miR-155靶向SOCS1和HDAC4调控肝脏糖异生的研究

Effective inhibition of gluconeogenesis is an important approach for type 2 diabetes mellitus (T2DM) treatment. Our previous studies revealed that the gain of miR-155 function led to the reduced hepatic and serum lipid levels, enhanced insulin sensitivity as well as improved glucose metabolism (PLoS Genetics，2016；PLoS One，2015). Our preliminary experiments showed that the liver-specific overexpression of miR-155 transgene in mice resulted in the increased hepatic glycogen synthesis, reduced G6Pase and PEPCK expression, and the inhibition of hepatic gluconeogenesis, suggesting the important roles of miR-155 in hepatic gluconeogenesis. Against this background, the present project plans to carry out the following studies: 1) miR-155 expression in fasted mice, STZ-induced diabetes mice and db/db mice will be determined by qRT-PCR; 2) the functions of miR-155 in hepatic gluconeogenesis will be completely examined by the combined use of miR-155 conditional transgenic mice and miR-155 conditional knockout mice; 3) the regulatory roles of miR-155 in hepatic gluconeogenesis will be evaluated by in vivo bioluminescent imaging; 4) the hypoglycemic effect of miR-155 agomir will be evaluated in db/db mice; 5) to explore whether the functions of miR-155 in the regulation of hepatic gluconeogenesis can be mediated by its target genes (i.e., SOCS1 or HDAC4); 6) During hepatic gluconeogenesis, we will explore how miR-155 affects AKT phosphorylation, and FoxO1 phosphorylation and deactylated modification. Finally, the expected findings will shed light on new mechanisms of hepatic gluconeogenesis, and provide a new potential target for the prevention and treatment of diabetes.

抑制肝糖异生亢进降血糖是T2DM治疗的重要手段。申请人发表的研究证实，miR-155具有降脂、降糖、提高胰岛素敏感性和改善葡萄糖代谢的功能。课题组前期研究发现小鼠肝脏转基因过表达miR-155可降低糖异生基因表达，抑制肝糖异生，提示miR-155在肝糖异生中起重要作用。基此，本项目拟进一步开展以下工作：1) 检测饥饿处理的小鼠和STZ诱导的糖尿病小鼠肝组织中miR-155表达水平；2) 综合运用转基因小鼠和基因敲除小鼠，阐明miR-155在肝糖异生中的作用；3) 活体生物发光成像技术评价miR-155对肝糖异生的调节作用；4)评价miR-155激动剂降糖功效；5) 确证miR-155对肝糖异生的调控作用可否由靶基因SOCS1或HDAC4介导；6) 确证肝糖异生中miR-155对AKT磷酸化、FoxO1磷酸化和乙酰化修饰的调控作用。预期成果将揭示新的糖异生调控机制，并为糖尿病防治提供新靶点。

我们发表的研究表明，miR-155正向调节胰岛素敏感性，具有治疗糖尿病的潜在应用，但是，它在调节肝糖异生和糖原生成中的作用及其潜在机制仍然未知。通过基因芯片和荧光定量PCR检测，我们发现miR-155转基因小鼠中肝脏糖异生、糖原代谢、糖酵解、葡萄糖转运蛋白、TCA循环和丙酮酸代谢途径中基因表达明显变化，表明在小鼠肝脏过表达miR-155转基因可诱导肝脏葡萄糖代谢改变。另外，我们发现在禁食和糖尿病小鼠的肝脏中miR-155的表达显著下调，表明miR-155可能在生理和病理条件下参与肝脏糖异生的调节。泛表达和肝脏特异过表达miR-155转基因的小鼠可出现低血糖，相反，miR-155基因敲除小鼠可导致肝脏葡萄糖过量产生和高血糖症。miR-155可负向调节肝糖异生基因的表达。此外，小鼠肝脏特异性过表达miR-155转基因可导致肝糖原储存增加，并伴随AKT和GSK-3β磷酸化增加，以及糖原合酶2(Gys2)和葡萄糖激酶(Gck)表达增强，进而改善小鼠葡萄糖耐量和提高胰岛素敏感性。miR-155通过抑制肝脏糖异生而导致低血糖，从机制上来讲这在很大程度上是通过直接抑制C/EBPβ表达来实现的。总之，我们的工作揭示了miR-155在调节肝脏糖异生和糖原生成方面以前未被重视的功能，这表明miR-155可能作为糖尿病的新型潜在治疗靶点。课题执行期间，在Nature communications, Journal of Cancer, Aging-US, Laboratory Investigation, Cell Death Discovery等上发表12篇SCI论文，另有1篇SCI论文待发表。在人才培养方面，共培养博士1人和硕士6人。

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