The Effect And Mechanism Of Long Non-coding RNA NEAT1 On Cellular Autophagy Of Hepatocellular Carcinoma

Objective: Hepatocellular Carcinoma(HCC)is the third most common cause of cancerrelated death worldwide.The early diagnosis of HCC is difficult,it has a rapid progression,and it is prone to clinical drug resistance and poor prognosis.Although surgery,radiotherapy and chemotherapy can delay survival rate of patients to some extent,most patients still cannot avoid metastasis and recurrence.Therefore,it is of great significance to elucidate the molecular mechanism of the development of HCC and explore the targets in the process of therapy of HCC.Long non-coding RNA(lnc RNA)plays an important role in the development of different tumors and its relationship with the development of HCC has been reported.NEAT1 is a new non-coding RNA,researchers had found its can participate in a variety of tumor growth,migration and the process of drug resistance.However,the biological role of NEAT1 in HCC is unclear.In this study,abnormal expression and poor survival rate were found through clinical data from TCGA.Autophagy is a self-protection mechanism of cells under stress,and proper activation of autophagy is conducive to cell growth and drug resistance.This study aims to explore the mechanism of NEAT1 in the process of autophagy and drug resistance of HCC and hopes to provide more theories to enrich the pathogenesis of HCC.Methods: 1.In this study,the expression of NEAT1 and autophagy-related genes in tumors and normal tissues were compared and survival analysis was performed by TCGA.PCR was used to detect the expression of RNA in cells.2.NEAT1 over-expressed HCC cell lines(Hep G2 and Huh7)were constructed,transfection efficiency was determined by Polymerase chain reaction,and cell proliferation and migration were evaluated by CCK8 assay,scratch assay and colony formation assay.3.The autophagy process and autophagyrelated genes were detected by Western Blot,Immunofluorescence and Transmission electron microscopy.5.Western blot assays was used to detected the activation of AKT/m TOR.6.FISH assays was performed to evaluate the location of NEAT1.7.Negative correlation of mi RNAs with ATG3 were analyzed by TCGA,PCR and Western blot assays was found that NEAT1 over-expression enhanced the ATG3 expression,dual luciferase gene reporter assays proved the binding between ATG3 m RNA and mi R-204.8.The binding between molecules was verified by Dual luciferase gene reporter assays,RNA immunoprecipitation and RNA pull down assays.Western blot assays proved that NEAT1 can regulate the expression of ATG3 via mi R-204.Results: 1.The analysis of TCGA found that the expression level of NEAT1 in HCC was up-regulated(P<0.05),and the expression of NEAT1 was negatively correlated with the survival rate of HCC patients with significantly statistical difference(P<0.01).The higher expression of NEAT1 in HCC cells was showed comparing with the normal(P<0.05).2.NEAT1 promotes the resistance of HCC cells to sorafenib(P<0.05).3.NEAT1 increased autophagy in HCC cells,with statistically significant differences(P<0.05).4.NEAT1 increased AKT/m TOR phosphorylation.5.NEAT1 is mainly distributed in the cytoplasm.6.After TCGA data was obtained,analysis of the data of tumors and the normal tissues showed the obvious difference in ATG3 expression(P<0.01),the high-expression of ATG3 showed poor survival rate(P<0.001),and the expression of ATG3 protein and m RNA increased after overexpression of NEAT1(P<0.05).7.According to the clinical information,analysis of TCGA database and the analysis of bioinformatics website,the possibility of ATG3 binding to mi R-204 was the highest,and the cytological experiment proved that the expression of ATG3 decreased after the overexpression of mi R-204.Luciferase assay demonstrated the binding of mi R-204 to ATG3(P<0.05).8.Overexpression of NEAT1 inhibited the expression of mi R-204(P<0.05).Dual luciferase gene reporter assays,RIP and RNA pull down proved that the binding of NEAT1 with mi R-204 was existed in cells.(P<0.05).Conclusion: NEAT1 is a novel oncogene for HCC that promotes the proliferation and migration of HCC cells and reduces the inhibitory effect of sorafenib.Mi R-204 can inhibit the expression of ATG3 and inhibit the autophagy of HCC cells mediated by ATG3.After overexpression of NEAT1,via sponging with mi R-204,the expression of ATG3 was upregulated,leading to the activation of autophagy in HCC.Therefore,NEAT1 may be a novel target in HCC.