miR-340-5p对BRCA1缺陷型卵巢癌细胞复制叉稳定性的影响  

Effect of miR-340-5p on replication fork stability in BRCA1-deficient ovarian cancer cells

作　　者：廖泽兰 裴凯歌 曹莹 王愚[1] LIAO Ze-lan;PEI Kai-ge;CAO Ying;WANG Yu(Department of Gynecology,Affiliated Hospital of Chengdu University,Chengdu 610000;Department of Gynecology,West China Second Hospital,Sichuan University,Chengdu 610000;Department of General Medicine,Affiliated Hospital of Chengdu University,Chengdu 610000,China)

机构地区：[1]成都大学附属医院妇科,四川成都610000 [2]四川大学华西第二医院妇科,四川成都610000 [3]成都大学附属医院全科医学科,四川成都610000

出　　处：《生物技术》2023年第5期592-598,共7页Biotechnology

摘　　要：[目的]探讨miR-340-5p对BRCA1缺陷型卵巢癌细胞复制叉稳定性的潜在机制。[方法]使用来自癌症基因组图谱(TCGA)卵巢癌数据集,将BRCA1突变肿瘤中的miR-340-5p表达与BRCA1野生型肿瘤进行比较。过表达或敲低miR-340-5p后,检测BRCA1缺陷型UWB1.289细胞中复制叉的稳定性、细胞增殖水平。UWB1.289细胞中敲低或过表达miR-340-5p后进行转录组测序并使用siRNA筛选鉴定miR-340-5p的靶向基因。[结果]miR-340-5p低表达与乳腺肿瘤中BRCA1缺乏相关(0.04±0.01 vs 0.15±0.04,P<0.05)。具有低表达miR-340-5p的BRCA1缺陷型肿瘤具有更高水平的基因组改变(0.37±0.05 vs 0.66±0.11,P<0.05)。过表达miR-340-5p后,BRCA1缺陷型UWB1.289细胞中复制叉不稳定性增强(1.12±0.23 vs 0.81±0.10,P<0.05)。相反,在UWB1.289细胞中同时过表达BRCA1和miR-340-5p并没有增强复制叉的不稳定性。此外,过表达或者敲低miR-340-5p后UWB1.289细胞的增殖水平分别下降或者上升(P<0.05),而在过表达BRCA1的UWB1.289细胞中无此现象。荧光素酶报告实验发现miR-340-5p靶向USP1 mRNA的3'端非翻译区(P<0.05)。过表达或者敲低miR-340-5p后,USP1的mRNA和蛋白水平分别下降或者上升(P<0.05)。敲低USP1或者过表达USP1时,UWB1.289细胞中复制叉不稳定性增强(P<0.05)。同时,敲低USP1后,UWB1.289细胞的增殖水平下降(P<0.05);过表达USP1后UWB1.289细胞的增殖水平上升(P<0.05),而在过表达BRCA1的UWB1.289细胞中无此现象。[结论]miR-340-5p能够促进BRCA1缺陷卵巢癌细胞复制叉的不稳定性,并抑制BRCA1缺陷细胞的存活。[Objective]To explore the potential mechanism of miR-340-5p regulating replication fork stability in BRCA1-deficient ovarian cancer cells.[Method]miR-340-5p expression in BRCA1-mutant tumors was compared to BRCA1 wild-type tumors using the ovarian cancer dataset from The Cancer Genome Atlas(TCGA).After overexpression or knockdown of miR-340-5p,the stability of replication fork and cell proliferation level in BRCA1-deficient UWB1.289 cells were detected.Transcriptome sequencing was performed after knockdown or overexpression of miR-340-5p in UWB1.289 cells and siRNA screening was used to identify the target genes of miR-340-5p.[Result]Low miR-340-5p expression was associated with BRCA1 deficiency in breast tumors(0.04±0.01 vs 0.15±0.04,P<0.05).BRCA1-deficient tumors with low expression of miR-340-5p had higher levels of genomic alterations(0.37±0.05 vs 0.66±0.11,P<0.05).Replication fork instability was enhanced in BRCA1-deficient UWB1.289 cells after overexpression of miR-340-5p(1.12±0.23 vs 0.81±0.10,P<0.05).In contrast,simultaneous overexpression of BRCA1 and miR-340-5p in UWB1.289 cells did not enhance replication fork instability.In addition,the proliferation level of UWB1.289 cells decreased after overexpression of miR-340-5p(1.67±0.06 vs 1.14±0.08,P<0.05);the proliferation level of UWB1.289 cells increased after knockdown of miR-340-5p(1.67±0.06 vs 2.15±0.08,P<0.05),while the proliferation level of UWB1.289 cells in the overexpression of BRCA1 in UWB1.289 cells did not.Luciferase reporter assays revealed that miR-340-5p targeted the untranslated region at the 3'end of USP1 mRNA(102.75±5.07 vs 13.55±1.87,P<0.05).The mRNA and protein levels of USP1 decreased after overexpression of miR-340-5p(0.76±0.07 vs 0.19±0.02,P<0.05);the mRNA and protein levels of USP1 increased after knockdown of miR-340-5p(0.76±0.07 vs 1.29±0.11,P<0.05).Replication fork instability was enhanced in UWB1.289 cells when USP1 was knocked down(1.15±0.23 vs 0.75±0.10,P<0.05).Replication fork stability was enhanced in UWB1.2

关 键 词：miR-340-5p BRCA1 卵巢癌 复制叉 USP1 UWB1.289 稳定性 增殖 

分 类 号：R737.31[医药卫生—肿瘤]