Effects Of Integrin-Linked Kinase Silencing By Sirna On Growth And Apoptosis Of Bladder Cancer BIU-87 Cells

Objective To construct the specific siRNA vectors of ILK gene and transfect bladder cancer BIU-87 cells. And study the effects of ILK gene silencing by siRNA on growth and apoptosis.Methods One siRNA vector specific to ILK gene and one non-homologous negative control vector were designed and constructed;then stably transfected into bladder cancer BIU-87 cells via Lipofectamine 2000; Stably expression cell lines are screened by G418, and were named BIU-87 si ILK and BIU-87 vector;Detected the interferential efficiency by RT-PCR , Westeron Blot and cell Immunofluorescence;The inhibitory effect on cell proliferation was assessed by Am-Blue and Flow Cytometry, cell apoptosis was assessed by Tunel Kit and Flow Cytometry, The expression level of p-Akt and p-GSK-3βwas assessed by cell Immunofluorescence;The expression level of Akt, p-Akt, GSK 3(α/β), p-GSK 3β,β-catenin, Caspase 3, Bax and Bcl-2 were assessed by Western Blot;The expression level of RI and location were assessed by laser Scanning Confocal。The BIU-87 cells, BIU-87 vector cells, and BIU-87 si ILK cells were respectively injected subcutaneously into the backs of the BALB/C nude mice. The time of tumor formation was recorded,and 30 days after injection, the mice were sacrificed ,and tumors were excised and weighed. The change of morphology was detected by HE dying;The density of tumor microvessel was detected by CD31 antibody;The expression of ILK, p-Akt,p-GSK-3β,β-catenin and RI protein were assessed by Immunohistochemistry;Xenograft tumor cell apoptosis was assessed by Tunel Kit .Results The siRNA ILK vectors were verified by enzyme digestion and DNA sequencing, the expression of mRNA and protein significantly decreased in bladder cancer BIU-87 cells by transfected siRNA ILK vectors compared with negative control, It is indicated that stably expression cell lines BIU-87 si ILK and BIU-87 vector were successfully obtained; The ability of cell proliferation stably decreased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells;cell apoptosis significantly increased in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells; The proliferation of BIU-87 si ILK cells was inhibited with G1 phase arresting, S phase reducing and G2-M phase delaying; The expression of p-Akt, p-GSK 3β,β-catenin及Bcl-2 significantly reduced in BIU-87 si ILK cells compared with BIU-87 vector and BIU-87 cells(P<0.05), The expression of Caspase 3 and Bax protein stably increased (P<0.05), but, The expression of Akt, GSK 3(α/β)do not significantly changed(P>0.05);Laser Scanning Confocal assay showed that ILK and RI could colocalized in cytoplasm and the expression of RI in transfected BIU-87 si LIK cells group markedly increase compared with the control cells(P<0.05)。xenograft tumor showed that the BIU-87 si ILK cells group significantly inhibited the growth of tumors compared with the other two control groups(P<0.05),The inhibiting rate of tumor was 64.73% or 66.73%(,P>0.05), HE staining showed significantly decrease in volume of tumor cells, in the ratio of nucleus to cytoplasm in the tumor of BIU-87 si ILK group, compared to the BIU-87 vector and BIU-87 group; BIU-87 si ILK group resulted in apparent inhibition of angiogenesis and much higher RI expression as well as lower ILK, p-Akt, p-GSK3βandβ-catenin expression in tumor tissue, whereas numerous small vessels and much higher ILK, p-Akt, p-GSK3βandβ-catenin expression were seen in tumor tissue of the control groups.Conclusion Specific siRNA of ILK expression vectors were constructed successfully, and significately inhibited proliferation and induced apoptosis of bladder cancer BIU-87 cells by regulated the expression of p-Akt, p-GSK-3β,β-catenin, Caspase 3, Bax and Bcl-2, Nude mouse experiment showed that ILK siRNA may significately inhibit BIU-87 cell growth in vivo,the mechanism of its may be ILK siRNA can decrease the expression of p-Akt, p-GSK-3βandβ-catenin.