目的 探讨敲低CD73的表达对人肺腺癌H1975细胞增殖、迁移、侵袭能力的影响及其具体机制.方法 选取人肺腺癌H1975细胞,将转染慢病毒HBLV-ZsGreen-PURO NC的人肺腺癌H1975细胞作为H1975-NC组,将转染3种不同位点敲低CD73表达的慢病毒HBLV-h-CD73短发夹RNA(shRNA)-ZsGreen-PURO的人肺腺癌H1975细胞分别作为H1975-shCD73-1组、H1975-shCD73-2组、H1975-shCD73-3组,以未转染人肺腺癌H1975细胞作为空白对照组.采用蛋白质印迹法(Western blot)选取敲低效果最明显的转染细胞进行后续实验,作为H1975-sh组.采用CCK8法、细胞划痕实验、Transwell侵袭实验分别检测细胞增殖、迁移及侵袭能力.采用West-ern blot检测上皮-间充质转化(EMT)标志蛋白[E-上皮钙黏素(E-cadherin)、波形蛋白(vimentin)]的相对表达量及磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(AKT)信号通路磷酸化蛋白表达比例.结果 Western blot检测结果显示,H1975-shCD73-2组(即H1975-sh组)细胞CD73的敲低效果最明显,选取该细胞进行后续实验.敲低CD73的表达后,转染不同时间,H1975-sh组细胞吸光度(OD)值均明显低于H1975-NC组(P﹤0.01),划痕面积愈合率均低于H1975-NC组(P﹤0.05),细胞侵袭数目均少于H1975-NC组(P﹤0.05).H1975-sh组细胞E-cadherin蛋白相对表达量高于H1975-NC组,vimentin蛋白相对表达量低于H1975-NC组,磷酸化PI3K(p-PI3K)/PI3K、磷酸化AKT(p-AKT)/AKT均低于H1975-NC组,差异均有统计学意义(P﹤0.05).结论 CD73能促进肺腺癌细胞的增殖、迁移和侵袭,其机制可能与PI3K/AKT信号通路和EMT过程有关.
Effect and mechanism of knocking down the expression of CD73on the proliferation,migration,invasion ability of human lung adenocarcinoma H1975 cells
Objective To investigate the effect and mechanism of knocking down the expression of CD73 on the pro-liferation,migration,invasion ability of human lung adenocarcinoma H1975 cells.Method Human lung adenocarcino-ma H1975 cells were selected,and human lung adenocarcinoma H1975 cells transfected with lentivirus HBLV-ZsGreen-PURO NC were selected as H1975-NC group.Human lung adenocarcinoma H1975 cells transfected with knocking down CD73 expression at three different sites of lentivirus HBLV-h-CD73 short hairpin RNA(shRNA)-ZsGreen-PURO were selected as H1975-SHCD73-1 group,H1975-SHCD73-2 group,and H1975-SHCD73-3 group,respectively.Non-trans-fected human lung adenocarcinoma H1975 cells were selected as blank control group.Western blot was used to select the transfected cells with the most significant knockdown effect for subsequent experiments,as H1975-sh group.The prolifer-ation,migration and invasion ability of the cells were detected by CCK8,cell scratch assay and Transwell invasion assay,respectively.Western blot was used to detect the relative expression levels of epithelial-mesenchymal transition(EMT)marker proteins(E-cadherin,vimentin)and the phosphorylated protein expression ratio of phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)signaling pathway.Result Western blot showed that the knocking down effect of CD73 in H1975-shCD73-2 cells(H1975-sh group)was the most obvious and these cells was selected for subsequent experi-ments.After knocking down the expression of CD73,at different transfection times,the optical density(OD)value of cells in H1975-sh group were significantly lower than those in H1975-NC group(P<0.01),the scratch area healing rate were lower than those in H1975-NC group(P<0.05),and the number of cell invasion were lower than those in H1975-NC group(P<0.05).The relative expression of E-cadherin protein in H1975-sh group was higher than that in H1975-NC group,the relative expression of vimentin protein was lower than that in H1975-NC group,phosphorylated-PI3K(p-PI3K)/PI3K and phosphorylated-AKT(p-AKT)/AKT were lower than those in H1975-NC group,and the differences were statistically significant(P<0.05).Conclusion CD73 can promote the proliferation,migration and invasion of H1975 lung adenocarcinoma cells,and the mechanism may be related to PI3K/AKT signal pathway and EMT process.
lung adenocarcinomaCD73epithelial-mesenchymal transitionphosphoinositide 3-kinaseprotein ki-nase B
万方数据
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