Role of miR-30d in insulin gene expression in pancreatic beta cells

描述（由申请人提供）： 我的长期目标是研究microRNAs（miRNAs）在调节β细胞功能中的作用，并阐明miRNAs在II型糖尿病发展中的作用。小RNA是小的非编码核糖核苷酸，其结合mRNA并且在哺乳动物中主要作为翻译抑制物起作用。miRNAs被认为在许多疾病中发挥作用，包括糖尿病。一些报道表明miRNA在胰岛素分泌和胰腺β细胞发育中的重要功能。我们最近在胰腺β细胞系MIN 6中进行了筛选，以鉴定响应葡萄糖浓度变化而丰度改变的miRNA。该筛选导致从MIN 6细胞中可检测的总共108种miRNA中鉴定出超过50种葡萄糖调节的miRNA。许多已鉴定的miRNA，包括miR-124 a、miR-107和miR-30 d，在高糖存在下上调。有趣的是，我们发现miR-30 d的过表达增加了胰岛素基因的转录。此外，通过过表达miR-30 d诱导胰岛素与β细胞特异性转录因子MafA的表达增加相关。这表明miR-30 d的推定靶基因可能是MafA/胰岛素基因表达的负调控因子。为了确定miR-30 d在胰岛素基因转录中的功能，我将：1）确定miR-30 d参与胰岛素基因转录调控的靶基因。2)表征miR-30 d在胰腺β细胞系和原代小鼠胰岛中的生物学功能。将通过siRNA敲低技术分析验证的靶标在胰岛素基因表达中的作用。此外，将表征miR-30 d对靶下游信号传导的影响。最后，将使用表达miR-30 d的重组腺病毒来证实miR-30 d在原代小鼠胰岛中的功能。这一发现将有助于我们了解胰岛素基因转录中miRNA的功能。 公共卫生相关性：开发新的方法来恢复胰腺β细胞的胰岛素产生和分泌是糖尿病研究的主要目标。MiR-30 d特别有趣，因为我们的初步结果表明它诱导葡萄糖刺激的胰岛素基因表达。我的目标是确定miR 30 d靶基因，并表征其在胰岛素生产中的功能。考虑到miR-30 d也诱导MafA表达，这种miRNA成为一种重要的靶点，可能有助于增强胰岛功能，从而有助于设计预防和治疗糖尿病的替代疗法。

DESCRIPTION (provided by applicant): My long-term goal is to study the role of microRNAs (miRNAs) in regulating beta-cell function and to elucidate the role of miRNAs in the development of type-II diabetes. MiRNAs are small noncoding ribonucleotides that bind mRNAs and function mainly as translational repressors in mammals. miRNAs have been implicated to play a role in many diseases, including diabetes. Several reports indicate an important function for miRNAs in insulin secretion and pancreatic beta cell development. We have recently carried out a screen in the pancreatic beta cell line MIN6 to identify miRNAs with altered abundance in response to changes in glucose concentrations. This screen resulted in identification of more than 50 glucose-regulated miRNAs from a total of 108 miRNAs detectable in MIN6 cells. Many of the identified miRNAs, including miR-124a, miR-107 and miR-30d were upregulated in the presence of high glucose. Interestingly, we found that overexpression of miR-30d increased insulin gene transcription. Moreover, the induction of insulin by overexpression of miR-30d is associated with increased expression of the beta-cell specific transcription factor MafA. This suggests that the putative target genes of miR-30d may be negative regulators of MafA/insulin gene expression. To determine the function of miR-30d in insulin gene transcription, I will: 1) Identify miR-30d target genes involved in regulation of insulin gene transcription. 2) Characterize the biological function of miR-30d in pancreatic beta cell lines and primary mouse islets. The verified targets will be analyzed for their role in insulin gene expression by siRNA knock-down techniques. Moreover, the effect of miR-30d on the target downstream signaling will be characterized. Finally, the function of miR-30d in primary mouse islets will be confirmed using a recombinant adenovirus expressing miR-30d. The finding from this proposal will contribute to our understanding of miRNA function in insulin gene transcription. PUBLIC HEALTH RELEVANCE: Developing new approaches to restore insulin production and secretion from pancreatic beta cells is a major goal in diabetes research. MiR-30d is particularly interesting since our preliminary results indicate that it induces glucose-stimulated insulin gene expression. My objective in this proposal is to identify miR30d target genes and to characterize their function in insulin production. Taking into account that miR-30d also induces MafA expression, this miRNA emerges as an important target that may be beneficial in enhancing islet function and may thus aid in the design of alternative therapies for the prevention and treatment of diabetes.