血清外泌体中mi R-642a-3p下调HK1对胰岛素分泌和儿童1型糖尿病进展的影响

Effect of down-regulation of HK1 by miR-642a-3p in serum exosomes on insulin secretion and progression of type 1 diabetes mellitus in children

目的:探讨血清外泌体中miR-642a-3p靶向HK1对1型糖尿病(T1DM)患儿胰岛β细胞增殖、凋亡及胰岛素分泌的影响。方法:GEO数据库筛选T1DM患儿血清中的差异表达miRNAs,Targetscan预测miRNAs的靶基因,双荧光素酶报告实验验证靶向关系。收集68例T1DM患儿外周血标本,分离并鉴定血清外泌体。高糖培养基处理胰岛β细胞。将转染后的外泌体与胰岛β细胞共培养并分组。采用qRT-PCR分别检测miR-642a-3p在外周血、血清外泌体中的表达水平。流式细胞术、CCK-8试验检测胰岛β细胞的凋亡和增殖情况。ELISA检测胰高血糖素样肽-1(GLP-1)和胰岛素分泌浓度。结果:相对于健康志愿者,T1DM患儿血清及血清外泌体中miR-642a-3p表达明显升高(均P<0.05)。HK1被证实为miR-642a-3p的靶点。相对于NC组,抑制血清外泌体中miR-642a-3p的表达后胰岛β细胞凋亡率降低,增殖活性增加,胰岛素分泌增多,GLP-1浓度上调(均P<0.05)。过表达miR-642a-3p能够提高胰岛β细胞凋亡率,减弱其增殖活性,使胰岛素分泌减少,抑制GLP-1表达(均P<0.05),从而进一步促进T1DM,但该作用被HK1部分挽救(均P<0.05)。结论:血清外泌体源性miR-642a-3p能够通过调控HK1抑制胰岛β细胞增殖,并促进其凋亡,从而促进儿童T1DM进展。血清外泌体源性miR-642a-3p有望在儿童T1DM的靶向治疗中发挥作用。

Objective:To explore the effects of serum exosomes containing miR-642a-3p on proliferation,apoptosis and insulin secretion of isletsβcells in type 1 diabetes mellitus(T1DM)children via targeting HK1.Methods:GEO database was used to screen differentially expressed miRNAs in T1DM children,Targetcan was used to predict target genes of miRNAs,dual luciferase reporter assay was used to confirm targeting relationship.Peripheral blood samples of 68 cases of T1DM children were collected,then serum exosomes were isolated and identified.Isletsβcells were treated by high glucose medium.Subsequently isletsβcells were cocultured with transfected exosomes and divided into different groups.qRT-PCR was used to detect expression of miR-642a-3p in peripheral blood and serum exosomes individually.Flow cytometry,CCK8 assay were employed to detect apoptosis and proliferation of isletsβcells.ELISA was used to determine expression of glucagon-like peptide-1(GLP-1)and insulin secretion concentration.Results:Compared with healthy control,miR-642a-3p expression in serum and serum exosomes was obviously increased in T1DM children(all P<0.05).HK1 was confirmed as a target of miR-642a-3p.Compared with NC group,inhibition of serum exosomal miR-642a-3p expression decreased apoptosis of isletsβcells,increased proliferative activity and insulin secretion,GLP-1 concentration was also up-regulated(all P<0.05).Overexpression of miR-642a-3p can increase apoptosis and decrease proliferative activity of isletsβcells,down-regulate insulin secretion and GLP-1 expression(all P<0.05),subsequently promoted T1DM progression,but this effects was partially saved by HK1(all P<0.05).Conclusion:Serum exosomal miR-642a-3p inhibited proliferation but promoted apoptosis of isletsβcells via regulating HK1,subsequently accelerate children T1DM.Serum exosomal miR-642a-3p is expected to play a role in targeted therapy of children’s T1DM.