摘要
目的探讨共济失调毛细血管扩张突变基因Rad3相关激酶(ATR)/沉默细胞周期检测点激酶1(CHK1)信号通路在卵巢上皮癌发生、发展过程中的作用。方法体外培养人卵巢上皮癌细胞OVCAR3,分为空白对照组(NC组)、siRNAsc组(转染siRNAsc的OVCAR3细胞)、siRNAATR组(转染siRNAATR的OVCAR3细胞)、VE822组(加入1μmol/L的ATR抑制剂VE822)、和siRNAATR+VE822组。采用siRNA技术和VE822干扰OVCAR3细胞中的ATR,通过实时荧光定量PCR(qRTPCR)和蛋白免疫印迹法(Western blot)检测细胞中ATR的表达确定干扰有效后,CCK8法检测各组细胞增殖抑制情况,流式细胞术(FCM)检测细胞周期和凋亡情况,qRTPCR检测卵巢上皮癌细胞中凋亡相关基因半胱氨酸的天冬氨酸蛋白水解酶3(Caspase3)、B细胞淋巴瘤2(Bcl2)、Bcl2相关X蛋白(Bax)mRNA的表达,蛋白免疫印迹法(Western blot)检测ATR/CHK1通路相关蛋白的表达。结果siRNA-ATR转染和VE-822干预后,与NC组和siRNA-sc组相比,siRNA-ATR组、VE-822组、siRNA-ATR+VE-822组OVCAR3细胞中ATR的mRNA[(1.55±0.12)、(1.51±0.13)、(0.71±0.11)、(0.73±0.12)、(0.49±0.09)]表达水平显著降低(P<0.05),细胞增殖抑制率[(0.00±0.00)%、(0.00±0.00)%、(32.84±1.08)%、(30.75±1.44)%、(43.90±1.57)%]、细胞G0/G1期的比例[(40.08±2.57)、(36.35±3.44)、(53.28±4.34)、(56.37±5.03)、(70.63±3.81)]、细胞凋亡率[(4.28±0.67)%、(5.35±0.94)%、(23.63±1.13)%、(24.57±1.20)%、(35.86±1.09)%]、Caspase-3、Bax mRNA表达明显升高(P<0.05),S期[(32.93±3.02)、(35.35±2.82)、(25.79±3.61)、(23.74±3.54)、(18.04±2.37)]和G2/M期[(26.99±2.84)、(28.30±2.72)、(20.93±3.01)、(19.98±2.87)、(11.33±2.11)]的细胞比例、Bcl-2 mRNA、ATR、p-CHK1/CHK1、细胞分裂周期蛋白25C(CDC25C)、细胞周期蛋白B1(cyclin B1)蛋白表达显著降低(P<0.05);与siRNA-ATR组相比,siRNA-ATR+VE-822组细胞中ATR的mRNA表达水平进一步降低(P<0.05),细胞增殖抑制率、细胞凋亡、Caspase-3、Bax mRNA表达进一步增�
Objective To discuss the role of ataxia telangiectasia and Rad3-related kinase(ATR)/check point kinase 1(CHK1)signaling pathway in the occurrence and development of epithelial ovarian cancer.Methods Human epithelial ovarian cancer cells OVCAR3 cells were cultured in vitro,siRNA and VE-822 were used to interfere with ATR in OVCAR3 cells,the effectiveness of interference with ATR expression was detected by real-time fluorescence quantitative PCR(qRT-PCR)and Western blot,the cell proliferation inhibition was detect by CCK-8 method,cell cycle and apoptosis were detected by flow cytometry(FCM),the mRNA expressions of Caspase-3,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)were detected by qRT-PCR,Western blot was used to detect the expression of ATR/CHK1 pathway related proteins.Results After siRNA-ATR transfection and VE-822 intervention,compared with those in NC group and siRNA-sc group,the expression level of ATR mRNA[(1.55±0.12),(1.51±0.13),(0.71±0.11),(0.73±0.12),(0.49±0.09)]in OVCAR3 cells in siRNA-ATR group,VE-822 group and siRNA-ATR+VE-822 group was significantly lower(P<0.05),the inhibition rate of cell proliferation[(0.00±0.00)%,(0.00±0.00)%,(32.84±1.08)%,(30.75±1.44)%,(43.90±1.57)%],ratio of G0/G1 phase[(40.08±2.57),(36.35±3.44),(53.28±4.34),(56.37±5.03),(70.63±3.81)],apoptosis rate[(4.28±0.67)%,(5.35±0.94)%,(23.63±1.13)%,(24.57±1.20)%,(35.86±1.09)%],expression of Caspase-3 and Bax mRNA were significantly higher(P<0.05),and the cell proportions in S phase[(32.93±3.02),(35.35±2.82),(25.79±3.61),(23.74±3.54),(18.04±2.37)]and G2/M phase[(26.99±2.84),(28.30±2.72),(20.93±3.01),(19.98±2.87),(11.33±2.11)],Bcl-2 mRNA,ATR,p-CHK1/CHK1,cell division cycle protein 25C(CDC25C)and cyclin B1 protein expression were significantly lower(P<0.05).Compared with those in siRNA-ATR group,the expression of ATR mRNA in siRNA-ATR+VE-822 group was further decreased(P<0.05),the inhibition rate of cell proliferation,apoptosis,expression of Caspase-3 and Bax mRNA were further increased(P<0.05),an
作者
李慰
翁立斌
黄慧
Li Wei;Weng Libin;Huang Hui(Department of Gynecology,People’s Hospital Affiliated to Ningbo University,Ningbo 315040,China;Department of Internal Medicine,Third Hospital,Haishu District,Ningbo 315171,China)
出处
《中华内分泌外科杂志》
CAS
2021年第6期612-617,共6页
Chinese Journal of Endocrine Surgery
基金
浙江省中医药科学研究基金项目(2017ZA130)。
