Effect Of Silencing Notch1Gene By RNA Interference On Proliferation, Apoptosis And Akt Signaling Pathway In Clear Renal Cell Carcinoma Cells

Objects To investigate the relationship between the expression ofNotch1,VEGF,MVD and the clinical and pathological manifestations of clearrenal cell carcinoma(CRCC).We further studied the changes of cellproliferation and apoptosis and the alteration of Akt signaling pathwayin786-0cells by applying RNA interference to silence Notch1gene.Methods (1)Tissue microarray cores were made from52paraffin-embeddedclear renal cell carcinomas samples and examed by immunohistochemistryfor Notch1, VEGF and MVD.Pericarcinous tissues and30normal renal tissueswere as controls.(2)The siRNA segments targeting Notch1gene weretransfected into786-0cells by Lipofectamine2000TM. Efficiency of siRNAsilencing Notch1gene was tested by Real-time PCR.(3)Cell prolifera-tion and apoptosis were measured via3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliu-mromide(MTT) and terminal deoxynucleoti-dyl transferase dUTP nick end labeling(TUNEL) methods respectively.(4)The expression of Notch1protein, Bcl-2,Pro-caspase3and T-Akt,p-Akt, p-mTOR, p-P70S6K in786-0cells were detected by Western Blot.Results (1) The expression of Notch1, VEGF in clear renal cell carcinomawas82.69%and92.30%,higher than that in pericarcinous（42.55%,40.42%）and normal renal tissues(16.67%,20.00%),P <0.01.MVD in clear renal cellcarcinoma was (64.24±12.35),higher than that in pericarcinous（32.48±11.74）and normal renal tissues（20.37±10.82）,P <0.01.Higherexpression of Notch1,VEGF,MVD are related to worse renal carcinoma cellFuhrman grade, tumour size and TNM staging. The expressions bewteen Notch1and MVD, VEGF and MVD were in positive correlation.(2) Notch1siRNAdownregulated Notch1mRNA and Notch1protein.(3)Notch1siRNA inhibitedcell proliferation and induced apoptosis in786-0cells.The expression of Bcl-2,Pro-caspase3was decreased by Notch1siRNA.(4) Notch1siRNAreduced the expression of p-Akt, p-mTOR and p-70S6K,while the alterationof T-Akt was not seen.Conclusions Over-expression of Notch1,VEGF and MVD might play importantrole in pathogenesis in clear renal cell carcinoma. Notch1and VEGF mightbe involved in regulation of tumor angiogenesis.Notch1,VEGF,MVD may beuseful as predictors for poor prognosis in clear renal cell carcinoma.Deplete of Notch1gene inhibits cell growth and induces apoptosis in786-0cell line.It inhibits the activation of the Akt signaling pathway bydephosphorylated p-Akt,p-mTOR and p-70S6K.Notch1might be a potentialgene target for anti-clear renal cell carcinoma therapy.