RAB3B Is Down-Regulated By MiR-92a And Contribute To Malignancy In Liver Cancer Cells

[Objective]As a malignancy,hepatocellular carcinoma(HCC)is one of the commonest solid cancer in world and the death rate just lower than gastric cancer and esophageal cancer.The development of HCC is associate with many reasons,but the major includes HBV and HCV infection,aflatoxin.Some studies in recent years show that many genes which had change of expression or activity can affect the development of cancer,like microRNAs,which are a class of small non-coding RNAs that are about22 nucleotides,can affect the development of liver tumor.MicroRNAs can regulate gene expression transcriptionally or post-transcriptionally,involve in cell growth,proliferation,differentiation and apoptosis.Recent studies show that a variety of miRNAs are involved in the regulation of cancer cells through changing target genes,which's function as on congeners or tumor suppressors.RAB3 B is a member of the RAS family and play an important role in intracellular signal transduction.Recent reports show that it may connected to the development of tumors.However,the effect of RAB3 B on the malignant behavior of liver cancer cells is not clear.This study discusses the role of RAB3 B in the development of hepatocellular carcinoma and its mechanism of upstream or downstream and providing a new perspective of the molecular diagnosis and treatment of liver cancer in the future.[Methods]First,we detected the miR-92 a expression level in human normal liver cell and cancer cell by real-time RT-PCR assay.Second,by using colon formation,MTT assay,wound healing assay and transwell migration and invasion assay to detect the effect of overexpress or blocked miR-92 a in both liver cancer cells QGY-7703 and HepG2.Besides,western blot were performed to investigate the expression difference of EMT related markers.At the same time,we predicted and screened the target gene of miR-92 a by bioinformatics method,we choosed RAB3 B as our final target gene.And we verified the direct target function by EGFP experiments.Through real-time RT-PCR and western blot are used to prove the direct regulation and the possibleregulatory mechanisms.Subsequently,RNA interfere technique was used to analyze the change of cell phenotype after target gene expression,and to study the specific function of target gene.Finally,we took the method of mass spectrometry analysis to select proteins which have interact with RAB3 B,detact the change of ATP in HCC cells which overwxpress RAB3 B.[Results]The real-time PCR results showed that the expression of miR-92 a is upregulated in hepatocellular carcinoma,overexpression of miR-92 a increased the migration and invasion significantly in QGY-7703 and HepG2 cells,accelerated cell proliferation and apoptosis,and the expression levels of E-cadherin,the molecular marker of epithelial cells,were decreased whereas the mesothelial cells marker Vimentin were increased.Through the experiment confirmed that RAB3 B play a target gene role of miR-92 a,and through deep study of the function of RAB3 B we found that overexpressed RAB3 B can promote cell migration and invasion,and can promote the process of EMT.Real-time PCR and Western blot experiments showed that overexpression or blocking of miR-92 a could decrease or increase the mRNA and protein expression levels of RAB3 B in HCC cells.By the interaction of protein spectrum analysis,we could find that RAB3 B may be associated with ATP synthesis and overexpressed RAB3 B can increase the production of ATP.[Conclusion]RAB3B may be closely associated with the occurrence of liver cancer development and be regulated by the miR-92 a.What's more,it can promote liver cancer cell proliferation and migration and invasion ability,participate in the process of EMT and promote the synthesis of ATP at the same time.