MiR-137 knockdown promotes the osteogenic differentiation of human adipose-derived stem cells via the LSD1/BMP2/SMAD4 signaling network

MicroRNAs are a group of endogenous regulators that participate in several cellular physiological processes. However, the role of miR-137 in the osteogenic differentiation of human adipose-derived stem cells (hASCs) has not been reported. This study verified a general downward trend in miR-137 expression during the osteogenic differentiation of hASCs. MiR-137 knockdown promoted the osteogenesis of hASCs in vitro and in vivo. Mechanistically, inhibition of miR-137 activated the bone morphogenetic protein 2 (BMP2)-mothers against the decapentaplegic homolog 4 (SMAD4) pathway, whereas repressed lysine-specific histone demethylase 1 (LSD1), which was confirmed as a negative regulator of osteogenesis in our previous studies. Furthermore, LSD1 knockdown enhanced the expression of BMP2 and SMAD4, suggesting the coordination of LSD1 in the osteogenic regulation of miR-137. This study indicated that miR-137 negatively regulated the osteogenic differentiation of hASCs via the LSD1/BMP2/SMAD4 signaling network, revealing a new potential therapeutic target of hASC-based bone tissue engineering.     

Osteoclast-derived miR-23a-5p-containing exosomes inhibit osteogenic differentiation by regulating Runx2

BackgroundSome microRNAs (miRNAs) are involved in osteogenic differentiation. In recent years, increasing evidences have revealed that exosomes contain specific miRNAs. However, the effect and mechanism of miR-23a-5p-containing exosomes in osteoblast remain largely unclear.MethodsWe extracted exosomes from RANKL-induced RAW 264.7 cells, and identified exosomes via transmission electron microscopy, western blot and flow cytometry analysis. In addition, exosome secretion was inhibited by GW4869 and Rab27a siRNAs. miR-23a-5p expression was analyzed by qRT-PCR, and the related protein levels were examined by western blot assay. Furthermore, the number and distribution of osteoclasts were detected by TRAP staining, and early osteogenesis was evaluated by ALP staining. Combination of YAP1 and Runx2 was verified by Co-IP assay, and the regulation of miR-23a-5p and Runx2 was measured by dual luciferase reporter assay.ResultsWe successfully extracted exosomes from RANKL-induced RAW 264.7 cells, and successfully verified exosomes morphology. We also indicated that miR-23a-5p was highly expressed in exosomes from RANKL-induced RAW 264.7 cells, and osteoclast-derived miR-23a-5p-containing exosomes inhibited osteoblast activity, while its inhibition weakened osteoclasts. In mechanism, we demonstrated that Runx2 was a target gene of miR-23a-5p, YAP interacted with Runx2, and YAP or Runx2 inhibited MT1DP expression. In addition, we proved that knockdown of MT1DP facilitated osteogenic differentiation by regulating FoxA1 and Runx2.ConclusionsWe demonstrated that osteoclast-derived miR-23a-5p-containing exosomes could efficiently suppress osteogenic differentiation by inhibiting Runx2 and promoting YAP1-mediated MT1DP. Therefore, we suggested miR-23a-5p in exosomes might provide a novel mechanism for osteoblast function.     

Myokine Irisin promotes osteogenesis by activating BMP/SMAD signaling via αV integrin and regulates bone mass in mice

