阿托伐他汀钙对人脐静脉内皮细胞增殖迁移的影响观察、关键靶基因及其相关lncRNA筛选

Effects of atorvastatin calcium on proliferation and migration of HUVECs and screening of hub mRNA and co-related lncRNA

目的观察阿托伐他汀钙对人脐静脉内皮细胞(HUVECs)细胞增殖及迁移的影响,筛选阿托伐他汀钙对HUVECs作用的关键基因和共相关长链非编码核糖核酸(lncRNA)。方法取对数生长期HUVECs细胞,分为一组、二组,一组、二组细胞分别加入5μmol/L的阿托伐他汀钙及等量PBS培养24 h。①阿托伐他汀钙对HUVECs细胞增殖及迁移的的影响观察。取两组细胞,使用流式细胞仪测算两组细胞周期、采用CCK-8法观察两组细胞增殖情况、采用划痕愈合实验观察两组细胞迁移情况。②阿托伐他汀钙对HUVECs作用的关键基因和共相关lncRNA筛选。取两组细胞,运用illumina平台检测两组细胞mRNA、lncRNA,使用R Studio软件中的limma包筛选|log2 Fold change|>1.5、校正P<0.05的差异表达mRNA和lncRNA,运用基因本体论(GO)、京都基因与基因组百科全书(KEGG)富集分析法分析差异表达mRNA的生物学功能。通过STRING数据库分析差异表达mRNA的蛋白互作用,使用CytoNCA插件筛选介数中心性最高的前10位关键基因,筛选与关键基因Pearson相关性系数>0.99的共表达lncRNA。采用实时荧光定量PCR(qPCR)法检测两组关键基因及其共表达lncRNA。结果与二组相比,一组细胞周期GO/G1期比例高、细胞增殖抑制,培养24 h细胞迁移面积小(P均<0.05)。与二组相比,一组细胞有1510个差异表达mRNA、744个差异表达lncRNA;差异表达mRNA的生物学功能涉及294个GO集及27条KEGG通路;关键基因共10个,与其共表达的lncRNA有130个,包括AC017101.1、AC125257.1及AC004846.1等。与二组相比,一组细胞HSP90AA1、CDK4、CCND1、PLK1、PCNA相对表达量降低,AC017101.1、AC125257.1、AC004846.1、AL390719.1相对表达量升高(P均<0.05)。结论阿托伐他汀钙能阻滞HUVECs的细胞周期于GO/G1期、抑制细胞的增殖和迁移。阿托伐他汀钙对HUVECs作用的关键基因有HSP90AA1、CDK4及CCND1等10个,共相关lncRNA有AC017101.1、AC125257.1及AC004846.1等130个。阿托伐他汀钙可能通过促进AC017101.1、AC125257.1及AC004846.1等共相关lncRNA表达,下调HUVECs细胞中HSP90AA1、CDK4及CCND1等关键基因表达,阻滞HUVECs的细胞周期、抑制细胞增殖及迁移。

Objective To observe the effects of atorvastatin calcium on the cell proliferation and migration of human umbilical vein endothelial cells(HUVECs),and to screen the hub mRNA and co-related long non-coding RNA(lncRNA)of atorvastatin calcium on HUVECs.Methods HUVECs in the logarithmic growth phase were divided into the group 1 and group 2.Cells in the group 1 and group 2 were treated with 5μmol/L atorvastatin calcium and the same amount of PBS for 24 h,respectively.①Observation of effects of atorvastatin calcium on the cell proliferation and migration of HUVECs:The cell cycle was measured by flow cytometry,the cell proliferation was observed by CCK-8 method,and the cell migration was observed by scratch healing test.②Screening of hub mRNAs and co-related lncRNAs in HUVECs treated with atorvastatin calcium:illumina platform was used to detect mRNA and lncRNA in cells of the two groups.Differentially expressed mRNAs and lncRNAs with|log2 Fold change|>1.5 and P adjusted<0.05 were screened using limma package in R Studio software.Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis were used to analyze the biological functions of differentially expressed mRNAs.The STRING database was used to analyze the protein interaction of differentially expressed mRNAs.The CytoNCA plug-in was used to screen the top 10 hub mRNAs with the highest mediation centrality,and the co-expressed lncRNAs with the hub mRNAs with Pearson correlation coefficient>0.99 were screened.Real-time fluorescence quantitative PCR(qPCR)was used to detect the hub mRNA and its coexpressed lncRNA in the two groups.Results Compared with the group 2,the proportion of cells in the G0/G1 phase of the group 1 was higher,the cell proliferation was inhibited,and the cell migration area was smaller after 24 h of culture(all P<0.05).Compared with the group 2,there were 1510 differentially expressed mRNAs and 744 differentially expressed lncRNAs in the group 1;the biological functions of differentially expressed mRNAs involved 294 GO sets and 27 KEGG pathways;there were 10 hub mRNAs and 130 lncRNAs co-expressed with them,including AC017101.1,AC125257.1,AC004846.1,etc.Compared with the group 2,the relative expression levels of HSP90AA1,CDK4,CCND1,PLK1,and PCNA in the group 1 were lower,and the relative expression levels of AC017101.1,AC125257.1,AC004846.1,and AL390719.1 were higher(all P<0.05).Conclusions Atorvastatin calcium can block the HUVECs in the G0/G1 phase and inhibit their proliferation and migration.There are 10 hub mRNAs such as HSP90AA1,CDK4 and CCND1,and 130 co-related lncRNAs such as AC017101.1,AC125257.1 and AC004846.1 in atorvastatin calcium treatment of HUVECs.Atorvastatin calcium may promote the expression of co-related lncrnas such as AC017101.1,AC125257.1 and AC004846.1 and down-regulate the expression of HSP90AA1,CDK4 and CCND1 mRNA in HUVECs.Thus,the cell cycle of HUVECs is arrested and cell proliferation and migration are inhibited.