Centre for Stem Cell Research, Universiti Tunku Abdul Rahman, Sungai Long, Kajang, Selangor DE, Malaysia.
Department of Preclinical Sciences, Faculty of Medicine and Health Sciences, Universiti Tunku Abdul Rahman, Sungai Long Campus, Bandar Sungai Long, Cheras, 43000, Kajang, Selangor DE, Malaysia.
Stem Cell Res Ther. 2017 Sep 29;8(1):214. doi: 10.1186/s13287-017-0666-3.
Introduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc (OSKM) is able to 'reprogram' somatic cells to become induced pluripotent stem cells (iPSCs). Several microRNAs (miRNAs) are known to enhance reprogramming efficiency when co-expressed with the OSKM factors. The primate-specific chromosome 19 miRNA cluster (C19MC) is essential in primate reproduction, development, and differentiation. miR-524-5p, a C19MC member, is highly homologous to the reprogramming miR-520d-5p; we also reported that miR-524-5p was expressed in iPSCs but not mesenchymal stem cells (MSCs). This study aimed to elucidate possible contributions of miR-524-5p to the reprogramming process.
A miR-524-5p precursor was introduced into human fibroblast HFF-1 in the presence of OSKM, and the relative number of embryonic stem cell (ESC)-like colonies that stained positively with alkaline phosphatase (AP) and Nanog were quantified to determine reprogramming efficiency. A miR-524-5p mimic was transfected to MSCs to investigate the effects of miR-524-5p on TP53INP1, ZEB2, and SMAD4 expression by real-time polymerase chain reaction (PCR) and Western blot. Direct gene targeting was confirmed by luciferase activity. A phylogenetic tree of TP53INP1 was constructed by the Clustal method. Contribution of miR-524-5p to cell proliferation and apoptosis was examined by cell counts, BrdU, MTT, and cell death assays, and pluripotency gene expression by real-time PCR.
Co-expressing the miR-524 precursor with OSKM resulted in a two-fold significant increase in the number of AP- and Nanog-positive ESC-like colonies, indicating a role for miR-524-5p in reprogramming. The putative target, TP53INP1, showed an inverse expression relationship with miR-524-5p; direct TP53INP1 targeting was confirmed in luciferase assays. miR-524-5p-induced TP53INP1 downregulation enhanced cell proliferation, suppressed apoptosis, and upregulated the expression of pluripotency genes, all of which are critical early events of the reprogramming process. Interestingly, the TP53INP1 gene may have co-evolved late with the primate-specific miR-524-5p. miR-524-5p also promoted mesenchymal-to-epithelial transition (MET), a required initial event of reprogramming, by directly targeting the epithelial-to-mesenchymal transition (EMT)-related genes, ZEB2 and SMAD4.
Via targeting TP53INP1, ZEB2, and SMAD4, miR-524-5p contributes to the early stage of inducing pluripotency by promoting cell proliferation, inhibiting apoptosis, upregulating expression of pluripotency genes, and enhancing MET. Other C19MC miRNAs may have similar reprogramming functions.
转录因子 Oct4、Sox2、Klf4 和 c-Myc（OSKM）的引入能够“重编程”体细胞成为诱导多能干细胞（iPSCs）。当与 OSKM 因子共表达时，几种 microRNAs（miRNAs）已知能够提高重编程效率。灵长类动物特有的 19 号染色体 miRNA 簇（C19MC）在灵长类动物的生殖、发育和分化中至关重要。miR-524-5p 是 C19MC 成员之一，与重编程 miR-520d-5p 高度同源；我们还报道 miR-524-5p 在 iPSCs 中表达，但不在间充质干细胞（MSCs）中表达。本研究旨在阐明 miR-524-5p 对重编程过程可能的贡献。
在存在 OSKM 的情况下，将 miR-524-5p 前体引入人成纤维细胞 HFF-1 中，并通过碱性磷酸酶（AP）和 Nanog 阳性染色的胚胎干细胞（ESC）样集落的相对数量来确定重编程效率。转染 miR-524-5p 模拟物以研究 miR-524-5p 对 TP53INP1、ZEB2 和 SMAD4 表达的影响，方法是实时聚合酶链反应（PCR）和 Western blot。通过荧光素酶活性证实了直接基因靶向。通过 Clustal 方法构建了 TP53INP1 的系统发育树。通过细胞计数、BrdU、MTT 和细胞死亡测定以及实时 PCR 检测 miR-524-5p 对细胞增殖和凋亡以及多能性基因表达的影响。
与 OSKM 共表达 miR-524 前体可使 AP 和 Nanog 阳性 ESC 样集落的数量增加两倍，表明 miR-524-5p 参与了重编程。假定的靶标 TP53INP1 与 miR-524-5p 呈反式表达关系；在荧光素酶测定中证实了对 TP53INP1 的直接靶向。miR-524-5p 诱导的 TP53INP1 下调增强了细胞增殖，抑制了细胞凋亡，并上调了多能性基因的表达，所有这些都是重编程过程的关键早期事件。有趣的是，TP53INP1 基因可能与灵长类动物特有的 miR-524-5p 共同进化而来。miR-524-5p 还通过直接靶向上皮-间充质转化（EMT）相关基因 ZEB2 和 SMAD4，促进间充质-上皮转化（MET），这是重编程的初始事件之一。
通过靶向 TP53INP1、ZEB2 和 SMAD4，miR-524-5p 通过促进细胞增殖、抑制细胞凋亡、上调多能性基因表达和增强 MET 来促进诱导多能性的早期阶段。其他 C19MC miRNAs 可能具有类似的重编程功能。
