自噬/Beclin1调节因子1通过Akt-Bad-Bcl-2通路降低乳腺癌化疗敏感性  

Ambra1 reduces the chemosensitivity of breast cancer cells via the Akt-Bad-Bcl-2 pathway

作　　者：孙蔚亮 罗婕[1] 梁俐[1] 王莉[1] 赵璧 SUN Wei-liang;LUO Jie;LIANG Li;WANG Li;ZHAO Bi(Department of Medical Oncology,Second Affiliated Hospital of Guangzi Medical University,Nanning 530007,China)

机构地区：[1]广西医科大学第二附属医院肿瘤内科,广西南宁530007

出　　处：《中华肿瘤防治杂志》2023年第1期20-29,35,共11页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(82160499);广西自然科学基金(2018GXNSFAA281002)。

摘　　要：目的检测自噬/Beclin1调节因子1(Ambra1)在乳腺癌中的表达及对化疗敏感性的影响,并探讨可能的机制。方法(1)临床标本测定:回顾性收集2018-03-01-2019-12-30在广西医科大学第二附属医院行手术切除的42例乳腺癌及癌旁组织标本,采用免疫组织化学方法检测Ambra1的表达。(2)细胞实验:通过Ambra1慢病毒表达载体(OE),使Ambra1在乳腺癌MCF-7和MDA-MB-231细胞高表达,以空载(Empty)作为对照。蛋白质印迹法分别检测细胞内Akt/pAkt、Bad/pBad^(s136)、Bcl-2和Bax的表达,定量逆转录聚合酶链反应(qRT-PCR)检测Akt mRNA表达;同时,细胞用紫杉醇(PTX)处理后,细胞计数盒8(CCK8)法和流式细胞术分别检测细胞存活和凋亡。为了研究pAkt的作用,细胞在感染OE的同时,使用GSK690693(GSK)抑制Akt磷酸化,蛋白质印迹法检测Bad/pBad^(s136)、Bcl-2和Bax表达,流式细胞术和CCK8法分别检测紫杉醇诱导的凋亡和细胞存活。结果(1)临床标本测定:Ambra1在乳腺癌组织中表达率为73.70%,高于正常乳腺组织(36.40%),差异有统计学意义,t=2.147,P=0.037。(2)细胞实验:感染了OE的MCF-7和MDA-MB-231细胞内Ambra1的相对表达量分别为1.07±0.09和0.63±0.04,高于Empty的0.25±0.03和0.22±0.02,差异有统计学意义,t值分别为8.801和10.132,均P<0.05。而且OE组细胞内Akt/pAkt、pBad^(s136)、Bcl-2蛋白,以及Akt mRNA表达均高于Empty组,而Bax蛋白表达低于Empty组,差异有统计学意义,MCF-7的t值分别为13.132、11.672、4.828、7.163、4.596和-3.321,MDA-MB-231的t值分别为4.239、9.966、4.048、7.456、3.479和-9.181,均P<0.05。PTX处理后,不同处理组间细胞凋亡率和存活率差异均有统计学意义(MCF-7的F值分别为291.608和19.110,均P<0.001;MDA-MB-231的F值分别为339.698和15.459,均P<0.001)。其中PTX+OE组细胞凋亡率均低于PTX组,而细胞存活率则高于PTX组,差异有统计学意义,均P<0.05。Ambra1高表达(OE+),有或无GSK时pAkt、pBad^(s136)、Bcl-2及Bax蛋白表达�Objective To determine the expression of Autophagy/Beclin 1 Regulator 1(Ambra1) in breast cancer and its effect on chemosensitivity and to explore the possible mechanism of its regulation of chemosensitivity.Methods(1) Determination of the clinical specimens: immunohistochemistry was used to determine the expression of Ambra1 in 42 cases of breast cancer and adjacent tissues that were surgically resected at Second Affiliated Hospital of Guangxi Medical University from March 1,2018 to December 30,2019.(2) Cell experiments: MDA-MB-231 and MCF-7 cells were infected with the lentiviral vector-Ambra1(OE) to overexpress Ambra1,and the empty vector(Empty) was used as a control.Then, western blotting was used to determine the expression of Akt/pAkt, Bad/pBad^(s136),Bcl-2 and Bax in cells with high Ambra1 expression, and quantitative reverse transcription polymerase chain reaction(qRT-PCR) was used to determine the expression of Akt mRNA.After the cells were treated with paclitaxel(PTX),cell survival and apoptosis were determined by cell counting kit 8(CCK8)assays and flow cytometry,respectively.To investigate the role of pAkt,GSK690693(GSK)was used to inhibit the phosphorylation of Akt when OE cells were infected.Then,western blotting was used to determine the expression of Bad/pBad^(s136),Bcl-2and Bax.In addition,after the cells were treated with OE+PTX±GSK,cell survival and apoptosis were determined by CCK8assays and flow cytometry.Results (1)Determination of the clinical specimens:the expression of Ambra1in breast cancer tissues was higher than that in normal breast tissues(73.70% vs36.40%),and the difference was statistically significant,t=2.147,P=0.037.(2)Cell experiments:the relative expressions of Ambra1in MCF-7and MDA-MB-231cells infected with OE were 1.07±0.09and 0.63±0.04,higher than0.25±0.03and 0.22±0.02in cells infected with Empty,the difference was statistically significant,t values were 8.801and 10.132,both P<0.05.In addition,the expressions of Akt/pAkt,pBad^(s136) and Bcl-2protein and Akt mRNA in OE g

关 键 词：自噬/Beclin1调节因子1 蛋白激酶B BAD 乳腺肿瘤 化疗敏感性 

分 类 号：R737.9[医药卫生—肿瘤]