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.     

miR-17-5p and miR-106a are involved in the balance between osteogenic and adipogenic differentiation of adipose-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) can differentiate into several distinct cell types, including osteoblasts and adipocytes. The balance between osteogenic and adipogenic differentiation is disrupted in several osteogenic-related disorders, such as osteoporosis. So far, little is known about the molecular mechanisms that drive final lineage commitment of MSCs. In this study, we revealed that miR-17-5p and miR-106a have dual functions in the modulation of human adipose-derived mesenchymal stem cells (hADSCs) commitment by gain- and loss-of-function assays. They could promote adipogenesis and inhibit osteogenesis. Luciferase reporter assay, western blot and ELISA suggested BMP2 was a direct target of miR-17-5p and miR-106a. Downregulation of endogeneous BMP2 by RNA interference suppressed osteogenesis and increased adipogenesis, similar to the effect of miR-17-5p and miR-106a upregulation. Moreover, the inhibitory effects of miR-17-5p on osteogenic and adipogenic differentiation of hADSCs could be reversed by BMP2 RNA interference. In conclusion, miR-17-5p and miR-106a regulate osteogenic and adipogenic lineage commitment of hADSCs by directly targeting BMP2, and subsequently decreased osteogenic TAZ, MSX2 and Runx2, and increased adipogenic C/EBPα and PPARγ.     

Could the effect of modeled microgravity on osteogenic differentiation of human mesenchymal stem cells be reversed by regulation of signaling pathways?

Microgravity (MG) results in a reduction in bone formation. Bone formation involves osteogenic differentiation from mesenchymal stem cells (hMSCs) in bone marrow. We modeled MG to determine its effects on osteogenesis of hMSCs and used activators or inhibitors of signaling factors to regulate osteogenic differentiation. Under osteogenic induction, MG reduced osteogenic differentiation of hMSCs and decreased the expression of osteoblast gene markers. The expression of Runx2 was also inhibited, whereas the expression of PPARgamma2 increased. MG also decreased phosphorylation of ERK, but increased phosphorylation of p38MAPK. SB203580, a p38MAPK inhibitor, was able to inhibit the phosphorylation of p38MAPK, but did not reduce the expression of PPARgamma2. Bone morphogenetic protein (BMP) increased the expression of Runx2. Fibroblast growth factor 2 (FGF2) increased the phosphorylation of ERK, but did not significantly increase the expression of osteoblast gene markers. The combination of BMP, FGF2 and SB203580 significantly reversed the effect of MG on osteogenic differentiation of hMSCs. Our results suggest that modeled MG inhibits the osteogenic differentiation and increases the adipogenic differentiation of hMSCs through different signaling pathways. Therefore, the effect of MG on the differentiation of hMSCs could be reversed by the mediation of signaling pathways.     

miR-335 orchestrates cell proliferation, migration and differentiation in human mesenchymal stem cells

In spite of the extensive potential of human mesenchymal stem cells (hMSCs) in cell therapy, little is known about the molecular mechanisms that regulate their therapeutic properties. We aimed to identify microRNAs (miRNAs) involved in controlling the transition between the resting and reparative phenotypes of hMSCs, hypothesizing that these miRNAs must be present in the undifferentiated cells and downregulated to allow initiation of distinct activation/differentiation programs. Differential miRNA expression analyses revealed that miR-335 is significantly downregulated upon hMSC differentiation. In addition, hMSCs derived from a variety of tissues express miR-335 at a higher level than human skin fibroblasts, and overexpression of miR-335 in hMSCs inhibited their proliferation and migration, as well as their osteogenic and adipogenic potential. Expression of miR-335 in hMSCs was upregulated by the canonical Wnt signaling pathway, a positive regulator of MSC self-renewal, and downregulated by interferon-γ (IFN-γ), a pro-inflammatory cytokine that has an important role in activating the immunomodulatory properties of hMSCs. Differential gene expression analyses, in combination with computational searches, defined a cluster of 62 putative target genes for miR-335 in hMSCs. Western blot and 3'UTR reporter assays confirmed RUNX2 as a direct target of miR-335 in hMSCs. These results strongly suggest that miR-335 downregulation is critical for the acquisition of reparative MSC phenotypes.     

