the mRNA and protein level of USP14 in GC cells were detected using the qRT-PCR and Western blot,respectively.After overexpressing or repressing USP14,the changes of cell phenotypes on cell growth,migration and invasion were measured using MTT assay,Transwell migration and invasion assay.Then the rescue experiment was performed to indentify the regulation between miR-320a and USP14.Finally,after overexpresing miR-320a or repressing USP14,the changes of apoptosis were detected by the Flow Cytometer,and the expressions of proteins involved in EMT were measured by Western blot.Results:Compared with normal gastric tissues and cells,miR-320a was down-expressed in both of the GC tissues and cells.According to the MTT assay,Transwell migration and invasion assay,overexpression of miR-320a suppressed the proliferation activity,migration and invasion capability in MGC-803 and BGC-823 cells.Subsequently,the EGFP reporter experiment,qRT-PCR and Western blot confirmed that miR-320a could directly bind to mRNA 3’UTR and negatively regulated the eandidate target gene USP14.Meanwhile,we found that proliferation activity,migration and invasion capability of MGC-803 and BGC-823 cells were reduced after knockdown of USP14.In contrast,overexpression of USP14 can rescue the changes of cell phenotypes caused by ectopic expression of miR-320a.Finally,Flow Cytometer results revealed that overexpression of miR-320a or knockdown of USP14 could promote apoptosis,and Western blot results showed that the expression level of E-cadherin was increased,while Vimentin and ICAM-1 were decreased dramatically.Conclusions:All the results demonstrated that miR-320a functions as a tumor suppressor that suppressed the proliferation activity,migration and invasion capability of GC cells through negative regulation of USP14.