腺病毒介导PKM2 RNAi对MCF7细胞影响的研究

Effects of PKM2 RNAi on the proliferation,invasion and adhesion of MCF7 mediated by recombinant adenovirus

目的丙酮酸激酶(pyruvate kinase,PK)是糖酵解的关键酶之一,其M2型同工酶(PKM2)高表达于乳腺癌等肿瘤中。本研究探讨PKM2RNAi对乳腺癌细胞MCF7系增殖、ATP产生、侵袭和细胞粘附能力的影响及其可能机制。方法通过介导PKM2shRNA构建重组腺病毒Ad-PKM2,用以感染人乳腺癌细胞系MCF7。病毒感染细胞24和48h后,收集细胞总蛋白行免疫印迹实验检测基因沉默的效果,CCK-8实验检测细胞增殖和粘附能力,Transwell实验检测细胞穿透人工基底膜能力,采用JC-1探针观察细胞线粒体活性并用试剂盒检测细胞ATP含量。结果免疫印迹结果表明,腺病毒成功介导MCF7细胞发生PKM2RNAi。病毒感染后细胞的增殖能力被抑制41.0%,P<0.05。穿过人工基底膜的细胞减少50.2%;细胞的粘附能力下降34.7%,P<0.05;细胞内的ATP含量从正常的90μmol/L下降至68.3μmol/L(P<0.05),而细胞线粒体活性并未受明显影响。结论在乳腺癌MCF7细胞中,抑制PKM2蛋白的表达可有效抑制细胞的增殖、侵袭和粘附能力,其机制可能是减少了细胞的ATP能量供应。

OBJECTIVE To observe the effects of PKM2 knockdown on the proliferation, invation, adhesion and ATP production of MCF7 cell,and investigate the mechanism. METHODS Recombinant adenovirus Ad-PKM2 infected human breast cancer cell line MCFT. After adenovirus infection for appropriate hours, cell total proteins were extracted to detect the consequence for gene knockdown. CCK-8 assay was employed to analyze the change of cell proliferation and adhesion. Ability of cell penetrating artificial basement membrane was observed by transwell assay. Probe JC-1 was used to test mitochondrial activity and ATP content was surveyed by kit. RESULTS Immunoblot indicated that PKM2 was knocked down successfully mediated by Ad-PKM2. After the virus infection, cell proliferation was inhibited by 41.0% percent（P〈0.05）. Totally 50.2% cells were inhibited to penetrate the artificial basement membrane, and the inhibitory rate of cell adhesion ability was 34.7M （P〈0.05）. Cellular ATP content dropped from 90. 0 μmol/L to 68. 3 μmol/L （P〈0.05）, but mitochondrial activity of cells were not affected obviously. CONCLUSION Proliferation, invasion and adhesion ability of breast cancer cell MCF7 can be effectively inhibited by means of PKM2 knockdown,and the mechanism may be the reduction of ATP energy supply.