核糖核酸干扰下调骨桥蛋白表达对胃癌细胞生物学行为的影响

Down-regulation of osteopontin by siRNA on the biological behavior of gastric cancer cell lines

目的探讨骨桥蛋白(OPN)下调对胃癌细胞株MKN28和SGC7901生物学行为的影响。方法参考相关文献,设计并合成OPN的小干扰RNA(siRNA),通过荧光标记测定2株细胞的转染效率。Western印迹法检测OPN蛋白下调情况。实时定量聚合酶链反应(RT-PCR)检测siRNA对OPN mRNA的下调率及随时间的变化。流式细胞仪检测转染前、后细胞周期和凋亡变化。四甲基偶氮唑蓝(MTT)法连续7d检测OPN干扰时肿瘤细胞增殖的变化,并采用混合模型进行统计分析。Trans- well实验检测转染前、后细胞的运动侵袭能力并行t检验进行分析。结果2株细胞中OPN siRNA的转染效率均在90%以上。干扰后2株细胞中OPN蛋白表达均下调。SGC7901细胞在siRNA转染后72hOPN mRNA表达下降最低,达47%;MKN28细胞在siRNA转染后48hOPN mRNA表达下降最低,达40%。OPN被干扰后,SGC7901和MKN28细胞的增殖能力明显减弱(混合模型分析与未干扰组比较,P<0.01)。OPN被干扰后MKN28和SGC7901细胞周期受到影响,其中SGC7901分裂期细胞比例由18.78%降至17.02%,MKN28分裂期细胞比例则由4.96%降至0.39%。2株细胞都未发现下调OPN后诱发细胞凋亡。OPN下调后2株肿瘤细胞的运动侵袭能力均下降,穿过人工基底膜的细胞数明显减少(SGC7901:t=5.172,P<0.01;MKN28:t=11.365,P<0.01)。结论siRNA能下调MKN28和SGC7901中OPN表达,OPN可能与MKN28和SGC7901细胞的增殖和运动侵袭相关。

Objective To explore the effects of down regulation of osteopontin （OPN） on the biological behavior of MKN28 and 8GC7901 cell lines. Methods OPN siRNA was designed according to the relevant literature and was transfected into the two cell lines. Fluorescent labeling was used to test the transfected efficiency. The down-regulation of OPN protein was measured by Western blot. Realtime PCR was used to test the ratio and time difference of down-regulation of OPN mRNA after siRNA transfection. The biological changes before and after OPN siRNA transfected into these two cell lines were tested by flow cytometry （to test cell cycle and apoptosis） and MTT method （to test the proliferation for the consecutive seven days） and the difference between OPN siRNA transfected or non-transfected cells was compared using mixed model. The capability of moving and invasion of cancer cells were tested by Transwell method and analyzed by t-test. Results The transfected efficiency of OPN siRNA were more than 90% in the two cell lines. OPN mRNA down-regulated to 47% at the 72th hour in SGC7901, while 40% at the 48th hour in MKN28. The expression of OPN protein was both downregulated after siRNA transfection in the two cell lines. The proliferation decreased after transfected withOPN siRNA both in MKN28 and SGC7901（ P 〈 0.01）. The cell cycle was interfered by transfection with OPN siRNA, the proportion of M phase cells decreased from 4.96% to 0.39% in MKN28 and from 18.78% to 17.02% in SGC7901. No apoptosis was induced after transfection in the two cell lines.Transwell experiment showed less cells moving through the artificial basement membrane after transfected with OPN siRNA （SGC7901： t = 5. 172, P 〈. 0.01; MKN28： t = 11. 365, P 〈. 0.01）. Conclusions The siRNA can down-regulated OPN, which may be a factor associated with proliferation, movement and invasion, in MKN28 and SGC7901 cell lines.