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

Abstract

Abstract:ObjectiveTo explore the effects of miR-451 on glycolysis and apoptosis of breast cancer cells by regulating the Rho/ROCK1 pathway.MethodsBreast 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.ResultsThe glucose intake of cells in the BC group, MN group, MI group and MM group were （14.22±2.36）×10⁵mg/h, （14.20±2.37）×10⁵mg/h, （21.55±2.43）×10⁵mg/h, （6.19±1.34）×10⁵mg/h （F=5.30,P<0.001）, respectively, and lactic acid production were （1.52±0.21）×10⁵μg/h, （1.53±0.22）×10⁵μg/h, （2.05±0.32）×10⁵μg/h, （0.54±0.12）×10⁵μg/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 （allP<0.05）. The glucose intake in the BC group, BL group and BF group were （14.22±2.36）×10⁵mg/h, （21.54±2.40）×10⁵mg/h, （6.20±1.35）×10⁵mg/h （F=5.33,P<0.001）, respectively. Lactic acid production were （1.52±0.21）×10⁵μg/h, （2.01±0.30）×10⁵μg/h, （0.55±0.12）×10⁵μg/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 （allP<0.05）. In the MF group, glucose intake was （3.21±0.89）×10⁵mg/h, lactic acid production was （0.33±0.04）×10⁵μg/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 （allP<0.05）. Dual luciferase report showed that miR-451 transfection significantly decreased the luciferase activity of ROCK1-3'-UTR-WT （0.59±0.03vs.1.01±0.05,t=17.64,P<0.001）, but had no significant effect on mutated genes （1.01±0.07vs.1.02±0.04,t=0.30,P=0.767）.ConclusionOverexpression 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

Feng Dongxu, Wu Wei, Gao Pingfa, Wang Jun, Shi Lijuan, Chen Dawei, Li Wenbing, Zhang Meifeng. Effects of miR-451 on glycolysis and apoptosis of breast cancer cells by regulating Rho/ROCK1 pathway[J]. Journal of International Oncology, 2023, 50(8): 449-456.

Figures/Tables13

引物	序列
miR-451	正向5'-CGTCATCAAACCGTTACCATTAC-3'
	反向5'-AAAGGTTGTTCTCCACTCTCTCTC-3'
U6	正向5'-GCTTCGGCAGCACATATACTAAAAT-3'
	反向5'-CGCTTCACGAATTTGCGTGTCAT-3'

细胞株	miR-451 mRNA相对表达量
HMEC	1.00±0.00
MCF7	0.41±0.02^a
HCC1937	0.69±0.03^ab
AU565	0.68±0.03^ab
SUM159	0.70±0.04^ab
F值	72.26
P值	<0.001

组别	糖酵解	凋亡率（%）	蛋白表达
BC组	14.22±2.36	1.52±0.21	10.13±1.35	2.31±0.46	2.51±0.41
MN组	14.20±2.37	1.53±0.22	10.16±1.37	2.32±0.41	2.52±0.42
MI组	21.55±2.43^a	2.05±0.32^a	5.36±1.24^a	2.95±0.35^a	3.05±0.33^a
MM组	6.19±1.34^ab	0.54±0.12^ab	28.47±2.56^ab	1.05±0.25^ab	1.15±0.13^ab
F值	5.30	3.28	6.36	2.86	2.43
P值	<0.001	0.008	<0.001	0.017	0.035

	糖酵解	凋亡率（%）	蛋白表达
BC组	14.22±2.36	1.52±0.21	10.13±1.35	2.31±0.46	2.51±0.41
BL组	21.54±2.40^a	2.01±0.30^a	5.34±1.22^a	2.94±0.45^a	3.08±0.42^a
BF组	6.20±1.35^ab	0.55±0.12^ab	28.44±2.54^ab	1.01±0.24^ab	1.13±0.12^ab
F值	5.33	3.28	6.45	2.40	2.38
P值	<0.001	0.008	<0.001	0.037	0.039

组别	糖酵解	凋亡率（%）	蛋白表达
MM组	6.19±1.34	0.54±0.12	28.47±2.56	1.05±0.25	1.15±0.13
BF组	6.20±1.35	0.55±0.12	28.44±2.54	1.01±0.24	1.13±0.12
MF组	3.21±0.89^a	0.33±0.04^a	38.01±2.87^a	0.55±0.14^a	0.51±0.10^a
F值	4.53	4.26	6.12	4.06	9.72
P值	0.001	0.002	<0.001	0.002	<0.001

质粒	miR-451-NC	miR-451	t值	P值
ROCK1-3′-UTR-WT	1.01±0.05	0.59±0.03	17.64	<0.001
ROCK1-3′-UTR-MUT	1.02±0.04	1.01±0.07	0.30	0.767

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