缺氧诱导血管内皮细胞中长链非编码RNALINC01116的表达及其功能的初步研究  

LINC01116 is induced in hypoxic vascular endothelial cells and regulates inflammatory reaction

作　　者：李满满[1] 谭小玲[2] 杨诚忠 罗羽莎 杨戎[1] 

机构地区：[1]重庆医科大学干细胞与组织工程研究室,400016 [2]陆军军医大学(第三军医大学)高原军事医学系高原生理学与高原生物学教研室,重庆400038

出　　处：《免疫学杂志》2018年第3期185-192,共8页Immunological Journal

基　　金：国家自然科学基金(81270108)

摘　　要：目的探讨缺氧条件下人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)中长链非编码RNA(long non-coding RNA,lnc RNA)LINC01116的表达及其在炎症分子表达调控中的作用。方法采用三气培养箱模拟缺氧环境(1%O_2),常氧对照组细胞于普通培养箱(21%O_2)中常规培养。采用实时荧光定量PCR检测LINC01116及各炎症分子的m RNA水平。Western blot检测缺氧诱导因子-1α(hypoxia-inducible factor 1 alpha,HIF-1α)蛋白水平,并用葡萄糖转运体-1(glucose transporter 1,GLUT-1)的转录表达水平反映HIF-1的转录活性。免疫荧光法检测核转录因子NF-κB(nuclear factor of kappa B,NF-κB)活性亚基p65在细胞中的分布,并计算p65入核的细胞比例。结果 q RT-PCR的结果显示,缺氧条件下,LINC01116在HUVEC中持续高表达,与常氧对照组相比,差异均具有统计学意义(P<0.01)。经脯氨酸羟化酶抑制剂(DMOG)和去铁酰胺(DFX)处理后,常氧培养HUVEC中HIF-1α蛋白表达和GLUT-1的转录表达增加,LINC01116表达增高,与溶剂对照组相比,差异均具有统计学意义(P<0.01)。经YC-1和小干扰RNA处理后,缺氧培养HUVEC中HIF-1α蛋白表达和GLUT-1的转录表达下降,LINC01116的表达显著下降,与各对照组相比,差异均具有统计学意义(P<0.01)。缺氧24 h时,HUVEC中炎症分子TNF-α、IL-1β、ICAM、E-SELECTIN的m RNA水平增加,干扰LINC01116表达后,上述炎症分子的m RNA水平显著降低(P<0.01)。免疫荧光的实验显示,缺氧24 h后,细胞核内p65的分布增加,而干扰LINC01116表达后,p65入核的细胞比例下降,与对照组相比,差异均具有统计学意义(P<0.05)。结论缺氧通过HIF-1途径诱导血管内皮细胞LINC01116的表达增加;LINC01116可以通过调控p65入核,调节内皮细胞炎症分子的表达,参与介导缺氧血管内皮细胞炎症反应。lncRNAs（long non-coding RNAs） are RNAs longer than 200 nucleotides that do not encode proteins, but essential for cellular activities. As yet, hypoxia-induced lnc RNAs and the function in vascular inflammation are still unknown. This study designed to explore the expression and function of LINC01116 in hypoxic human umbilical vein endothelial cell（HUVEC）. Hypoxic cells were incubated in a culture chamber with 1%O2, 5%CO2 and 94%N2, while the cells cultured in the chamber with air and 5%CO2 were served as normoxic controls.q RT-PCR, si RNA transfection, Western blotting and immunochemistry analysis were used in this study. Data showed LINC01116 were significantly upregulated in hypoxic HUVEC（P〈0.01）; HIF-1α protein and GLUT-1 mRNA were increased in normoxic cells withdimethyloxalylglycine（DMOG） or desferrioxamine（DFX） treatment, while LINC01116 was upregulated.When hypoxic cells was treated with YC-1 or transfected with siRNA for HIF-1α, the levels of HIF-1α protein and GLUT-1 mRNA were decreased, while hypoxia-induced LINC01116 expression would attenuated（P〈0.01）. In LINC01116-deficient hypoxic HUVECs,hypoxia-induced upregulation of inflammatory factors（TNF-α, IL-1β, ICAM, E-SELECTIN） were remarkably decreased. Meanwhile, hypoxia-induced nuclear translocation of p65 was reduced when LINC01116 expression was inhibited by si RNA. In conclusion, hypoxia upregulates LINC01116 expression in HUVEC in HIF-1-dependent way. LINC01116 takes part in hypoxia-induced inflammatory reaction through regulating inflammatory factors expression and the activity of NF-κB pathway.

关 键 词：LINC01116 缺氧诱导因子-1 核转录因子-ΚB 炎症分子 血管内皮细胞 缺氧 

分 类 号：R392.12[医药卫生—免疫学]