LINC00926通过募集ELAVL1促进低氧诱导的人脐静脉血管内皮细胞焦亡

LINC00926 promotes pyroptosis of hypoxia-induced human umbilical vein vascular endothelial cells by recruiting ELAVL1

目的探讨长链非编码RNA LINC00926对低氧诱导的人脐静脉血管内皮细胞(HUVECs)焦亡的调控作用及分子机制。方法低氧(5%O_(2))处理HUVECs细胞体外模拟冠心病。实验将HUVECs细胞分为常氧对照组(Normal)、转染空载质粒组(OENC)、转染LINC00926过表达质粒组(OE-LINC00926)、低氧处理组(Hypoxia)、Hypoxia+OE-NC对照组、Hypoxia+OELINC00926组、Hypoxia+si-Ctrl对照组、Hypoxia+si-ELAVL1组、Hypoxia+si-ELAVL1+OE-LINC00926组。应用RT-qPCR及Western blot检测LINC00926和ELAVL1的表达情况。采用CCK-8法检测细胞增殖情况,ELISA法检测炎性因子IL-1β水平,Western blot检测焦亡相关蛋白caspase1、cleaved-caspase1和NLRP3的蛋白表达水平。应用RIP实验验证LINC00926与ELAVL1的结合情况。结果与Normal组相比,低氧处理上调了HUVECs中LINC00926的mRNA表达和ELAVL1的蛋白表达(P<0.05),但不影响ELAVL1的mRNA表达。与Normal组相比,过表达LINC00926可抑制HUVECs细胞增殖(P<0.05),提高了HUVECs中炎性因子IL-1β水平(P<0.05)以及焦亡相关蛋白(caspase1、cleaved-caspase1和NLRP3)的表达(P<0.05)。在低氧处理的HUVECs中,过表达LINC00926可以进一步提高低氧处理上调的ELAVL1蛋白水平。RIP实验结果证实了LINC00926与ELAVL1蛋白的结合关系。在低氧诱导的HUVECs中,敲低ELAVL1可降低IL-1β水平和焦亡相关蛋白(caspase1、cleavedcaspase1、NLRP3)的表达(P<0.05),而LINC00926过表达可部分逆转敲低ELAVL1的影响。结论在低氧处理的HUVECs中,LINC00926通过募集ELAVL1促进HUVECs细胞焦亡。

Objective To investigate the regulatory role of the long non-coding RNA LINC00926 in pyroptosis of hypoxiainduced human umbilical vein vascular endothelial cells(HUVECs)and explore the molecular mechanism.Methods HUVECs were transfected with a LINC00926-overexpressing plasmid(OE-LINC00926),a siRNA targeting ELAVL1,or both,followed by exposure to hypoxia(5%O_(2))or normoxia.The expression of LINC00926 and ELAVL1 in hypoxia-treated HUVECs was detected using real-time quantitative PCR(RT-qPCR)and Western blotting.Cell proliferation was detected using Cell Counting Kit-8(CCK-8),and the levels of IL-1βin the cell cultures was determined with ELISA.The protein expression levels of pyroptosis-related proteins(caspase-1,cleaved caspase-1 and NLRP3)in the treated cells were analyzed using Western blotting,and the binding between LINC00926 and ELAVL1 was verified with RNA immunoprecipitation(RIP)assay.Results Exposure to hypoxia obviously up-regulated the mRNA expression of LINC00926 and the protein expression of ELAVL1 in HUVECs,but did not affect the mRNA expression of ELAVL1.LINC00926 overexpression in the cells significantly inhibited cell proliferation,increased IL-1βlevel and enhanced the expressions of pyroptosis-related proteins(all P<0.05).LINC00926 overexpression further up-regulated the protein expression of ELAVL1 in hypoxia-exposed HUVECs.The results of RIP assay confirmed the binding between LINC00926 and ELAVL1.ELAVL1 knockdown significantly decreased IL-1βlevel and the expressions of pyroptosis-related proteins in hypoxia-exposed HUVECs(P<0.05),while LINC00926 overexpression partially reversed the effects of ELAVL1 knockdown.Conclusion LINC00926 promotes pyroptosis of hypoxia-induced HUVECs by recruiting ELAVL1.