Alkbh1‐mediated DNA N6‐methyladenine modification regulates bone marrow mesenchymal stem cell fate during skeletal aging

ObjectivesDNA N6‐methyladenine (N6‐mA) demethylase Alkbh1 participates in regulating osteogenic differentiation of mesenchymal stem cell (MSCs) and vascular calcification. However, the role of Alkbh1 in bone metabolism remains unclear.Materials and MethodsBone marrow mesenchymal stem cells (BMSCs)‐specific Alkbh1 knockout mice were used to investigate the role of Alkbh1 in bone metabolism. Western blot, qRT‐PCR, and immunofluorescent staining were used to evaluate the expression of Alkbh1 or optineurin (optn). Micro‐CT, histomorphometric analysis, and calcein double‐labeling assay were used to evaluate bone phenotypes. Cell staining and qRT‐PCR were used to evaluate the osteogenic or adipogenic differentiation of BMSCs. Dot blotting was used to detect the level of N6‐mA in genomic DNA. Chromatin immunoprecipitation (Chip) assays were used to identify critical targets of Alkbh1. Alkbh1 adeno‐associated virus was used to overexpress Alkbh1 in aged mice.ResultsAlkbh1 expression in BMSCs declined during aging. Knockout of Alkbh1 promoted adipogenic differentiation of BMSCs while inhibited osteogenic differentiation. BMSC‐specific Alkbh1 knockout mice exhibited reduced bone mass and increased marrow adiposity. Mechanistically, we identified optn as the downstream target through which Alkbh1‐mediated DNA m6A modification regulated BMSCs fate. Overexpression of Alkbh1 attenuated bone loss and marrow fat accumulation in aged mice.ConclusionsOur findings demonstrated that Alkbh1 regulated BMSCs fate and bone‐fat balance during skeletal aging and provided a potential target for the treatment of osteoporosis.     

大鼠骨髓间充质干细胞的原代培养及鉴定

摘要 目的：研究大鼠BMSCs（骨髓间充质干细胞）原代培养与纯度鉴定的方法。方法：无菌环境中,从SD大鼠股骨与胫骨端采集骨髓,先行酶消化,利用全骨髓细胞悬液贴壁法对提取BMSCs实施传代培养,选取生长良好的第3代细胞进行鉴定;对BMSCs实施成脂与成骨诱导分化,同时经由油红O（ORO）与茜素红（ARS）染色法对诱导分化效果加以鉴定;借助流式细胞术（FCM）对CD34、CD44与CD90这3类BMSCs表面标志物的表达展开分析。结果：BMSCs是长梭状贴壁细胞,生长状态为纤维细胞样漩涡状;在第3代BMSCs传代期间,其第1-3 d发展至生长潜伏期,呈较慢速的生长;第3-5 d发展至对数生长期,呈高速生长;待至第7 d长速增殖最大,速度停止上升进入平缓期;BMSCs成骨、成脂诱导结束后,对其诱导分化鉴定发现：细胞出现明显形态学变化,通过ORO对脂肪染色,细胞显示橘红色;待成骨诱导培养结束,通过ARS对钙盐染色,显示红色,且出现矿化结节沉积,说明BMSCs具有良好的成骨、成脂分化能力;FCM测定发现：CD34表达呈阴性（1.09 %）,CD90（96.8 %）与CD44（92.4 %）皆呈阳性,与BMSCs表型相符。结论：经由全骨髓黏附培养技术有效分离BMSCs,且完成培养。     

过表达MicroRNA-21通过PTEN/PI3K/AKT信号通路调控人退变髓核细胞自噬的研究

目的:探讨过表达mi R-21通过PTEN/PI3K/AKT通路对人退变髓核细胞自噬的影响。方法:构建稳定过表达mi R-21 mimic人退变髓核细胞,转染无意义序列作为mi R-21 mimic control组,采用RT-qPCR检测转染效率;利用MDC荧光染色法观察细胞自噬泡;Western-Blot检测细胞自噬相关蛋白LC3和P62的表达以及PTEN/PI3K/AKT信号通路中关键蛋白PTEN、PI3K及AKT的表达水平。结果:RT-qPCR结果表明mi R-21 mimic转染成功且效率较高,与mi R-21 mimic control组及空白细胞对照组相比,差异显著(P0.05)。荧光显微镜观察MDC染色情况,mi R-21 mimic组的细胞中几乎没有发现自噬体,而mi R-21 mimic control组以及空白对照组细胞中自噬体均较多,与前者相比差异均明显,具有统计学意义(P0.05)。mi R-21 mimic组细胞中LC3-II/LC3-I表达量的比值均显著低于mi R-21 mimic control组及空白对照组细胞(P0.05);而P62在mi R-21 mimic组细胞中表达量显著高于mi R-21 mimic control组及空白细胞对照组,具有统计学意义(P0.05)。mi R-21 mimic组中PTEN蛋白的表达水平较低,与另外两组相比具有统计学意义(P0.05);磷酸化的PI3K(p-PI3K)和AKT(p-Akt)在mi R-21 mimic组中均明显高于mi R-21 mimic control组和空白细胞对照组,差异具有统计学意义(P0.05)。结论:mi R-21可以通过靶向沉默PTEN,促进PI3K和AKT发生磷酸化,进而使PTEN/PI3K/AKT信号通路被激活,最终抑制人椎间盘退变髓核细胞的自噬。     

