Department of Medicine, Major in Medical Genetics, Graduate School, Hanyang University, Seoul, Korea.
Department of Neurosurgery, Seoul National University Boramae Medical Center, 20 Boramae-ro 5-gil, Dongjak-gu, Seoul, 07061, Korea.
J Orthop Surg Res. 2024 Nov 6;19(1):730. doi: 10.1186/s13018-024-05163-3.
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.
我们研究了组蛋白去乙酰化酶 (HDACs) 在人骨髓间充质干细胞 (MSCs) 成骨分化中的 miR-2861 调节作用，以及 miR-2861 通过 CRISPR 激活 (CRISPRa) 的结合位点。将 miR-2861 转染到人 MSCs 中，并分析其对成骨分化的影响。使用无催化活性的 Cas12a，CRISPRa 系统诱导内源性 miRNA 的靶向过表达，并抑制包含功能性 miRNA 靶位的报告基因的荧光素酶活性。miR-2861 转染到 MSCs 中，通过降低 HDAC1、4 和 5 基因的表达，增强成骨分化。miR-2861 对人类 HDAC5 的抑制机制尚未完全阐明。为此，分析了 HDAC5 mRNA 序列，在 3'非翻译区 (3'-UTR) 中鉴定出一个假定的灵长类特异性 miR-2861 结合位点。应用 CRISPRa 验证该假定的结合位点，发现内源性 miR-2861 的增加会抑制含有新型 miR-2861 结合位点的报告基因的表达。miR-2861 转染到人 MSCs 中增强了成骨分化。在 3'-UTR 中，miR-2861 结合位点介导了 HDAC5 的抑制，miR-2861 通过抑制 HDAC5 促进成骨分化，通过一个灵长类特异性 miRNA 结合位点。因此，miR-2861 与 CRISPRa 方法相结合，可能成为促进成骨的良好生物材料。
