mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway.

To study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin. We isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone. Up-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin. miR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

Qi JL ，Zhang ZD ，Dong Z ，Shan T ，Yin ZS ... -  《Journal of Orthopaedic Surgery and Research》

Circular RNA AFF4 modulates osteogenic differentiation in BM-MSCs by activating SMAD1/5 pathway through miR-135a-5p/FNDC5/Irisin axis.

Liu C ，Liu AS ，Zhong D ，Wang CG ，Yu M ，Zhang HW ，Xiao H ，Liu JH ，Zhang J ，Yin K ... -  《Cell Death & Disease》

lncRNA IGF2-AS promotes the osteogenic differentiation of bone marrow mesenchymal stem cells by sponging miR-3,126-5p to upregulate KLK4.

Osteoporosis (OP) is a bone disease characterized by reduced amount and quality of bone. This study was designed to explore the role and mechanism of lncRNA IGF2-AS in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Human lncRNA and miRNA microarray analyses were performed to measure the differential expression levels of lncRNAs and miRNAs in undifferentiated and osteogenically differentiated BMSCs. lncRNA IGF2-AS, miR-3,126-5p, and KLK4 levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Osteogenic differentiation of BMSCs was assessed by alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS). Protein levels of osterix (Osx), osteocalcin (OCN), and runt-related transcription factor 2 (RUNX2) were examined by RT-PCR and western blot assays. The binding relationship between miR-3,126-5p and lncRNA IGF2-AS or KLK4 was predicted by TargetScan (http://www.targetscan.org/vert_72/) and then verified with a dual-luciferase reporter assay. lncRNA IGF2-AS and KLK4 were highly expressed and miR-3,126-5p was weakly expressed in osteogenically differentiated BMSCs. Moreover, lncRNA IGF2-AS overexpression enhanced the osteogenic differentiation of BMSCs. In contrast, lncRNA IGF2-AS knockdown showed the opposite trend. Moreover, miR-3,126-5p overexpression abolished the lncRNA IGF2-AS-mediated osteogenic differentiation of BMSCs. lncRNA IGF2-AS functions as a sponge of miR-3,126-5p to regulate KLK4 expression. lncRNA IGF2-AS enhances the osteogenic differentiation of BMSCs by modulating the miR-3,126-5p/KLK4 axis, suggesting a promising therapeutic target for bone-related diseases.

Tang JZ ，Zhao GY ，Zhao JZ ，Di DH ，Wang B ... -  《-》

Circular RNA-FK501 binding protein 51 boosts bone marrow mesenchymal stem cell proliferation and osteogenic differentiation via modulating microRNA-205-5p/Runt-associated transcription factor 2 axis.

Osteogenesis is the key process of bone homeostasis differentiation. Numerous studies have manifested that circular RNA (circRNA) is a critical regulator of osteogenesis. The research was to explore circRNA-mediated mechanisms in osteogenesis. Bone marrow mesenchymal stem cells (BMSCs) were cultured and induced to osteogenic differentiation (OD). Then, oe-circ-FKBP5, oe-NC, si-circ-FKBP5, si-NC, miR-205-5p mimic, mimic NC, miR-205-5p inhibitor, inhibitor NC, sh-RUNX2, or sh-NC were transfected into BMSCs. Alkaline phosphatase (ALP) activity was detected by ALP staining, cell mineralization was detected by alizarin red staining, cell proliferation was detected by CCK-8, and cell apoptosis was detected by flow cytometry. Then, the expression of circ-FKBP5, miR-205-5p, RUNX2 and osteogenic marker genes was detected by RT-qPCR, and the expression of RUNX2 protein was detected by Western blot. Finally, the targeting relationship between miR-205-5p and circ-FKBP5 or RUNX2 was verified by bioinformation website analysis and dual luciferase reporter gene detection. Circ-FK501 binding protein 51 (FKBP5) was distinctly elevated during OD of BMSCs. Elevated circ-FKBP5 boosted the proliferation and OD, as well as expression of osteogenic marker genes while reduced apoptosis of BMSCs. Down-regulation of circ-FKBP5 inhibited BMSCs proliferation, OD and osteogenic marker gene expression, and promoted apoptosis of BMSCs. Subsequently, circ-FKBP5 combined with miR-205-5p and constrained miR-205-5p expression. Silenced miR-205-5p boosted proliferation, OD, and expression of osteogenic marker genes and suppressed apoptosis of BMSCs. However, up-regulation of miR-205-5p inhibited BMSC proliferation, OD and osteogenic marker gene expression, and promoted apoptosis. Additionally, miR-205-5p targeted Runt-associated transcription factor 2 (RUNX2). Repression of RUNX2 turned around the effect of circ-FKBP5 overexpression on BMSCs. In brief, circ-FKBP5 boosted BMSC proliferation and OD by mediating the miR-205-5p/RUNX2 axis.

Shen Y ，Jiang B ，Luo B ，Jiang X ，Zhang Y ，Wang Q ... -  《Journal of Orthopaedic Surgery and Research》

MiR-210 promotes bone formation in ovariectomized rats by regulating osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells through downregulation of EPHA2.

In osteoporosis, the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) is disrupted. The osteogenic differentiation of bone marrow MSCs (BMSCs) is important for improving osteoporosis. The aim of this study was to explore the role and molecular mechanism of miR-210 in the balance of osteogenic/adipogenic differentiation of BMSCs in postmenopausal osteoporosis. Postmenopausal osteoporosis rat models were constructed by ovariectomy (OVX). BMSCs were isolated from the femur in rats of Sham and OVX groups. MiR-210 was overexpressed and suppressed by miR-210 mimics and inhibitor, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA expression of miR-210, ephrin type-A receptor 2 (EPHA2), alkaline phosphatase (ALP), osterix (OSX), osteocalcin (Bglap), Runt-related transcription factor 2 (Runx2), peroxisome proliferator activated receptor gamma, and fatty acid binding protein 4 (FABP4) in each group of rat femoral tissues or BMSCs. Western blot was applied to detect the protein expression level of EPHA2 in rat femoral tissues and cells. Alizarin red S staining and oil red O staining were performed to assess the osteogenic and adipogenic differentiation of BMSCs, respectively. In addition, the targeting relationship between miR-210 and EPHA2 was verified by a dual luciferase gene reporter assay. The expression of miR-210 was significantly reduced in femoral tissues and BMSCs of OVX rats, and its low expression was associated with reduced bone formation. The osteogenic differentiation was enhanced in OVX rats treated with miR-210 mimic. Overexpression of miR-210 in transfected BMSCs was also found to significantly promote osteogenic differentiation and even inhibit adipogenic differentiation in BMSCs, while knockdown of miR-210 did the opposite. Further mechanistic studies showed that miR-210 could target and inhibit the expression of EPHA2 in BMSCs, thus promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs. MiR-210 promotes osteogenic differentiation and inhibits adipogenic differentiation of BMSCs by down-regulating EPHA2 expression. As it plays an important role in the osteogenic/adipogenic differentiation of osteoporosis, miR-210 can serve as a potential miRNA biomarker for osteoporosis.

Ren L ，Zhu X ，Tan J ，Lv X ，Wang J ，Hua F ... -  《Journal of Orthopaedic Surgery and Research》