METTL3在同型半胱氨酸诱导小鼠胰岛β细胞自噬中的作用

Role of METTL3 in homocysteine-induced autophagy in mouse islet beta cells

背景:高同型半胱氨酸(homocysteine,Hcy)血症与胰岛β细胞功能密切相关,但其具体分子机制尚不完全明确。目的:探讨METTL3在Hcy诱导小鼠胰岛β细胞自噬中的作用。方法:取第3,4代小鼠胰岛β细胞进行实验。①细胞模型建立和分组:对照组细胞不加入Hcy,Hcy组细胞加入浓度为100µmol/L Hcy干预48h;②按Lipofectamine^(TM) 2000说明书将过表达质粒Ad-METTL3及si-METTL3转染小鼠胰岛β细胞,设计3种不同干扰片段,PCR验证、筛选出干扰效率最好的干扰片段;③转染后实验分组:对照组、Hcy组、Ad-NC(阴性对照)+Hcy组、Ad-METTL3+Hcy组、si-NC(阴性对照)+Hcy组和si-METTL3+Hcy组;④采用qRT-PCR及Western blot检测细胞中METTL3及细胞自噬相关蛋白LC3Ⅱ/Ⅰ、p62的表达;ELISA法测定胰岛素水平来评价胰岛β细胞胰岛素分泌能力;分别过表达和干扰METTL3后检测细胞自噬相关蛋白及胰岛素水平。结果与结论:①与对照组相比,Hcy组自噬相关蛋白LC3Ⅱ/Ⅰ的表达水平升高(P<0.05),而p62表达明显降低(P<0.05),胰岛素分泌能力明显下降(P<0.05);②与对照组相比,Hcy组中METTL3蛋白及mRNA水平表达均降低(P<0.05);③胰岛β细胞中沉默METTL3后,Hcy进一步上调了细胞中LC3Ⅱ/Ⅰ的表达(P<0.05),而p62表达显著下降(P<0.05),细胞中胰岛素水平增加(P<0.05);过表达METTL3后,Hcy则使LC3Ⅱ/Ⅰ表达显著降低且p62表达则增高(P<0.05);④结论:METTL3参与了Hcy诱导的胰岛β细胞自噬调控,对胰岛素的分泌发挥着调控作用。

BACKGROUND:Hyperhomocysteinemia is closely related to the function of isletβcells,but its specific molecular mechanism is not fully understood.OBJECTIVE:To investigate the role of N6 methyltransferase-like 3(METTL3)in homocysteine(Hcy)-induced autophagy of mouse isletβcells.METHODS:The 3rd and 4th generation mouse isletβcells were taken for the experiment.(1)Cell modeling and grouping:cells in control group were not treated with Hcy,while those in homocysteine group were treated with 100μmol/L Hcy for 48 hours.(2)The mouse isletβ-cells were transfected with the plasmids overexpressing Ad-METTL3 and si-METTL3 according to the instructions of LipofectamineTM 2000.Three different interfering fragments were designed,and the one with the best interfering efficiency was verified and screened by PCR.(3)After transfection,the cells were divided into control group,Hcy group,Ad-NC(negative control)+Hcy group,Ad-METTL3+Hcy group,si-NC(negative control)+Hcy group and si-METTL3+Hcy group.(4)qRT-PCR and western blot were used to detect the expression levels of METTL3 and autophagy-related proteins LC3II/I and p62 in cells.Insulin level was determined by ELISA to evaluate insulin secretion capacity of islet cells.Autophagy-related proteins and insulin level were detected after overexpression and interference with METTL3.RESULTS AND CONCLUSION:Compared with the control group,the expression level of LC3II/I was increased(P<0.05),the expression of p62 was significantly reduced(P<0.05),and the insulin secretion capacity was significantly decreased(P<0.05)in the Hcy group.Compared with the control group,the protein and mRNA levels of METTL3 were reduced in the Hcy group(P<0.05).After METTL3 silencing in isletβcells,Hcy further upregulated the expression of LC3II/I(P<0.05),significantly dowregulated the expression of p62(P<0.05),and increased the insulin level(P<0.05).After overexpression of METTL3,Hcy significantly decreased the LC3II/I expression and increased the p62 expression in isletβcells(P<0.05).To conclude,METTL3 is involved in the Hcy-induced autophagy regulation of isletβcells and plays a role in the regulation of insulin secretion.