异槲皮苷介导miRNA-29调控靶基因改善胰岛素抵抗的作用机制

Improving insulin resistance plays a key role on therapy type 2 diabetes mellitus. Overexpression is related to the development and progression of type 2 diabetes mellitus, which the mechanisms are still unclear. At our prophase studies, we confirmed that isoquercitrin ameliorated insulin resistance by regulation the expression of gene and protein on PI3K/AKT signaling pathway, and downregulation miR-29 expression. Through the target genes prediction and related bioinformatics analysis, the data provided PIK3R1 and PTEN genes contained the binding site of miR-29 in 3’UTR.At present, the mechanisms of isoquercitrin improving insulin resistance by regulating miR-29 had not been reported. We proposed a hypothesis that isoquercitrin ameliorate insulin resistance by miR-29-mediated regulation the target genes PIK3R1 and PTEN. We will perform the experiments from various aspects, such as molecular, cell, tissue and animal, and so on, to identify the effect of isoquercitrin regulation on miR-29;to explore the mechanism of isoquercitrin regulation target genes PIK3R1,PTEN by miR-29-mediated,and to determine the target genes of miR-29 are PIK3R1 and PTEN. Our researchs will utilize a innovation views which miRNA regulate the target gene to study the mechanisms of isoquercitrin improving insulin resistance on regulation target genes via miR-29 mediated. It will provide a new ways for prevention and therapy type 2 diabetes mellitus.

改善胰岛素抵抗是治疗2型糖尿病的关键。miR-29过表达与2型糖尿病发生发展有密切关系，但机制尚未清楚。课题组前期研究证明异槲皮苷可调控PI3K/AKT信号通路基因、蛋白表达，改善胰岛素抵抗；并下调胰岛素抵抗细胞miR-29的表达；生物信息学预测miR-29与PIK3R1/PTEN-3’UTR碱基序列互补配对。目前异槲皮苷调控miR-29改善胰岛素抵抗的机制尚未见有报道。我们提出假说：异槲皮苷通过介导miR-29调控靶基因PIK3RI、PTEN，改善胰岛素抵抗。本课题拟从分子、细胞、组织及动物水平等多层次明确异槲皮苷调控miR-29作用；探讨异槲皮苷介导miR-29调控PIK3R1、PTEN靶基因机制；确定miR-29的靶基因是PIK3R1、PTEN。本研究将通过miRNA调控靶基因这一新观点探讨异槲皮苷介导miR-29调控靶基因改善胰岛素抵抗的作用机制，为2型糖尿病的预防与治疗提供新思路

胰岛素抵抗（IR）是 2型糖尿病（T2DM ）发生发展的关键因素，且miR-29在T2DM中过表达。课题组前期研究证明异槲皮苷可改善IR，下调miR-29的表达，机制尚未明确。课题组（1）利用生物信息学法预测PIK3R1、PTEN与miR-29a的靶向关系。（2）构建PIK3R1（WT/Mut）质粒，用双荧光素酶报告基因检测法、Western Blot法、ELISA和qRT-PCR等方法验证PIK3R1、PTEN与miR-29a-3p的靶向关系；结果表明：共转染miR-29a-3pmimic、PIK3R1WT质粒时，荧光强度减小而PIK3R1 Mut质粒荧光强度无改变；（3）miR-29a-3pmimic/inhibitor质粒并分别转染IRHepG2细胞；结果显示：转染miR-29a-3pmimic细胞中PIK3R1、PTENmRNA、蛋白明显降低，GSK3β明显升高；转染miR-29a-3pinhibitor中PIK3R1明显升高，PTEN、GSK3β明显降低；（4）在异槲皮苷干预miR-29a-3pmimic转染IRHepG2细胞，显示：IR+20IS+mimics组中PIK3R1、p-AKT/AKT、p-GSK3β/GSK3β，GLUT4蛋白表达增高，PTEN、FOXO1蛋白表达降低；葡萄糖消耗量实验显示：IR+ Isoquectrin+miR-29a-3pmimics组的葡萄糖消耗量明显减少；（5）miR-29a-3p过表达腺病毒质粒尾静脉注射db/db小鼠，异槲皮苷灌胃给药14天，异槲皮苷干预组小鼠空腹血糖明显降低、空腹胰岛素明显升高；给药组HE染色胰岛轮廓相对清晰，形状规则；异槲皮苷各剂量灌胃组小鼠的 miR-29a-3p 表达下降；异槲皮苷给药组小鼠PIK3R1，AKT、GLUT4表达量增加，GSK3β表达下降；因此，本研究从体外、体内实验结果证明了：1.PIK3RI、PTEN与miR-29a-3p有靶向关系；2.异槲皮苷可通过降低miR-29a-3p的表达调控PI3KR1、PTEN、AKT、GSK3β、GLUT4的表达，从而增加机体对葡萄糖的利用，改善IR，为T2MD的预防与治疗提供新思路。