The Role And Mechanism Of PTBP1 In Occurrence And Progress Of Renal Cell Carcinoma

Objective: This study aims to investigate the role of PTBP1 in occurrence and progress of renal cell carcinoma,and explore its molecular mechanism.Methods: The expression of PTBP1 in normal renal tissues and renal cell cancer tissues was tested by immunohistochemistry;Control-shRNA and PTBP1-shRNA were transfected into RCC cell line ACHN and 786-O by lentivirus transfection to build PTBP1 stable knockdown cells,then Control vector and HIF-1? plasmid were also transfected into ACHN and 786-O cells.Effects of PTBP1-shRNA or HIF-1? plasmid on the expression of PTBP1 protein,the protein levels of VEGFA and HIF-1? proteins were detected by Western Blot in two kind of RCC cells.The effects of PTBP1 on cell proliferation of ACHN and 786-O cells were measured by cell proliferation assays.Transwell cell migration and invasion assays were performed to investigate the migration and invasion abilities of ACHN and 786-O cells after PTBP1 knockdown or HIF-1? over-expression.Angiogenesis formation assay was used to detect the angiogenesis abilities of RCC cell lines after PTBP1 gene knockdown or HIF-1? over-expression.To generate the model of metastasis in vivo,BALB/c nude mice were randomly divided into two groups consisting of 6 mice each,Luc-786-O-shPTBP1 and Luc-786-O-shControl cells were administered to BALB/c nude mice by tail vein injected.After 6 weeks,bioluminescence imaging was used to measure the growth of the metastatic tumor.Results: 1.Renal cell carcinoma tissue microarray immunohistochemical analysis showed that the expression of PTBP1 in RCC was significantly higher than that in normal renal tissue(P<0.05).The positive expression of PTBP1 in tumor diameter >7cm was significantly higher than that in ? 7cm(P<0.001).The expression of PTBP1 in stage III and IV was significantly higher than that in stage I and II(P < 0.001).Data showed that PTBP1 staining was dramatically increased in tumor with metastasis compared with that without metastasis(P<0.001).Our data revealed that high PTBP1 staining was positively correlated with poor 5-year overall survival and disease-specific patient survival(P<0.001).2.Western Blot showed that expression level of PTBP1 protein in ACHN and 786-O cells was significantly decreased by shRNA silencing when compared with Control-shRNA group(P<0.001).Furthermore,the protein levels of VEGFA and HIF-1? were decreased after PTBP1 silence,and the expression of HIF-1? and VEGFA proteins was restored after the transfection of HIF-1? plasmid.3.Cell proliferation assay showed that the proliferation of ACHN and 786-O cells was significantly decreased after transfected with PTBP1-shRNA,compared with Control-shRNA group(P<0.05).4.Cell migration assay showed that compared with Control-shRNA group,the migration ability of ACHN and 786-O cells was significantly decreased after PTBP1 knockdown,and it was restored after transfection of HIF-1? over expression plasmid.5.Cell invasion assay proved that compared with Control-shRNA group,the invasion ability of ACHN and 786-O cells was significantly decreased after transfection of PTBP1-shRNA,and the invasion ability of cells was recovered after transfection of HIF-1? over expression plasmid.6.Angiogenesis formation assay suggested that the ability of angiogenesis in ACHN and 786-O cells was significantly decreased after PTBP1 knockdown,and the ability of angiogenesis in cells was restored after transfection of HIF-1? over expression plasmid.7.Animal assay indicated that compared to the Luc-786-O-shControl group,Luc-786-O-shPTBP1 group significantly decreased the luminescence detected of metastatic nodules,and the difference has statistical significance.Conclusions: 1.PTBP1 is highly expressed in renal carcinoma and renal cell carcinoma cells,and the high expression of PTBP1 is positively correlated with tumor size,TNM stage,M stage and poor prognosis.2.PTBP1 knockdown can inhibit the proliferation and metastasis of renal cell carcinoma cells in vitro.3.PTBP1 regulates the proliferation and metastasis of renal cell carcinoma cells through the HIF-1? signaling pathway.4.PTBP1 knockdown can repress lung metastasis of RCC cells in vivo.