LncRNA VPS9D1-AS1 Promotes The Proliferation Of Hepatocellular Carcinoma Cells Through HuR/CDK4 Axis

Background: Hepatocellular carcinoma(HCC)is the third most common cause of cancer-related death.Lack of early diagnosis and delayed drug development are the main reasons for the low 5-year survival rate.Therefore,in order to further explore the molecular mechanism of HCC occurrence and development,it is important to search for early diagnostic markers and new therapeutic targets for HCC research.Long noncoding RNA(Lnc RNAs)is a type of RNA molecules with transcription length over 200 nt and no protein-coding function.Lnc RNA can regulate gene expression at multiple levels of transcription initiation,transcription and post-transcription,thus affecting the occurrence and development of tumors.In the existing studies on colon cancer and prostate cancer,lnc RNA VPS9D1 antisense RNA1(lnc RNA VPS9D1-AS1)plays a significant promoting role.Whether Lnc RNA VPS9D1-AS1 plays a similar regulatory role in HCC remains to be studied.Methods: The expression of VPS9D1-AS1 in HCC and normal liver tissue samples was compared by using microarray data from NCBI GEO(GSE65485)database and TCGA(Cancer Genome Atlas)database.80 pairs of HCC tumors and paracancer tissues were collected.The relative expression level of VPS9D1-AS1 in tissues was detected by q PCR.Small-interfering RNA(si RNA)specific for VPS9D1-AS1 has been synthesized in vitro and is used to knock down VPS9D1-AS1.The pc DNA-VPS9D1-AS1 overexpression fusion plasmid was constructed using pc DNA3.0 vector,and VPS9D1-AS1 overexpression cell line was established.Proliferation of knockdown and overexpressed cell lines was detected by CCK8 and colony formation.Stable VPS9D1-AS1 knockdown cell lines were constructed and subcutaneously implanted into BALB/c nude mice to evaluate the role of lnc RNA VPS9D1-AS1 as a regulator of HCC occurrence.The subcellular localization of VPS9D1-AS1 was analyzed by cell component q PCR.RIP experiments were performed to screen the interacting proteins and substrate targeting molecules of VPS9D1-AS1,and further molecular biology and phenotypic experiments were performed to verify the molecular mechanism of VPS9D1-AS1 regulating cell proliferation.Results: Database analysis showed that VPS9D1-AS1 was highly expressed in HCC cells.In 80 pairs of hepatocellular carcinoma tissues collected in our study,the level of VPS9D1-AS1 was significantly higher than that of paracancer tissues.The increased expression of VPS9D1-AS1 was associated with larger tumor size and later TNM stage.The higher the expression level of VPS9D1-AS1 in HCC patients,the worse the survival prognosis.VPS9D1-AS1 knockdown and overexpressed Hep G2 and SMMC-7721 cell lines were successfully constructed by transfection of small interfering RNA and overexpressed plasmid.Knockdown of VPS9D1-AS1 decreased the proliferation rate of cell lines and the activity of colony formation.In contrast,overexpressed cell lines showed increased proliferation and colony formation rate.In the tumorigenesis experiment of nude mice,the tumorigenesis ability of VPS9D1-AS1 stable knockdown cell line was weakened.The tumor volume growth and tumor weight were significantly decreased within 15 days.The expression level of KI67 was significantly decreased.Molecular mechanism studies have shown that VPS9D1-AS1 expression is localized in the cytoplasm of HCC.VPS9D1-AS1 forms a complex with Hu R in HCC cells,and CDK4 m RNA is the binding substrate of Hu R.By knockdown Hu R,it was found that the m RNA stability and expression level of CDK4 in HCC decreased,while the knockdown of VPS9D1-AS1 could further strengthen the knockdown effect of Hu R.In addition,VPS9D1-AS1 knockdown inhibited the binding of Hu R to CDK4 m RNA.These results suggest that VPS9D1-AS1 plays a synergistic role with Hu R in maintaining the stability and expression of CDK4 m RNA in cells.Further functional experiments showed that VPS9D1-AS1 overexpression could enhance CDK4 expression and promote cell proliferation,while inhibition of Hu R expression could inhibit the CDK4 expression and cell proliferation phenotype induced by VPS9D1-AS1 overexpression.Conclusion: In this study,high expression of lnc RNA VPS9D1-AS1 was found in tumors.VPS9D1-AS1 binds to Hu R in HCC cells,synergically regulates the expression of CDK4,a cell proliferation regulatory protein,and affects cell proliferation ability.VPS9D1-AS1 is a potential diagnostic and therapeutic target.