MicroRNA-27b靶向CBFB抑制人骨髓間充質干細胞向增生性軟骨細胞的分化

2020年9月，吉林大學基礎醫學院病理學系病理生物學教育部重點實驗室（The Key Laboratory of Pathobiology, Ministry of Education, Department of Pathology, College of Basic Medical Sciences, Jilin University, Changchun 130021, China）Guangfan Chi1 and Yulin Li1研究團隊在《Stem Cell Research & Therapy》發表了論文，標題為：“MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic

chondrocytes ”

“MicroRNA-27b靶向CBFB抑制人骨髓間充質干細胞向增生性軟骨細胞的分化”

Abstract

Background：Human bone marrow-derived mesenchymal stem cells (hBMSCs) have chondrocyte differentiation potential and are considered to be a cell source for cell-transplantation-mediated repair of cartilage defects, including those associated with osteoarthritis (OA). However, chondrocyte hypertrophic differentiation is a major obstacle for the application of hBMSCs in articular cartilage defect treatment. We have previously shown that microRNA-27b (miR-27b) inhibits hypertrophy of chondrocytes from rat knee cartilage. In this study, we investigated the role of miR-27b in chondrocyte hypertrophic differentiation of hBMSCs.

Methods：Chondrogenic marker and microRNA expression in hBMSC chondrogenic pellets were evaluated using RT-qPCR and immunohistochemistry. The hBMSCs were transfected with miR-27b before inducing differentiation. Gene and protein expression levels were analyzed using RT-qPCR and western blot. Coimmunoprecipitation was used to confirm interaction between CBFB and RUNX2. Luciferase reporter assays were used to demonstrate that CBFB is a miR-27b target. Chondrogenic differentiation was evaluated in hBMSCs treated with shRNA targeting CBFB. Chondrogenic hBMSC pellets overexpressing miR-27b were implanted into cartilage lesions in model rats; therapeutic effects were assessed based on histology and immunohistochemistry.

Results：The hBMSCs showed typical MSC differentiation potentials. During chondrogenic differentiation, collagen 2 and 10 (COL2 and COL10), SOX9, and RUNX2 expression was upregulated. Expression of miR-140, miR-143, and miR-181a increased over time, whereas miR-27b and miR-221 were downregulated. Cartilage derived from hBMSC and overexpressing miR-27b exhibited higher expression of COL2 and SOX9, but lower expression of COL10, RUNX2, and CBFB than did the control cartilage. CBFB and RUNX2 formed a complex, and CBFB was identified as a novel miR-27b target. CBFB knockdown by shRNA during hBMSC chondrogenic differentiation led to significantly increased COL2 and SOX9 expression and decreased COL10 expression. Finally, miR-27b-overexpressing hBMSC chondrogenic pellets had better hyaline cartilage morphology and reduced expression of hypertrophic markers and tend to increase repair efficacy in vivo.

Conclusion：MiR-27b plays an important role in preventing hypertrophic chondrogenesis of hBMSCs by targeting CBFB and is essential for maintaining a hyaline cartilage state. This study provides new insights into the mechanism of hBMSC chondrocyte differentiation and will aid in the development of strategies for treating cartilage injury based on hBMSC transplantation.

摘要

背景：人骨髓間充質干細胞(hBMSCs)具有軟骨細胞分化潛能，被認為是細胞移植介導的軟骨缺損修復的細胞來源，包括與骨關節炎(OA)相關的軟骨缺損。然而，軟骨細胞增生性分化是hBMSCs應用于關節軟骨缺損治療的主要障礙。我們之前已經證明，microRNA-27b (miR-27b)抑制大鼠膝關節軟骨軟骨細胞的肥大。在這項研究中，我們研究了miR-27b在hBMSCs軟骨細胞增生性分化中的作用。

方法：采用RT-qPCR和免疫組織化學方法評估hBMSC軟骨顆粒中軟骨標志物和microRNA的表達。誘導分化前轉染miR-27b。采用RT-qPCR和western blot分析基因和蛋白表達水平。用共免疫沉淀法確定CBFB與RUNX2的相互作用。熒光素酶報告基因檢測證實CBFB是miR-27b的靶標。用靶向CBFB的shRNA治療hBMSCs，評估其軟骨分化。將過表達miR-27b的成軟骨hBMSC微球植入模型大鼠軟骨病變;根據組織學和免疫組織化學評估治療效果。

結果：hBMSCs表現出典型的MSC分化潛能。在軟骨分化過程中，膠原2和10 (COL2和COL10)、SOX9和RUNX2的表達上調。隨著時間的推移，miR-140、miR-143和miR-181a的表達增加，而miR-27b和miR-221的表達下調。過表達miR-27b的hBMSC衍生軟骨與對照軟骨相比，COL2和SOX9的表達較高，COL10、RUNX2和CBFB的表達較低。CBFB和RUNX2形成復合物，CBFB被鑒定為新的miR-27b靶點。hBMSC軟骨分化過程中shRNA敲低CBFB導致COL2和SOX9表達顯著升高，COL10表達顯著降低。最后，過表達mir -27b的hBMSC軟骨顆粒在體內具有更好的透明軟骨形態和減少增生性標志物的表達，并傾向于提高修復效果。

結論：MiR-27b通過靶向CBFB在阻止hBMSCs的增生性軟骨形成中發揮重要作用，并且對于維持透明軟骨狀態至關重要。本研究為hBMSC軟骨細胞分化機制提供了新的見解，并將有助于制定基于hBMSC移植治療軟骨損傷的策略。

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