Long Noncoding RNA NEAT1 Upregulates Survivin And Facilitates Gallbladder Cancer Progression By Sponging MicroRNA-335

Background:Gallbladder cancer(GBC)is the most common malignant tumor of the biliary tract in the world,with no precise molecular-targeted treatments.Long noncoding RNAs(lncRNAs)have shown a disorder of expression in tumors which are closely related to the development and progression of tumors.MicroRNAs(miRs,miRNAs)play a key role in the development of many cancers,acting as oncogenes or tumor suppressor genes.Recently,it was found that the expression of microRNA(miR)-335 was significantly down-regulated in GBC,with an unclear molecular mechanism.The association between the aberrant expression of lncRNA NEAT1 and progression of GBC is still unknown,and the mechanism through which lncRNA NEAT1 exerts its oncogenic action needs further verification.Objective:(1)To describe the mechanism of miR-335 downregulation in GBC.(2)To clarify the possible signal pathways through which lncRNA NEAT1 regulates Survivin.(3)To screen and establish effective combined targeted gene therapy strategies aiming at Survivin and its regulatory factors.Methods:(1)Collect human gallbladder cancer specimens(n=25)and establish cell lines of GBC-SD and SCG-996.(2)Complete the extraction of RNA,and qRT-PCR was used to detect the expression of miR-335.(3)Western blot was used to detect the expression of Survivin in GBC cells.(4)Complete the transfection of GBC cell lines and the construction and infection of lentiviral vectors.(5)The database of bioinformatics was used to predict target genes which was furtherly verified with luciferase reporter gene experiments.(6)Cell Counting Kit-8(CCK-8)assays and colony formation assay were used to detect cell proliferation.(7)Transwell assay was used to detect cell invasion and metastasis.(8)Tumor xenograft experiment and IHC analysis were used to detect the effect of miR-335 for GBC cells in vivo.(9)Statistical Analysis:Results were presented as means±SD.Statistical analysis was performed with GraphPad Prism,version 5.0(GraphPad Software Inc.,San Diego,CA,USA),using Mann-Whitney test for comparison of two groups and one-way ANOVA for two or more groups followed by Tukey post hoc test.The analysis of correlation between factors was performed by Spearman's correlation coefficient rank test.Result:(1)The expression of lncRNA NEAT1and Survivin were up-regulated in GBC tissues,in contrast to the expression of miR-335.(2)Overexpression of miR-335 could inhibit the proliferation,migration and invasion of GBC cells.(3)LncRNA NEAT1 and miR-335 have a common binding site,and Survivin is a direct downstream target gene of miR-335.(4)Knockdown of lncRNA NEAT1 suppressed the expression of Survivin because lncRNA NEAT1 could competitively "sponge" miR-335.(5)Knockdown of lncRNA NEAT 1 could inhibite the proliferation and invasion of GBC cell in vitro as well as the growth of GBC tumor in vivo.(6)MiR-335 promoted the proliferation and metastasis of GBC cells through the NEAT 1/miR-335/Survivin axis.(7)The elevated level of LncRNA NEAT1 was found to be negatively correlated with miR-335 levels and positively correlated with Survivin.Conclusion:Overexpression of miR-335 could inhibit the proliferation and invasion of GBC cells.LncRNA NEAT1 could promote the expression of Survivin by competitively sponging miR-335,thus promoting the proliferation and invasion of GBC cell in vitro as well as the growth of GBC tumor in vivo..Moreover,the elevated level of LncRNA NEAT1 was found to be negatively correlated with miR-335 levels and positively correlated with Survivin.