miR-24-3p靶向HAP1基因对STZ诱导胰岛β细胞凋亡的影响

Effects of miR-24-3p targeting HAP1 gene on apoptosis of pancreaticβ-cell induced by STZ

目的探讨微小RNA-24-3p(miR-24-3p)靶向亨廷顿蛋白相关蛋白(HAP)1基因表达对链脲佐菌(STZ)诱导胰岛β细胞凋亡的影响。方法培养小鼠胰岛β细胞NIT,分别将anti-miR-NC、anti-miR-24-3p、pcDNA、pcDNA-HAP1转染入NIT细胞,加入STZ诱导细胞;不做任何处理为Con组。采用实时荧光定量聚合酶链反应(qRT-PCR)检测miR-24-3p及HAP1 mRNA表达水平;采用蛋白免疫印迹(Western印迹)实验检测HAP1及B细胞淋巴瘤(Bcl)-2、Bcl-2相关X蛋白(Bax)、活化的半胱氨酸蛋白酶(cleaved-caspase)-3蛋白表达水平。双荧光素酶报告实验验证miR-24-3p的靶基因。流式细胞术检测各组细胞凋亡情况。结果STZ处理后胰岛β细胞中miR-24-3p相对表达量明显高于Con组(P<0.05),而HAP1 mRNA及蛋白表达均明显降低(P<0.05);STZ可增加胰岛β细胞凋亡率,抑制miR-24-3p表达可降低胰岛β细胞凋亡率,还可上调抗凋亡蛋白Bcl-2表达,下调促凋亡蛋白Bax、cleaved-caspase-3表达;HAP1过表达与抑制miR-24-3p表达均可抑制STZ诱导的胰岛β细胞凋亡;HAP1是miR-24-3p的靶基因,miR-24-3p可负向调节HAP1表达;抑制HAP1表达可促进胰岛β细胞凋亡。结论miR-24-3p表达可通过抑制靶基因HAP1表达进而促进STZ诱导的胰岛β细胞凋亡。

Objective To investigate the effect of microRNA-24-3 p(miR-24-3 p)-targeted Huntingtin-related protein 1(HAP1) gene expression on apoptosis of pancreatic β-cells induced by streptozotocin(STZ).Methods Mouse islet β-cell NIT was cultured, and anti-miR-NC, anti-miR-24-3 p, pcDNA and pcDNA-HAP1 were transfected into NIT cells and added to STZ-induced cells. Real-time quantitative polymerase chain reaction(qRT-PCR) was used to detect the expression of miR-24-3 p and HAP1 mRNA. Western blot was used to detect HAP1 and Bcl-2, Bax and cleaved-caspase-3 proteins. The dual luciferase reporter assay validated the target gene of miR-24-3 p. Apoptosis of each group was detected by flow cytometry.Results The expression of miR-24-3 p in pancreatic β-cell was significantly higher than that in the control group(P<0.05), while the expressions of HAP1 mRNA and protein were significantly decreased(P<0.05). STZ could increase the apoptosis rate of pancreatic β-cell, inhibit the expression of miR-24-3 p, decrease the apoptosis rate of pancreatic β-cells, up-regulate the expression of anti-apoptotic protein Bcl-2, and down-regulate the pro-apoptotic proteins expressions of Bax and cleaved-caspase-3. The effect of HAP1 over-expression on pancreatic β-cell apoptosis was the same as that of suppressing miR-24-3 p expression. HAP1 was a target gene of miR-24-3 p, and miR-24-3 p negatively regulated HAP1 expression. Inhibition of HAP1 expression could promote pancreatic β-cell apoptosis. Conclusions miR-24-3 p expression could promote STZ-induced apoptosis of pancreatic β-cells by inhibiting the expression of target gene HAP1.