咪达唑仑上调微RNA-98-5p影响乳腺癌细胞的增殖和凋亡研究

Midazolam up-regulates microRNA-98-5p and affects the proliferation and apoptosis of breast cancer cells

目的:探讨咪达唑仑对人乳腺癌细胞MDA-MB-231增殖及凋亡的影响及作用机制。方法:将MDA-MB-231细胞按照随机数字表法分为对照组(A组),咪达唑仑10、30、50 mg/L浓度组(分别为B组、C组、D组),干扰微RNA-98-5p (microRNA-98-5p, miR-98-5p)表达+50 mg/L咪达唑仑处理组(E组),干扰miR-98-5p表达阴性对照+50 mg/L咪达唑仑处理组(F组),miR-98-5p过表达阴性对照组(G组)及miR-98-5p过表达组(H组),每组设置6个复孔。四甲基偶氮唑盐(methyl thiazolyl tetrazolium, MTT)法检测MDA-MB-231细胞存活率,流式细胞术检测细胞周期与细胞凋亡率,实时荧光定量PCR (real-time fluorescent quantitative PCR, RT-qPCR)法检测各组MDA-MB-231细胞中miR-98-5p表达情况,Western blot法检测MDA-MB-231细胞周期蛋白(Cyclin Dl)、B淋巴细胞瘤-2 (B cell lymphoma-2, Bcl-2)、半胱氨酸蛋白酶-3 (cysteine protease-3, caspase-3)蛋白水平。结果:与A组比较,B组、C组、D组MDA-MB-231细胞存活率、S期及G 2/M期细胞比例、Cyclin Dl蛋白水平、Bcl-2蛋白水平明显降低( P<0.05),B组、C组、D组MDA-MB-231细胞miR-98-5p相对表达量、G 0/G 1期细胞比例、细胞凋亡率、caspase-3蛋白水平明显升高( P<0.05)。与B组比较,C组、D组细胞存活率、S期及G 2/M期细胞比例、Cyclin Dl蛋白水平、Bcl-2蛋白水平明显降低( P<0.05),miR-98-5p相对表达量、G 0/G 1期细胞比例、细胞凋亡率、caspase-3蛋白水平明显升高( P<0.05)。与C组比较,D组细胞存活率、S期及G 2/M期细胞比例、Cyclin Dl蛋白水平、Bcl-2蛋白水平明显降低( P<0.05),miR-98-5p相对表达量、G 0/G 1期细胞比例、细胞凋亡率、caspase-3蛋白水平明显升高( P<0.05)。与D组比较,E组细胞存活率、S期及G 2/M期细胞比例、Cyclin Dl蛋白水平、Bcl-2蛋白水平明显升高( P<0.05),miR-98-5p相对表达量、G 0/G 1期细胞比例、细胞凋亡率、caspase-3蛋白水平明显降低( P<0.05)。F组与D组miR-98-5p相对表达量、G 0/G 1期和S期细胞比例、细胞存活率、细胞凋亡率、Cyclin Dl蛋白水平、Bcl-2蛋白水平、caspase-3蛋白水平差异无统计学意义( P>0.05),F组G 2/M期细胞比例低于D组( P<0.05)。A组与G组细胞存活率、细胞凋亡率、细胞周期分布及Cyclin Dl、Bcl-2、caspase-3蛋白水平差异无统计学意义( P>0.05)。与G组比较,H组细胞存活率、S期及G 2/M期细胞比例、Cyclin Dl蛋白水平、Bcl-2蛋白水平明显降低( P<0.05),细胞凋亡率、G 0/G 1期细胞比例及caspase-3蛋白水平明显升高( P<0.05)。 结论:咪达唑仑通过上调miR-98-5p表达进而抑制乳腺癌细胞增殖,促进其凋亡。

Objective To investigate the effect and mechanism of midazolam on the proliferation and apoptosis of human breast cancer cell line MDA-MB-231.Methods According to the random number table method,MDA-MB-231 cells were divided into the following groups:a control group(group A),midazolam groups at the doses of 10,30 and 50 mg/L(groups B,C,and D,respectively),an interfering microRNA-98-5p(microRNA-98-5p,miR-98-5p)expression+50 mg/L group(group E),an interference miR-98-5p expression negative control+50 mg/L midazolam group(group F),a miR-98-5p overexpression negative control group(group G)and a miR-98-5p overexpression group(group H),with six replicated wells per group.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the survival rate of MDA-MB-231 cells,while flow cytometry was used to detect cell cycle and apoptosis.The expression of miR-98-5p in MDA-MB-231 cells was detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR),while the levels of Cyclin Dl,B cell lymphoma-2(Bcl-2),and cysteine protease-3(caspase-3)in MDA-MB-231 cells were detected by Western blot.Results Compared with group A,the survival rate of MDA-MB-231 cells,the proportion of cells in the S phase and G2/M phase,and the levels of Cyclin Dl and Bcl-2 significantly decreased in groups B,C,and D(P<0.05),while the relative expression of miR-98-5p,the proportion of cells in the G0/G1 phase,the apoptotic rate,and the expression of caspase-3 significantly increased in groups B,C,and D(P<0.05).Compared with group B,the survival rate of cells,the proportion of cells in the S phase and G2/M phase,the levels of Cyclin Dl and Bcl-2 significantly decreased in groups C and D(P<0.05),while the relative expression of miR-98-5p,the proportion of cells in the G0/G1 phase,the apoptotic rate,and the expression of caspase-3 significantly increased in groups C and D(P<0.05).Compared with group C,the survival rate of cells,the proportion of cells in the S phase and G2/M phase,the levels of Cyclin Dl and Bcl-2 significantly decreased in group D(P<0.05),while the relative expression of miR-98-5p,the proportion of cells in the G0/G1 phase,the apoptotic rate,and the expression of caspase-3 significantly increased in group D(P<0.05).Compared with group D,the survival rate of cells,the proportion of cells in the S phase and G2/M phase,the levels of Cyclin Dl and Bcl-2 significantly increased in group E(P<0.05),while the relative expression of miR-98-5p,the proportion of cells in the G0/G1 phase,the apoptotic rate and the expression of caspase-3 protein significantly decreased in group E(P<0.05).There was no statistical difference in the relative expression of miR-98-5p,the proportion of cells in the G0/G1 phase and S phase,cell survival rate,the apoptotic rate,and the expression of Cyclin Dl,Bcl-2,caspase-3 between group F and group D(P>0.05).The proportion of the G2/M phase decreased in group F,compared with those in group D(P<0.05).There was no statistical difference in cell survival rate,apoptosis rate,cell cycle distribution,and the expression of Cyclin Dl,Bcl-2,and caspase-3 between group A and group G(P>0.05).Compared with group G,the survival rate of cells,the proportion of cells in the S phase and G2/M phase,and the expression of Cyclin Dl and Bcl-2 significantly decreased in group H(P<0.05),while the apoptotic rate,the proportion of cells in the G0/G1 phase and the expression of caspase-3 expression significantly increased(P<0.05).Conclusions Midazolam can up-regulate the expression of miR-98-5p to inhibits the proliferation of breast cancer cells and promotes apoptosis.