Irisin is well-known to contribute to bone homeostasis due to its bidirectional regulation on osteogenesis and osteoclastogenesis. However, the mechanisms of irisin involved in mesenchymal stem/stromal cells (MSCs)-derived osteogenesis are still under investigated. Fibronectin type III domain-containing protein 5 (FNDC5) is the precursor protein of irisin, compare with wild type (WT) littermates, FNDC5^-/- mice lost bone mass significantly, collectively evidenced by the decrease of bone mineral density (BMD), impaired bone formation and reduced N-terminal propertied of type I procollagen (P1NP) in sera. Meanwhile, the bone resorbing of FNDC5^-/- mice has enhanced accompanied by increased tartrate phosphatase (TRAP) staining cells morphologically and cross-Linked C-telopeptide of type 1 collagen (CTX) level in sera. In vitro study showed that lack of irisin impeded the MSC-derived osteogenesis of FNDC5^-/- mice. The addition of irisin promote the osteogenesis of WT and irisin-deficient MSCs, by activating αV integrin-induced ERK/STAT pathway, subsequently enhancing bone morphogenetic protein 2 (BMP2) expression and BMP/SMAD signaling activation. Taken together, these findings further indicate that irisin regulates bone homeostasis. Moreover, irisin promotes MSC-derived osteogenesis by binding to αV integrin and activating BMP/SMAD signaling consequently. Thus, irisin may be a promising therapeutic target for osteoporosis and bone defects.     

Long non-coding RNA DANCR modulates osteogenic differentiation by regulating the miR-1301-3p/PROX1 axis

The balance of osteoblasts and marrow adipocytes from bone marrow mesenchymal stem cells (BM-MSCs) maintains bone health. Under aging or other pathological stimuli, BM-MSCs will preferentially differentiate into marrow adipocytes and reduce osteoblasts, leading to osteoporosis. Long non-coding RNA differentiation antagonizing non-protein coding RNA (DANCR) participates in the osteogenic differentiation of human BM-MSCs, but the mechanism by which DANCR regulates the osteogenic differentiation of human BM-MSCs has not been fully explained. We observed that DANCR and prospero homeobox 1 (PROX1) were downregulated during osteogenic differentiation of human BM-MSCs, while miR-1301-3p had an opposite trend. DANCR overexpression decreased the levels of alkaline phosphatase, RUNX2, osteocalcin, Osterix in BM-MSCs after osteogenic induction, but DANCR silencing had the opposite result. Moreover, DANCR sponged miR-1301-3p to regulate PROX1 expression. miR-1301-3p overexpression reversed the suppressive role of DANCR elevation on the osteogenic differentiation of human BM-MSCs. Also, PROX1 elevation abolished the promoting role of miR-1301-3p overexpression on the osteogenic differentiation of human BM-MSCs. In conclusion, DANCR suppressed the osteogenic differentiation of human BM-MSCs through the miR-1301-3p/PROX1 axis, offering a novel mechanism by which DANCR is responsible for the osteogenic differentiation of human BM-MSCs.

     

MiRNA-1271-5p regulates osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by targeting forkhead box O1 (FOXO1)

Forkhead box O1 (FOXO1) is a key regulator of osteogenesis. The aim of this study was to identify the mechanisms of microRNAs (miRNAs) targeting FOXO1 in osteogenic differentiation of human bone marrow mesenchymal stem cells (hMSCs). Three miRNA target prediction programs were used to search for potential miRNAs that target FOXO1. Quantitative real-time polymerase chain reaction was conducted to detect the expression of miR-1271-5p and FOXO1 during osteogenic differentiation. Target gene prediction and screening, luciferase reporter assay was used to verify the downstream target gene of miR-1271-5p. The expression levels of FOXO1 and Runx2 were detected by RT-qPCR and Western blot analysis. Alkaline phosphatase (ALP) activity and matrix mineralization were detected by biochemical methods. The expression levels of Runx2, ALP, and osteocalcin were detected by RT-qPCR. Our results showed that miR-1271-5p was downregulated during osteogenic induction. And the expression levels of miR-1271-5p were higher in osteoporotic tissues than that in adjacent nonosteoporotic tissues. The expression levels of FOXO1 were lower in osteoporotic tissues than that in adjacent nonosteoporotic tissues. And a negative correlation was found between miR-1271-5p and FOXO1 in osteoporotic tissues. Overexpression of miR-1271-5p downregulated FOXO1 and inhibited osteogenic differentiation in hMSCs. Overexpression of miR-1271-5p downregulated the expression of osteogenic markers and reduced ALP activity. In addition, ectopic expression of FOXO1 reversed the effect of miR-1271-5p on osteogenic differentiation. In conclusion, miR-1271-5p functioned as a therapeutic target of osteogenic differentiation in hMSCs by inhibiting FOXO1, which provides valuable insights into the use of miR-1271-5p as a target in the treatment of osteoporosis and other bone metabolic diseases.     