Ghrelin联合三氧化二砷对骨髓间充质干细胞增殖与成骨分化的影响

该文主要探究Ghrelin对三氧化二砷(As2O3)导致的骨髓间充质干细胞(BMSCs)增殖和成骨分化的影响。BMSCs设为对照组、As2O3组、Ghrelin组和联合(As2O3+Ghrelin)组。MTT法检测细胞增殖能力;成骨诱导的第7天和第14天,Real-time PCR及Western blot分别检测成骨相关因子OPN、ALP、RUNX2的mRNA及蛋白表达;第21天,茜素红染色分析钙盐沉积情况。结果显示,细胞增殖能力Ghrelin组>对照组>联合组>As2O3组。与对照组比,As2O3组各因子表达均显著下调(P<0.05),Ghrelin组第14天OPN蛋白表达无显著变化,其余因子均上调(P<0.05);联合组与As2O3组比,第14天OPN基因表达和第7天ALP蛋白表达无显著差异,其余均显著上调(P<0.05)。钙盐沉积:Ghrelin组>对照组>联合组>As2O3组。提示0.5μmol/L As2O3抑制BMSCs增殖和成骨分化,600 ng/mL Ghrelin增强细胞增殖和成骨分化;且Ghrelin能减弱As2O3导致的BMSCs增殖和成骨分化抑制作用。     

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis.MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively.ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN.ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.     

MiR-19b-3p accelerates bone loss after spinal cord injury by suppressing osteogenesis via regulating PTEN/Akt/mTOR signalling

Rapid and extensive bone loss, one of the skeletal complications after spinal cord injury (SCI) occurrence, drastically sacrifices the life quality of SCI patients. It has been demonstrated that microRNA (miRNA) dysfunction plays an important role in the initiation and development of bone loss post-SCI. Nevertheless, the effect of miR-19b-3p on bone loss after SCI is unknown and the accurate mechanism is left to be elucidated. The present work was conducted to explore the role of miR-19b-3p/phosphatase and tensin homolog deleted on chromosome ten (PTEN) axis on osteogenesis after SCI and further investigates the underlying mechanisms. We found that miR-19b-3p level was increased in the femurs of SCI rats with decreased autophagy. The overexpression of miR-19b-3p in bone marrow mesenchymal stem cells (BMSCs) targeted down-regulation of PTEN expression, facilitated protein kinase B (Akt) and mammalian target of rapamycin (mTOR) phosphorylation, and thereby suppressing BMSCs osteogenic differentiation via autophagy. Besides, the inhibiting effects of miR-19b-3p on osteogenic differentiation of BMSCs could be diminished by autophagy inducer rapamycin. Meanwhile, bone loss after SCI in rats was also reversed by antagomir-19b-3p treatment, suggesting miR-19b-3p was an essential target for osteogenic differentiation via regulating autophagy. These results indicated that miR-19b-3p was involved in bone loss after SCI by inhibiting osteogenesis via PTEN/Akt/mTOR signalling pathway.     

LncRNA SNHG1 modulates adipogenic differentiation of BMSCs by promoting DNMT1 mediated Opg hypermethylation via interacting with PTBP1

Recent evidence indicates that the abnormal differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) plays a pivotal role in the pathogenesis of osteoporosis. LncRNA SNHG1 has been found to be associated with the differentiation ability of BMSCs. In this study, we aimed to elucidate the role of lncRNA SNHG1 and its associated pathway on the differentiation of BMSCs in osteoporosis. Mice that underwent bilateral ovariectomy (OVX) were used as models of osteoporosis. Induced osteogenic or adipogenic differentiation was performed in mouse BMSCs. Compared to sham animals, lncRNA SNHG1 expression was upregulated in OVX mice. Also, the in vitro expression of SNHG1 was increased in adipogenic BMSCs but decreased in osteogenic BMSCs. Moreover, overexpression of SNHG1 enhanced the adipogenic capacity of BMSCs but inhibited their osteogenic capacity as determined by oil red O, alizarin red, and alkaline phosphatase staining, while silencing of SNHG1 led to the opposite results. LncRNA SNHG1 interacting with the RNA‐binding polypyrimidine tract‐binding protein 1 (PTBP1) promoted osteoprotegerin (Opg) methylation and suppressed Opg expression via mediating DNA methyltransferase (DNMT) 1. Furthermore, Opg was showed to regulate BMSC differentiation. Knockdown of SNHG1 decreased the expressions of adipogenic related genes but increased that of osteogenic related genes. However, the knockdown of Opg partially reversed those effects. In summary, lncRNA SNHG1 upregulated the expression of DNMT1 via interacting with PTBP1, resulting in Opg hypermethylation and decreased Opg expression, which in turn enhanced BMSC adipogenic differentiation and contributed to osteoporosis.     

