国际肿瘤学杂志

国际肿瘤学杂志 ›› 2023, Vol. 50 ›› Issue (8): 449-456.doi: 10.3760/cma.j.cn371439-20221129-00087

• 论著 • 上一篇    下一篇

miR-451通过调控Rho/ROCK1信号通路对乳腺癌细胞糖酵解及凋亡的影响

冯东旭1, 吴炜1(), 高平发1, 王军2, 施丽娟2, 陈大伟2, 李文兵2, 张美峰2   

  1. 1 上海健康医学院附属崇明医院甲乳外科，上海 202150
    2 上海健康医学院附属崇明医院普外科，上海 202150
  • 收稿日期:2022-11-29 修回日期:2023-06-21 出版日期:2023-08-08 发布日期:2023-10-24
  • 通讯作者: 吴炜 E-mail:wuweixinhuayiyuan@163.com

Effects of miR-451 on glycolysis and apoptosis of breast cancer cells by regulating Rho/ROCK1 pathway

Feng Dongxu1, Wu Wei1(), Gao Pingfa1, Wang Jun2, Shi Lijuan2, Chen Dawei2, Li Wenbing2, Zhang Meifeng2   

  1. 1 DeparTMent of Thyroid and Breast Surgery, Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai 202150, China
    2 DeparTMent of General Surgery, Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences, Shanghai 202150, China
  • Received:2022-11-29 Revised:2023-06-21 Online:2023-08-08 Published:2023-10-24
  • Contact: Wu Wei E-mail:wuweixinhuayiyuan@163.com

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摘要：

目的 探究miR-451通过调控Rho/ROCK1信号通路对乳腺癌细胞糖酵解及凋亡的影响。方法 将乳腺癌MCF7细胞分为乳腺癌细胞（BC）组、乳腺癌细胞+miR-451-NC（MN）组、乳腺癌细胞+miR-451 inhibitor（MI）组、乳腺癌细胞+miR-451 mimic（MM）组、乳腺癌细胞+溶血磷脂酸（BL）组、乳腺癌细胞+法舒地尔（BF）组、乳腺癌细胞+miR-451 mimic+法舒地尔（MF）组。用葡萄糖摄取检测试剂盒和乳酸检测试剂盒检测细胞糖酵解，DAPI染色法检测细胞凋亡，蛋白质印迹法检测Rho/ROCK1通路蛋白表达，双荧光素酶报告实验证实miR-451和Rho/ROCK1的相互作用。结果 BC组、MN组、MI组、MM组细胞葡萄糖摄取量分别为（14.22±2.36）×105 mg/h、（14.20±2.37）×105 mg/h、（21.55±2.43）×105 mg/h、（6.19±1.34）×105 mg/h（F=5.30，P<0.001），乳酸生成量分别为（1.52±0.21）×105 μg/h、（1.53±0.22）×105 μg/h、（2.05±0.32）×105 μg/h、（0.54±0.12）×105 μg/h（F=3.28，P=0.008），凋亡率分别为（10.13±1.35）%、（10.16±1.37）%、（5.36±1.24）%、（28.47±2.56）%（F=6.36，P<0.001），Rho蛋白相对表达量分别为2.31±0.46、2.32±0.41、2.95±0.35、1.05±0.25（F=2.86，P=0.017），ROCK1蛋白相对表达量分别为2.51±0.41、2.52±0.42、3.05±0.33、1.15±0.13（F=2.43，P=0.035），MN组和MI组、MN组和MM组、MI组和MM组间差异均有统计学意义（均P<0.05）。BC组、BL组、BF组细胞葡萄糖摄取量分别为（14.22±2.36）×105 mg/h、（21.54±2.40）×105 mg/h、（6.20±1.35）×105 mg/h（F=5.33，P<0.001），乳酸生成量分别为（1.52±0.21）×105 μg/h、（2.01±0.30）×105 μg/h、（0.55±0.12）×105 μg/h（F=3.28，P=0.008），凋亡率分别为（10.13±1.35）%、（5.34±1.22）%、（28.44±2.54）%（F=6.45，P<0.001），Rho蛋白相对表达量分别为2.31±0.46、2.94±0.45、1.01±0.24（F=2.40，P=0.037），ROCK1蛋白相对表达量分别为2.51±0.41、3.08±0.42、1.13±0.12（F=2.38，P=0.039），3组间两两比较差异均有统计学意义（均P<0.05）。MF组细胞葡萄糖摄取量为（3.21±0.89）×105 mg/h，乳酸生成量为（0.33±0.04）×105 μg/h，凋亡率为（38.01±2.87）%，Rho蛋白相对表达量为0.55±0.14，ROCK1蛋白相对表达量为0.51±0.10，MM组、BF组、MF组3组间差异均有统计学意义（F=4.53，P=0.001；F=4.26，P=0.002；F=6.12，P<0.001；F=4.06，P=0.002；F=9.72，P<0.001），MF组与BF组间差异均有统计学意义（均P<0.05）。双荧光素酶报告结果显示，转染miR-451后可显著降低ROCK1-3′-UTR-WT的荧光素酶活性（0.59±0.03比1.01±0.05，t=17.64，P<0.001），但对突变基因无显著影响（1.01±0.07比1.02±0.04，t=0.30，P=0.767）。结论 过表达miR-451可显著抑制乳腺癌细胞糖酵解，并促进乳腺癌细胞凋亡，其机制可能与抑制Rho/ROCK1信号通路有关。

