LncRNA Unigene56159 Promotes The Migration And Invasion In Hepatocellular Carcinoma Cells

【Objective】HCC(hepatocellular carcinoma,HCC)is one of the most common prevalent cancers worldwide.High recurrence and metastasis are the most important factors affecting prognosis and mortality.Various factors and epigenetic changes are closely associated with hepatocarcinogenesis,including long non-coding RNAs(lnc RNAs).Lnc RNAs are a class of more than 200 nt endogenous RNA molecules,with no protein coding capacity and can regulate gene expression at multiple aspects,involved in various physiological and pathological processes.Massive evidence have revealed that amounting lnc RNAs are involved in the progress of human diseases,particularly in human cancers.To investigate the role of lnc RNAs in the development of HBV-related HCC,we identified a new highly expressed lnc RNA-Unigene56159 in HBV positive HCC tissues via deep sequencing.The present study aims to explore the regulatory mechanism of aberrently expression of Unigne56159 in HBV positive HCC cells,and identify the effects of Unigene56159 on cell growth,migration and invasion ability in vitro.Furthermore,the molecular biological mechanisms and possible regulatory networks were investigated.This study will help us enrich our understanding of HBV induced-lnc RNA,and provide novel insights and new theoretical basis for the therapeutic strategies of HBV-related HCC.【Methods】First,RT-qPCR assay was used to detect the expression level of Unigene56159 in HBV negative and HBV positive HCC tissues and hepatoma cell lines.Then Hep G2 and Huh7 cells were transiently transfected with HBx to examine the expression level of Unigene56159.Transwell migration and invasion assays,colony formation assay and MTT assay were carried out to analyze the effects of Unigene56159 on cell migration and invasion ability,cell growth in vitro.Besides,we assesed the protein levels of E-cadherin and vimentin which involved in EMT process by Western blot.Bioinformatics was used to predict the candidate miRNAs for Unigene56159,and EGFP fluorescent reporter assay and RT-qPCR were used to confirm that Unigene56159 was a direct target of miR-140-5p and negatively regulated by it.Moreover,we examined the effects of miR-140-5p on HCC cell migration and invasion and screened the target gene Slug for miR-140-5p.Then the results showed that Slug is directly targeted by miR-140-5p.In addition,the m RNA and protein levels of Slug were evaluated through RT-qPCR and Western blot with the up-regulation or down-regulation of Unigene56159.Moreover,the EGFP fluorescent reporter assay was used to explore the miR-140-5p dependency of Unigene56159-mediated regulation.【 Results 】 Compared with HBV negative HCC tissues,Unigene56159 was up-regulated in HBV positive HCC tissues by RT-qPCR assay.Moreover,Unigene56159 expression level in Hep G2.2.15 cells was significantly higher than that in Hep G2 cells.Unigene56159 was also found to be up-regulated in Huh7 cells after transfected with p HBV1.3.Unigene56159 was up-regulated in Hep G2 and Huh7 cells after transfected with HBx plasmid,indicating that HBx induced the high expression of Unigene56159.A series of assays showed that Unigene56159 can promote the migration and invasion ability through promoting the progress of EMT in HCC,while it had no effect on cell growth and viability of Hep G2 and Hep G2.2.15 cells.Bioinformatics was used to predict the miRNAs for Unigene56159,EGFP fluorescent reporter assay and RT-qPCR were used to prove that Unigene56159 is a direct target of miR-140-5p and negatively regulated by miR-140-5p.Moreover,we found that miR-140-5p inhibited the migration and invasion ability in HCC cells.The candidate genes that targeted by miR-140-5p were predicted by bioinformatics,and verified by the EGFP fluorescent reporter assay,RT-qPCR and Western blot.Further experiments showed that the m RNA and protein levels of Slug were positively regulated by Unigene56159.Additionally,we found that Unigene56159 can act as a ce RNA to regulate the expression level of Slug in a miR-140-5p-dependent manner.【Conclusions】 In the present study,we identified a significantly up-regulated novel lnc RNA-Unigene56159 in HBV positive HCC tissues,which was induced by HBx protein.Unigene56159 acts as an oncogene,which promoting the migration and invasion ability of hepatocellular carcinoma cells by through affecting EMT progress.Further exploration showed that Unigene56159 competed with Slug for miR-140-5p.