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.     

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

Bone marrow-derived mesenchymal stem cells (BM-MSCs), the common progenitor cells of adipocytes and osteoblasts, have been recognized as the key mediator during bone formation. Herein, our study aim to investigate molecular mechanisms underlying circular RNA (circRNA) AFF4 (circ_AFF4)-regulated BM-MSCs osteogenesis. BM-MSCs were characterized by FACS, ARS, and ALP staining. Expression patterns of circ_AFF4, miR-135a-5p, FNDC5/Irisin, SMAD1/5, and osteogenesis markers, including ALP, BMP4, RUNX2, Spp1, and Colla1 were detected by qRT-PCR, western blot, or immunofluorescence staining, respectively. Interactions between circ_AFF4 and miR-135a-5p, FNDC5, and miR-135a-5p were analyzed using web tools including TargetScan, miRanda, and miRDB, and further confirmed by luciferase reporter assay and RNA pull-down. Complex formation between Irisin and Integrin αV was verified by Co-immunoprecipitation. To further verify the functional role of circ_AFF4 in vivo during bone formation, we conducted animal experiments harboring circ_AFF4 knockdown, and born samples were evaluated by immunohistochemistry, hematoxylin and eosin, and Masson staining. Circ_AFF4 was upregulated upon osteogenic differentiation induction in BM-MSCs, and miR-135a-5p expression declined as differentiation proceeds. Circ_AFF4 knockdown significantly inhibited osteogenesis potential in BM-MSCs. Circ_AFF4 stimulated FNDC5/Irisin expression through complementary binding to its downstream target molecule miR-135a-5p. Irisin formed an intermolecular complex with Integrin αV and activated the SMAD1/5 pathway during osteogenic differentiation. Our work revealed that circ_AFF4, acting as a sponge of miR-135a-5p, triggers the promotion of FNDC5/Irisin via activating the SMAD1/5 pathway to induce osteogenic differentiation in BM-MSCs. These findings gained a deeper insight into the circRNA-miRNA regulatory system in the bone marrow microenvironment and may improve our understanding of bone formation-related diseases at physiological and pathological levels.Subject terms: Stem cells, Diseases     

Long noncoding RNA TCL6 binds to miR-106a-5p to regulate hepatocellular carcinoma cells through PI3K/AKT signaling pathway

Long noncoding RNAs (lncRNAs) have been reported to dysregulate and involve in the pathology of hepatocellular carcinoma (HCC). Nonetheless, the functional role of lncRNA T cell leukemia/lymphoma 6 (TCL6) and its underlying mechanism in HCC remain unclear. Herein, we analyzed the expression of TCL6 and elucidated its mechanistic involvement in HCC. Bioinformatics analyses indicated TCL6 was evidently downregulated in HCC tissues compared with normal controls. TCL6 was downregulated while microRNA-106a-5p (miR-106a-5p) was upregulated in HCC cell lines. Moreover, knockdown or overexpression of TCL6 significantly raised or diminished the expression level of miR-106a-5p in HCC cells, similar to the effect of miR-106a-5p on TCL6 expression. Functionally, TCL6 inhibited the proliferative, migratory, and invasive potentials of HCC cells as analyzed by cell counting kit-8, scratch wound healing, and transwell assays, respectively. Conversely, miR-106a-5p exerted an opposite effect on the proliferative, migratory, and invasive potentials of HCC. RNA immune precipitation and luciferase reporter assays revealed TCL6 directly bound to miR-106a-5p and luciferase reporter assay verified phosphatase and tensin homolog (PTEN) was a target gene of miR-106a-5p. Mechanistically, TCL6 knockdown evidently reduced PTEN expression at both messenger RNA and protein levels, and miR-106a-5p inhibitor partially rescued this reduction effect in HCC cells. Additionally, western blot assays demonstrated miR-106a-5p downregulation or TCL6 overexpression promoted the protein level of PTEN, and suppressed the phosphorylation level of AKT, the protein level of phosphatidylinositol 3-kinase (PI3K). Collectively, these results revealed TCL6 as a tumor-suppressive lncRNA regulates PI3K/AKT signaling pathway via directly binding to miR-106a-5p in HCC. This mechanism provides a theoretical basis for HCC pathogenesis and a potential therapeutic strategy for HCC treatment.     

