LncRNA Kcnq1ot1/miR-214-3p/caspase-1通路调控高糖处理的心脏成纤维细胞焦亡

LncRNA Kcnq1ot1/miR-214-3p/caspase-1 Pathway Regulates Pyroptosis of High Glucose-Treated Cardiac Fibroblasts

目的:探讨长链非编码RNA Kcnq1ot1在高糖处理的心脏成纤维细胞中调控焦亡的作用及具体机制.方法:培养C57BL/6乳鼠原代心脏成纤维细胞,分别用5.5 mM和30 mM葡萄糖培养,用免疫荧光、qRT-PCR和western blot方法检测NLRP3、cas-pase-1和IL1β的表达.高糖处理的成纤维细胞抑制Kcnq1ot1,检测caspase-1的表达.生物信息学和荧光素酶报告基因检测验证与Kcnq1ot1和caspase-1存在共同互补结合位点的microRNA.应用qRT-PCR和western blot方法检测高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p的表达,以及过表达或干扰miR-214-3p后caspase-1的表达水平.高糖诱导的细胞单独干扰Kc-nq1ot1或同时抑制Kcnq1ot1和miR-214-3p,检测caspase-1、NLRP3和IL-1β的表达水平.结果:高糖诱导的成纤维细胞中焦亡激活,Kcnq1ot1表达明显升高;抑制Kcnq1ot1后caspase-1表达显著下调.生物信息学和荧光素酶报告基因检测发现miR-214-3p与Kcnq1ot1和caspase-1存在共同互补结合位点.高糖诱导的心脏成纤维细胞干扰Kcnq1ot1后miR-214-3p表达升高;过表达miR-214-3p后caspase-1表达降低,抑制miR-214-3p后caspase-1表达升高.同时抑制Kcnq1ot1和miR-214-3p可逆转干扰Kc-nq1ot1对caspase-1的降低作用.结论:干扰Kcnq1ot1能够通过抑制miR-214-3p/caspase-1信号转导通路,抑制高糖诱导的心脏成纤维细胞焦亡.

Objective:To investigate the role and mechanism of long non-coding RNA Kcnq1ot1 in regulating pyroptosis in high glucose-treated cardiac fibroblasts.Methods:Primary cardiac fibroblasts of C57BL/6 mice were divided into the control group(5.5 m M glucose)and the high glucose group(HG group,30 m M glucose).The expression levels of NLRP3,caspase-1 and IL-1βwere detected by immunofluorescence,qRT-PCR and western blot.Si-Kcnq1ot1 was transfected into high glucose-treated cardiac fibroblasts and the expression levels of caspase-1 were detected.Bioinformatics and luciferase assay were used to forecast the microRNAs that have the common complementary binding sites with both Kcnq1ot1 and caspase-1.The expression of miR-214-3p after silencing Kcnq1ot1 and the expression of caspase-1 after transfection of miR-214-3p mimimics or AMO-214-3p in high glucose-treated cardiac fibroblasts were detected by qRT-PCR and western blot.The HG-treated fibroblasts were respectively transfected with si-NC+AMO-NC,si-Kcnq1ot1+AMO-NC and si-Kcnq1ot1+AMO-214-3p.The expression levels of caspase-1,NLRP3,IL-1βwere detected.Results:Pyroptosis was activated and the expression of Kcnq1ot1 was elevated in high glucose-treated primary cardiac fibroblasts.Caspase-1 was downregulated after silencing Kcnq1ot1.Bioinformatics and luciferase assay reveal that miR-214-3p has common complementary binding sites with both Kcnq1ot1 and caspase-1.Mi R-214-3p was increased after inhibiting Kcnq1ot1 in HG-treated cardiac fibroblasts,while caspase-1 was decreased after overexpression of miR-214-3p and was increased after inhibiting miR-214-3p.Reverse experiments showed that Kcnq1ot1 regulated caspase-1 through miR-214-3p.Conclusions:Silencing Kcnq1ot1 can alleviate high glucose-induced cardiac fibroblast pytoptosis by inhibiting miR-214-3p/caspase-1 signal pathway.