miR-199a-3p通过调控BRCA1影响小细胞肺癌细胞对阿帕替尼敏感性的研究  

Study on miR-199a-3p regulating BRCA1 to influence the sensitivity of small cell lung cancer cells to apatinib

作　　者：张静 李唯源 胡国志 郭艺航 ZHANG Jing;LI Weiyuan;HU Guozhi;GUO Yihang(Department of Oncology,Tangshan Central Hospital,Tangshan,Hebei 063000,China)

机构地区：[1]河北省唐山中心医院肿瘤科,河北唐山063000

出　　处：《检验医学与临床》2025年第6期760-767,共8页Laboratory Medicine and Clinic

基　　金：河北省青年科技课题(20181033)。

摘　　要：目的探讨miR-199a-3p通过调控人乳腺癌易感基因1(BRCA1)影响小细胞肺癌(SCLC)细胞对阿帕替尼敏感性的机制。方法选择人SCLC细胞系H446、H1688以及正常支气管上皮细胞BEAS-2B进行培养,构建阿帕替尼耐药SCLC细胞系(H446/APA和H1688/APA),将H446/APA和H1688/APA细胞分为未转染和转染(转染的核酸分子包括miR-199a-3p mimics、miR-NC、sh-BRCA1、sh-NC)。采用反转录实时荧光定量聚合酶链反应(qRT-PCR)检测正常支气管上皮细胞BEAS-2B、H446、H1688、H446/APA、H1688/APA及转染miR-199a-3p mimics、转染miR-NC的H446/APA、H1688/APA细胞中miR-199a-3p表达水平。采用蛋白质印迹法检测正常支气管上皮细胞BEAS-2B、H446、H1688、H446/APA、H1688/APA及转染不同核酸分子的H446/APA、H1688/APA细胞中BRCA1蛋白表达水平。采用噻唑蓝溴化四唑(MTT)法测定H446、H1688、H446/APA、H1688/APA及转染不同核酸分子的H446/APA、H1688/APA细胞对阿帕替尼的半数最大抑制浓度(IC 50)值,测定转染不同核酸分子的H446/APA、H1688/APA细胞活力。通过集落形成测定分析转染不同核酸分子的H446/APA和H1688/APA细胞集落数。通过流式细胞术检测转染不同核酸分子的H446/APA和H1688/APA细胞的凋亡率。通过Transwell实验测定转染不同核酸分子的H446/APA、H1688/APA细胞的迁移和侵袭情况。通过双荧光素酶报告基因检测系统测定荧光素酶报告基因载体的荧光素酶活性,分析miR-199a-3p与BRCA1之间的联系。结果与正常支气管上皮细胞BEAS-2B相比,人SCLC细胞系H446、H1688、H446/APA、H1688/APA细胞中miR-199a-3p表达水平均降低(P<0.05),而BRCA1蛋白表达水平均升高(P<0.05)。与对应的亲代细胞相比,H446/APA和H1688/APA细胞中miR-199a-3p表达水平均降低(P<0.05),并且BRCA1蛋白表达水平均升高(P<0.05)。转染不同核酸分子的同一细胞系(H446/APA、H1688/APA)中:转染miR-199a-3p mimics的细胞中miR-199a-3p表达水平均明显高于转染miR-NC的细Objective To investigate the mechanism by which miR-199a-3p regulates breast cancer susceptibility gene 1(BRCA1)to affect the sensitivity of small cell lung cancer(SCLC)cells to apatini b.Methods Human SCLC cell lines(H446,H1688)and normal bronchial epithelial cells(BEAS-2B)were cultured.Apatinib-resistant SCLC cell lines(H446/APA and H1688/APA)were established.The resistant cells were divided into untransfected and transfected cells(transfected with miR-199a-3p mimics,miR-NC,sh-BRCA1 or sh-NC).Reverse transcription quantitative real-time polymerase chain reaction(qRT-PCR)was used to measure miR-199a-3p expression levels in BEAS-2B,H446,H1688,H446/APA,H1688/APA,and miR-199a-3p mimics-transfected,miR-NC-transfected H446/APA and H1688/APA cells.Western blotting was performed to detect BRCA1 protein expression in normal bronchial epithelial cells BEAS-2B,H446,H1688,H446/APA,H1688/APA,and H446/APA,H1688/APA cells transfected with different nucleic acid molecules.The half maximal inhibitory concentration(IC 50)values of apatinib were determined by thiazolyl blue tetrazolium bromide(MTT)assay in H446,H1688,H446/APA,H1688/APA and H446/APA,H1688/APA cells transfected with different nucleic acid molecules to determine the cell viability of H446/APA,H1688/APA cell transfected with different nucleic acid molecules.The number of colonies of H446/APA and H1688/APA cells transfected with different nucleic acid molecules was analyzed by colony formation assay.The apoptosis rate of H446/APA and H1688/APA cells transfected with different nucleic acid molecules was detected by flow cytometry.Migration and invasion of H446/APA and H1688/APA cells transfected with different nucleic acid molecules were determined by Transwell assay.The luciferase activity of luciferase reporter gene vectors was determined by dual luciferase reporter gene assay system to analyze the association between miR-199a-3p and BRCA1.Results Compared with BEAS-2B cells,miR-199a-3p expression levels in H446,H1688,H446/APA and H1688/APA cells were significantly r

关 键 词：miR-199a-3p 小细胞肺癌 阿帕替尼 化疗耐药 敏感性 机制 

分 类 号：R734.2[医药卫生—肿瘤]