小RNA干扰骨桥蛋白抑制人U251细胞的体外生长与侵袭

Effect of RNA interference targeting osteopontin on the growth and invasion of human glioma cell line U251

目的：研究慢病毒载体介导的骨桥蛋白（OPN） RNA干扰对人U251神经胶质瘤细胞体外生长和侵袭的影响,并探讨其对神经胶质瘤生长、侵袭的可能机制.方法：应用本实验室已构建并鉴定的OPN特异性短发卡RNA慢病毒表达载体（LV-OPN shRNA）感染U251神经胶质瘤细胞.RT-PCR检测干扰前、后U251细胞OPN mRNA的表达;免疫印迹法检测干扰前、后U251细胞OPN、MMP-2、MMP-9、uPA蛋白表达;MTT法检测细胞增殖能力的变化;Transwell体外侵袭实验检测细胞侵袭能力的变化;细胞划痕实验观察细胞迁移能力的变化.结果：与未感染组、空载体感染组相比,LV-OPN shRNA感染组OPN mRNA、OPN蛋白及MMP-2、MMP-9、uPA蛋白表达均明显下降;LV-OPN shRNA感染组细胞增殖、侵袭能力及迁移能力也明显下降.结论：慢病毒载体介导的骨桥蛋白RNA干扰在体外能抑制U251细胞OPN mRNA和蛋白表达, 进而抑制U251细胞在体外的生长及侵袭,因此可以推测OPN是促进胶质瘤增殖和侵袭的基因之一,为胶质瘤的治疗提供新的靶点.

Objective： To study the effect of the lentivirus-mediated RNA interference targeting osteopontin （OPN） on the growth of human glioma ceil line U251, so as to explore the possible mechanism on their growth and invasion. Methods： Specific shRNA lentiviral vector targeting osteopontin was already constructed and identified, and then infected into U251 cells. The mRNA expression of OPN was examined by RT-PCR and the protein expressions of OPN, MMP-2, MMP-9 and uPA were examined by immunoblotting. The effects of LV-OPN shRNA on proliferation and invasion of the infected cells were investigated by MTT assay and transwell experiments respectively. The migration was observed by cell scratching model in vitro. Results： After transfeetion with LV-OPN shRNA, OPN expression in U251 cells was significantly inhibited at both mRNA and protein levels compared with the non-transfected and empty vector transfected U251 cells. The protein expressionss of MMP-2, MMP-9 and uPA were significantly inhibited. The proliferation, invasion and migration were also inhibited. Conclusion： OPN expression in LV-OPN shRNA U251 cells is significantly inhibited, the proliferation and invasion is evidently inhibited,which implies that OPN is one of the genes to promote the proliferation and invasion of glioma, and it may provide a new target for the treatment of glioma.