MiR-92a-3p_R+1和miR-92a-1-5p在ox-LDL诱导大鼠VSMC表型转化和增殖中的作用

Effect of miR-92a-3p_R+1 and miR-92a-1-5 in ERK signaling pathway of ox-LDL induced phenotypic transformation and proliferation of VSMC in rats

目的探讨miR-92a-3p_R+1和miR-92a-1-5p在氧化型低密度脂蛋白(ox-LDL)诱导大鼠血管平滑肌细胞(VSMC)表型转化和增殖中的作用及其与ERK1/2信号通路的相关性。方法分离、培养大鼠VSMC,以ox-LDL(50 mg/L)诱导VSMC,采用ERK1/2特异性抑制剂U0126(10μmol/L)阻断ox-LDL(50 mg/L)诱导VSMC的ERK1/2信号激活,MicroRNA微阵列分析VSMC的miR-92a-3p_R+1和miR-92a-1-5p表达,CCK-8法和Brdu流式细胞术检测细胞增殖;免疫荧光法检测VSMC收缩表型标志蛋白SM22α的变化;Western blot检测VSMC的ERK1/2通路信号激活情况(ERK、p-ERK)、表型标志蛋白SM22α、细胞周期相关蛋白(PCNA、cyclin D1、p21、p27)的表达情况。结果ox-LDL诱导下,VSMC的miR-92a-3p_R+1和miR-92a-1-5p表达明显上调,VSMC的ERK1/2磷酸化水平明显增加,SM22α的表达降低,同时细胞周期相关蛋白PCNA、cyclin D1高表达,p21、p27低表达;ERK1/2通路特异性抑制剂U0126干预后,ERK1/2磷酸化水平受抑制,相应的VSMC miR-92a-3p_R+1和miR-92a-1-5p表达明显下调(P<0.05),VSMC增殖显著下降,SM22α的表达上调(P<0.05),提示VSMC由合成表型转化为收缩表型,并下调PCNA、cyclin D1的表达,上调p21、p27蛋白的表达(P<0.05),说明其表型转化和增殖明显受抑制。结论miR-92a-3p_R+1和miR-92a-1-5p在ox-LDL诱导VSMC表型转化和增殖中起重要作用,并与ERK1/2信号通路密切相关。

Aim To investigate the effect of miR-92 a-3 p_R+1 and miR-92 a-1-5 p on phenotypic transformation and proliferation of rat vascular smooth muscle cells(VSMC)induced by oxidized low-density lipoprotein(ox-LDL)in the ERK 1/2 signaling pathway.Methods VSMC isolated from rats,and ox-LDL(50 mg/L)was used to induce VSMC.ERK1/2 inhibitors U0126(10μmol/L)was used for intervention.The miRNA microarray profiling was performed using small RNA sequencing analysis.CCK-8 method and Brdu flow cytometry were used to detect VSMC proliferation.Immunofluorescence assay was performed to detect the expression of SM22αprotein in VSMC.Western blot was used to detect the expression changes of ERK1/2 pathway signal molecules(ERK,p-ERK),phenotype marker protein SM22αand proliferation associated proteins such as PCNA,cyclin D1,p21 and p27.Results Under ox-LDL induction,theexpressions of miR-92 a-3 p_R+1 and miR-92 a-1-5 p in VSMC were significantly up-regulated.After the intervention of ERK1/2 inhibitors U0126,the phosphorylation level of ERK1/2 was inhibited,the corresponding VSMC miR-92 a-3 p_R+1 and miR-92 a-1-5 p expression significantly lowered(P<0.05).Therefore,the study speculated that ERK1/2 signaling pathway may affect the phenotypic transformation and proliferation of VSMC by regulating the expression of miR-92 a.After inhibiting the ERK1/2 signaling pathway,the proliferation of VSMC was significantly reduced,and the expression of SM22αwas up-regulated(P<0.05).The expression of PCNA and cyclin D1 was down-regulated and the expression of p21 and p27 proteins were up-regulated(P<0.05).This indicated that the phenotypic transformation and proliferation were significantly inhibited.Conclusion Mir-92 a-3 p_R+1 and miR-92 a-1-5 p play important roles in the ERK1/2 signaling pathway that ox-LDL induces phenotypic transformation and proliferation of VSMC.