INK4基因座中反义非编码RNA对乳腺癌细胞增殖能力及顺铂敏感性的影响  

Effects of antisense non-coding RNA in the INK4 locus on the proliferation ability and cisplatin sensitivity of breast cancer cells

作　　者：韩一菲 张芳芳 王靖楠 邹炎 应莉 谢依天 李娜[1] HAN Yifei;ZHANG Fangfang;WANG Jingnan;ZOU Yan;YING Li;XIE Yitian;LI Na(Department of Pathology,Xinxiang Medical University,Xinxiang 453003,Henan Province,China;Sanquan college of Xinxiang Medical University,Xinxiang 453000,Henan Province,China)

机构地区：[1]新乡医学院病理学系,河南新乡453003 [2]新乡医学院三全学院,河南新乡453000

出　　处：《新乡医学院学报》2025年第2期93-98,106,共7页Journal of Xinxiang Medical University

基　　金：河南省教育厅高校重点科研项目(编号:24A310010);新乡医学院大学生创新创业计划项目(编号:202410472052)。

摘　　要：目的探讨INK4基因座中反义非编码RNA(ANRIL)对乳腺癌细胞增殖能力及顺铂敏感性的影响及其作用机制。方法采用实时荧光定量聚合酶链反应(RT-qPCR)技术检测乳腺癌组织标本及配对的癌旁组织、人乳腺癌细胞株MCF-7、MDA-MB-231、SKBR3以及人正常乳腺上皮细胞株MCF-10A中ANRIL的表达;将对数生长期的MDA-MB-231细胞随机分为si-NC组(转染对照的空载siRNA)、si-ANRIL组(转染ANRIL干扰片段)、si-ANRIL-anti-NC组(共转染ANRIL的干扰片段和miR-7-5p的空载体)、si-ANRIL-anti-miR-7-5p组(转染ANRIL的干扰片段和miR-7-5p的干扰片段)、miR-NC组(转染对照miRNA的空载体)、miR-7-5p mimic组(转染miR-7-5p的过表达载体)、miR-7-5p+ANRIL-WT转染组(共转染miR-7-5p mimics和ANRIL-WT)、miR-NC+ANRIL-WT转染组(共转染mimics Control和ANRIL-WT)、miR-7-5p+ANRIL-MUT转染组(共转染miR-7-5p mimics和ANRIL-MUT)和miR-NC+ANRIL-WUT转染组(共转染mimics Control和ANRIL-MUT)。采用细胞计数试剂盒-8法检测乳腺癌细胞的体外增殖能力,克隆形成实验检测细胞对顺铂的敏感性,双荧光素酶报告实验和RNA免疫沉淀(RIP)实验检测ANRIL和miR-7-5p的靶向结合关系。结果乳腺癌组织中ANRIL mRNA相对表达量显著高于癌旁组织(P<0.05);MDA-MB-231、MCF-7和SKBR3细胞中ANRIL mRNA的相对表达量显著高于正常乳腺上皮细胞(P<0.05);MDA-MB-231细胞中ANRIL mRNA的表达量显著高于MCF-7和SKBR3细胞(P<0.05)。培养24、48、72 h时,si-ANRIL组细胞存活率显著低于si-NC组(P<0.05)。与si-NC组相比,si-ANRIL组细胞克隆形成数目显著降低(P<0.05)。与miR-NC+ANRIL-WT转染组相比,miR-7-5p+ANRIL-WT转染组细胞荧光素酶活性显著降低(P<0.05);miR-NC+ANRIL-MUT转染组与miR-7-5p+ANRIL-MUT转染组细胞荧光素酶活性比较差异无统计学意义(P>0.05)。miR-7-5p mimic组细胞中ANRIL富集量显著高于miR-NC组(P<0.05)。在培养24、48、72 h时,si-ANRIL-anti-miR-7-5p组MDA-MB-231细胞存活率显著高于si-Objective To explore the effects of antisense non-coding RNA in the INK4 locus(ANRIL)on the proliferation ability and cisplatin sensitivity of breast cancer cells and relevant mechanisms.Methods The real-time quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of ANRIL in breast cancer tissue specimens,paired paracancerous tissues,human breast cancer cell lines(MCF-7,MDA-MB-231,and SKBR3),and the human normal breast epithelial cell line MCF-10A.The MDA-MB-231 cells in the logarithmic phase were divided into the si-NC group(transfected with control empty siRNA),si-ANRIL group(transfected with interfering fragments of ANRIL),si-ANRIL-anti-NC group(co-transfected with interfering fragments of ANRIL and empty vectors of miR-7-5p),si-ANRIL-anti-miR-7-5p group(transfected with interfering fragments of ANRIL and miR-7-5p),miR-NC group(transfected with empty vectors of control miRNA),miR-7-5p mimic group(transfected with overexpression vectors of miR-7-5p),miR-7-5p+ANRIL-WT transfection group(co-transfected with miR-7-5p mimics and ANRIL-WT),miR-NC+ANRIL-WT transfection group(co-transfected with mimics control and ANRIL-WT),miR-7-5p+ANRIL-MUT transfection group(co-transfected with miR-7-5p mimics and ANRIL-MUT),and miR-NC+ANRIL-WUT transfection group(co-transfected with mimics control and ANRIL-MUT).The proliferation ability of breast cancer cells in vitro was detected by the cell counting kit-8(CCK-8)assay,and the sensitivity of these cells to cisplatin was detected by the clonal formation assay.The targeted binding relationship between ANRIL and miR-7-5p was detected by the dual-luciferase reporter assay and the RNA immunoprecipitation(RIP)assay.Results The expression level of ANRIL mRNA in breast cancer tissues was significantly higher than that in paracancerous tissues(P<0.05);the expression level of ANRIL mRNA in MDA-MB-231,MCF-7,and SKBR3 cells was significantly higher than that in normal breast epithelial cells(P<0.05);the expression level of ANRIL mRNA in MDA-MB-231 cells was signific

关 键 词：INK4基因座中反义非编码RNA 微RNA-7-5p 乳腺癌 生物学行为 顺铂敏感性 

分 类 号：R730[医药卫生—肿瘤]