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-H2 DCFDA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 of α-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/L α-MSH group. The cells were stained with CM-H2 DCFDA to detect cell antioxidant capacity. The optimal concentration of α-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. 5 μmol/L α-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 the α-MSH treatment group;53 lncRNAs were markedly up-regulated and 6 lncRNAs were down-regulated in the α-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 polymerase Ⅱ promoter. Conclusions Under hyperglycemia and hyperlipidemia, the influence of α-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.
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