LOC103691336/miR-138-5p/BMPR2 axis modulates Mg-mediated osteogenic differentiation in rat femoral fracture model and rat primary bone marrow stromal cells

Intramedullary stabilization is frequently used to treat long bone fractures. Since implant removal can become technically very challenging with the potential to cause further tissue damage, biodegradable materials are emerging as alternative options. Magnesium (Mg)-based biodegradable implants have a controllable degradation rate and good tissue compatibility, which makes them attractive for musculoskeletal research. Herein, the degradation of Mg and steel implants, the pathological characteristics and osteoblast differentiation in mice femora were examined. To investigate the molecular mechanism, we analyzed the differentially expressed long noncoding RNAs (lncRNAs) and messenger RNAs (mRNAs) in Mg-implanted or stain-steel-implanted callus tissues. lncRNA LOC103691336 was upregulated in Mg-implanted tissues and most relevant to BMPR2, a kinase receptor of BMPs with an established role in osteogenesis. The knockdown of LOC103691336 attenuated Mg-mediated osteogenic differentiation. Furthermore, miR-138-5p, previously reported to inhibit osteogenic differentiation, could bind to LOC103691336 and BMPR2 in bone marrow stromal cells (BMSCs). LOC103691336 competed with BMPR2 for miR-138-5p binding in BMSCs to attenuate the inhibitory effect of miR-138-5p on BMPR2 expression. Finally, the effect of LOC103691336 knockdown on Mg-mediated osteogenic differentiation could be attenuated by miR-138-5p inhibition. In conclusion, we provided a novel mechanism of Mg implants mediating the osteogenesis differentiation and demonstrated that Mg implants may be promising for improving fracture healing.     

miR-146a-5p circuitry uncouples cell proliferation and migration,but not differentiation,in human mesenchymal stem cells

Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton’s jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared with BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed and has high abundance in WJ-MSCs. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration, whereas overexpression of miR-146a-5p in BM-MSCs did not influence their osteogenic and adipogenic potentials. Chemokine (C-X-C motif) ligand 12 (CXCL12), together with SIKE1, which is an I-kappa-B kinase epsilon (IKKε) suppressor, is a direct target of miR-146a-5p in MSCs. Knockdown of miR-146a-5p resulted in the down-regulation of nuclear factor kappa-B (NF-κB) activity, which is highly activated in WJ-MSCs and is known to activate miR-146a-5p promoter. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. These findings suggest that miR-146a-5p is critical to the uncoupling of motility and proliferation of MSCs. Our miRNome data also provide a roadmap for further understanding MSC biology.     

过表达SCD1对骨髓间质干细胞成骨分化的影响及其基因表达谱变化研究

目的:研究固醇辅酶A去饱和酶1(SCD1)过表达后对骨髓间质干细胞(BM-MSCs)成骨分化作用的影响,并利用基因芯片技术分析基因表达谱的变化。方法:利用已构建成功的SCD1慢病毒转染BM-MSCs,采用RT-PCR及C14技术检测SCD1在BM-MSCs中过表达情况及其活性。成骨诱导培养BM-MSCs后,采用Western blot和茜素红染色技术检测骨钙素(OC)等相关成骨指标,进一步运用全基因芯片检测过表达SCD1对BM-MSCs成骨分化表达谱的影响。结果:SCD1在BM-MSCs中成功过表达,过表达组SCD1活性明显高于对照组。成骨诱导7天、14天时,过表达组中的碱性磷酸酶(APL)活性和骨钙素水平均明显高于对照组(P<0.05)。成骨诱导一周、两周时,过表达组的碱性磷酸酶染色和茜素红染色均多于对照组。基因表达芯片的结果显示,过表达SCD1改变骨髓间质干细胞表达谱,检测出差异基因2896个。基因通路分析提示干扰素通路为表达差异最显著通路(P<0.05)。结论:过表达SCD1可以促进BM-MSCs的成骨分化,可能通过作用于干扰素通路影响成骨分化功能。这一发现可能为骨折愈合提供重要的思路和潜在治疗策略,值得深入研究。     

CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression

Adipose-derived mesenchymal stem cells (ADSCs) are promising candidate for regenerative medicine to repair non-healing bone defects due to their high and easy availability. However, the limited osteogenic differentiation potential greatly hinders the clinical application of ADSCs in bone repair. Accumulating evidences demonstrate that circular RNAs (circRNAs) are involved in stem/progenitor cell fate determination, but their specific role in stem/progenitor cell osteogenesis, remains mostly undescribed. Here, we show that circRNA-vgll3 originating from the vgll3 locus markedly enhances osteogenic differentiation of ADSCs; nevertheless, silencing of circRNA-vgll3 dramatically attenuates ADSC osteogenesis. Furthermore, we validate that circRNA-vgll3 functions in ADSC osteogenesis through a circRNA-vgll3/miR-326-5p/integrin α5 (Itga5) pathway. Itga5 promotes ADSC osteogenic differentiation and miR-326-5p suppresses Itga5 translation. CircRNA-vgll3 directly sequesters miR-326-5p in the cytoplasm and inhibits its activity to promote osteogenic differentiation. Moreover, the therapeutic potential of circRNA-vgll3-modified ADSCs with calcium phosphate cement (CPC) scaffolds was systematically evaluated in a critical-sized defect model in rats. Our results demonstrate that circRNA-vgll3 markedly enhances new bone formation with upregulated bone mineral density, bone volume/tissue volume, trabeculae number, and increased new bone generation. This study reveals the important role of circRNA-vgll3 during new bone biogenesis. Thus, circRNA-vgll3 engineered ADSCs may be effective potential therapeutic targets for bone regenerative medicine.Subject terms: Epigenetics, Stem-cell research     

MiR-21协同BMP9促进间充质干细胞C3H10T1/2成骨分化

目的:探讨miR-21与BMP9之间的关系,明确miR-21在BMP9诱导间充质干细胞成骨分化中的作用。方法:(1)Ad-BMP9感染C3H10T1/2细胞,Real-time-PCR检测miR-21表达。RT-PCR检测ALP的表达。(2)MiR-21转染C3H10T1/2细胞,Real-time-PCR检测miR-21和BMP9表达。(3)MiR-21和BMP9-CM处理C3H10 T1/2细胞,ALP活性和染色实验检测C3H10 T1/2细胞早期成骨能力。茜素红S染色实验检测钙盐沉积情况。(4)MiR-21和BMP9-CM处理C3H10 T1/2细胞,Real-time-PCR检测成骨分化相关因子ALP,OCN的表达。(5)MiR-21和BMP9-CM处理C3H10T1/2细胞,Western blot检测p-Smad1/5蛋白水平的表达。结果:(1)BMP9暂时降低miR-21的表达。MiR-21也可以暂时降低BMP9的表达。(2)MiR-21可以协同BMP9增强ALP和钙盐沉积。(3)MiR-21协同BMP9增加了p-Smad1/5蛋白水平的表达。结论:MiR-21与BMP9存在相互关系,两者可以互相调节表达。MiR-21可以协同BMP9促进间充质干细胞C3H10T1/2细胞成骨分化,这一过程与增强BMP9/Smad信号的激活程度有关。     

