The Role Of LncRNA PVT1 In Renal Cell Carcinoma And Its Mechanism

PurposeTo investigate the role of LncRNA PVT1 in renal cell carcinoma and its mechanism and explore the significance of PVT1 as a biomarker in the prognosis of renal cell carcinoma.MethodsTCGA RNA-seq expression data of renal clear cell carcinoma were analyzed and the IncRNA PVT1 was picked out.PVT1 expression was detected by quantitative PCR in RCC tissues and cell lines.GSEA analysis on KEGG pathway was performed according to PVT1 expression.Cell proliferation assay,Edu,cell colony formation,flow cytometry,Scratch assay,Transwell migration assay and invasion assay were performed on PVT1 knockdown and overexpression renal cell lines to investigate the effect of PVT1 on proliferation,migration and invasion of renal carcinoma cells.Western blot was used to detect the expression of the cell cycle and EMT related proteins.Nude mice bearing subcutaneous tumor assay was performed to detect the tendency of ectopic tumorigenesis of PVT1 overexpressed or interfered renal carcinoma cell lines.The clinical data were used to analyze the effect of PVT1 on the prognosis and tumor grade and stage.ResultsWe found that PVT1 was upregulated in clear cell renal carcinoma(ccRCC)tissues and cells.The GSEA revealed that genes associated with cell cycle was remarkably enriched in higher PVT1 tumors versus lower tumors.Further experiments revealed that knockdown of PVT 1 remarkably inhibited ccRCC cell proliferation and cell invasion.Western blots indicate that P21,P16 and E-cadherin proteins were upregulated,while Phospho-Rb,CDK6,CCND2,N-cadherin and vimentin were downregulated after PVT1 knocked down while PVT1 overexpression reversed these changes.Last,in vivo study revealed that PVT1 promotes xenograft tumor growth in nude mice.Kaplan-Meier curve and Cox regression analysis showed that high expression of PVT1 was associated with poor overall survival and disease-free survival in renal cell carcinoma patients,PVT1 expression was correlated with clinicopathological factors.ConclusionPVT1 is highly expressed in renal clear cell carcinoma and promotes the proliferation,migration and invasion of renal cell carcinoma and expedites the formation of heterotopic tumor in nude mice.High expression of PVT1 was associated with poor overall survival and disease-free survival in renal cell carcinoma patients.The inhibition of PVT1 expression may be a promising strategy for ccRCC targeted therapy.PurposeTo investigate the possibility of PVT1 and its splicing transcript PVT1ΔE4 acting as ceRNA in renal cell carcinoma and regulating cell proliferation and invasion through interacting with miR200s.MethodsWe analysed the RNA-seq and miRNA-seq of ccRCC in TCGA database to find miRNAs negatively correlated with PVT1 expression and tried to pick out those with binding sites to PVT1 at the same time.Insights of the mechanism of competitive endogenous RNAs(ceRNAs)were gained through luciferase assays and RNA binding protein immunoprecipitation(RIP)and rescue experiments.Correlation between PVT1 and BMI1,ZEB1,ZEB2 and E-cadherin as well as correlation between miR-200c and PVT1,BMI1,ZEB1 and ZEB2 in 50 paired ccRCC tumor tissues were determined with RT-PCR.Last,the expression of PVT1ΔE4 in renal cancer tissues was checked by PCR,and the rescue experiments were also performed to evaluate its ability to binding miR-200s.ResultsmiR-20b/106b-5p/203a and miR-200s are qualified potential miRNAs that can bind PVT1.Further experiments revealed that PVT1 act as a ceRNA to sponge miR-200s especially miR-200c to regulate BMI1,ZEB1 and ZEB2 expression.MTS assays,colony formation and Transwell migration and invasion assay indicated that miR-200c significantly inhibited the proliferation and invasion of renal cancer cells.Besides,we found a positive correlation between PVT1 and BMI1,ZEB1 and ZEB2 while a negtive correlation between miR-200c and PVT1,BMI1,ZEB1 and ZEB2 in our 50 paired renal cancer tissues and their normal counterparts through RT-PCR.Last,a noval transcript of PVT1 with its exon 4 skipped was found,which had the same function as the full length transcript but exhibited a higher expression level than that of the full length transcript.As the 4th exon does not contain the binding site of miR-200s,it can also binding with miR-200s to act as a ceRNA to regulate BMI1,ZEB1 and ZEB2 expression.ConclusionTaken together,our research demonstrates that PVT1 and its splicing variant PVT1ΔE4 could promote renal cancer cell proliferation and invasion partially by competitively binding the miR-200 family and regulating the expression of BMI1,ZEB1 and ZEB2.