RNAi沉默Skp2抑制胃癌细胞SGC7901侵袭作用的实验研究

Study of Small Interfering RNA Silencing Skp2 Inhibiting Invasion of Gastric Carcinoma Cell Line SGC7901

目的研究RNA干扰(RNAi)沉默S期激酶相关蛋白2(Skp2)对胃癌细胞侵袭的影响。方法以胃癌细胞株SGC7901为实验对象,构建、并筛选出以Skp2基因为靶向的稳定表达载体,导入胃癌细胞,筛选获得稳定转染细胞株。采用侵袭实验检测侵袭能力的变化;明胶酶谱法观察基质金属蛋白酶(MMP)2、MMP-9蛋白的活性;Western Blotting检测p27、p21、Sp1和Kazal基序逆向诱导半胱氨酸丰富蛋白(RECK)的表达。结果侵袭实验显示:与对照组相比,实验组侵袭能力显著下降,侵袭细胞数显著减少(P<0.05);明胶酶实验显示:实验组中MMP-2、MMP-9活性降低;Western Blotting显示:实验组细胞p21、p27和RECK蛋白表达显著增强,Sp1蛋白表达下调(P<0.05)。结论通过Skp2-shRNA特异性沉默Skp2基因可下调Sp1,增强RECK表达,降低胃癌细胞在体外的侵袭能力。

Objective To study the effect of Skp2-shRNA on inhibiting the invasion of gastric carcino- ma cell line SGC7901. Methods We designed the stable-expression vector which targeted the Skp2 gene for degradation. The stably transfectant cells were constructed by screening. Invasion assays were used to deteet the number of invaded tumor eells; a gelatin-zymography assay was performed to test the activities of MMP-2 and MMP-9 ; Western blotting assays were used to analyze the expressions of p27, p21, Spl and RECK（ reversion-inducing cysteine rich protein with Kazal motif） proteins. Results Compared with the control group,invasion assays showed the invaded numbers of Skp2-depleted ceils were signifieantly reduced （ P 〈 0.05 ）. A galatin-zymography assay showed that the activities of MMP-2 and-9 were inhibited in the Skp2-depleted cells; Western blotting assays showed that the expressions of p27, p21 and RECK proteins were elevated, and the expression of Spl was reduced, in the Skp2-deleted cells （P 〈 0.05 ）. Conclusion Skp2-shRNA silencing Skp2 could inhibit the ex vivo invasion of gastric carcinoma ceils through reducing Spl and increasing RECK.