The lncRNA SNHG15/miR-18a-5p axis promotes cell proliferation in ovarian cancer through activating Akt/mTOR signaling pathway

Here, we report the expression pattern, function and regulatory mechanism of SNHG15 together with miR-18a-5p micro RNA in ovarian cancer (OC) for the first time. We recruited 20 patients and took normal ovarian tissues and ovarian tumor tissues from them. We used cell culture, transfection, in vivo tumor xenograft assay, and multiple types of detection assays to investigate the expression and regulation of long noncoding RNA (lncRNA) SNHG15/miR-18a-5p in ovarian tissues and cells. Results: We found that the messenger RNA expression level of SNHG15 was significantly higher and miR-18 was decreased in ovarian cancer tissues and in OC cells. Functional experiments showed that SNHG15 overexpression potentiated the migration and invasion of OC cells, while SNHG15 inhibition reduced the tumor proliferation, which was restored via overexpression of miR-18a. SNHG15 was found to directly target and suppress the expression of miR-18a. Our results illustrate the possible molecular mechanism of lncRNA SNHG15/miR-18a-5p functions in cell proliferation in OC. SNHG15/miR-18a promoted the progression of OC cells via the protein kinase B/mammalian target of rapamycin signaling pathway.     

miR-140-5p suppresses BMP2-mediated osteogenesis in undifferentiated human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have self-renewal and differentiation capabilities but the regulatory mechanisms of MSC fate determination remain poorly understood. Here, we aimed to identify microRNAs enriched in hMSCs that modulate differentiation commitments. Microarray analysis revealed that miR-140-5p is commonly enriched in undifferentiated hMSCs from various tissue sources. Moreover, bioinformatic analysis and luciferase reporter assay validated that miR-140-5p directly represses bone morphogenic protein 2 (BMP2). Furthermore, blocking miR-140-5p in hMSCs increased the expression of BMP signaling components and critical regulators of osteogenic differentiation. We propose that miR-140-5p functionally inhibits osteogenic lineage commitment in undifferentiated hMSCs.     

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.     

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.     

MiR-125a-5p抑制3T3-L1前体脂肪细胞分化

MicroRNAs(miRNAs) 是一类在脂肪组织发育中发挥重要作用的小非编码RNA. 为探明miR-125a-5p在3T3-L1前体脂肪细胞中的作用,采用实时qPCR检测了miR-125a-5p在小鼠各组织及3T3-L1前体脂肪细胞分化过程中的表达|使用经化学修饰的miR-125a-5p模拟物agomir及抑制剂antagomir转染3T3-L1前体脂肪细胞,采用实时qPCR 和 Western印迹检测成脂标志基因Pparγ和aP2的表达,油红O染色观察脂肪细胞脂质积累. 结果显示,miR-125-5p在小鼠脂肪组织中高丰度表达,在3T3-L1前体脂肪细胞分化过程中表达下降.过表达miR-125a-5p,与对照组相比,成脂标志基因Pparγ和aP2在mRNA和蛋白质水平均明显下降|油红O染色及定量结果显示脂质积累减少. 抑制剂处理结果显示,Pparγ和aP2在mRNA和蛋白质水平均有不同程度上升,但油红O染色及定量结果差异不显著. 以上结果表明,miR-125a-5p在脂肪细胞分化中发挥负调控作用.     

