国际麻醉学与复苏杂志   2022, Issue (6): 0-0
    
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
张永慧, 焦丽, 唐凯1()
1.河南省洛阳市洛龙区妇幼保健院
Midazolam up‑regulates microRNA‑98‑5p and affects the proliferation and apoptosis of breast cancer cells
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摘要：

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G2/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、G0/G1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G2/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、G0/G1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G2/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、G0/G1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G2/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、G0/G1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、G0/G1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G2/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G2/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、G0/G1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。
关键词： 咪达唑仑； 人乳腺癌细胞； 增殖； 凋亡； 微RNA‑98‑5p
Abstract:

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.
Key words: Midazolam; Human breast cancer cells; Proliferation; Apoptosis; MicroRNA‑98‑5p