MicroRNAs(miRNAs)是干细胞功能的关键调节因子,包括自我更新和分化。在这项研究中,我们旨在鉴定在人结肠上皮终末分化过程中上调的miRNA,并阐明它们在干细胞特性的机制控制中的作用。
“隐窝底部”(EPCAM/CD44/CD66alow)和“隐窝顶部”(EPCAM/CD44neg/CD66ahigh)上皮细胞来自8个原发性结肠标本(6人,2只小鼠)通过流式细胞术纯化,并分析335种miRNA的差异表达。在结肠癌RNA-seq数据库(n=439名患者)中,测试了在“隐窝顶部”(终末分化)上皮细胞中显示最高上调的miRNA与生存结果的正相关性和相关性。评估了具有最强的“隐窝顶部”表达谱的两个miRNA下调自我更新效应子和抑制结肠癌细胞体外增殖的能力,正常结肠上皮细胞的体外类器官形成和患者来源的异种移植物(PDX)的体内致瘤性。
六个miRNA(miR-200a,miR-200b,miR-200c,miR-203,miR-210,miR-345)在“隐窝顶部”细胞中上调,并且在结肠癌中的表达呈正相关。具有最高相互相关系数的三种miRNA的过表达(miR-200a,miR-200b,miR-200c)与提高生存率相关。前两个过表达的miRNA(miR-200c,miR-203)协同合作抑制BMI1的表达,BMI1是干细胞群体自我更新的关键调节因子,在抑制增殖方面,结肠上皮细胞的类器官形成和致瘤性。
在结肠上皮,终末分化与miR-200c和miR-203的协同上调相关,它们共同抑制BMI1并禁用上皮细胞的扩增能力。
MicroRNAs (miRNAs) are key regulators of stem cell functions, including self-renewal and differentiation. In this study, we aimed to identify miRNAs that are upregulated during terminal differentiation in the human colon epithelium, and elucidate their role in the mechanistic control of stem cell properties.
\"Bottom-of-the-crypt\" (EPCAM+/CD44+/CD66alow) and \"top-of-the-crypt\" (EPCAM+/CD44neg/CD66ahigh) epithelial cells from 8 primary colon specimens (6 human, 2 murine) were purified by flow cytometry and analyzed for differential expression of 335 miRNAs. The miRNAs displaying the highest upregulation in \"top-of-the-crypt\" (terminally differentiated) epithelial cells were tested for positive correlation and association with survival outcomes in a colon cancer RNA-seq database (n = 439 patients). The two miRNAs with the strongest \"top-of-the-crypt\" expression profile were evaluated for capacity to downregulate self-renewal effectors and inhibit in vitro proliferation of colon cancer cells, in vitro organoid formation by normal colon epithelial cells and in vivo tumorigenicity by patient-derived xenografts (PDX).
Six miRNAs (miR-200a, miR-200b, miR-200c, miR-203, miR-210, miR-345) were upregulated in \"top-of-the-crypt\" cells and positively correlated in expression among colon carcinomas. Overexpression of the three miRNAs with the highest inter-correlation coefficients (miR-200a, miR-200b, miR-200c) associated with improved survival. The top two over-expressed miRNAs (miR-200c, miR-203) cooperated synergistically in suppressing expression of BMI1, a key regulator of self-renewal in stem cell populations, and in inhibiting proliferation, organoid-formation and tumorigenicity of colon epithelial cells.
In the colon epithelium, terminal differentiation associates with the coordinated upregulation of miR-200c and miR-203, which cooperate to suppress BMI1 and disable the expansion capacity of epithelial cells.