微小RNA-374-5p对大鼠垂体泌乳素腺瘤细胞系 MMQ增殖、凋亡、侵袭的影响及其机制  

Effects of miR-374-5p on cell proliferation,apoptosis,and migration of rat pituitary tumor cell line MMQ

作　　者：王大维 李振业[1] 高华[1] WANG Dawei;LI Zhenye;GAO Hua(Beijing Neurosurgical Institute,Beijing 100070,China)

机构地区：[1]北京市神经外科研究所,北京100070 [2]首都医科大学第八临床医学院

出　　处：《山东医药》2020年第9期1-5,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81601205)。

摘　　要：目的观察微小RNA-374-5p(miR-374-5p)对大鼠垂体泌乳素腺瘤细胞系MMQ增殖、凋亡、侵袭的影响,并探讨其作用机制。方法将对数生长期MMQ细胞分为A、B、C、D组。A组加入常规培养基,B组加入miR-374-5p类似物,C组加入miR-374-5p抑制剂,D组加入miR-374-5p类似物和miR-374-5p抑制剂。各组培养0、24、48、72 h时,酶标仪在490 nm波长处检测各组细胞光密度值(OD值,以OD值表示细胞增殖能力)。各组细胞培养72 h时,离心收集细胞,加入Annexin V-FITC和PI染色,流式细胞仪检测Annexin V染色阳性细胞百分数和PI染色阳性细胞百分数(以Annexin V染色阳性细胞百分数和PI染色阳性细胞百分数表示细胞凋亡能力)。各组细胞培养72 h时,采用实时定量PCR法检测各组细胞侵袭相关基因[E-钙黏蛋白(E-cadherin)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、N-钙黏蛋白(N-cadherin)、蜗牛家族转录阻遏物1(Snail)、波形蛋白(Vimentin),以侵袭相关基因mRNA的相对表达量表示细胞侵袭能力]及垂体肿瘤转化基因(PTTG)mRNA。将野生型(WT)PTTG荧光素酶载体和突变型(mut)PTTG荧光素酶载体分别与microRNA-NC或miR-374-5p类似物共转染至MMQ细胞,记为NC-WT组、NC-mut组、WT组、mut组,转染72 h后,使用双荧光素酶报告基因检测系统检测细胞上清液中荧光素酶活性。结果与A、D组相比,培养24、48、72 h时,B组细胞增殖能力显著降低(P均<0.05),C组细胞增殖能力显著升高(P均<0.05)。A、B、C、D组Annexin V染色阳性细胞百分数分别为3.7%±1.4%、17.6%±5.3%、3.4%±1.5%、4.5%±1.4%,PI染色阳性细胞百分数分别为2.4%±0.9%、11.2%±3.7%、2.5%±1.2%、2.7%±0.9%,其中B组Annexin V染色阳性细胞百分数和PI染色阳性细胞百分数与A、C、D组相比,P均<0.05。与A、D组相比,B组细胞E-cadherin mRNA、PTTG mRNA相对表达量显著降低(P均<0.05),N-cadherin mRNA相对表达量显著升高(P均<0.05)。NC-WT组、NC-mut组、WT组、muObjective To study the effects of microRNA-374-5p(miR-374-5p)on the cell proliferation,apoptosis,and migration of rat pituitary tumor cell line MMQ,and to explore its mechanism.Methods MMQ cells in the logarithmic growth phase were divided into groups A,B,C and D.MMQ cells in the group A were cultured in cell culture medium,and group B was added with miR-374-5p mimic,group C with miR-374-5p inhibitor,and group D with miR-374-5p mimic and miR-374-5p inhibitor.After 0,24,48 and 72 hours of treatment,microplate reader was used to detect the optical density value of each group at 490 nm(OD value meant the ability of cell proliferation in each group).After 72-hour treatment,cells were collected by centrifugation for Annexin V-FITC and PI staining.Flow cytometry was used for detecting the percent of Annexin positive cells and PI positive cells which indicated the apoptosis level in each group.Meanwhile,the mRNA levels of PTTG,E-cadherin,matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),Ncadherin,Snail,and Vimentin were measured by real-time PCR.We separately cotransfected the wild type(WT)pituitary tumor-transforming gene(PTTG)luciferase vector and mutant(mut)PTTG luciferase vector with microRNA-NC or miR-374-5p mimic into MMQ cells,which were named as NC-WT group,NC-mut group,WT group,and mut group,respectively.Dual luciferase reporter gene assay was used to measure the optical density value after 72-hour transfection.Results Compared with groups A and D,the cell proliferation decreased in the group B and increased in the group C at 24,48,and 72 h(both P<0.05).The percentages of Annexin V-positive cells in the groups A,B,C,and D were 3.7%±1.4%,17.6%±5.3%,3.4%±1.5%,and 4.5%±1.4%,respectively,the percentages of PI-positive cells were 2.4%±0.9%,11.2%±3.7%,2.5%±1.2%,and 2.7%±0.9%,respectively;there was statistical difference between group B and groups A,C and D(all P<0.05).Compared with groups A and D,the mRNA levels of PTTG and Ncadherin of group B decreased and the mRNA level of E-cadherin incr

关 键 词：微小RNA 微小RNA-374-5p MMQ细胞 泌乳素腺瘤 垂体肿瘤转化基因 细胞增殖 细胞凋亡 E-钙黏蛋白 N-钙黏蛋白 

分 类 号：R736.4[医药卫生—肿瘤]