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Analysis of the role of tumor suppressor miRNA in intestinal stem cells

抑癌miRNA在肠道干细胞中的作用分析

基本信息

批准号：
22790221
负责人：
OUCHI Yasuo
金额：
$ 2.5万
依托单位：
Chubu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2010
资助国家：
日本
起止时间：
2010 至 2011
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-22790221/
关键词：
miRNA 腸管上皮幹細胞幹細胞腸管系腫瘍 Let-7 Lin28

项目摘要

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. The self-renewing epithelium of the small intestine is ordered into crypts and villi.In this study, we focused on the expression and role of tumor suppressor miRNA, Let-7, in mouse small intestine. As a result, we found expression of Let-7 family miRNAs were down-regulated in the crypt region. While expression of Lin28A, which is strong inhibitor of Pri-and Pre-let-7 processing, was only detected at crypt region, Lin28A-mediated inhibition of Let-7 was speculated.To examine the processing of Let-7 in vivo, we use Let-7-GFP transgenic mice, in which processing of pri-miRNA were visualized by GFP expression. We found that processing of pri-let-7 was inhibited in crypt region. To examine the role of Lin28A-mediated down-regulation of Let-7 in intestinal stem cells, we use in vitro and in vivo analysis. Interestingly, we found that forced expression of Lin28A in rat crypt cell-derived cell line, IEC-6 cells, enhanced cell proliferation, migration activity and decreased contact inhibition of the cells. We observed that overexpression of Lin28 in IEC-6 cells potently depletes levels of multiple mature miRNAs in the let-7 family, leading to a concomitant increase in protein abundance of c-Myc, a let-7 target.To investigate Lin28A function in vivo, we generated a transgenic mouse strain that expresses Lin28A in intestinal epithelium cells. Since some of the Lin28A transgenic mouse displays adenocarcinoma in small intestine, we are now examining the molecular mechanism of the phenotype.Taken together, our results suggested that crypt specific Lin28A expression regulates cell proliferation of intestinal stem cells, and may contribute to malignant progression of intestinal tumors.

肠上皮是成年哺乳动物自我更新最快的组织。小肠的自我更新上皮有序排列成隐窝和绒毛。在本研究中，我们重点研究了肿瘤抑制miRNA Let-7在小鼠小肠中的表达及其作用。结果，我们发现Let-7家族mirna在隐窝区表达下调。Lin28A是强抑制Pre-let-7加工的基因，仅在隐窝区表达，推测Lin28A介导了Let-7的抑制作用。为了研究Let-7在体内的加工过程，我们使用了Let-7-GFP转基因小鼠，通过GFP表达可视化了pri-miRNA的加工过程。我们发现prilet -7的加工在隐窝区受到抑制。为了研究lin28a介导的Let-7下调在肠道干细胞中的作用，我们使用了体外和体内分析。有趣的是，我们发现在大鼠隐窝细胞衍生细胞系IEC-6细胞中强制表达Lin28A可增强细胞增殖、迁移活性并降低细胞的接触抑制。我们观察到，在IEC-6细胞中，Lin28的过度表达可能会耗尽let-7家族中多个成熟mirna的水平，导致let-7靶点c-Myc蛋白丰度的增加。为了研究Lin28A在体内的功能，我们在肠上皮细胞中产生了表达Lin28A的转基因小鼠品系。由于一些Lin28A转基因小鼠在小肠中显示出腺癌，我们现在正在研究这种表型的分子机制。综上所述，我们的研究结果表明，隐窝特异性Lin28A表达调节肠道干细胞的细胞增殖，并可能促进肠道肿瘤的恶性进展。