Preliminary Study On The Affect And The Potential Mechanism Of Overexpression Of LncRNA TRERNA1 On The Sensitivity Of Hepatocellular Carcinoma Cells To Sorafenib

Background: Hepatocellular carcinoma(HCC)is one of the increasingly serious malignant tumors worldwide.Its morbidity and mortality are the second in cancer varieties.There are some problems including limited curative effect and poor prognosis in HCC.Especially in the middle and advanced stage of hepatocellular carcinoma,surgical treatment generally has some great troubles.Sorafenib is a first-line clinical drug for advanced hepatocellular carcinoma,and a multi-kinase inhibitory and multi-target oral drug.Hepatitis B virus(HBV)is the main inducer of hepatocellular carcinoma.HBx(hepatitis B virus X protein)is an important multifunctional regulatory protein encoded by the HBV genome,with considered as the main inducing factor for the formation of hepatocellular carcinoma.Relevant researches have demonstrated that protein HBx of HBV can induce high expression of lncRNA in hepatocellular carcinoma.Based on previous researches in the laboratory,large quantities of lncRNAs induced by HBx were screened by high throughput LncRNA chip.It was discovered that LncRNA TRERNA1 was highly expressed in hepatocellular carcinoma cells and was thought to be associated with the occurrence and development of hepatocellular carcinoma.Objective: Investigating the specific affect of HBx-induced lncRNA TRERNA1 on the proliferation and sensitivity of hepatocellular carcinoma cells to sorafenib as well as further exploring the possible signaling pathways involved in the affect of lncRNA TRERNA1 on sorafenib sensitivity.Methods: Firstly,the expression level of lncRNA TRERNA1 in 110 patients with hepatocellular carcinoma and adjacent cancers was detected by RNA in situ hybridization technique,to analyze the correlation between the expression level of TRERNA1 and theoccurrence and development of HCC.The correlation between TRERNA1 expression and prognosis of HCC was analyzed by pathological data.The over-expression TRERNA1 cell model in HepG2 cells was constructed by using plasmid pcDNA3.1 vector,and stable low-expression TRERNA1 cell model in HepG2.215 cells(which can express HBV genome)was constructed by using lentiviruses vector with transfected siRNA of TRERNA1.Then,we use CCK-8 method and plate cloning experiment to analyzed the effects of TRERNA1 on the proliferation of hepatocellular carcinoma cells.Furthermore,the sensitivity of hepatocellular carcinoma cells to sorafenib(a chemotherapeutic drug for hepatocellular carcinoma)and the correlation between TRERNA1 and the survival rate of hepatocellular carcinoma cells were analyzed by CCK-8 assay.Secondly the cancer-related signaling pathways and genes affected by TRERNA1 knockdown were screened by RNA-seq sequencing analysis,and to verify the results by Western Blot experiment.Fifteen genes related to Wnt signaling pathway induced by TRERNA1 knockdown were analyzed by tumor gene mapping database(TCGA),to screene out Genes associated with hepatocellular carcinom.RNA-seq results were verified by qPCR in over-expressed TRERNA1 HepG2 cells and HepG2.215 knockdown TRERNA1.Western blot was used to detect the expression of β-catenin protein in the nucleus of HepG2 cells over-expressing TRERNA1 and HepG2.215 cells under-expressing TRERNA1,in order to determine whether TRERNA1 is involved in regulating Wnt signaling pathway.Western Blot was also used to detect the expression of classical cancer-related target proteins downstream of Wnt signaling pathway,and to further determine which related proteins regulated by TRERNA1 through Wnt signaling pathway to affect the susceptibility of sorafenib.Results:1)RNA in situ hybridization analysis demonstrated that the expression level of TRERNA 1 in hepatocellular carcinoma tissues was higher than that in paracancerous tissues(P =0.0294)2)The results of five-year survival data analysis illustrated that the expression level of TRERNA1 was significantly correlated with the prognostic survival rate of HCC patients(P=0.0084).The prognostic survival time of HCC patients with high expression of TRERNA1 was less than that of patients with low expression level of TRERNA1.3)Cell proliferation experiments in constructed cells with over-expression and knockdown of TRERNA1 demonstrated that TRERNA1 could promote the cell proliferation and foci formation of HCC cells.4)The affect of TRERNA1 on the sensitivity of sorafenib to HCC cells demonstrated that TRERNA1 could effectively reduce the sensitivity of hepatocellular carcinoma cell to sorafenib.5)RNA-seq data of HepG2.215 cells knocked down TRERNA1 and analysis of its effect on cancer-related signaling pathways illustrated that knockdown TRERNA1 could significantly promote up-regulation of Wnt signaling pathway-related genes(15 genes).6)RNA-seq was further validated by TCGA database and qPCR experiments.The results illustrated that the knockdown of TRERNA1 in HepG2.215 cells could increase the expression level of Wnt signaling pathway antagonists SFRP1 and SFRP5(secreted frizzled-related protein 1,5).7)Western blot results demonstrated that the overexpression of TRERNA1 could increase the protein level of β-catenin in the nucleus and effectively promote the up-regulation of Wnt signaling pathway,thus promoting the expression of c-JUN、 c-myc、CD44 and cyclinD1,which were the downstream target molecules of wnt.Conclusion1.Overexpression of TRERNA1 is associated with poor prognosis of HCC2.Overexpression of TRERNA1 promotes cell proliferation of HCC cells.3.TRERNA1 can activate the Wnt signaling pathway by inhibiting the expression of SFRP1 and SFRP5.c-JUN,c-myc,CD44 and cyclinD1 proteins can promote the proliferation of hepatocellular carcinoma cells and reduce the sensitivity to sorafenib.