Expression And Roles Of NEDD4L In Clear Cell Renal Cell Carcinoma

Objective:To study the expression of gene NEDD4L in clear cell renal cell carcinoma and its effects on the biological function of clear cell renal cell carcinoma cell line such as growth and migration.To investigate the relevant mechanism of NEDD4L in clear cell renal cell carcinoma,it provides a theoretical basis and experimental basis in diagnose and treat clear cell renal cell carcinoma.Methods:1.Using TCGA data mining to analyze the expression of NEDD4L in clear cell renal cell carcinoma and adjacent normal tissues,and to explain the relationship between the expression of NEDD4L and the survival of its patients.2.Using immunohistochemical staining to detect the expression of NEDD4L protein in 25 pairs of clear cell renal cell carcinoma tissue and adjacent normal tissues.3.Using qRT-PCR to analysis the expression of NEDD4L mRNA in 50 pairs of clear cell renal cell carcinoma and adjacent normal tissues,and using chi-square test to analysis the relationship between the clinical pathological features and the expression of NEDD4L in clear cell renal cell carcinoma.4.Western Blot was used to detect the expression of NEDD4L in human normal kidney cells 293T and clear cell renal cell carcinomacells ACHN,A498,Caki-1and 786-0.Lentiviral infection was used to introduce NEDD4L overexpression vector and empty vector into human renal clear cell carcinoma cell line 786-0 and ACHN,and construction of NEDD4L overexpression and no-load control stable cell lines.And the expression of NEDD4L will be detected by qRT-PCR and Western Blot.5.The effect of overexpression of NEDD4L on the proliferation of 786-0 and ACHN cells was detected by WST-1 proliferation assay and plate colony formation assay.Using Transwell in vitro migration model and cell scratch assay to detect the migration ability of 786-0 and ACHN cell with overexpression of NEDD4L.6.The nude mice were inoculated subcutaneously with established stable NEDD4L and empty control cell lines ACHN NC and ACHN NEDD4L,and the effect of NEDD4L overexpression on the growth of human renal clear cell carcinoma was observed in nude mice.Results:1.TCGA data mining revealed that NEDD4L was significantly lower in clear cell renal cell carcinoma tissue than normal tissue adjacent,and its patients with low NEDD4L expression had lower overall survival than patients with high NEDD4L expression.2.In 25 pairs of clear cell renal cell carcinoma tissues and adjacent normal tissues,Immunohistochemical staining showed thatthe NEDD4L protein expression of clear cell renal cell carcinoma was significantly lower than that in adjacent normal tissues.3.In 50 pairs of clear cell renal cell carcinoma tissues and adjacent normal tissues,compared with adjacent normal tissues,the expression of NEDD4L mRNA was significantly lower in renal clear cell carcinoma tissues(P<0.001),and as the NEDD4L mRNA expression decreased,the higher the clinical histopathological grade(P<0.05).4.The results of Western Blot showed that NEDD4L had a low expression level in human clear cell carcinoma cellsACHN,A498,Caki-1 and 786-0.ACHN and 786-0 were infected with NEDD4L overexpression vector and no-load lentivirus and screened with puromycin to construct a stable over-expressed NEDD4L and no-load control cell lines:786-0 NEDD4L,786-0 NC,ACHN NEDD4L,ACHN NC.qRT-PCR showed that the NEDD4L mRNA expression of 786-0 NEDD4L was significantly higher than that of 786-0 NC(P<0.001);the NEDD4L mRNA expression of ACHN NEDD4L was significantly higher than that of ACHN NC(P<0.01).Western Blot identification showed that the NEDD4L protein expression of 786-0 NEDD4L was significantly higher than that of 786-0 NC(P<0.0001);the NEDD4L protein expression of ACHN NEDD4L was significantly higher than that of ACHN NC(P<0.001).5.Observe the growth and migration ability of stable renal cell carcinoma stable cell lines.The results of WST-1 proliferation assay showed that after 48 hours of culture the proliferation rate of 786-0 NC group was significantly higher than that of 786-0 NEDD4L group(P<0.001);the proliferation rate of ACHN NC group cells was significantly higher than that of ACHN NEDD4L group(P<0.01),it's suggesting that overexpression of NEDD4L can significantly reduce the cell proliferation ability of 786-0 and ACHN.The results of plate cloning experiments further showed that the number of clones in the 786-0 NC group was significantly more than that in the 786-0 NEDD4L group(P<0.001);the number of clones in the ACHN NC group was significantly more than that in the ACHN NEDD4L group(P<0.001),it's suggesting that overexpression of NEDD4L can significantly reduce the cell colony-forming ability of 786-0 and ACHN.Transwell migration experiments showed that 24 hours after the cells were transfected into the Transwell chamber,the number of cells migrated in the 786-0 NC group was significantly more than that in the 786-0 NEDD4L group(P<0.01);The number of cells migration in the ACHN NC group was significantly more than that in the ACHN NEDD4L group(P<0.01).The cell scratch test results showed that after 12 hours of scratching in the 786-0 cell line,the migration distance of the 786-0 NC group was significantly longer than that of the 786-0 NEDD4L group(P<0.01).After 24 hours of scratching in the ACHN cell line,the migration distance of the ACHN NC group was much more longer than that of the ACHN NEDD4L group(P<0.001).Transwell migration assays and cell scratch assays have shown that NEDD4L overexpression in clear cell renal cell carcinoma can significantly reduce cell migration.6.About 10 days after cell inoculation,the site where ACHN NC cells were inoculated can touch the solid mass.However,there were 2 sites where ACHN NEDD4L cells were inoculated and solid masses could not be touched,and small solid masses could be touched in about 16 days after cell inoculation.Anatomically killed nude mice,cut the tumor and measure.The tumor volume in the ACHN NC group was significantly greater than that in the ACHN NEDD4L group(P<0.05).Tumor formation experiments in nude mice have shown that overexpression of NEDD4L can reduce the tumorigenicity of ACHN cells in nude mice and inhibit tumor growth in vivo.Conclusion:1.NEDD4L protein and mRNA were low expressed in clear cell renal cell carcinoma.The low expression of NEDD4L mRNA was related to the pathological grade of clear cell renal cell carcinoma.2.The low expression of NEDD4L was significantly positively correlated with the overall survival rate of patients with clear cell renal cell carcinoma.3.NEDD4L inhibits the growth and migration of renal clear cell carcinoma cells,and inhibits the ability of renal clear cell carcinoma cells to grow in nude mice and the growth of transplanted tumors.