Mechanism Of Long Non-coding RNA WDFY3-AS2 Regulating The Progression Of Clear Cell Renal Cell Carcinoma

Objective: 1)To investigate the role of lncRNA WDFY3-AS2 in the biological behaviors of renal clear cell carcinoma(cc RCC),including proliferation,growth,invasion and migration and subcutaneous tumor proliferation ability in NOD-scid mice,respectively;2)To explore the molecular mechanism of lncRNA WDFY3-AS2 regulating the progression of cc RCC.Methods: 1)The relative expression levels of lncRNA WDFY3-AS2 m RNA in renal and paracancer tissues were analyzed by The Cancer Genome Atlas(TCGA)database.Kaplan-Meier Plotter was used to analyze the relationship between lncRNA WDFY3-AS2 and overall survival(OS)of patients with renal cancer.2)Quantitative real-time fluorescence polymerase chain reaction(q RT-PCR)was used to detect the relative expression of lncRNA WDFY3-AS2 in 101 pairs of cc RCC tissues and adjacent tissues,as well as the relative expression of lncRNA WDFY3-AS2 in cc RCC cell lines.3)Data of 101 patients acquired by q RT-PCR were collected to analyze the correlation between expression and clinicopathologic features of patients with cc RCC.4)RNA in situ hybridization(ISH)and fluorescence in situ hybridization(FISH)were used to determine its localization in cells.5)Lentiviral vectors were used to construct 786-O and 769-P cc RCC lines overexpressing lncRNA WDFY3-AS2,and the effects of lncRNA WDFY3-AS2 on the proliferation,migration,invasion and tumori genetic ability of cc RCC cells were investigated through in vitro and in vivo experiments.6)Bioinformatics was used to predict potential target genes that might interact with lncRNA WDFY3-AS2.Double luciferase assay and q RT-PCR were used for confirmation.Meanwhile,lentiviral vectors were used to construct cc RCC cell lines that overexpressed target mi RNAs.The effects of lncRNA WDFY3-AS2 on proliferation,migration and invasion of cc RCC cells were investigated in vitro.7)Bioinformatics was used to predict the downstream target genes of target mi RNAs,which were finally verified by q RT-PCR and Western Blot experiments.Results: 1)TCGA database analysis showed that lncRNA WDFY3-AS2 expression level in renal cell carcinoma was lower than that in normal paracancer tissue,and Kaplan-Meier survival curve suggested that the higher lncRNA WDFY3-AS2 expression level in renal cell carcinoma patients,the higher OS was(p< 0.001).2)lncRNA WDFY3-AS2 showed low expression in cc RCC tissues and cells(P<0.01).3)lncRNA WDFY3-AS2 expression in cc RCC patients was significantly correlated with earlier pathological stage and Fuhrman grade(P<0.001).4)The results of RNA in ISH and FISH experiments showed that lncRNA WDFY3-AS2 was mainly localized in the cytoplasm.5)CCK-8 assay,cell scratch assay,transwell invasion assay and tumor formation assay in NOD-scid mice all proved that overexpression of lncRNA WDFY3-AS2 could significantly inhibit proliferation(P<0.01),invasion(P<0.01),migration(P<0.01)and tumor formation ability(P<0.01)of cc RCC.6)TCGA data analysis showed that lncRNA WDFY3-AS2 might act by targeting mi R-222 and mi R-222 was negatively correlated with lncRNA WDFY3-AS2 expression.Bioinformatics software Lnc Base Predicted v.2 to predict the possible interaction sites of lncRNA WDFY3-AS2 and mi R-222.Double luciferase assay results showed that lncRNA WDFY3-AS2 could directly bind to mi R-222.7)The downstream target gene TP53 of mi R-222 was screened by bioinformatics analysis.q RT-PCR and Western Blot showed that overexpression of mi R-222 could significantly inhibit the expression of TP53.Conclusion: 1)lncRNA WDFY3-AS2 was down-regulated in renal cell carcinoma tissues,and its expression level was correlated with the clinical stage and malignant grade of RCC.Patients with renal cell carcinoma with high expression of lncRNA WDFY3-AS2 had higher survival rate;2)Overexpression of lncRNA WDFY3-AS2 inhibited the proliferation,migration and invasion of renal cancer cells and the tumorigenic ability of NOD-scid mice,suggesting that lncRNA WDFY3-AS2 could be a prognostic biomarker for cc RCC;3)lncRNA WDFY3-AS2 negatively regulates mi R-222 through molecular sponging;4)mi R-222 can negatively regulate the expression of TP53.