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)×10^(5) mg/h、(14.20±2.37)×10^(5) mg/h、(21.55±2.43)×10^(5) mg/h、(6.19±1.34)×10^(5) mg/h(F=5.30, P<0.001), 乳酸生成量分别为(1.52±0.21)×10^(5) μg/h、(1.53±0.22)×10^(5) μg/h、(2.05±0.32)×10^(5) μg/h、(0.54±0.12)×10^(5) 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)×10^(5) mg/h、(21.54±2.40)×10^(5) mg/h、(6.20±1.35)×10^(5) mg/h(F=5.33, P<0.001), 乳酸生成量分别为(1.52±0.21)×10^(5) μg/h、(2.01±0.30)×10^(5) μg/h、(0.55±0.12)×10^(5) 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)×10^(5) mg/h, 乳酸生成量为(0.33±0.04)×10^(5) 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。
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)×10^(5) mg/h,(14.20±2.37)×10^(5) mg/h,(21.55±2.43)×10^(5) mg/h,(6.19±1.34)×10^(5) mg/h(F=5.30,P<0.001),respectively,and lactic acid production were(1.52±0.21)×10^(5)μg/h,(1.53±0.22)×10^(5)μg/h,(2.05±0.32)×10^(5)μg/h,(0.54±0.12)×10^(5)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)×10^(5) mg/h,(21.54±2.40)×10^(5) mg/h,(6.20±1.35)×10^(5) mg/h(F=5.33,P<0.001),respectively.Lactic acid production were(1.52±0.21)×10^(5)μg/h,(2.01±0.30)×10^(5)μg/h,(0.55±0.12)×10^(5)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)×10^(5) mg/h,lactic acid production was(0.33±0.04)×10^(5)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.
作者
冯东旭
吴炜()
高平发
王军
施丽娟
陈大伟
李文兵
张美峰
Feng Dongxu;Wu Wei;Gao Pingfa;Wang Jun;Shi Lijuan;Chen Dawei;Li Wenbing;Zhang Meifeng(Department of Thyroid and Breast Surgery,Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences,Shanghai 202150,China;Department of General Surgery,Chongming Hospital Affiliated to Shanghai University of Medicine and Health Sciences,Shanghai 202150,China)
出处
《国际肿瘤学杂志》
CAS
2023年第8期449-456,共8页
Journal of International Oncology
