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MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes.

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.

SUBMITTER: Lv S

PROVIDER: S-EPMC7488425 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes.

Lv Shuang S Xu Jinying J Chen Lin L Wu Haitao H Feng Wei W Zheng Yangyang Y Li Pengdong P Zhang Haiying H Zhang Lihong L Chi Guangfan G Li Yulin Y

Stem cell research & therapy 20200911 1

<h4>Background</h4>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 chondro ...[more]

PMID: 32917285

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Project description:ObjectiveAberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP-13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA.MethodsChondrocytes were stimulated with interleukin-1beta (IL-1beta) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)-based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription-PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF-kappaB was evaluated using specific inhibitors.ResultsIn IL-1beta-stimulated OA chondrocytes, 42 microRNA were down-regulated, 2 microRNA were up-regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3'-untranslated region (3'-UTR) of MMP-13 mRNA complementary to the seed sequence of microRNA-27b (miR-27b). Increased expression of MMP-13 correlated with down-regulation of miR-27b. Overexpression of miR-27b suppressed the activity of a reporter construct containing the 3'-UTR of human MMP-13 mRNA and inhibited the IL-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b.ConclusionOur data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-induced activation of signal transduction pathways associated with the expression of MMP-13 down-regulated the expression of miR-27b. Thus, miR-27b may play a role in regulating the expression of MMP-13 in human chondrocytes.

Project description:The production of a clinically useful engineered cartilage is an outstanding and unmet clinical need. High-throughput RNA sequencing provides a means of characterizing the molecular phenotype of populations of cells and can be leveraged to better understand differences among source cells, derivative engineered tissues, and target phenotypes. In this study, small RNA sequencing is utilized to comprehensively characterize the microRNA transcriptomes (miRNomes) of native human neonatal articular cartilage and human bone marrow-derived mesenchymal stem cells (hBM-MSCs) differentiating into cartilage organoids, contrasting the microRNA regulation of engineered cartilage with that of a promising target phenotype. Five dominant microRNAs are upregulated during cartilage organoid differentiation and disproportionately regulate transcription factors: miR-148a-3p, miR-140-3p, miR-27b-3p, miR-140-5p, and miR-181a-5p. Two microRNAs that dominate the miRNomes of hBM-MSCs, miR-21-5p and miR-143-3p, persist throughout the differentiation process and may limit the ability of these cells to differentiate into an engineered cartilage resembling target native articular cartilage. By using predictive bioinformatics tools and antagomir inhibition, these persistent microRNAs are shown to destabilize the mRNA of genes with known or potential roles in cartilage biology including FGF18, TGFBR2, TET1, STOX2, ARAP2, N4BP2L1, LHX9, NFIA, and RPS6KA5. These results shed light on the extent to which only a few microRNAs contribute to the complex regulatory environment of hBM-MSCs for engineered tissues. Impact statement MicroRNAs are emerging as important controlling elements in the differentiation of human bone marrow-derived mesenchymal stem cells (hBM-MSCs). By using a robust bioinformatic approach and further validation in vitro, here we provide a comprehensive characterization of the microRNA transcriptomes (miRNomes) of a commonly studied and clinically promising source of multipotent cells (hBM-MSCs), a gold standard model of in vitro chondrogenesis (hBM-MSC-derived cartilage organoids), and an attractive in vivo target phenotype for clinically useful engineered cartilage (neonatal articular cartilage). These analyses highlighted a specific set of microRNAs involved in the chondrogenic program that could be manipulated to acquire a more robust articular cartilage-like phenotype. This characterization provides researchers in the cartilage tissue engineering field a useful atlas with which to contextualize microRNA involvement in complex differentiation pathways.

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MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes.

Publications

MicroRNA-27b targets CBFB to inhibit differentiation of human bone marrow mesenchymal stem cells into hypertrophic chondrocytes.

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