CRISPR 激活发现了一个新的 miR-2861 结合位点,它能促进人类间充质干细胞的成骨。

IF 2.8 3区 医学 Q1 ORTHOPEDICS
Seong-Ho Park, Jungwoo Kim, Hee-Jin Yang, Ju Yeon Lee, Chi Heon Kim, Junho K Hur, Sung Bae Park
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引用次数: 0

摘要

我们通过 CRISPR 激活(CRISPRa)研究了 miR-2861 在人类间充质干细胞(MSCs)成骨细胞分化过程中对组蛋白去乙酰化酶(HDACs)的调控以及 miR-2861 的结合位点。研究人员将 miR-2861 转染到人间充质干细胞中,并分析了其对成骨细胞分化的影响。CRISPRa 系统使用无催化活性的 Cas12a,诱导了内源性 miRNA 的靶向过表达,并抑制了含有功能性 miRNA 靶位点的报告基因的荧光素酶活性。将 miR-2861 植入间充质干细胞可降低 HDAC1、4 和 5 基因的表达,从而促进成骨细胞的分化。miR-2861 在人体中抑制 HDAC5 的机制尚未完全阐明。为此,研究人员分析了 HDAC5 mRNA 序列,并在 3' 非翻译区(3'-UTR)发现了一个假定的灵长类特异性 miR-2861 结合位点。应用 CRISPRa 验证了推测的结合位点,发现内源性 miR-2861 的增加会抑制包含新型 miR-2861 结合位点的报告基因的表达。向人类间充质干细胞输送 miR-2861 能促进成骨细胞分化。在 3'-UTR 中,HDAC5 的抑制是由 miR-2861 结合位点介导的,miR-2861 通过灵长类特异的 miRNA 结合位点抑制 HDAC5,从而促进成骨细胞分化。因此,采用CRISPRa方法制备的miRNAmiR-2861可能是一种良好的成骨增强生物材料。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
CRISPR activation identifies a novel miR-2861 binding site that facilitates the osteogenesis of human mesenchymal stem cells.

We investigated the regulation of histone deacetylases (HDACs) by miR-2861 in the osteoblastic differentiation of human mesenchymal stem cells (MSCs) and miR-2861 binding site by CRISPR activation (CRISPRa). Transfection of miR-2861 into human MSCs was performed and the effect on osteoblast differentiation was analyzed. Using catalytically inactive Cas12a, the CRISPRa system induced targeted overexpression of endogenous miRNA and repressed the luciferase activities of reporters that contained functional miRNA target sites. The delivery of miR-2861 into MSCs enhanced osteoblast differentiation by decreased expressions of the HDAC1, 4 and 5 genes. The mechanism of HDAC5 repression by miR-2861 in humans has not been fully elucidated. To this end, the HDAC5 mRNA sequence was analyzed and a putative primate-specific miR-2861 binding site was identified in the 3' untranslated region (3'-UTR). CRISPRa was applied to validate the putative binding site and an increase in endogenous miR-2861 was found to repress the expression of a reporter that contained the novel miR-2861 binding site. The delivery of miR-2861 to human MSCs enhanced osteoblast differentiation. In the 3'-UTR, the HDAC5 repression was mediated by the miR-2861 binding site, and miR-2861 promoted osteoblast differentiation via the inhibition of HDAC5 through a primate-specific miRNA binding site. Therefore, miRNAmiR-2861 with the CRISPRa methods might be a good biomaterial for osteogenesis augmentation.

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来源期刊
CiteScore
4.10
自引率
7.70%
发文量
494
审稿时长
>12 weeks
期刊介绍: Journal of Orthopaedic Surgery and Research is an open access journal that encompasses all aspects of clinical and basic research studies related to musculoskeletal issues. Orthopaedic research is conducted at clinical and basic science levels. With the advancement of new technologies and the increasing expectation and demand from doctors and patients, we are witnessing an enormous growth in clinical orthopaedic research, particularly in the fields of traumatology, spinal surgery, joint replacement, sports medicine, musculoskeletal tumour management, hand microsurgery, foot and ankle surgery, paediatric orthopaedic, and orthopaedic rehabilitation. The involvement of basic science ranges from molecular, cellular, structural and functional perspectives to tissue engineering, gait analysis, automation and robotic surgery. Implant and biomaterial designs are new disciplines that complement clinical applications. JOSR encourages the publication of multidisciplinary research with collaboration amongst clinicians and scientists from different disciplines, which will be the trend in the coming decades.
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