Long Noncoding RNA NEAT1 Promotes Cell Proliferation And Invasion By Regulating HnRNP A2 Expression In Hepatocellular Carcinoma Cells

Objectives:The purpose of this study is to detect expressing level of nuclear enriched abundant transcript 1(NEAT1)in hepatocellular carcinoma(HCC)to find the association between NEAT1 expression level.We aimed to determine the roles,and functional mechanisms of NEAT1 in the proliferation and invasion of HCC using small interfering RNA(si-RNA)against NEAT1.We further explore the molecular mechanism of long noncoding RNA nuclear enriched abundant transcript 1(IncRNA NEAT1)-heterogeneous nuclear ribonucleoprotein A2(hnRNP A2)regulation in HCC progression.By using RNA Immunoprecipitation(RIP),whether RNA binding proteins involve into this mechanism will be explored.Methods:Total RNA was extracted from tissues and cell lines using Trizol-Chloroform reagent.The expression of each RNA was detected by RT-qPCR and normalized to that of GAPDH.HCC cell lines HepG2 and SMMC-7721 were transfected using si-RNA NEAT1(100nM)and Lipofectamine 2000.The effect of NEAT1-knockdown was detected by RT-qPCR.To focus on the function and potential molecular mechanism of NEAT1 in HCC,we profiled its gene expression pattern by gene sequencing and detected proliferation and invasion in a NEAT 1-knockdown HCC cell lines(n=2).Selected RNA binding proteins(Starbase 2.0)were verified by RIP assay.Targeting sites were verified by RNA pull-down assay.Western-Blotting assay was performed to detect hnRNP A2 expression after si-RNA(100nM)NEAT1 or U2AF65 treatment.Cells stably transduced with retroviral vectors expressing T7-tagged hnRNP A2 cDNA to overexpress hnRNP A2.Proliferation and invasion of each group were detect by CCK8 count assay(10?l/2000 cells)and transwell assay(2×105/welll).Results:We found that NEAT1 was up-regulated in HCC tissues and cell lines(p<0.001).Knockdown of NEAT1 altered global gene expression patterns in HCC cells(229 genes up-regulated,148 genes down-regulated,fold change>2,p<0.05).NEAT1-knockdown inhibited cell proliferation(p<0.01),migration and invasion(p<0.001)in HCC cell lines.U2AF65 interacted with NEAT1 and hnRNP A2 mRNA(enrichment fold changes compared with IgG group:82.659 for NEAT1 and 53.527 for hnRNP A2 mRNA).NEAT 1-knockdown leaded down-regulation of hnRNP A2(p<0.001).This regulation might be associated with the NEAT1-U2AF65 protein complex.Overexpression of hnRNP A2 rescued the proliferation and invasion in HCC cells that express low levels of NEAT1(p<0.01).Conclusions:long noncoding RNA NEAT1 is an oncogene that,when down-regulated,inhibits HCC cell proliferation and invasion as well as HCC tumor growth in xenograft models.These effects strongly correlated with altered hnRNP A2 expression,which is also regulated by NEAT1.