The recombinant adenoviruses expressing miR-22 (Ad-miR-22) was constructed and the effect of Ad-miR-22 on insulin signal pathway and glucose uptake in HepG2 cells was analyzed. MiR-22 gene was amplified by PCR from human hepatocytes and cloned into the pAdTrack-CMV vector to generate the shuttle plasmid pAdT-22. The positive colonies were confirmed by PCR and sequencing. The resultant shuttle plasmid was linearized with Pme I, followed by co-transformation into competent BJ5183 cells containing an adenoviral backbone plasmid (pAdEasy-1) to create the recombinant plasmid pAd-miR-22. After digested with Pac I, the linearized pAd-miR-22 was transfected into 293A packaging cell line to generate recombinant adenoviruses Ad-miR-22. HepG2 cells were infected with Ad-miR-22 or control Ad-GFP (adenoviruses expressing green fluorescent protein), and then the miR-22 expression levels were analyzed by qPCR. The result shows that adenovirus-mediated overexpression of miR-22 significantly decreased insulin-induced glucose uptake in HepG2 cells. Moreover, overexpression of miR-22 markedly decreased insulin-induced phosphorylation of GSK-3β. miR-22 also increased the mRNA levels of gluconeogenic genes in HepG2 cells. Furthermore, Western blotting results indicate that the protein expression of SIRT1 decreased in Ad-miR-22 infected HepG2 cells as compared with Ad-GFP infected HepG2 cells. In summary, overexpressing of miR-22 significantly increased gluconeogenesis while decreased glucose uptake in HepG2 cells. The effect of miR-22 on glucose metabolism may be mediated by SIRT1.
文中构建了miR-22 重组腺病毒Ad-miR-22，分析了其对HepG2 细胞胰岛素信号通路及葡萄糖摄取的抑制作用。通过PCR 方法，扩增了miR-22 的前体及侧翼序列，酶切后克隆至腺病毒穿梭载体pAdTrack-CMV 中，构建穿梭质粒pAdT-22，经PCR 及测序鉴定。穿梭质粒经PmeⅠ线性化后，直接转化含有腺病毒骨架载体的感受态细胞BJ5183，产生重组腺病毒质粒Ad-miR-22，最后经PacⅠ线性化后转染包装细胞系293A。重组腺病毒经过3 轮扩增后感染HepG2 细胞，通过荧光定量PCR 检测miR-22 表达水平。通过葡萄糖摄取实验观察Ad-miR-22对HepG2 细胞葡萄糖摄取的影响。采用Western blotting 检测Ad-miR-22 对HepG2 细胞SIRT1 在蛋白质水平的表达及GSK-3β 磷酸化水平的影响。采用荧光定量PCR 检测miR-22 对PEPCK 及G6Pase 等基因在mRNA 水平表达的影响。结果表明，重组腺病毒Ad-miR-22 感染显著增加HepG2 细胞miR-22 表达水平。此外，Ad-miR-22 显著抑制胰岛素诱导的HepG2 葡萄糖摄取，并通过下调GSK-3β 磷酸化抑制胰岛素信号通路的激活。Ad-miR-22 反转胰岛素对糖异生关键酶表达的抑制作用，并下调SIRT1 基因在蛋白质水平的表达。综上所述，构建了miR-22的重组腺病毒，发现其显著增加糖异生，抑制HepG2 细胞葡萄糖摄取，该作用可能与miR-22 调节SIRT1 在蛋白质水平的表达有关。.
Keywords: SIRT1; gene therapy; gluconeogenesis; glucose uptake; miR-22; recombinant adenoviruses.