Hcy促血管平滑肌细胞增殖迁移相关的miRNAs筛选研究

Screening of miRNAs Associated with Hcy Promoting Proliferation and Migration of Vascular Smooth Muscle Cells

目的明确同型半胱氨酸(homocysteine,Hcy)对人源血管平滑肌细胞(vascular smooth muscle cell,VSMC)增殖迁移的影响以及分析Hcy干预下VSMC增殖迁移相关差异miRNAs,为探讨Hcy促VSMC增殖迁移的分子机制提供依据。方法体外培养VSMC,分为Control组和100μmol·L^(-1) Hcy组。采用CCK-8法测量VSMC的增殖活性;采用划痕实验分析VSMC在0 h和48 h的迁移情况。高通量测序分析各组VSMC中差异化表达的miRNAs,采用miRanda和TargetScan软件进行靶基因预测,使用BLAST软件将预测靶基因序列与GO和KEGG数据库比对,分析靶基因的功能,获得与VSMC增殖和迁移相关的靶基因信息。通过RT-qPCR对其中表达上调的miRNAs进行验证。结果Hcy干预能增加VSMC的增殖活力(P<0.01),且VSMC的划痕面积减少(P<0.01)。两组细胞共检测到576个miRNAs,其中已知miRNAs 404个,新预测miRNAs 172个。与Control组相比,Hcy组存在88个差异表达的miRNAs,其中表达上调的20个,表达下调的68个。与VSMC增殖和迁移相关的miRNAs有20个,表达上调的包括miRNA39、miRNA206和miRNA212-5p,表达下调的包括miRNA35等17个。RT-qPCR结果显示,Hcy组miRNA212-5p的表达上调(P<0.05)。结论Hcy致VSMC增殖迁移过程中存在差异表达的miRNAs,miRNA212-5p可能参与了Hcy对VSMC的作用。

Objective To determine the effects of homocysteine(Hcy)on proliferation and migration of human vascular smooth muscle cell(VSMC)and analyze the differences related to the proliferation and migration of VSMC under the intervention of Hcy.Methods VSMC was cultured in vitro and divided into Control group and 100μmol·L^(-1) Hcy intervention group.The proliferative activity of VSMC was measured by CCK-8 method.The migration of VSMC at 0 h and 48 h was analyzed by scratch test.The differentiated expression of miRNAs in VSMC in each group was analyzed by high-throughput sequencing,target gene prediction was performed using miRanda and TargetScan software,and the function of target genes was analyzed by BLAST software compared with GO and KEGG databases to obtain the target gene information related to VSMC proliferation and migration.The up-regulated miRNAs were verified by RT-qPCR.Results Hcy intervention could significantly increase the proliferative activity of VSMC(P<0.01),and the scratch area of VSMC was significantly reduced(P<0.01),suggesting that 100μmol·L^(-1) Hcy intervention could promote the proliferation and migration of VSMC,which was consistent with the results of previous studies.In terms of mechanism,a total of 576 miRNAs were detected in both groups of cells,of which 404 were known and 172 were newly predicted.Compared with the Control group,there were 88 differentially expressed miRNAs in the Hcy group,of which 20 were up-regulated and 68 were down-regulated.Among them,there were 20 miRNAs related to VSMC proliferation and migration,including miRNA39,miRNA206,and miRNA212-5p whose expression was up-regulated,and 17 miRNA35 whose expression was down-regulated.The results of RT-qPCR showed that the expressions of miRNA212-5p was significantly increased in the Hcy group(P<0.05).Conclusion There are differentially expressed miRNAs in the proliferation and migration of VSMC induced by Hcy.miRNA212-5p may be involved in the effect of Hcy on VSMC.