α-黑素细胞刺激素作用后高糖高脂下视网膜血管内皮细胞差异表达基因分析

Differentially expressed genes analysis of retinal vascular endothelial cells under hyperglycemia and hyperlipidemia induced byα-melanocyte-stimulating hormone

目的探讨α-黑素细胞刺激素(α-MSH)对高糖高脂刺激下视网膜血管内皮细胞中mRNA及长链非编码RNA(lncRNA)表达的影响。方法对恒河猴视网膜血管内皮细胞系(RF/6A)进行培养,将细胞分为正常对照组、模型对照组、0.1μmol/Lα-MSH处理组、0.5μmol/Lα-MSH处理组和1.0μmol/Lα-MSH处理组。各组细胞进行CM-H2DCFDA染色,检测细胞抗氧化能力,筛选α-MSH的最佳工作浓度。正常对照组、模型对照组和α-MSH处理组细胞处理后24 h,收集各组细胞提取总RNA,经高通量RNA测序(RNA-seq)构建RNA测序文库并进行生物信息学分析。结果0.5μmol/Lα-MSH组细胞荧光强度明显低于模型对照组,差异有统计学意义(P<0.05),选择0.5μmol/L为α-MSH的最佳作用浓度并进行后续实验。RNA-seq结果显示,与模型对照组相比,α-MSH处理组中243个mRNAs表达下调,81个mRNAs表达上调,53个lncRNAs表达上调,6个lncRNAs表达下调。生物信息学分析结果显示,α-MSH处理组表达下调的基因中主要富集的通路包括转化生长因子-β(TGF-β)信号通路、局部黏附通路和细胞外基质(ECM)受体相互作用通路,主要参与的生物过程为调节小三磷酸鸟苷酶(small GTPase)介导的信号转导。α-MSH处理组中表达上调的共表达lncRNA主要富集通路包括ECM受体互作通路、缺氧诱导因子-1(HIF-1)通路等,主要参与轴突导向、RNA聚合酶Ⅱ启动子转录的正向调节等生物过程。结论α-MSH处理后,高糖、高脂刺激的视网膜血管内皮细胞主要表现为lncRNA表达上调及mRNA表达下调,提示α-MSH可能通过上调lncRNA的表达来下调下游基因的mRNA表达.

Objective To investigate the effect ofα-melanocyte-stimulating hormone(α-MSH)on the expression of mRNA and long noncoding RNA(lncRNA)in retinal vascular endothelial cells stimulated by hyperglycemia and hyperlipidemia.Methods The simian retinal vascular endothelial cells(RF/6A)were cultured and divided into normal control group,model control group,0.1μmol/Lα-MSH group,0.5μmol/Lα-MSH group and 1.0μmol/Lα-MSH group.The cells were stained with CM-H 2 DCFDA to detect cell antioxidant capacity.The optimal concentration ofα-MSH was screened.The cells from normal control group,model control group andα-MSH treatment group were collected at 24 hours after treatment,the total RNA was extracted,the cDNA library was constructed,and the high throughput RNA sequencing(RNA-seq)was carried out with bioinformatics analysis to analyze the expression profiling of mRNA and lncRNA.Results The fluorescence intensity of cells in 0.5μmol/Lα-MSH group was significantly lower than that in model control group(P<0.05).α-MSH of 0.5μmol/L was chosen as the optimal concentration for subsequent experiments.Compared with the model control group,243 mRNAs were significantly down-regulated,while 81 mRNAs were up-regulated in theα-MSH treatment group;53 lncRNAs were markedly up-regulated and 6 lncRNAs were down-regulated in theα-MSH treatment group.Bioinformatics analysis showed that the major enrichment pathways of the down-regulated genes were transforming growth factor-β(TGF-β)signaling pathway,focal adhesion signaling pathway and extracellular matrix(ECM)receptor interactions pathways,and the main biological process involved was the regulation of small GTPase-mediated signal transduction.The co-expression gene enrichment pathways of differentially expressed lncRNA included ECM receptor interaction and hypoxia inducible factor-1(HIF-1)pathway,et al.These pathways were mainly involved in the biological processes,such as axon guidance and positive regulation of transcription from RNA polymeraseⅡpromoter.Conclusions Under hyperglycemia and hyperlipidemia,the influence ofα-MSH on the transcriptome of the retinal vascular endothelial cells manifests the downregulation of mRNA and upregulation of lncRNA.α-MSH may upregulate the lncRNA expression,which downregulates the downstream mRNA expression.