miR-29c在乙型肝炎并发糖尿病中的作用研究

The incidence of diabetes mellitus in the patients with hepatitis B is significantly higher than that in the normal population. However, the pathogenesis of hepatitis B complicated by diabetes remains unclear. Emerging studies have shown that microRNAs play a crucial role in the pathogenesis of diabetes mellitus. Our previous research revealed that lysophosphatidic acid (LPA) involves in the regulation of insulin secretion, and hepatitis B virus (HBV) pre-S2 protein activated the promoter of lysophospholipase A-1 (LYPLA1) which can hydrolyze LPA. Bioinformatics analysis indicated that LYPLA1 and the PI3K of insulin signaling pathway are the targeting genes of miR-29c. And gene-chip data and quantitative PCR analysis displayed that HBV can down regulate significantly the expression of miR-29c. These results of studies and analysis suggest that it might be one of the nosogenesis of hepatitis B complicated by diabetes that HBV impairs insulin secretion and signaling via affecting the expression of miR-29c targeting LYPLA1 and PI3K. In this project, we intend to investigate the regulation of miR-29c on insulin secretion and signaling in HBV infection and elucidate the molecular mechanism of the regulation by using the HBV transgenic mice and the expression vectors of HBV X protein and pre-S2 protein which are the major HBV trans regulators, and thus provide experimental basis for being clear and definite of the role of miR-29c in the pathogenesis of the diabetes complication of hepatitis B.

乙型肝炎患者糖尿病发病率远高于正常人群，但乙型肝炎并发糖尿病的机制仍不明确。新近的研究表明，miRNAs在糖尿病发病中具有重要作用。我们的前期研究发现，溶血磷脂酸（LPA）参与胰岛素分泌调节，乙型肝炎病毒（HBV）前S2蛋白激活水解LPA的溶血磷脂酶A1（LYPLA1）基因启动子。生物信息学分析指出，LYPLA1和胰岛素信号转导通路的PI3K是miR-29c的靶基因。基因芯片数据和定量PCR分析揭示，HBV可显著下调miR-29c表达。这些研究和分析结果提示，HBV通过影响靶向LYPLA1和PI3K 的miR-29c表达，导致胰岛素分泌和信号转导受损可能是乙型肝炎并发糖尿病发病的一种机制。本项目拟应用HBV转基因小鼠和HBV主要反式调节蛋白表达载体研究HBV感染下miR-29c对胰岛素分泌和信号转导的调节作用，阐明其分子机制，为明确miR-29在乙型肝炎并发糖尿病中的作用提供实验依据。

乙型肝炎病毒（HBV）感染和糖尿病均为主要的全球性公共卫生问题。乙型肝炎患者糖尿病发病率显著高于正常人群，但乙型肝炎并发糖尿病的机制仍不明确。本项目研究HBV反式调节蛋白HBx及Pre-S2对胰岛素分泌细胞（NIT），胰岛素靶细胞（AML12、C2C12和3T3-1）miR-29c的表达，NIT胰岛素分泌以及AML12葡萄糖摄取的影响；miR-29c对PI3K和溶血磷脂酶A1（LYPLA1）的靶向作用；过表达miR-29c对NIT胰岛素分泌的影响，以及溶血磷脂酸（LPA）受体拮抗剂KI16425对过表达miR-29c NIT胰岛素分泌的影响；HBV转基因小鼠和rAAV8-1.3HBV注射HBV小鼠的高脂饲养高糖血症/肥胖症模型的葡萄糖稳态；鸭乙肝模型的病毒肝内外组织感染及其对胰岛素分泌和葡萄糖稳态的影响。结果显示，HBx及Pre-S2显著降低NIT、AML12、C2C12和3T3-1的miR-29c表达，并显著降低NIT的胰岛素分泌和AML12的葡萄糖摄取；miR-29c靶向沉默LYPLA1基因，降低LYPLA1 mRNA水平；过表达miR-29c上调NIT胰岛素分泌，而KI16425增强miR-29c上调NIT胰岛素分泌的作用；HBV转基因小鼠和rAAV8-1.3HBV注射HBV小鼠高糖血症/肥胖症糖耐量实验中血糖显著升高；鸭乙肝模型有除肝组织感染外，肾、胰和骨骼肌等肝外组织也有感染，并在糖耐量实验中血糖显著升高，但胰岛素水平无显著变化。这些研究结果表明，HBV感染通过其反式调节蛋白HBx及Pre-S2下调miR-29c表达和胰岛素分泌，而miR-29c靶向沉默LYPLA1，影响LPA信号，可能在HBV感染下胰岛素分泌调控发挥重要作用。本项目研究结果为阐明乙型肝炎并发糖尿病发病机制提供了进一步的理论与实验依据。