骨桥蛋白基因对大肠癌细胞增殖、侵袭及Akt磷酸化的影响

Effects of osteopontin gene on prolireation and invasion and Akt phosphorylation of human corectal cancer cell

目的探讨骨桥蛋白(osteopontin,OPN)基因对大肠癌细胞侵袭的影响及机制。方法以人大肠癌Colo 205细胞为模型,应用OPN基因小干扰RNA(small interfering RNA,siRNA)转染处理后,采用荧光实时定量PCR检测OPN基因mRNA水平,采用软琼脂集落培养试验检测癌细胞的锚着不依赖性增殖能力,采用Boyden小室模型方法检测癌细胞侵袭能力,采用蛋白质印迹检测癌细胞Akt磷酸化水平。结果 OPN转染组癌细胞OPN mRNA和蛋白水平明显被抑制,且与时间和浓度相关。与空白对照组比较,OPN转染组细胞所形成的软琼脂集落数和穿膜细胞数明显减少,且呈浓度依赖性。蛋白质印迹检测发现,OPN转染组细胞Akt磷酸化水平明显下调。结论 OPN基因转染可明显下调大肠癌细胞的增殖和恶性侵袭能力,下调Akt磷酸化水平,是OPN基因转染下调癌细胞的增殖和恶性侵袭能力重要机制之一。

Objective To study the effects of osteopontin（OPN） gene on invasion and it′s mechanism of human colon cancer cell.Methods After human colon cancer Colo 205 cell were transfected by OPN small interfering RNA（siRNA）,the expression of OPN mRNA and protein were exmined by real-time PCR and western blot respectively,and anchorage-independent growth was exmined by clon formation in soft agar,and invasion ability was determined by boyden chamber model,and Akt phosphorylation was evaluated by Western blot assay.Results The level of mRNA and level of OPN of group transfectd with siRNA decreased mard in a time-dependent and dose-dependent manner.Compared with control group,both colony formation in soft agar and cells traversed membrane reduced significantly in a dose-dependent manner.The results from western blot showed that there were significant phenomenon of inhibition,which was in a dose-dependent manner.Conclusion Knock-downing OPN with siRNA can inhibit and invasion ability of colon cancer through induing Akt phosphorylation.