Long noncoding RNA C21orf121/bone morphogenetic protein 2/microRNA-140-5p gene network promotes directed differentiation of stem cells from human exfoliated deciduous teeth to neuronal cells

Previous studies have revealed that long noncoding RNA (lncRNA) and microRNA play a crucial role in autism, which is a childhood neurodevelopmental disorder with complicated genetic origins. Hence, the study concerns whether lncRNA C21orf121/bone morphogenetic proteins 2 (BMP2)/miR-140-5p gene network affects directed differentiation of stem cells from human exfoliated deciduous teeth (SHED) to neuronal cells in rats with autism. Autism models were successfully established. The neuron cells that differentiated from SHED cell were identified. The expression of lncRNA C21orf121, miR-140-5p, BMP2, Nestin, βIII-tubulin, and microtubule-associated protein 2 (MAP2) and the expression of neuron-specific enolase (NSE) were examined. Besides, the gap junction (GJ) function of SHED, the intracellular free Ca ²⁺ concentration, and the social behavior and repetitive stereotyped movements of rats in autism were detected. The target relationship between lncRNA C21orf121 and miR-140-5p and that between miR-140-5p and BMP2 were also verified. Firstly, we successfully isolated SHED and identified the differentiated neurons of SHED. Besides, the expression of BMP2, MAP2, Nestin, βIII-tubulin, NSE positive rate, GJ function, and intracellular free Ca ²⁺ concentration were increased with the upregulation of C21orf121 and downregulation of miR-140-5p, and accumulated time of repetitive stereotyped movements decreased and the frequency of social behavior increased. The results indicate that lncRNA C21orf121 as a competing endogenous RNA competes with BMP2 binding to miR-140-5p, thereby promoting SHED to differentiate into neuronal cells via upregulating BMP2 expression.     

The promising effects of BMP2 transfected mesenchymal stem cells on human osteosarcoma

Selective targeting of transfected mesenchymal stem cells (MSCs) carrying specific antioncogenes to the tumor was suggested as a treatment option. Bone morphogenetic protein-2 (BMP2) was shown to inhibit the proliferation and aggressiveness of osteosarcoma (OS) cells. Here, we aimed to assess the homing efficiency of intraperitoneally administered hMSCs transfected with BMP2 to the tumoral site and their effects on OS using an orthotopic xenograft murine model. Orthotopic xenograft murine model of OS in six-week-old female NOD/SCID mice using 143B cells was established. hMSCs transfected with BMP2 (BMP2⁺hMSC) were used. In vivo experiments performed on four groups of mice that received no treatment, or intraperitoneally administered BMP2, hMSCs, and BMP2⁺hMSCs. Histopathological and immunohistochemical studies were used to evaluate the pathological identification and to assess the dimensions and necrotic foci of the tumor, the features of lung metastases, and immunostaining against p27, Ki-67, and caspase-3 antibodies. The osteogenic differentiation markers BMP2, BMP4, COL1A1, OPN, OCN and PF4 evaluated using RT-PCR. The tumor dimensions in the hMSCs group were significantly higher than those of the remaining groups (p < 0.01). The number of metastatic foci in the BMP2⁺hMSCs group was significantly lower than those of the other groups (p < 0.01). The current results showed that the intraperitoneal route could be efficiently used for targeting hMSCs to the tumoral tissues for effective BMP2 delivery. In this study, the effects of BMP2 transfected hMSCs on human OS and metastasis were promising for achieving osteogenic differentiation and reduced metastatic process.     

microRNA expression profiles and the potential competing endogenous RNA networks in NELL-1-induced human adipose-derived stem cell osteogenic differentiation

