Duan Jing, Qian Xian-Ling, Li Jun, Xiao Xing-Hua, Lu Xiang-Tong, Lv Lin-Chen, Huang Qing-Yun, Ding Wen, Zhang Hong-Yan, Xiong Li-Xia
Department of Pathophysiology, Medical College, Nanchang University, 461 Bayi Road, Nanchang 330006, China.
Department of Pathology, Second Affiliated Hospital, Nanchang University, No. 1 Mingde Road, Nanchang 330006, China.
Int J Endocrinol. 2019 Aug 28;2019:5219782. doi: 10.1155/2019/5219782. eCollection 2019.
Diabetes is a progressive metabolic disease characterized by hyperglycemia. Functional impairment of islet cells can occur to varying degrees. This impairment can initially be compensated for by proliferation and metabolic changes of cells. Cell division control protein 42 (Cdc42) and the microRNA (miRNA) miR-29 have important roles in -cell proliferation and glucose-stimulated insulin secretion (GSIS), which we further explored using the mouse insulinoma cell line MIN6.
Upregulation and downregulation of miR-29a and Cdc42 were accomplished using transient transfection. miR-29a and Cdc42 expression was detected by real-time PCR and western blotting. MIN6 proliferation was detected using a cell counting kit assay. GSIS under high-glucose (20.0 mM) or basal-glucose (5.0 mM) stimulation was detected by enzyme-linked immunosorbent assay. The miR-29a binding site in the Cdc42 mRNA 3'-untranslated region (UTR) was determined using bioinformatics and luciferase reporter assays.
miR-29a overexpression inhibited proliferation ( < 0.01) and GSIS under high-glucose stimulation ( < 0.01). Cdc42 overexpression promoted proliferation ( < 0.05) and GSIS under high-glucose stimulation ( < 0.05). miR-29a overexpression decreased Cdc42 expression ( < 0.01), whereas miR-29a downregulation increased Cdc42 expression ( < 0.01). The results showed that the Cdc42 mRNA 3'-UTR is a direct target of miR-29a . Additionally, Cdc42 reversed miR-29a-mediated inhibition of proliferation and GSIS ( < 0.01). Furthermore, miR-29a inhibited -catenin expression ( < 0.01), whereas Cdc42 promoted -catenin expression ( < 0.01).
By negatively regulating Cdc42 and the downstream molecule -catenin, miR-29a inhibits MIN6 proliferation and insulin secretion.
糖尿病是一种以高血糖为特征的进行性代谢疾病。胰岛β细胞功能损害可不同程度发生。这种损害最初可通过β细胞的增殖和代谢变化得到代偿。细胞分裂控制蛋白42（Cdc42）和微小RNA（miRNA）miR-29在β细胞增殖和葡萄糖刺激的胰岛素分泌（GSIS）中起重要作用，我们使用小鼠胰岛素瘤细胞系MIN6对此进行了进一步研究。
通过瞬时转染实现miR-29a和Cdc42的上调和下调。通过实时PCR和蛋白质印迹法检测miR-29a和Cdc42的表达。使用细胞计数试剂盒检测MIN6细胞增殖。通过酶联免疫吸附测定法检测高糖（20.0 mM）或基础葡萄糖（5.0 mM）刺激下的GSIS。使用生物信息学和荧光素酶报告基因测定法确定Cdc42 mRNA 3'-非翻译区（UTR）中的miR-29a结合位点。
miR-29a过表达抑制高糖刺激下的细胞增殖（P<0.01）和GSIS（P<0.01）。Cdc42过表达促进高糖刺激下的细胞增殖（P<0.05）和GSIS（P<0.05）。miR-29a过表达降低Cdc42表达（P<0.01），而miR-29a下调增加Cdc42表达（P<0.01）。结果表明，Cdc42 mRNA 3'-UTR是miR-29a的直接靶标。此外，Cdc42逆转了miR-29a介导的对细胞增殖和GSIS的抑制作用（P<0.01）。此外，miR-29a抑制β-连环蛋白表达（P<0.01），而Cdc42促进β-连环蛋白表达（P<0.01）。
通过负调控Cdc42和下游分子β-连环蛋白，miR-29a抑制MIN6细胞增殖和胰岛素分泌。