NT-3 promotes osteogenic differentiation of mouse bone marrow mesenchymal stem cells by regulating the Akt pathway

Objectives:To investigate the effect of neurotrophin-3 (NT-3) on osteogenic/adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs).Methods:Osteogenic differentiation was detected by alkaline phosphatase (ALP) staining and alizarin red staining (ARS). Adipogenic differentiation was detected by oil red O (ORO) staining. The expression of bone-related genes (Runx2, Osterix, OCN, ALP) and lipogenic genes (FABP4, PPAR, CEBP, LPL) was detected by real-time quantitative polymerase chain reaction (real-time qPCR). The expression of p-Akt and Akt protein was detected by Western blot assay.Results:ALP staining and ARS staining showed that the overexpression of NT-3 could promote the differentiation into osteoblasts, while knockdown of NT-3 could inhibit that. Real-time qPCR showed that the overexpression of NT-3 could increase the expression of osteoblast genes, while knockdown of NT-3 could inhibit that. ORO staining showed that the overexpression of NT-3 could inhibit the differentiation into adipogenesis, while knockdown of NT-3 can promote that. Real-time qPCR showed that the overexpression of NT-3 could reduce the expression of lipogenic genes. while knockdown NT-3 could increase that. In addition, the overexpression of NT-3 increased p-Akt/Akt levels significantly, while knockdown NT-3 reduced that significantly.Conclusion:NT-3 could promote the differentiation of mouse BMSCs into osteoblasts and inhibit their differentiation into adipogenesis.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Bone marrow-derived mesenchymal stem cells repair severe acute pancreatitis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling

BackgroundSevere acute pancreatitis (SAP) is associated with high morbidity and mortality. Bone marrow mesenchymal stem cells (BMSCs) have shown obvious protective effect on SAP. However, little is known about the underlying mechanism. The objective of this study is to unravel the role and regulatory mechanism of miR-181a-5p in BMSCs-mediated pancreatic repair.MethodsBMSCs were isolated from Sprague-Dawley rats and characterized by flow cytometry and Oil Red O staining. Sodium taurocholate- and caerulein-induced models were used as SAP models in vivo and in vitro, respectively. Pancreatic injury were evaluated by H&E and histopathological analysis, as well as by measuring levels of amylase, lipase and cytokines. qRT-PCR and western blotting were performed to detect the level of miR-181a-5p and the protein levels of PTEN/Akt, respectively. ELISA was conducted to detect the levels of TNF-α, IL-1β, IL-6, angiopoietin, IL-4, IL-10 and TGF-β1. The apoptotic rate of AR42 J cells was quantitated by concurrent staining with Annexin-V-FITC and PI.ResultsBMSCs significantly attenuated pancreatic injury in SAP rats by reducing inflammatory infiltration and necrosis, and this effect was abolished by CXCR4 agonist AMD3100. ADM3100 exhibited more severe pancreatic injury and decreased miR-181a-5p levels in the pancreas and serum compared to SAP group. Overexpression of miR-181a-5p in BMSCs (BMSCs-miR-181a-5p) markedly potentiated the protective effect of BMSCs by reducing histological damage and levels of amylase and lipase. Moreover, BMSCs-miR-181a-5p dramatically reduced levels of angiopoietin, TNF-α, IL-1β and IL-6, but induced the levels of IL-4 and IL-10. In caerulein-treated AR42 J cells, co-culturing of BMSCs-miR-181a-5p alleviated caerulein-induced increase of amylase and lipase, and apoptosis via PTEN/Akt/TGF-β1 signaling.ConclusionBMSCs alleviate SAP and reduce inflammatory responses and apoptosis by secreting miR-181a-5p to target PTEN/Akt/TGF-β1 signaling. Hence, BMSCs-miR-181a-5p could serve as potential therapeutic target for SAP.     

