2024 年第 11 期 第 19 卷

依托咪酯通过下调含WW结构域的E3泛素蛋白连接酶2表达抑制非小细胞肺癌A549细胞增殖并诱导其凋亡的实验研究

Experimental study of etomidate inhibiting proliferation and inducing apoptosis in non-small cell lung cancer A549 cells through down-regulation of recombinant WW domain containing E3 ubiquitin protein ligase 2 expression

作者：何元段晓飞党莎杰王培

英文作者：He Yuan Duan Xiaofei Dang Shajie Wang Pei

单位：陕西省肿瘤医院麻醉科，西安710061

英文单位：Department of Anesthesiology Shaanxi Provincial Cancer Hospital Xi′an 710061 China

关键词：非小细胞肺癌；依托咪酯；含WW结构域的E3泛素蛋白连接酶2；磷酸酶与张力蛋白同源物/磷脂酰肌醇-3-激酶/蛋白激酶B通路；增殖；凋亡

英文关键词：Non-smallcelllungcancer;Etomidate;RecombinantWWdomaincontainingE3ubiquitinproteinligase2;Apoptosisandphosphataseandtensinhomologdeletedonchromosometen/phosphatidylinositol-3-kinase/proteinkinaseBpathway;Proliferation;Apoptosis

• 摘要：

• 目的 探讨依托咪酯（ETO）对非小细胞肺癌(NSCLC)A549细胞增殖和凋亡的影响及含WW结构域的E3泛素蛋白连接酶2（WWP2）在其中发挥的作用机制。方法 分别采用0、1、2和3 mg/L的ETO处理人正常肺上皮细胞系BESA-2B和人NSCLC细胞系A549，细胞计数试剂盒法检测细胞活力；将A549细胞随机分为对照组、ETO（3 mg/L）组、ETO+NC组和ETO+WWP2-OE组，后2组分别于ETO处理后转染空载体pcDNA3.1-NC或WWP2过表达载体pcDNA3.1-WWP2，集落形成试验检测细胞增殖能力；TUNEL染色检测细胞凋亡；实时荧光定量聚合酶链反应法检测各组细胞中WWP2的mRNA表达；STITCH数据库选择与ETO直接相互作用的蛋白质；蛋白质印迹法检测各组细胞中WWP2、增殖、凋亡和磷酸酶与张力蛋白同源物(PTEN)/磷脂酰肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)通路相关蛋白表达。结果 ETO以剂量依赖性方式显著降低A549细胞活力（F=147.923，P＜0.001）；而对正常肺上皮BESA-2B细胞活力无影响（F=1.427，P=0.126）。不同浓度（0、1、2、3 mg/L）ETO处理A549细胞后，集落形成数量逐渐减少［0、1、2、3 mg/L ETO组分别为（898±38）、（785±48）、（635±36）、（388±20）个］，细胞凋亡率逐渐升高［0、1、2、3 mg/L ETO组分别为（2.23±0.65）%、（7.63±0.35）%、（13.24±0.47）%、（18.93±0.36）%］，呈剂量依赖性（F=218.732、352.786，均P＜0.001）。STITCH数据库预测ETO可通过上调WWP2蛋白表达和下调PTEN蛋白表达与WWP2和PTEN相互作用。与对照组相比，ETO组细胞中WWP2的mRNA和蛋白表达降低，集落形成数量减少，细胞凋亡率升高，增殖细胞核抗原（PCNA）、细胞增殖抗原Ki67、B淋巴细胞瘤基因2(Bcl-2)和PTEN蛋白表达降低，Bcl-2相关X蛋白（Bax）和含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)蛋白表达升高，磷酸化PI3K（p-PI3K）/PI3K和磷酸化Akt（p-Akt）/Akt比值降低（均P＜0.01）；与ETO+NC组比较，ETO+WWP2-OE组细胞中WWP2的mRNA和蛋白表达升高，集落形成数量增多，细胞凋亡率降低，PCNA、Ki67、Bcl-2和PTEN蛋白表达增多，Bax和Cleaved caspase-3蛋白表达减少，p-PI3K/PI3K和p-Akt/Akt比值升高（均P＜0.01）。结论 ETO可抑制A549细胞增殖并促进细胞凋亡，其作用机制可能与调控WWP2的下调和PTEN/PI3K/Akt通路的激活有关。

• Objective To investigate the effects of etomidate (ETO) on the proliferation and apoptosis of non-small cell lung cancer (NSCLC) A549 cells and the mechanism in which recombinant WW domain containing E3 ubiquitin protein ligase 2 (WWP2) plays a role. Methods The human normal lung epithelial cell line BESA-2B and the human NSCLC cell line A549 were treated with ETO at 0, 1, 2 and 3 mg/L, respectively, and cell viability was detected by the cell counting kit method; the A549 cells were randomly divided into the control group, the ETO (3 mg/L) group, the ETO+NC group and the ETO+WWP2-OE group, and the latter two groups were transfected with empty vector pcDNA3.1-NC or WWP2 overexpression vector pcDNA3.1-WWP2 after ETO treatment, respectively. Colony formation assay was performed to detect cell proliferation. TUNEL staining was performed to detect apoptosis. Quantitative real-time polymerase chain reaction was performed to detect mRNA expression of WWP2 in the cells of each group; and proteins directly interacting with ETO were selected from STITCH database. Western blot detected WWP2, proliferation, apoptosis and phosphatase and tensin homolog deleted on chromosome ten (PTEN) / phosphatidylinositol-3-kinase (PI3K) / protein kinase B (Akt) pathway related protein expression in each group of cells. Results ETO decreased A549 cell viability in a dose-dependent manner (F=147.923, P＜0.001), whereas it had no effect on the viability of normal lung epithelial BESA-2B cells (F=1.427, P=0.126). The number of colony formation was progressively reduced［0, 1, 2, 3 mg/L ETO group were (898±38), (785±48), (635±36), (388±20)］ and apoptosis was progressively increased ［0, 1, 2, 3 mg/L ETO group were (2.23±0.65)%, (7.63±0.35)%, (13.24±0.47)%, (18.93±0.36)%］ in a dose-dependent manner after ETO treatment(0, 1, 2, 3 mg/L) of A549 cells (F=218.732 and 352.786, both P＜0.001).The STITCH database predicted that ETO could interact with WWP2 and PTEN by upregulating WWP2 protein expression and downregulating PTEN protein expression. Compared with the control group, cells in the ETO group showed decreased mRNA and protein expression of WWP2, decreased number of colony formation, increased apoptosis rate, decreased proliferating cell nuclear antigen (PCNA), cell proliferation antigen Ki67, B-cell lymphoma-2 (Bcl-2) and PTEN protein expression, increased Bcl-2 associated X protein (Bax) and cysteinyl aspartate specific proteinase 3 (Cleaved caspase-3) protein expression, and decreased phospho-PI3K (p-PI3K)/PI3K and phospho-Akt (p-Akt)/Akt ratios (all P＜0.01); compared with the ETO+NC group, cells in the ETO+WWP2-OE group showed elevated mRNA and protein expression of WWP2, increased number of colony formation, decreased apoptosis rate, increased expression of PCNA, Ki67, Bcl-2 and PTEN proteins, decreased expression of Bax and Cleaved caspase-3 proteins, and decreased p-PI3K/ PI3K and p-Akt/Akt ratios were elevated (all P＜0.01). Conclusion ETO inhibits proliferation and promotes apoptosis in A549 cells, and its mechanism of action may be related to the regulation of WWP2 down-regulation and activation of PTEN/PI3K/Akt pathway.