The Biological Function And Mechanism Of LncRNA ADAMTS9-AS2 In Hepatocellular Carcinoma

Background and Objective:Hepatocellular carcinoma(HCC)is the most common type of liver cancer,nearly half of the deaths occur in China every year.In recent years,comprehensive treatment strategies including local therapy,molecular targeted therapy and immunotherapy have made some progress,but the overall efficacy of the treatment is still not optimistic.Long non-coding RNAs(LncRNAs)is involved in the regulation of biological behavior of cancer cells and has attracted more and more attention.ADAMTS9 is a member of the ADAMTS family and has been confirmed as a tumor suppressor gene in a variety of tumors.LncRNA A disintegrin and metallo-proteinase with thrombospondin type 1 motifs 9 antisense RNA 2(LncRNA ADAMTS9-AS2)is antisense transcripts of ADAMTS9,as well as contiguous gene of ADAMTS9.The biological function and mechanism of lncRNA ADAMTS9-AS2 in hepatocellular carcinoma are still unclear.The purpose of this study was to explore the expression of lncRNA ADAMTS9-AS2 in HCC cells and their biological function and mechanism,so as to provide potential targets for accurate diagnosis and treatment.Methods:1.Expression of lncRNA ADAMTS9-AS2 in HCC tissues and survival curves of HCC patients were retrieved from UALCAN database;the expression levels of lncRNA ADAMTS9-AS2 in human HCC cell lines(HepG2,MHCC97-H,Hep3B2.1-7,SMCC-7721)and normal liver cell line MIHA were detected by RT-qPCR.2.HCC cell lines(HepG2 and MHCC97-H)with significant abnormal expression of lncRNA ADAMTS9-AS2 were selected as the research objects.The cell lines with gene overexpression and knockdown were constructed by pcDNA3.1 plasmid transfection and siRNA interference,respectively.CCK8 cell proliferation assay,wound healing test and Transwell invasion test were used to detect biological functions of the cells.3.HCC cell lines(HepG2 and MHCC97-H)with significant abnormal expression of lncRNA ADAMTS9-AS2 were selected as the research objects.Overexpressed and knockdown cell lines were constructed by pcDNA3.1 plasmid transfection and siRNA interference respectively to detect the expression of p-AKT,AKT,PIK3CB,p-mTOR and mTOR,the key proteins of PI3K-AKT pathway.The markers of autophagy(LC3-?/?,BECN1 and SQSTM1),apoptosis-related proteins(Bax and Bcl-2)and ADAMTS9 were detected.Bioinformatics was used to predict and screen miRNAs binding to lncRNA ADAMTS9-AS2 and ADAMTS9,and dual luciferin reporter assay,RT-qPCR and Western blot were used to study the regulation mechanism of ceRNA.4.Establish a transplanted tumor model in nude mice,observe the effects of lncRNA ADAMTS9-AS2 and its binding miRNA on the growth of transplanted tumors in nude mice.The expressions of PI3K-Akt pathway key proteins,autophagy and apoptotic proteins in the transplanted tumor tissues were detected by RT-qPCR and Western blot.Results:1.By searching UALCAN database,LncRNA ADAMTS9-AS2 was low expressed in HCC tissues,and the survival rate of Asian people was significantly improved when the expression of ADAMTS9-AS2 was upregulated.The expression of ADAMTS9-AS2 was significantly lower in HepG2 and MHCC97-H cell lines,and its expression was positively correlated with the expression of AD AMTS 9.2.In HepG2 and MHCC97-H HCC cell lines,the upregulated expression of lncRNA ADAMTS9-AS2 inhibited the proliferation,migration and invasion activities of HCC cells in vitro.3.In HCC cell lines in vitro,lncRNA ADAMTS9-AS2 expression was up-regulated,PI3K/Akt/mTOR pathway activity was inhibited,autophagy was promoted,and apoptosis of HCC cells was induced.miR-93 and miR-23b were screened out by bioinformatics prediction.miR-93 can bind to lncRNA ADAMTS9-AS2 and ADAMTS9,and down-regulate the expression of lncRNA ADAMTS9-AS2 and ADAMTS9.RT-qPCR and Western blot analysis showed that the expression of lncRNA ADAMTS9-AS2 and ADAMTS9 were significantly down-regulated when miR-93 was overexpressed.When miR-93 and lncRNA ADAMTS9-AS2 were overexpressed at the same time,the expression of ADAMTS9 was significantly down-regulated compared to the upregulated lncRNA ADAMTS9-AS2 only.4.The transplanted model of HCC in nude mice was successfully constructed.Compared with the blank control group,the tumors of the overexpression ADAMTS9-AS2 group grew slowly and the smallest;the overexpressed miR-93 group had the largest tumors and the fastest growth.When both ADAMTS9-AS2 and miR-93 were overexpressed,the tumor growth rate was slower than that of the control group and faster than that of the ADAMTS9-AS2 group.LncRNAADAMTS9-AS2 was upregulated in nude mice,inhibiting the activation of PI3K-Akt pathway and promoting autophagy and apoptosis of cells,while miR-93 had the opposite effect.Conclusions:LncRNA ADAMTS9-AS2 can inhibit PI3K-AKT pathway activity by up-regulating expression,and meanwhile,through the ceRNA mechanism,it can compete with miR-93 to up-regulate ADAMTS9 expression,promote autophagy and apoptosis of HCC cells,and inhibit the proliferation,migration and invasion of HCC cells.LncRNA ADAMTS9-AS2 plays a role as a tumor suppressor gene in HCC,which may become a potential biomarker for accurate diagnosis of HCC and a target for treatment.