MiR-26b-3p regulates osteoblast differentiation via targeting estrogen receptor α

BackgroundUnderstanding of the molecular mechanisms of miRNAs involved in osteoblast differentiation is important for the treatment of bone-related diseases.MethodsMC3T3-E1 cells were induced to osteogenic differentiation by culturing with bone morphogenetic protein 2 (BMP2). After transfected with miR-26b-3p mimics or inhibitors, the osteogenic differentiation of MC3T3-E1 cells was detected by ALP and ARS staining. Cell viability was analyzed by MTT. The expressions of miR-26b-3p and osteogenic related markers and signaling were examined by qPCR and western blot. Direct binding of miR-26b-3p and ER-α were determined by dual luciferase assay.ResultsmiR-26b-3p was significantly down-regulated during osteoblast differentiation. Overexpression of miR-26b-3p inhibited osteoblast differentiation, while inhibition of miR-26b-3p enhanced osteoblast differentiation. Further studies demonstrated miR-26b-3p inhibited the expression of estrogen receptor α (ER-α) by directly targeting to the CDS region of ER-α mRNA. Overexpression of ER-α rescued the suppression effects of miR-26b-3p on osteoblast differentiation, while knockdown of ER-α reversed the upregulation of osteoblast differentiation induced by knockdown of miR-26b-3p.ConclusionOur study demonstrates that miR-26b-3p suppresses osteoblast differentiation of MC3T3-E1 cells via directly targeting ER-α.     

mTORC1 signaling is essential for neurofibromatosis type I gene modulated osteogenic differentiation of BMSCs

Neurofibromatosis type I (NF1), which is caused by mutations in the NF1 gene, is a common autosomal dominant genetic disease leading to skeletal abnormalities. Both NF1 gene and mammalian target of rapamycin complex 1 (mTORC1) signaling are associated with the osteogenic differentiation of bone marrow stem cells (BMSCs). In this study, we hypothesized that mTORC1 signaling is involved in NF1-modulated osteoblast differentiation of BMSCs. Human BMSCs were cultured in an osteogenic induction medium. The expression of NF1 was either inhibited or overexpressed by transfecting NF1 with a specific small interfering RNA (siRNA) or pcDNA3.0 plasmid, respectively. In addition, an mTORC1 signaling inhibitor and agonist were used to investigate the effects of mTORC1 on NF1-modulated osteogenic differentiation of BMSCs. The results indicated that inhibiting the expression of NF1 with siRNA significantly decreased the mRNA levels of NF1, whereas overexpressing the expression of NF1 with pcDNA3.0 plasmid significantly increased the mRNA levels of NF1 at days 3, 7, 14 and 21 after culture. We observed reduced osteogenic differentiation and cell proliferation in the NF1-siRNA group and enhanced osteogenic differentiation and cell proliferation of BMSCs in the NF1-pcDNA3.0 group. The activity of mTORC1 signaling (p-mTORC1, p-S6K1, and p-4EBP1) was significantly upregulated in the NF1-siRNA group and significantly inhibited in the NF1-pcDNA3.0 group, 7 and 14 days after culture. The effects of NF1-siRNA and NF1- pcDNA3.0 on osteogenic differentiation of BMSCs and cell proliferation were reversed by mTORC1 inhibitor and agonist, respectively. In conclusion, NF1 modulates osteogenic differentiation and cell proliferation of human BMSCs and mTORC1 signaling is essential for this process.     

Comparison of gingiva‐derived and bone marrow mesenchymal stem cells for osteogenesis

Presently, bone marrow is considered as a prime source of mesenchymal stem cells; however, there are some drawbacks and limitations. Compared with other mesenchymal stem cell (MSC) sources, gingiva‐derived mesenchymal stem cells (GMSCs) are abundant and easy to obtain through minimally invasive cell isolation techniques. In this study, MSCs derived from gingiva and bone marrow were isolated and cultured from mice. GMSCs were characterized by osteogenic, adipogenic and chondrogenic differentiation, and flow cytometry. Compared with bone marrow MSCs (BMSCs), the proliferation capacity was judged by CCK‐8 proliferation assay. Osteogenic differentiation was assessed by ALP staining, ALP assay and Alizarin red staining. RT‐qPCR was performed for ALP, OCN, OSX and Runx2. The results indicated that GMSCs showed higher proliferative capacity than BMSCs. GMSCs turned more positive for ALP and formed a more number of mineralized nodules than BMSCs after osteogenic induction. RT‐qPCR revealed that the expression of ALP, OCN, OSX and Runx2 was significantly increased in the GMSCs compared with that in BMSCs. Moreover, it was found that the number of CD90‐positive cells in GMSCs elevated more than that of BMSCs during osteogenic induction. Taking these results together, it was indicated that GMSCs might be a promising source in the future bone tissue engineering.