LncRNA SNHG12 promotes proliferation and epithelial mesenchymal transition in hepatocellular carcinoma through targeting HEG1 via miR-516a-5p

Hepatocellular carcinoma (HCC) is the most common cancer and its prognosis is poor due to metastasis and recurrence. EMT is associated with metastasis. A deep understanding of regulatory mechanism of EMT is critical. LncRNA is involved in regulation of various biological processes including EMT. This study aimed to investigate the regulatory signal axis among lncRNA SNHG12, miR-516a-5p and the target gene HEG1 during EMT. Cell cycle and apoptosis were analyzed by flow cytometry. Tumorigenesis was analyzed by clone formation assay. Wound healing assay and transwell assay was performed to detect migration and invasion, respectively. Interaction among SNHG12, miR-516a-5p and HEG1 were analyzed by dual luciferase assay and RIP assay. We also detected expression of RNA and protein by QPCR and western blotting. Finally, tumor growth was analyzed by tumorigenesis assay in vivo. Ki-67 and HEG1 level in tumor tissues was analyzed by IHC. SNHG12 and HEG1 were upregulated, miR-516a-5p was downregulated in HCC cell lines. SNHG12 could interact with and inhibit miR-516a-5p. MiR-516a-5p could interact with HEG1 and inhibit HEG1 expression. Knock down SNHG12 inhibited proliferation, migration, invasion, EMT and promoted apoptosis of HCC cells. Such effects were antagonized by inhibiting miR-516a-5p. SNHG12 overexpression lead to opposite results. Similar results were observed in mice. SNHG12 could promote EMT in HCC through targeting and inhibiting miR-516a-5p, which eventually upregulated HEG1 expression, in both cell and mice.     

Exosomal microRNA-155-5p from PDLSCs regulated Th17/Treg balance by targeting sirtuin-1 in chronic periodontitis

The mechanism of local inflammation and systemic injury in chronic periodontitis is complicated, in which and exosomes play an important role. In our study, we found that T helper cell 17 (Th17)/regulatory T cell (Treg) balance is destabilized in the peripheral blood of patients with periodontitis, with upregulated Th17 or downregulated Treg, respectively. Porphyromonas gingivalis lipopolysaccharide (LPS) was used to simulate the inflammatory microenvironment of chronic periodontitis. The exosomes were extracted from periodontal ligament stem cells (PDLSCs) in LPS-induced periodontitis environment, which inversely effected on CD4+ T cells under normal and inflammatory conditions. Furthermore, compared with exosomes from normal PDLSCs, lower expression of microRNA-155-5p (miR-155-5p) and higher expression of Sirtuin-1 (SIRT1) were observed in exosomes from LPS-stimulated PDLSCs. Exosomes from PDLSCs alleviated inflammatory microenvironment through Th17/Treg/miR-155-5p/SIRT1 regulatory network. This study aimed to find the “switching” factors that affected the further deterioration of periodontitis to maximally control the multiple downstream damage signal factors to further understand periodontitis and find new targets for its treatment.     

MiRNA-199a-5p positively regulated RANKL-induced osteoclast differentiation by target Mafb protein

MicroRNAs are involved in osteoclast differentiation. Although miR-199a-5p plays an important role in many different systems and diseases, its function during osteoclastogenesis remains unclear. In this study, we investigated the function and the target gene of miR-199a-5p in osteoclast differentiation. The in vitro data showed that miR-199a-5p was significantly upregulated after the stimulation by receptor activator of nuclear factor kappa-B ligand in macrophages and RAW 264.7 cells. After transfection of miR-199a-5p mimic, the messenger RNA expression level of nuclear factor of activated T-cells cytoplasmic 1, tartrate-resistant acid phosphatase (TRAP), and receptor activator of nuclear factor kappa-B was significantly increased in RAW 264.7 cells and the number of TRAP-positive cells was also increased. MiR-199a-5p inhibitor showed the complete opposite outcome which brought additional proof to our finding. Overexpression of miR-199a-5p led to downregulation of Mafb protein. The luciferase activity was obviously repressed when WT-pGL3-Mafb and miR-199a-5p mimics were cotransfected into 293 T cells and the inhibitors cotransfected demonstrated reverse result. MiR-199a-5p overexpressed during osteoclast differentiation and positively regulated osteoclast formation in vitro by target Mafb.     

