Effects of circular RNA_0000231 on proliferation and migration of vascular smooth muscle cells
Objective To investigate the effects of circular RNA(circRNA)_0000231 on the pro-liferation and migration of vascular smooth muscle cells(VSMCs)and its regulatory mechanism.Methods Four-week-old male C57BL/6J mice with ApoE knockout(ApoE--)were used(24 mice were purchased from the Basic Research Institute of Peking Union Medical College,Beijing).The mice were randomly di-vided into four groups according to the digital table method:the normal diet group(normal group)was giv-en ordinary feed as a blank control;The high-fat diet group(high fat group)was given a high-fat diet to es-tablish an atherosclerosis(AS)mouse model;the high-fat diet+empty vector adenovirus group(Ad-sh-nc group)was injected with adenovirus carrying an empty vector through the tail vein on the basis of a high-fat diet;the high-fat diet+circRNA_0000231 adenovirus knockdown group(Ad-sh-circ)was injected with circRNA_0000231 adenovirus knockdown on the basis of a high-fat diet.After 12 weeks,the aortas of the mice were taken to detect the expression of circRNA_0000231,and the degree of atherosclerosis was detec-ted by vascular oil red O staining.Mouse aortic smooth muscle cells(MASMCs)were cultured to evaluate the effect of circRNA_0000231 on their phenotypes.The medium(containing 10％FBS)was added,shak-en,and placed in a 37 ℃ incubator.RNA pull-down assay and dual luciferase reporter gene assay were used to verify the downstream regulatory target genes of circRNA_0000231.In addition,the effects of cir-cRNA_0000231 on the proliferation and migration of MASMCs were detected by methyl thiazolyl tetrazolium(MTT)colorimetry and Transwell assay.Unpaired t-test or nonparametric test was used for comparison be-tween groups,and one-way analysis of variance was used for multiple comparisons of three or more groups.Results The expression of circRNA_0000231 in the plaque tissue of AS model mice was higher than that of normal mice(1.03±0.06 vs.4.98±1.37,t=7.042,P＜0.01);oil red O staining of blood vessels showed that the area of atherosclerotic lesions in the experimental group was lower than that in the control group[(0.59±0.11)cm2 vs.(0.34±0.07)cm2,t=4.694,P＜0.01],and the proportion of plaques in the experimental group was lower than that in the control group[(46.02±10.14)cm2 vs.(21.05±8.81)cm2,t=4.552,P＜0.01].Knockdown of circRNA_0000231 can increase the expression of mi-croRNA(miR)-630(0.99±0.03 vs.3.77±0.19,t=24.824,P＜0.01),while overexpression of cir-cRNA_0000231 can inhibit the expression of miR-630(0.99±0.12 vs.0.41±0.05,t=7.695,P＜0.01).MTT results showed that knockdown of circRNA_0000231 inhibited the proliferation of MASMCs(1.55±0.09 vs.0.89±0.11,t=7.787,P＜0.01).Transwell and scratch experiments showed that knocking down circRNA_0000231 weakened the migration ability of MASMCs[(149.33±8.02)vs.(72.67±4.04),t=14.785,P＜0.01;(100.33±8.50)vs.(60.67±6.03),t=6.591,P＜0.01].Western blotting results showed that knocking down circRNA_0000231 inhibited the expression of PCNA,MMP-2 and MMP-9 in MASMCs(0.780±0.025 vs.0.410±0.015,t=21.377,P＜0.01;0.790±0.021 vs.0.381±0.009,t=31.506,P＜0.01;0.820±0.013 vs.0.410±0.008,t=47.385,P＜0.01).Overexpression of miR-630 can inhibit the expression of Zeste enhancer homolog 2(EZH2)(1.020±0.056 vs.0.260±0.035,t=20.085,P＜0.01),and knockdown of miR-630 promotes the expression of EZH2(1.010±0.035 vs.3.690±0.251,t=18.356,P＜0.01).Conclusion Silencing circRNA_0000231 can inhibit the proliferation and migration of vascular smooth muscle cells.
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