小干扰RNA抑制胸腺生成素基因的表达对肺癌A549细胞增殖和凋亡的影响  

Proliferation inhibition and apoptosis promotion induced by deletion of TMPO in A549

作　　者：董向阳[1] 李文静[2] 翟波 Dong Xiangyang;Li Wenjing;Zhai Bo(Thoracic and Cardiac Surgery Department,Children's Hospital Affiliated to Zhengzhou University/Henan Children's Hospital/Zhengzhou Children′s Hospital,Zhengzhou 450018,China;Endocrinology and Hereditary Metabolites Department,Children′s Hospital Affiliated to Zhengzhou University/Henan Children′s Hospital/Zhengzhou Children′s Hospital,Zhengzhou 450018,China)

机构地区：[1]郑州大学附属儿童医院、河南省儿童医院、郑州儿童医院胸心外科,450018 [2]郑州大学附属儿童医院、河南省儿童医院、郑州儿童医院内分泌遗传代谢科,450018

出　　处：《中华肿瘤杂志》2019年第10期742-747,共6页Chinese Journal of Oncology

基　　金：河南省科技攻关计划项目(162102310224).

摘　　要：目的探讨小干扰RNA(siRNA)抑制胸腺生成素(TMPO)基因的表达对肺癌A549细胞增殖和凋亡的影响及其作用机制。方法采用脂质体转染法将TMPO siRNA转染至人肺癌A549细胞,将转染siRNA control作为阴性对照组。转染后48h,采用实时荧光定量聚合酶链式反应(RT-PCR)和Western blot检测转染后细胞中TMPO的表达水平,四甲基偶氮唑蓝(MTT)法检测细胞的增殖情况,流式细胞术检测细胞周期和细胞的凋亡情况,Western blot检测细胞中PCNA、cleaved caspase-3、Notch1和mTOR的表达水平。结果RT-PCR检测结果显示,空白对照组、阴性对照组和转染组细胞中TMPO mRNA的表达水平分别为1.01±0.11、0.99±0.10和0.36±0.04。Western blot检测结果显示,空白对照组、阴性对照组和转染组细胞中TMPO蛋白的表达水平分别为0.27±0.02、0.29±0.03和0.08±0.10;转染组细胞中TMPO mRNA和蛋白的表达水平均低于空白对照组和阴性对照组(均P<0.05)。MTT检测结果显示,空白对照组、阴性对照组和转染组细胞在转染24 h时的吸光度(A)值分别为0.35±0.04、0.37±0.04和0.34±0.03,转染48 h时A值分别为0.47±0.06、0.46±0.08和0.37±0.04,转染72 h时A值分别为0.75±0.08、0.73±0.07和0.49±0.05,转染96 h时A值分别为1.09±0.07、1.06±0.08和0.56±0.06;转染组细胞在转染48、72、96 h的A值均低于空白对照组和阴性对照组(均P<0.05)。流式细胞术检测结果显示,空白对照组、阴性对照组和转染组中G0/G1期细胞比例分别为(62.55±2.03)%、(61.24±3.15)%和(47.35±2.44)%,S期细胞比例分别为(17.12±1.31)%、(17.70±2.01)%和(20.81±2.06)%,G2/M期细胞比例分别为(20.33±1.43)%、(21.06±1.52)%和(31.84±2.76)%,转染组G0/G1期细胞比例均低于空白对照组和阴性对照组(均P<0.05),G2/M期细胞比例均高于空白对照组和阴性对照组(均P<0.05)。转染组细胞的凋亡率为(34.10±2.69)%,明显高于空白对照组[(2.96±0.03)%]和阴性对照组[(3.01±0.04)%,均P<0.05]。WesObjective To investigate the effect of thymopoietin (TMPO) gene deleted by small interfering RNA (RNAi) on the proliferation and apoptosis of lung cancer cell A549 and its mechanism. Methods TMPO siRNA was transfected into A549 cells by lipofection. The transfected siRNA control was used as a negative control, and the parent cells were used as blank control. Forty-eight hours later, the expression of TMPO in the transfected cells was detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR) and Western blot. Cell proliferation was detected by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2H tetrazolium bromide (MTT) assay, cell cycle and apoptosis were detected by flow cytometry, the protein levels of proliferating cell nuclear antigen (PCNA), cleaved caspase-3, notch receptor 1 (Notch1) and mammalian rapamycin target protein (mTOR) were detected by Western blot analysis. Results The results of RT-PCR and Western blot showed that the expression levels of TMPO mRNA in the blank control group, the negative control group and TMPO siRNA transfected group were (1.01±0.11),(0.99±0.10),(0.36±0.04), respectively, the protein levels were (0.27±0.02),(0.29±0.03),(0.08±0.10), respectively. The expression levels of TMPO mRNA and protein in the transfected group were significantly lower than those in the blank control and negative control group (P<0.05). The results of MTT assay showed that the OD values of the blank control group, the negative control group and the transfected group were (0.35±0.04),(0.37±0.04) and (0.34±0.03) at 24 h of transfection, respectively. The OD values at 48 h were (0.47±0.06),(0.46±0.08),(0.37±0.04), the OD values at 72 h were (0.75±0.08),(0.73±0.07),(0.49±0.05), respectively, and the OD values at 96 h were (1.09±0.07),(1.06±0.08),(0.56±0.06). The proliferation abilities of the transfected cells at 48, 72, 96 h were significantly lower than those of the blank control and the negative control group (P<0.05). The results of flow cytometry showed that the propo

关 键 词：肺肿瘤 TMPO基因 细胞增殖 细胞凋亡 Notch1/mTOR信号通路 

分 类 号：R734[医药卫生—肿瘤]