The Impact Of Biological Behaviors Of SiRNA Targeting Against OPN Gene On Human Bladdle Cancer Cell Line T24 By Mediated By Lentivirus

PartⅠConstruction and identification of a Lentivirus expression vector coding for OPN-RNAiObjective: To construct a Lentivirus expression vector coding for the short hairpin RNA (shRNA) targeting OPN mRNA.Methods: Four plasmid expression vectors coding for siRNA targeting OPN gene sequence were constructed.The recombinant plasmids were identified by PCR and sequencing, and then transfected stably into 293T cells respectively. The targeting OPN gene silencing effect was detected by Western blotting, then judging effectiveness of RNAi . We choose target point of which OPN-RNAi effectively silences OPN gene the most in 293T cells for the package.pGC-LV ,pHelper 1.0 and pHelper 2.0 were used for the package of OPN-shRNA vector.The viral purity,identity,titer of Lentivirus viral stock were analyzed.Results :The expected bands were amplified from the plasmids coding for shRNA by PCR,then sequencing confirmed that the OPN-shRNA plasmids were successfully constructed. Transfection of 293T cells with shRNA plasmids resulted in an inhibition of OPN protein expressions respectively. The target point of KD1,KD2,KD3 and KD4 have been successfully knockdown, The titer of OPN -shRNA was approximately 2×109 TU/ml.Conclusions : The Lentivirus viral vector of OPN-shRNA is successfully constructed and prepared. PartⅡStudy of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vitroObjective: To observe the inhibitory effects of OPN-siRNA mediated by Lentivirus on human pancreatic carcinoma cells cultured in vitro.Methods: OPN-shRNA was transfected into human bladdle cancer cell line T24 and the efficiency of transfection was detected. Then RT-PCR, and Western blotting were used to detect OPN mRNA and protein expression. While MTT assay was used to determine proliferation rate of OPN. Detection of apoptosis was manipulated by the means of Annexin V-FITC/PI double staining. Detection of invasion was manipulated by the means of Transwell cabin.Results: OPN-shRNA gene has been integrated into T24 cells and the transfection efficiency detected was above 90%.The characteristics of the OPN-RNAi cells were studied.Compared with the Negative Control cells and Control cell,the OPN-shRNA cells showed that: (1) the growth rate of OPN-shRNA cells was diminished; (2) The ability of OPN-shRNA cell's invasion and migration diminished; (3) Western blot analysis also showed that the products of OPN gene have decreased in OPN-RNAi cells.Conclusions:OPN-shRNA can dramatically inhibit products of OPN, growth ,invasion and promote apoptosis of human bladdle cancer in vivo.PartⅢresearch of the impact of biological behaviors on human bladdle cancer cell line T24 by OPN-shRNA in vivoObjective: To research inhibition of biological behaviors of human bladdle cancer such as growth,metastasis and invasion by OPN-shRNA in vivo.Methods Human T24 carcinoma transplanted subcutaneously in nude mouse were established firstly. OPN-shRNA,negative-shRNA and physiological saline were intratumorally injected with 500ug in subcutaneous xenograft model, inhibitory effect of tumor growth was observed,OPN protein expressions as well as value of VEGF were detected by immunohistochemical SP method.Results:The inhibitory effect of tumor growth and metastasis of tumor were observed. Growth rate of subcutaneous xenograft significantly decreased in OPN-RNAi group,the tumor's size and weight of experimental group were significantly lesser than those of negative control group and blank control group.For experimental group, expressions of OPN and VEGF proteins were weaker than those of control groups (for OPN, H= 11.2049,P=0.0037; for VEGF, H=9.5697, P=0.0084).Conclusions: OPN-shRNA can dramatically inhibit growth, and reduce neovascularization of human bladdle cance carcinoma in vivo.