miR-29家族小RNA对SPARC/ AMPK通路的调控在胰岛素抵抗发生中的作用及机制

Secreted protein acidic and rich in cysteine (SPARC) is a key player in the pathologies associated with obesity and type 2 diabetes. In our previous study, we found that SPARC is an AMPK-interacting protein. Overexpression of SPARC enhanced AMPK phosphorylation and increased Glut4 expression, however, the molecular mechanisms by which it mediates these effects are largely unknown. This project is aimed at understanding the Effect of the interaction of SPARC and AMPK on regulating glucose and lipid metabolism and its molecule mechanism in cells and animal models. This study will determine(1) the effect of the interaction of SPARC and AMPK on insulin signal molecules and adipo-cytokines. (2)The effect of knockdown of SPARC on Glut4 and adipo-cytokines in adipocytes.(3)we will perform experiments using normal regular mice (wild type) and gene manipulated mice (knockout mice) to study the influence of SPARC on blood glucose, plasma lipid profile, Glut4 and adipo-cytokines in in muscle, liver and adipose tissue. (4) We will then investigate the expression of SPARC in muscle, liver and adipose tissue in db/db mice. SPARC could be a convergence point for insulin and AMPK signaling, making it an attractive molecule for prospective pharmacological or genetic manipulation. microRNAs (miRNAs) are small noncoding RNAs acting as translational repressors that play a major role in the control of cell proliferation and survival. These regulatory molecules bind to the 3′ untranslated region of target mRNAs, leading to translational inhibition and/or messenger degradation. Using in miRNA target prediction programs, we prioritized candidate that SPARC is one of miR-29 target genes. In this project, we assess the effects of miR-29 family on glucose metabolism and insulin resistance through SPARC/AMPK signal pathway.

富含半胱氨酸的酸性分泌蛋白（SPARC）是近来发现的影响胰岛素抵抗和2型糖尿病的重要因子。本项目的前期研究，在国际上首先发现并报道SPARC是AMPK相互作用蛋白质，SPARC/AMPK通路是影响胰岛素抵抗和糖尿病的重要因素。我们利用生物信息学方法分析发现SPARC受到miR-29家族(miR-29abc)调控，本课题拟采用过表达或干涉（抑制、敲除）等方法，通过导入化学合成的小分子miR-29s mimics或拮抗miR-29s 的antagomir，观察靶基因SPARC mRNA及编码蛋白表达以及细胞、动物水平的表型变化。从而揭示miR-29家族对SPARC基因表达量改变以及对SPARC/AMPK信号网络调控整合效应，最终证明胰岛素抵抗过程中miR-29s和靶基因SPARC之间存在相互制约关系，SPARC/AMPK信号网络的miR-29s调控失衡与胰岛素抵抗发生密切相关。

miR-29是正常代谢中的重要调节因子，可能代表2型糖尿病和相关代谢疾病的新的治疗靶点。在本研究中，使用TargetScan，我们发现SPARC是miR-29的靶标。miR-29和SPARC mRNA之间的相互作用是由3'非翻译区（UTR）中的种子结构域介导的。所有三个miR-29家族成员都可以针对SPARC。我们进行荧光素酶报告测定，在这里我们可以看到miR-29模拟物的过度表达显著降低了SPARC 3'UTR报道的基因活性，表明miR-29a/b/c可以直接靶向SPARC的3'UTR序列。我们探讨了mir-29s是否在葡萄糖代谢中发挥作用，以及mir-29s是否直接作用于SPARC。我们还研究了mir-29s对糖代谢的影响及如何关联SPARC。我们发现，高表达mir-29s减少葡萄糖的摄取和GLUT4水平；miR-29直接针对SPARC，导致编码的SPARCmRNA和蛋白水平降低；在3T3-L1脂肪细胞中，SPARC的敲减降低葡萄糖的摄取和GLUT4蛋白水平在；mir-29s减少葡萄糖的摄取和GLUT4水平；我们调查了miR-29a与SPARC对葡萄糖代谢的相关性，结果显示，mir-29s家族对葡萄糖代谢的负向调节是通过抑制SPARC的作用而实现的。miR-29对葡萄糖代谢的影响是通过抑制SPARC而发挥作用。 我们检查了SPARC基因敲除小鼠肌肉组织中GLUT4和AMPK的表达水平。我们发现，GLUT4的表达和AMPK磷酸化水平降低。miR-29a在组织中的表达水平均高于野生型组织。SPARC基因敲除小鼠与野生型相比，miR-29a水平的升高与SPARC基因沉默对3T3-L1脂肪细胞的影响一致。空腹血糖、空腹胰岛素水平，和SPARC基因敲除小鼠HOMA-IR显著高于野生型小鼠。SPARC基因敲除小鼠的胰岛数量减少和胰岛面积减小。因此，我们的结论是，SPARC基因敲除小鼠受损的葡萄糖代谢中，mir29与SPARC/AMPK有密切相关。