关键词: 乳腺肿瘤, miR-451, Rho/ROCK1通路, 糖酵解, 细胞凋亡

Abstract: Objective To explore the effects of miR-451 on glycolysis and apoptosis of breast cancer cells by regulating the Rho/ROCK1 pathway. Methods Breast cancer MCF7 cells were divided into breast cancer cells （BC） group, breast cancer cells + miR-451-NC （MN） group, breast cancer cells + miR-451 inhibitor （MI） group, breast cancer cells + miR-451 mimic （MM） group, breast cancer cells + lysophosphatidic acid （BL） group, breast cancer cells + fasudil （BF） group, and breast cancer cells + miR-451 mimic + fasudil （MF） group. Glucose uptake detection kit and lactate detection kit were used to detect cell glycolysis, DAPI staining was used to detect cell apoptosis, Western blotting was used to detect Rho/ROCK1 pathway protein expression, and double luciferase reporting assay was used to confirm the interaction between miR-451 and Rho/ROCK1. Results The glucose intake of cells in the BC group, MN group, MI group and MM group were （14.22±2.36）×105 mg/h, （14.20±2.37）×105 mg/h, （21.55±2.43）×105 mg/h, （6.19±1.34）×105 mg/h （F=5.30, P<0.001）, respectively, and lactic acid production were （1.52±0.21）×105 μg/h, （1.53±0.22）×105 μg/h, （2.05±0.32）×105 μg/h, （0.54±0.12）×105 μg/h （F=3.28, P=0.008）, respectively. The apoptosis rates were （10.13±1.35）%, （10.16±1.37）%, （5.36±1.24）%, （28.47±2.56）% （F=6.36, P<0.001）, respectively. The expressions of Rho protein were 2.31±0.46, 2.32±0.41, 2.95±0.35, 1.05±0.25 （F=2.86, P=0.017）, respectively. The expressions of ROCK1 protein were 2.51±0.41, 2.52±0.42, 3.05±0.33, 1.15±0.13 （F=2.43, P=0.035）, and there were statistically significant differences between the MN and MI groups, MN and MM groups, MI and MM groups （all P<0.05）. The glucose intake in the BC group, BL group and BF group were （14.22±2.36）×105 mg/h, （21.54±2.40）×105 mg/h, （6.20±1.35）×105 mg/h （F=5.33, P<0.001）, respectively. Lactic acid production were （1.52±0.21）×105 μg/h, （2.01±0.30）×105 μg/h, （0.55±0.12）×105 μg/h （F=3.28, P=0.008）, respectively. The apoptosis rates were （10.13±1.35）%, （5.34±1.22）%, （28.44±2.54）% （F=6.45, P<0.001）. The expressions of Rho protein were 2.31±0.46, 2.94±0.45, 1.01±0.24 （F=2.40, P=0.037）, respectively, and the expressions of ROCK1 protein were 2.51±0.41, 3.08±0.42 and 1.13±0.12, respectively （F=2.38, P=0.039）. The pairwise comparisons among the three groups were statistically significant （all P<0.05）. In the MF group, glucose intake was （3.21±0.89）×105 mg/h, lactic acid production was （0.33±0.04）×105 μg/h, apoptosis rate was （38.01±2.87）%, Rho protein expression was 0.55±0.14, and ROCK1 protein expression was 0.51±0.10. There were statistically significant differences among the MM group, BF group and MF group （F=4.53, P=0.001； F=4.26, P=0.002； F=6.12, P<0.001； F=4.06, P=0.002； F=9.72, P<0.001）, and there were statistically significant differences between the MF group and BF group （all P<0.05）. Dual luciferase report showed that miR-451 transfection significantly decreased the luciferase activity of ROCK1-3'-UTR-WT （0.59±0.03 vs. 1.01±0.05, t=17.64, P<0.001）, but had no significant effect on mutated genes （1.01±0.07 vs. 1.02±0.04, t=0.30, P=0.767）. Conclusion Overexpression of miR-451 can significantly inhibit glycolysis of breast cancer cells and promote apoptosis of breast cancer cells, the mechanism of which may be related to inhibition of Rho/ROCK1 pathway.

Key words: Breast neoplasms, miR-451, Rho/ROCK1 pathway, Glycolysis, Apoptosis