长链非编码RNA-MEG3在人心脏微血管内皮细胞缺氧损伤中的作用  

The role of long non-coding RNA-MEG3 in ischemia-reperfusion injury of human cardiac microvascular endothelial cells

作　　者：黄静聪 李亮 卢子瑄 HUANG Jingcong;LI Liang;LU Zixuan(Department of Emergency,Seventh People’s Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 200137,China)

机构地区：[1]上海中医药大学附属第七人民医院急诊科,200137

出　　处：《河北医药》2024年第4期511-515,共5页Hebei Medical Journal

基　　金：上海市卫生健康委员会科研课题(编号:202040188)。

摘　　要：目的研究长链非编码RNA-MEG3在人心脏微血管内皮细胞(HCMEC)缺氧损伤中的作用。方法将HCMEC分为对照组、缺氧/复氧(H/R)组及缺氧/复氧联合MEG3敲减(H/R+siMEG3)组。荧光定量PCR检测MEG3及炎性因子的表达情况[白介素1β(IL-β)、IL-6、IL-8、IL-10、肿瘤坏死因子(TNF-α)];CCK-8检测HCMEC细胞的增殖活性;流式细胞术分析细胞凋亡情况;Western blotting检测PI3K/AKT/eNOS信号通路表达变化,ELISA检测一氧化氮(NO)、超氧化物歧化酶(SOD)、血管内皮生长因子(VEGF)和活性氧(ROS)表达水平。结果利用qPCR检测对照组及H/R组细胞MEG3表达,结果发现:H/R组MEG3相对表达倍数[(6.87±0.239)倍]较对照组MEG3表达倍数(1.00±0.026)倍显著升高(n=3,t=42.32,P<0.0001),提示MEG3可能在心脏微血管内皮细胞IRI中起到重要作用。H/R组细胞与对照组比较,细胞增殖活性显著减弱(P<0.01);而H/R+siMEG3组与H/R组比较,细胞增殖活性进步受到抑制(P<0.01)。H/R组较对照组PI3K、AKT及eNOS磷酸化水平显著减低,而H/R+siMEG3组细胞较H/R组磷酸化水平更低(P<0.05)。H/R组与对照组比较,NO、SOD及VEGF显著减低,而ROS水平升高(P<0.05);而H/R+siMEG3组与H/R组比较,NO、SOD及VEGF水平进步下降,ROS升高显著。利用qPCR检测各处理组细胞相关炎症基因表达,结果发现:H/R组较对照组基因表达显著升高(P<0.05);H/R+SiMEG3组较H/R组基因表达进一步升高(P<0.01)。结论长链非编码RNA-MEG3在心脏微血管内皮细胞H/R损伤过程中起到重要保护作用,靶向提升MEG3水平有望成为减缓心脏微血管IRI的潜在治疗靶点。Objective To study the role of long non-coding RNA maternally expressed gene 3(lncRNA MEG3)in ischemia-reperfusion injury(IRI)of human cardiac microvascular endothelial cells(HCMECs).Methods HCMECs were treated with blank control,induced with hypoxia and reoxygenation(H/R),orinduced with H/Rfollowed by transfection of si-MEG3.Relative levels of lncRNA MEG3 and inflammatory factors,including the interleukin beta(IL-β),IL-6,IL-8,IL-10 and tumoRnecrosis factor alpha(TNF-α)were detected by quantitative real-time polymerase chain reaction(qRT-PCR).Cell proliferation and apoptosis were detected by CCK-8 assay and flow cytometry,respectively.Protein expressions of key proteins in the phosphatidylinositol 3-kinase/protein kinase B/endothelial nitric oxide synthase(PI3K/Akt/eNOS)signaling pathway were detected by Western blot.The enzyme-linked immunosorbent assay(ELISA)was performed to measure relative levels of nitric oxide(NO),superoxide dissolving enzyme(SOD),vascular endothelial growth factor (VEGF)and reactive oxygen species(ROS).Results qRT-PCRdata showed that the relative level of lncRNA MEG3 in H/R-induced HCMECs was significantly higher than that of controls[6.87±0.239]times vs[1.00±0.026]times,n=3,t=42.32,P<0.0001),suggesting the role of lncRNA MEG3 in IRI of HCMECs.Cell proliferation was significantly inhibited in H/R-induced HCMECs than that of controls,which was further inhibited in H/R-induced HCMECs with knockdown of lncRNA MEG3(P<0.01).Protein expressions of p-PI3K,p-AKT and p-eNOS were significantly lower in H/R-induced HCMECs than those of controls,which were further downregulated in H/R-induced HCMECs with knockdown of lncRNA MEG3(P<0.05).Significantly decreased NO,SOD and VEGF levels and increased ROS level were detected in H/R-induced HCMECs than those of controls,which were more pronounced in H/R-induced HCMECs with knockdown of lncRNA MEG3(P<0.05).Significantly increased mRNA levels of inflammatory factors were detected in H/R-induced HCMECs than those of controls,which were significantly higher

关 键 词：心脏微血管细胞内皮细胞 MEG3 ENOS NO SOD ROS 炎症因子 

分 类 号：R543[医药卫生—心血管疾病]