Circ_0022382/let-7a-5p/PI3K/AKT/mTOR轴促进乳腺癌细胞增殖和迁移  

Circ_0022382/let-7a-5p/PI3K/AKT/mTOR axis promotes the proliferation and migration of breast cancer cells

作　　者：刘伟[1] 张俊 张佳雯 叶雨 诸健琴 虞绮雯 李涛 孙晓春[1] 陈华标 LIU Wei;ZHANG Jun;ZHANG Jiawen;YE Yu;ZHU Jianqin;YU Qiwen;LI Tao;SUN Xiaochun;CHEN Huabiao(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 202013;Department of Clinical Laboratory,Affiliated Hospital of Yangzhou University,Yangzhou Jiangsu 225012;School of Medicine,Ningbo University,Ningbo Zhejiang 315211,China)

机构地区：[1]江苏大学医学院,江苏镇江212013 [2]扬州大学附属医院检验科,江苏扬州225012 [3]宁波大学医学院,浙江宁波315211

出　　处：《江苏大学学报(医学版)》2025年第2期145-153,共9页Journal of Jiangsu University:Medicine Edition

摘　　要：目的:研究circ_0022382对乳腺癌细胞增殖和迁移的影响及可能的作用机制。方法:①筛选乳腺癌中高表达的circRNA,首先分别对乳腺癌circRNA相关GEO数据集(GSE165884)和miRNA相关GEO数据集(GSE45498)进行差异分析,然后通过ENCORI数据库预测差异表达的circRNA所结合的miRNA,最后通过韦恩图对circRNA结合的miRNA和GEO数据库预测的miRNA取交集,得到交集的miRNA及其对应的circRNA。②分别予以收敛引物和发散引物对乳腺癌细胞MDA-MB-231来源的互补DNA(cDNA)和基因组DNA(gDNA)进行PCR,并通过琼脂糖凝胶电泳实验对PCR产物进行分离以验证circ_0022382的环形结构。③实时荧光定量PCR(qRT-PCR)分别检测乳腺癌细胞MDA-MB-231和MCF-7及正常乳腺上皮细胞MCF-10A,乳腺癌组织和癌旁组织中circ_0022382表达水平。④在MDA-MB-231细胞和MCF-7细胞中分别转染si-NC、si-circ_0022382,采用qRT-PCR检测转染效率,分别采用克隆形成实验和划痕愈合实验检测乳腺癌细胞增殖和迁移;qRT-PCR检测let-7a-5p的表达水平;在si-circ_0022382组中分别转染let-7a-5p inhibitor NC、let-7a-5p inhibitor,克隆形成实验和划痕愈合实验检测乳腺癌细胞增殖和迁移。⑤通过京都基因与基因组百科全书(KEGG)富集分析let-7a-5p的下游信号通路;在MDA-MB-231细胞和MCF-7细胞中分别转染let-7a-5p NC、let-7a-5p mimic,蛋白质印迹法和细胞荧光免疫法检测PI3K/AKT/mTOR信号通路相关蛋白表达,蛋白质印迹法检测let-7a-5p inhibitor和si-circ_0022382共转染后乳腺癌细胞p-AKT表达。结果:①GSE165884差异分析得到37个高表达的circRNA,GSE45498差异分析得到6个低表达的miRNA,37个高表达的circRNA中,circ_0022382对应的miRNA和6个低表达的miRNA有交集基因let-7。②乳腺癌细胞MDA-MB-231来源的cDNA既可以通过发散引物又可以通过收敛引物来扩增,而gDNA只能通过收敛引物来扩增。③与MCF-10A细胞相比,circ_0022382在乳腺癌细胞MDA-MB-231和MCF-7Objective:To study the effects of circ_0022382 on the proliferation and migration of breast cancer cells and the possible underlying mechanism.Methods:①Screening for circRNAs that are highly expressed in breast cancer.Firstly,the differential analysis of the circRNA-related GEO dataset(GSE165884)and miRNA-related GEO dataset(GSE45498)of breast cancer were carried out,respectively,and then the miRNAs bound by the differentially expressed circRNAs were predicted by the ENCORI database,and finally the intersecting miRNAs and the miRNAs predicted by the GEO database were obtained by the Venn diagram.②The complementary DNA(cDNA)and genomic DNA(gDNA)derived from MDA-MB-231 of breast cancer cells were PCR with convergent primers and divergent primers,respectively,and the PCR products were separated by agarose gel electrophoresis to verify the circular structure of the circ_0022382.③Real-time quantitative PCR(qRT-PCR)was used to detect circ_0022382 expression in breast cancer cells MDA-MB-231 and MCF-7 and normal breast epithelial cell MCF-10A,as well as in breast cancer tissues and adjacent tissues,respectively.④MDA-MB-231 cells and MCF-7 cells were transfected with si-NC and si-circ_0022382,qRT-PCR was used to detect transfection efficiency,clone formation assay and scratch healing assay were used to detect the proliferation and migration of breast cancer cells.qRT-PCR was used to detect the expression level of let-7a-5p.The si-circ_0022382 group was transfected with let-7a-5p inhibitor NC and let-7a-5p inhibitor,respectively,and the proliferation and migration of breast cancer cells were detected by clone formation assay and scratch healing assay.⑤The downstream signaling pathway of let-7a-5p was analyzed by Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.MDA-MB-231 cells and MCF-7 cells were transfected with let-7a-5p NC and let-7a-5p mimic,respectively,and the expression of PI3K/AKT/mTOR signaling pathway-related proteins was detected by Western blotting and cellular immunofluorescence,and the expr

关 键 词：circ_0022382 let-7a-5p PI3K/AKT/mTOR信号通路 增殖 迁移 

分 类 号：R737.9[医药卫生—肿瘤]