LncRNA GIHCG promoted the proliferation and migration of renal cell carcinoma through regulating miR-499a-5p/XIAP axis

BackgroundOur previous study demonstrated that lncRNA GIHCG is upregulated in renal cell carcinoma (RCC) and that knockdown of lncRNA GIHCG suppresses the proliferation and migration of RCC cells. However, the mechanism of lncRNA GIHCG in RCC needs further exploration.MethodsThe proliferation, cell cycle, migration, and apoptosis of RCC cells were tested using CCK-8, flow cytometry, wound healing and Annexin-V/-FITC/PI flow cytometry assays, respectively. Dual-luciferase reporter and RNA pull-down or RNA immunoprecipitation assays (RIPs) were performed to analyze the interactions among lncRNA GIHCG, miR-499a-5p and XIAP. A tumour xenograft study was conducted to verify the function of lncRNA GIHCG in RCC development in vivo.ResultsKnockdown of lncRNA GIHCG inhibited cell proliferation and migration and induced G0/G1 arrest while promoting apoptosis. Overexpression of lncRNA GIHCG led to the opposite results. LncRNA GIHCG sponged miR-499a-5p and downregulated its expression in RCC cells. MiR-499a-5p overexpression suppressed RCC cell growth. MiR-499a-5p targeted XIAP and inhibited its expression. LncRNA GIHCG knockdown reduced the growth of tumour xenografts in vivo and the expression of XIAP while increasing miR-499a-5p levels.ConclusionLncRNA GIHCG accelerated the development of RCC by targeting miR-499a-5p and increasing XIAP levels.     

Long noncoding RNA linc00467 plays an oncogenic role in hepatocellular carcinoma by regulating the miR-18a-5p/NEDD9 axis

Increasing evidence has shown that numerous long noncoding RNAs (lncRNAs) play critical roles in tumorigenesis. Herein, we investigated the biological role of lncRNA linc00467 in the cancer biology of hepatocellular carcinoma (HCC). We observed that linc00467 was upregulated in HCC tissues and cells. Silencing of linc00467 using small interfering RNA interference significantly inhibited the growth and motility of HCC cells, and increased cell apoptosis through regulating the Bcl-2/Bax axis and the caspase cascade, suggesting that linc00467 exerted oncogenic functions in the progression of HCC. Moreover, we found that linc00467 could target miR-18a-5p, and NEDD9 was a target for miR-18a-5p in HCC cells. Furthermore, either the miR-18a-5p inhibitor or upregulation of NEDD9 could recover the inhibitory effects caused by silencing of linc00467. In conclusion, our data highlighted the oncogenic role of linc00467 in HCC progression by regulating the miR-18a-5p/NEDD9 axis.     

DANCR contributed to hepatocellular carcinoma malignancy via sponging miR-216a-5p and modulating KLF12

Long noncoding RNA (lncRNA) differentiation antagonizing nonprotein coding RNA (DANCR) has been identified as an oncogene in several cancers. However, the biological function and role of DANCR in hepatocellular carcinoma (HCC) remain unclear. Our current study aimed to investigate the detailed mechanism of DANCR in HCC. We found that DANCR was significantly upregulated in HCC cell lines in comparison to LO2 cells. Then, we observed that knockdown of DANCR could greatly inhibit Huh7 and HepG2 cell proliferation. In addition, HCC cell apoptosis was increased by silence of DANCR and meanwhile, cell cycle progression was blocked in G1 phase. Apart from these, downregulation of DANCR repressed HCC cell migration and invasion ability obviously. As predicted by the bioinformatics analysis, microRNA-216a-5p (miR-216a-5p) could serve as a direct target of DANCR. MiR-216a-5p has been reported to be involved in many cancers. Here, the correlation between miR-216a-5p and DANCR was confirmed using dual-luciferase reporter assay and radioimmunoprecipitation assay. Subsequently, Kruppel-like factor 12 (KLF12) exerts an important role in different tumor types. KLF12 can function as a downstream target of miR-216a-5p. Finally, the in vivo experiments were used and the data proved that DANCR also strongly suppressed HCC tumor growth in vivo via targeting miR-216a-5p and KLF12. In conclusion, our study indicated that DANCR might provide a new perspective for HCC treatment.     

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.