Studies have indicated that Nel-like molecule-1 (NELL-1) was an osteoblast-specific cytokine and some specific microRNAs (miRNAs) could serve as competing endogenous RNA (ceRNA) to partake in osteogenic differentiation of human adipose-derived stem cells (hASCs). The aim of this study was to explore the potential functional mechanisms of recombinant human NELL-1 protein (rhNELL-1) during hASCs osteogenic differentiation. rhNELL-1 was added to osteogenic medium to activate osteogenic differentiation of hASCs. High-throughput RNA sequencing (RNA-Seq) was performed and validated by real-time quantitative polymerase chain reaction. Gene ontology functional annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed to detect the functions of differentially expressed miRNAs and genes. Coding-noncoding gene co-expression network and ceRNA networks were constructed to predict the potential regulatory role of miRNAs. A total of 1010 differentially expressed miRNAs and 1762 differentially expressed messenger RNAs (mRNAs) were detected. miRNA-370-3p, bone morphogenetic protein 2 (BMP2), and parathyroid hormone like hormone (PTHLH) were differentially expressed during NELL-1-induced osteogenesis. Bioinformatic analyses demonstrated that these differentially expressed miRNAs and mRNAs enriched in Rap1 signaling pathway, PI3K-Akt signaling pathway, p53 signaling pathway, Glucagon signaling pathway, and hypoxia-inducible factor-1 signaling pathway, which were important pathways related to osteogenic differentiation. In addition, miRNA-370-3p and has-miR-485-5p were predicted to interact with circ0001543, circ0002405, and ENST00000570267 in ceRNA networks. Based on the gain or loss of functional experiments by transfection, the results showed that miR-370-3p was a key regulator in osteogenic differentiation by targeting BMP2 and disturbing the expression of PTHLH, and participated in NELL-1-stimulated osteogenesis. The present study provided the primary data and evidence for further exploration on the roles of miRNAs and ceRNAs during NELL-1-induced ossification of hASCs.     

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。     

Subchronic nandrolone administration reduces cardiac oxidative markers during restraint stress by modulating protein expression patterns

lncRNAs are an emerging class of regulators involved in multiple biological processes. MEG3, an lncRNA, acts as a tumor suppressor, has been reported to be linked with osteogenic differentiation of MSCs. However, limited knowledge is available concerning the roles of MEG3 in the multilineage differentiation of hASCs. The current study demonstrated that MEG3 was downregulated during adipogenesis and upregulated during osteogenesis of hASCs. Further functional analysis showed that knockdown of MEG3 promoted adipogenic differentiation, whereas inhibited osteogenic differentiation of hASCs. Mechanically, MEG3 may execute its role via regulating miR-140-5p. Moreover, miR-140-5p was upregulated during adipogenesis and downregulated during osteogenesis in hASCs, which was negatively correlated with MEG3. In conclusion, MEG3 participated in the balance of adipogenic and osteogenic differentiation of hASCs, and the mechanism may be through regulating miR-140-5p.     

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.     

A set of microRNAs mediate direct conversion of human umbilical cord lining-derived mesenchymal stem cells into hepatocytes

In a previous study, we elucidated the specific microRNA (miRNA) profile of hepatic differentiation. In this study, we aimed to clarify the instructive role of six overexpressed miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424 and miR-542-5p) during hepatic differentiation of human umbilical cord lining-derived mesenchymal stem cells (hMSCs) and to test whether overexpression of any of these miRNAs is sufficient to induce differentiation of the hMSCs into hepatocyte-like cells. Before hepatic differentiation, hMSCs were infected with a lentivirus containing a miRNA inhibitor sequence. We found that downregulation of any one of the six hepatic differentiation-specific miRNAs can inhibit HGF-induced hepatic differentiation including albumin expression and LDL uptake. Although overexpression of any one of the six miRNAs alone or liver-enriched miR-122 cannot initiate hepatic differentiation, ectopic overexpression of seven miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424, miR-542-5p and miR-122) together can stimulate hMSC conversion into functionally mature induced hepatocytes (iHep). Additionally, after transplantation of the iHep cells into mice with CCL4-induced liver injury, we found that iHep not only can improve liver function but it also can restore injured livers. The findings from this study indicate that miRNAs have the capability of directly converting hMSCs to a hepatocyte phenotype in vitro.     

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.