Down-regulation of Noggin and miR-138 coordinately promote osteogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate to osteocytes under suitable conditions. In recent years, micro-nucleotides have been progressively used to modulate gene expression in cells due to the consideration of safety. Our present study aimed to investigate whether co-delivery of Noggin-siRNA and antimiR-138 enhances the osteogenic effect of MSCs. Using a murine MSC line, C3H/10T1/2 cells, the delivery efficiency of Noggin-siRNA and antimiR-138 into MSCs was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). Cell phenotype and proliferation capacity was assessed by flow cytometry and MTT assay respectively. The osteogenesis of MSCs was tested by Alkaline Phosphatase (ALP) staining, qRT-PCR, and western blot analyses. Our results demonstrated that the expression of Noggin and miR-138 were significantly silenced in MSCs by Noggin-siRNA and/or antimiR-138 delivery, while the phenotype and proliferation capacity of MSCs were not affected. Down-regulation of Noggin and miR-138 cooperatively promoted osteogenic differentiation of MSCs. The ALP positive cells reached about 83.57?±?10.18%. Compared with single delivery, the expression of osteogenic related genes, such as Alp, Col-1, Bmp2, Ocn and Runx2, were the highest in cells with co-delivery of the two oligonucleotides. Moreover, the protein level of RUNX2, and the ratios of pSMAD1/5/SMAD1/5 and pERK1/2/ERK1/2 were significantly increased. The activation of Smad, Erk signaling may constitute the underlying mechanism of the enhanced osteogenesis process. Taken together, our study provides a safe strategy for the clinical rehabilitation application of MSCs in skeletal deficiency.     

Upregulation of miR-135b Is Involved in the Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived from Multiple Myeloma Patients

Previous studies have demonstrated that mesenchymal stem cells from multiple myeloma (MM) patients (MM-hMSCs) display a distinctive gene expression profile, an enhanced production of cytokines and an impaired osteogenic differentiation ability compared to normal donors (ND-hMSCs). However, the underlying molecular mechanisms are unclear. In the present study, we observed that MM-hMSCs exhibited an abnormal upregulation of miR-135b, showing meanwhile an impaired osteogenic differentiation and a decrease of SMAD5 expression, which is the target of miR-135b involved in osteogenesis. By gain and loss of function studies we confirmed that miR-135b negatively regulated hMSCs osteogenesis. We also found that MM cell-produced factors stimulated ND-hMSCs to upregulate the expression of miR-135b. Importantly, treatment with a miR-135b inhibitor promoted osteogenic differentiation in MM-hMSCs. Finally, we observed that MM cell-derived soluble factors could induce an upregulation of miR-135b expression in ND-hMSCs in an indirect coculture system and the miR-135b expression turned to normal level after the removal of MM cells. Collectively, we provide evidence that miR-135b is involved in the impaired osteogenic differentiation of MSCs derived from MM patients and might therefore be a promising target for controlling bone disease.     

Total flavonoids of Herba Epimedii improves osteogenesis and inhibits osteoclastogenesis of human mesenchymal stem cells

IntroductionIn China Herba Epimedii is one of the most common herbs that could be prescribed for treating osteoporosis. It is known to increase the overall mineral content, therefore, to promote bone formation and to increase lumbar bone mineral density (BMD). The present study was aimed at investigating the effect of flavonoids of Herba Epimedii (HEF) on osteogenesis in human MSCs.MethodsThe human bone marrow-derived MSCs (BM-MSCs) were isolated and their osteogenic differentiation was evaluated by their alkaline phosphatase (ALP) activities and level of mineralization. After treating with total flavonoids during osteogenic differentiation process, differential mRNA expression was examined by RT-PCR.ResultsThe total time needed for osteogenic differentiation of BM-MSCs was significantly shortened by adding HEF. Up-regulation of mRNA expression by HEF was observed for several marker genes and osteogenic regulators. HEF was also found to inhibit osteoclastogenesis of MSCs by enhancing the ratio OPG/RANKL.ConclusionsOur study demonstrated that the HEF could improve osteogenic differentiation and inhibit the osteoclast differentiation of BM-MSCs concurrently.