Effect and mechanism of knocking down the expression of CD73on the proliferation,migration,invasion ability of human lung adenocarcinoma H1975 cells
肖文华 1黄真 2刘凤娟 3张弘 4苏红 5孙荣丽4
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作者信息
1. 青岛大学青岛医学院,山东 青岛 266042
2. 康复大学青岛中心医院(青岛市中心医院)院长办公室,山东 青岛 266042
3. 康复大学青岛中心医院(青岛市中心医院)药物临床试验中心一期病房,山东 青岛 266042
4. 康复大学青岛中心医院(青岛市中心医院)呼吸与危重症医学科,山东 青岛 266042
5. 康复大学青岛中心医院(青岛市中心医院)基层医疗科,山东 青岛 266042
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摘要
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.
Abstract
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.
关键词
肺腺癌/CD73/上皮-间充质转化/磷脂酰肌醇3激酶/蛋白激酶B
Key words
lung adenocarcinoma/CD73/epithelial-mesenchymal transition/phosphoinositide 3-kinase/protein ki-nase B
维普
万方数据