The Study Of LncRNA TRERNA1 Up-regulated By HBx On Promoting Tumor Progression And Poor Prognosis In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is a malignant tumor disease with high recurrence rate and high mortality.The X protein(HBx)encoded by HBV(Hepatitis B virus)plays various biological roles in the process of proliferation and metastasis of hepatocarcinoma through genetic aberrations and disorders,epigenetic modification,non-coding RNA(nc RNA)regulation.Long non-coding RNA(lncRNA)defined as being longer than 200 nucleotidesis that does not encode protein.It may participate in the biological processes of various tumors by means of signal,decoy,guide or scaffold.The results of lncRNA in HCC suggest that lncRNA can affect the proliferation,metastasis,apoptosis and other functions of HCC.HBx can also participate in the development of liver cancer by regulating the expression of related molecules,especially epigenetic regulation.Further exploration of lncRNAs induced by HBx and its mechanism of action in the malignant progression of tumors will contribute to the effective diagnosis and targeted therapeutic strategies in HCC.The differentially expressed lncRNAs and m RNAs induced by HBx in HCC cells were screened using Human Lnc RNA Array and Human m RNA Array analysis.TRERNA1 was considered as a candidate because of its relatively high expression level after transfection with HBx using Lnc RNA Array.The regulation of HBx to lncRNA TRERNA1 was detected by HBx plasmid transfection and RNAi.Quantitative Real-time PCR(q-PCR)assay showed a negative correlation between TRERNA1 and HBx in liver cancer tissues and cells.The effect of TRERNA1 on cell migration and invasion was analyzed by wound healing assay,transwell migration assay and matrigel invasion assay.Metastatic model of tail vein injection and H&E staining in nude mice were used to detect the effect of TRERNA1 on metastasis in vivo.The potential regulatory mechanisms of TRERNA1 to its target gene CDH1 were explored by q PCR and WB assays.RIP and IP assay were employed to detect the physical association among TRERNA1,EHMT2 and SNAI1.The binding of EHMT2 to CDH1 promoter region was analyzed by Ch IP assay.Q-PCR was used to detect the expression of CDH1 in clinical cases and evaluate the relationship between CDH1 and TRERNA1.RNA in situ hybridization was used to assess the prognosis of TRERNA1 in HCC cases.The biological functions and signaling pathways that TRERNA1 may participate in were analyzed by GO and KEGG Pathway.CCK-8 assay,plate colony formation,flow cytometry and xenograft mice tumorigenicity assay were used to assess the proliferation of HCC cells.Cell cycle associated proteins were detected by western blot.CCK-8 assay was performed to determine the impact of TRERNA1 expression on the sensitivity of HCC cells to sorafenib.At the same time,western blot was used to detect the effect of TRERNA1 on Raf-MEK-ERK phosphorylation pathway.Through the present study,the results showed as following:1)LncRNA TRERNA1 was up-regulated by HBx,and there was a dose-dependent manner between TRERNA1 expression and HBx expression.2)The expression of TRERNA1 was not only positively correlated with tumor metastasis and staging grade,but also with poor prognosis in HCC cases.3)In vitro and in vivo,increased TRERNA1 expression promoted cell migration and invasion.TRERNA1 also promoted the proliferation of hepatoma cells and the tumorigenicity of xenografts in nude mice.4)High expression of TRERNA1 reduced the sensitivity of sorafenib to HCC cells,it was associated with poor prognosis of HCC.5)Combined with GO and KEGG Pathway analysis,the differential genes regulated by TRERNA1 were involved in cell proliferation and cell cycle.6)As a scaffold,TRERNA1 repressed CDH1 expression via recruiting EHMT2/SNAI1 complex to di-methylate H3K9 of CDH1 promoter region.In the meantime,TRERNA1 regulated its neighboring gene SNAI1 as an enhancer-like lncRNA to promote HCC migration and invasion.7)TRERNA1 inhibited the activity of p21 protein and enhanced the expression of cell cycle regulatory molecules Cyclin D1,CDK2 and CDK4,which accelerated cell cycle progression of G1/S phase and promoted the proliferation of HCC.8)TRERNA1 reduced the sensitivity of sorafenib to HCC cells by activating the phosphorylated Raf-MEK-ERK signaling pathway.Taken together,we have revealed for the first time that lncRNA TRERNA1 induced by HBx inhibits the expression of CDH1 by recruiting the EHMT2/SNAI1 complex to promote HCC metastasis.TRERNA1 reduces the sensitivity of sorafenib to HCC cells by activating the phosphorylated Raf-MEK-ERK signaling pathway.The study of lncRNA TRERNA1 will help reveal HCC progression and evaluate the efficacy of HCC-targeted drug therapy,which will provide new research strategies for the diagnosis and treatment of HCC patients.