miR-101-3p通过靶向EIF4G2逆转卵巢癌SKOV3/DDP细胞对顺铂耐药性及其机制研究  

The role and mechanism of miR-101-3p reverses cisplatin resistance of ovarian cancer SKOV3/DDP cells by targeting EIF4G2

作　　者：于歌 冯晓玲[3] 牛明[4] Yu Ge;Feng Xiaoling;Niu Ming(Institute of Traditional Chinese Medicine,Heilongjiang University of Chinese Medicine,Harbin 150006,China;Department of Gynecology,Harbin Medical University Cancer Hospital,Harbin 150081,China;Department of Gynecology,the First Affiliated Hospital,Heilongjiang University of Chinese Medicine,Harbin 150040,China;Department of Breast Surgery,Harbin Medical University Cancer Hospital,Harbin 150081,China)

机构地区：[1]黑龙江中医药大学药学院,哈尔滨150006 [2]哈尔滨医科大学附属肿瘤医院妇科,哈尔滨150081 [3]黑龙江中医药大学附属第一医院妇科,哈尔滨150040 [4]哈尔滨医科大学附属肿瘤医院乳腺外科,哈尔滨150081

出　　处：《国际免疫学杂志》2022年第5期449-455,共7页International Journal of Immunology

基　　金：黑龙江省博士后基金(LBH-Z19215);黑龙江省中医药管理局基金(ZHY2020-173)。

摘　　要：目的探讨microRNA-101-3p(miR-101-3p)在卵巢癌顺铂(cisplatin,DDP)耐药中的作用及其机制。方法购买miR-101-3p模拟物(miR-101-3p mimic)、miR-101-3p抑制剂(miR-101-3p-in)、及其各自的阴性对照RNA(miR-NC mimic和miR-NC inhibitor)。应用定量逆转录聚合酶链反应(real-time reverse transcription-PCR,qRT-PCR)和western blotting检测miR-101-3p和B淋巴瘤momlv插入区1同源物(eukaryotic translation initiation factor 4 gamma 2,EIF4G2)在卵巢癌SKOV3细胞和DDP耐药SKOV3/DDP细胞中的表达水平。用miR-101-3p模拟物和miR-101-3p模拟阴性对照转染SKOV3/DDP细胞。采用CCK-8法检测miR-101-3p模拟物转染后细胞对DDP的敏感性。流式细胞仪检测转染对细胞凋亡的影响。将EIF4G2野生型和突变型荧光素酶报告质粒与miRNA-101-3p模拟物或miRNA-101-3p NC共转染,用双荧光素酶报告系统分析荧光素酶活性。结果CCK-8法检测结果显示,miR-101-3p在SKOV3/DDP细胞中的表达水平显著低于SKOV3细胞,EIF4G2在SKOV3/DDP细胞中的表达水平显著高于SKOV3细胞。过表达miR-101-3p能够显著提高miR-101-3p mimic组中SKOV3/DDP细胞对DDP的敏感性,其IC50值为8μmol/L和64μmol/L,miR-101-3p mimic可抑制SKOV3/DDP细胞活性;AO/EB实验验证过表达miR-101-3p后miR-101-3p mimic能够明显诱导SKOV3/DDP细胞凋亡。Western blotting检测结果表明,miR-101-3p mimic可显著增加B细胞淋巴瘤2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(associated X protein,Bax)及裂解型半胱天冬酶-3的表达水平(t=3.57、3.95、4.17,P值均<0.05)。与miR-NC mimic相比,过表达miR-101-3p后能够明显降低EIF4G2的表达(t=4.15,P<0.05);荧光素酶活性检测数据显示,miR-101-3p的过表达会抑制野生型EIF4G2的荧光素酶活性,但不抑制突变型EIF4G23’UTR的荧光素酶活性。结论EIF4G2是miR-101-3p的直接作用靶点,MiR-101-3p可能通过靶向调控EIF4G2,调节卵巢癌对DDP的耐药性。Original Article The role and mechanism of miR-101-3p reverses cisplatin resistance of ovarian cancer SKOV3/DDP cells by targeting EIF4G2 Yu Ge,Feng Xiaoling,Niu Ming Published 2022-09-05 Cite as Int J Immunol,2022,45(5):449-455.DOI:10.3760/cma.j.issn.1673-4394.2022.05.002 Abstract Objective To investigate the role and mechanism of microRNA-101-3p(miR-101-3p)in cisplatin(DDP)resistance of ovarian cancer.Methods miR-101-3p mimic(miR-101-3p mimic),miR-101-3p inhibitor(miR-101-3p-in),and their respective negative control RNAs(miR NC mimic and miR NC inhibitor)have been purchased.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)and Western blotting were applied to detect the expression levels of miR-101-3p and eukaryotic translation initiation factor 4 gamma 2(EIF4G2)in ovarian cancer SKOV3 cells and DDP-resistant SKOV3/DDP cells.SKOV3/DDP cells were transfected with miR-101-3p mimic and miR-101-3p mimic negative control.The expression of EIF4G2 was detected by real time reverse transcription-PCR(qRT-PCR)and Western blotting,and the sensitivity of cells to DDP after transfection with miR-101-3p mimic was detected by CCK-8 assay.The effect of transfection on the apoptosis was detected via flow cytometry.EIF4G2 wild-type and mutant-type luciferase reporter plasmids were co-transfected with miRNA-101-3p mimic or miRNA-101-3p NC,and luciferase activity was analyzed by dual-luciferase reporter system.Results The results of CCK-8 method show that the miR-101-3p expression level in SKOV3/DDP cells was significantly lower than that in SKOV3 cells,while the expression level of EIF4G2 in SKOV3/DDP cells was significantly higher than that in SKOV3 cells.Overexpression of miR-101-3p significantly increased the sensitivity of SKOV3/DDP cells to DDP in the miR-101-3p mimic group with an IC50 of 8μmol/L and 64μmol/L.miR-101-3p mimic could inhibit the activity of SKOV3/DDP cells.AO/EB experiment verified that miR-101-3p mimic could significantly induce apoptosis of SKOV3/DDP cells after overexpression of miR-10

关 键 词：miR-101-3p EIF4G2 卵巢癌 顺铂 细胞耐药 凋亡 

分 类 号：R737.31[医药卫生—肿瘤]