摘要
目的探讨microRNA-181b(miR-181b)能否通过靶向调控NDRG2参与2型糖尿病胰岛素分泌的调节。方法选用小鼠胰岛β细胞进行体外传代培养,构建NDRG2野生型(NDRG2^(WT)-1uc)与突变型(NDRG2^(MUT)-1uc)荧光素酶报告基因质粒并进行转染,转染结束后进行双荧光素酶报告分析。构建NDRG2过表达和敲低质粒并转染小鼠胰岛β细胞,检测NDRG2 mRNA和蛋白表达、胰岛素分泌量。未转染的小鼠胰岛β细胞培养48 h后采用实时荧光定量聚合酶链反应检测miR-181b和NDRG2 mRNA的表达。采用葡萄糖刺激的胰岛素释放试验检测5 mmol/L和20 mmol/L葡萄糖条件下的小鼠胰岛β细胞分泌的胰岛素。结果(1)荧光素酶报告基因实验检测发现,各组相对荧光强度比较,差异有统计学意义(P<0.05)。miR-181b模拟物可以明显抑制NDRG2^(WT)-luc的3’端非翻译区的表达,但对NDRG2-MUT组和Control组无明显影响;miR-181b inhibitor可以加强NDRG2^(WT)-luc的3’-端非翻译区的表达,但对NDRG2-MUT组和Control组无明显影响。(2)过表达组NDRG2 mRNA和蛋白相对表达量高于过表达对照组(P<0.05);在5 mmol/L葡萄糖条件下,过表达组与过表达对照组胰岛素分泌量比较,差异无统计学意义(P>0.05);在20 mmol/L葡萄糖条件下,过表达对照组胰岛素分泌量高于过表达组(P<0.05)。敲低组NDRG2 mRNA和蛋白相对表达量低于敲低对照组(P<0.05);在5 mmol/L葡萄糖条件下,敲低组与敲低对照组胰岛素分泌量比较,差异无统计学意义(P>0.05);在20 mmol/L葡萄糖条件下,敲低组胰岛素分泌量高于敲低对照组(P<0.05)。(3)5 mmol/L葡萄糖组与对照组的miR-181b和NDRG2 mRNA相对表达量比较,差异无统计学意义(P>0.05);20 mmol/L葡萄糖组miR-181b相对表达量高于对照组和5 mmol/L葡萄糖组(P<0.05),NDRG2 mRNA相对表达量低于对照组和5 mmol/L葡萄糖组(P<0.05)。(4)5 mmol/L葡萄糖条件下,Control组、miR-181b mimics组、miR-181b inhibitor组胰岛素分泌量比较,差异无统计学意义(P>0.05)。在20 mmol/L葡萄糖条件下,miR-181b mimics组胰岛素分泌量高于Control组(P<0.05),miR-1b1 inhibitor组胰岛素分泌量低于Control组和miR-181b mimics组(P<0.05)。结论miR-181b可以通过靶向抑制NDRG2的表达来提高胰岛素的分泌。
Objective To explore whether microRNA181-b(miR-181b)could target on NDRG2 to regulate the insulin secretion in type 2 diabetes mellitus.Methods MIN6 cells were cultured in vitro and NDRG2 wild-type(NDRG2^(WT)-1uc)and mutant(NDRG2^(MUT)-1uc)luciferase reporter gene plasmids were constructed and transfected.After transfection,the dual luciferase reporter assay was performed.The NDRG2 overexpression and knockdown plasmids were constructed and transfected into MIN6 cells.The mRNA and protein expressions of NDRG2 and the insulin secretion were detected.After 48-hour culture of MIN6 cells without transfection,the mRNA expressions of miR-181b and NDRG2 were detected.The glucose stimulated insulin release assay was performed under the condition of 5 mmol/L and 20 mmol/L of glucose to detect the insulin secretion.Results The luciferase reporter assay showed that the relative fluorescence intensity was different among the groups(P<0.05).The miR-181b mimics could significantly inhibit the expression of the 3’-untranslated region of NDRG2^(WT)-luc,but had no effect on NDRG2^(MUT)-1uc and control plasmids.On the contrary,miR-181b inhibitor could enhance the expression of the 3’-untranslated region of NDRG2^(WT)-luc,but had no effect on NDRG2^(MUT)-1uc and control plasmids.The overexpression of NDRG2 significantly increased the mRNA and protein expressions of NDRG2(P<0.05).With a glucose concentration of 5 mmol/L,there was no significant difference in the insulin secretion between the overexpression group and the control group(P>0.05).When the glucose concentration was set at 20 mmol/L,the insulin secretion was significantly reduced in the overexpression group(P<0.05).The knockdown of NDRG2significantly decreased the mRNA and protein expressions of NDRG2(P<0.05).With a glucose concentration of 5mmol/L,there was no significant difference in the insulin secretion between the knockdown group and the control group(P>0.05).When the glucose concentration was set at 20 mmol/L,the insulin secretion was significantly elevated in the knockdown group(P<0.05).The relative mRNA expressions of miR-181b and NDRG2 were not different between the 5 mmol/L glucose group and the control group(P>0.05).In contrast,the relative mRNA expression of miR-181b in the 20 mmol/L glucose group was higher than that of the control group and the 5 mmol/L glucose group(P<0.05),while the relative m RNA expression of NDRG2 in the 20 mmol/L glucose group was lower than that of the control group and the 5 mmol/L glucose group(P<0.05).Under the condition of 5 mmol/L of glucose,there was no difference in the insulin secretion among the control group,miR-181b mimics group and miR-181b inhibitor group(P>0.05).When the glucose concentration was 20 mmol/L,the insulin secretion was greater in the miR-181b mimics group than that in the control group(P<0.05),whereas the insulin secretion was lower in the miR-181b inhibitor group compared with the control group and the miR-181b mimics group(P<0.05).Conclusions The miR-181b can improve the insulin secretion by targeted inhibition of NDRG2 expression.
作者
曾维新
云川
李晓燕
李大伟
Wei-xin Zeng;Chuan Yun;Xiao-yan Li;Da-wei Li(Department of Endocrinology,The First Affiliated Hospital of Hainan Medical College,Haikou,Hainan 570102,China)
出处
《中国现代医学杂志》
CAS
北大核心
2022年第10期30-35,共6页
China Journal of Modern Medicine
基金
海南省卫生健康行业科研项目(No:20A200012)。
