Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/?-catenin axis.
ABSTRACT: OBJECTIVES:It is of profound significance for clinical bone regeneration to clarify the specific molecular mechanism from which we found that osteogenic differentiation of adipose-derived stem cells (ADSCs) will be probably promoted by exosomes. MATERIALS AND METHODS:By means of lentiviral transfection, miR-130a-3p overexpression and knockdown ADSCs were constructed. Alizarin Red S was used to detect the calcium deposits, and qPCR was used to detect osteogenesis-related genes, to verify the effect of miR-130a-3p on the osteogenic differentiation of ADSCs. CCK-8 was used to detect the effect of miR-130a-3p on the proliferation of ADSCs. The target binding between miR-130a-3p and SIRT7 was verified by dual-luciferase reporter gene assay. Furthermore, the role of Wnt signalling pathway in the regulation of ADSCs osteogenesis and differentiation by miR-130a-3p was further verified by detecting osteogenic-related genes and proteins and alkaline phosphatase activity. RESULTS:(a) Overexpression of miR-130a-3p can enhance the osteogenic differentiation of ADSCs while reducing protein and mRNA levels of SIRT7, a target of miR-130a-3p. (b) Our study further found that overexpression of miR-130a-3p leads to down-regulation of SIRT7 expression with up-regulation of Wnt signalling pathway-associated protein. (c) Overexpression of miR-130a-3p inhibited proliferation of ADSCs, while knockdown promoted it. CONCLUSIONS:The obtained findings indicate that exosomal miR-130a-3p can promote osteogenic differentiation of ADSCs partly by mediating SIRT7/Wnt/?-catenin axis, which will hence promote the application of exosomal microRNA in the field of bone regeneration.
Project description:Human adipose-derived stem cells (ADSCs) can release exosomes; however, their specific functions remain elusive. In this study, we verified that exosomes derived from osteogenically differentiated ADSCs can promote osteogenic differentiation of ADSCs. Furthermore, in order to investigate the importance of exosomal microRNAs (miRNAs) in osteogenic differentiation of ADSCs, we used microarray assays to analyze the expression profiles of exosomal miRNAs derived from undifferentiated as well as osteogenically differentiated ADSCs; 201 miRNAs were upregulated and 33 miRNAs were downregulated between the two types of exosomes. Additionally, bioinformatic analyses, which included gene ontology analyses, pathway analysis, and miRNA-mRNA-network investigations, were performed. The results of these analyses revealed that the differentially expressed exosomal miRNAs participate in multiple biological processes, such as gene expression, synthesis of biomolecules, cell development, differentiation, and signal transduction, among others. Moreover, we found that these differentially expressed exosomal miRNAs connect osteogenic differentiation to processes such as axon guidance, MAPK signaling, and Wnt signaling. To the best of our knowledge, this is the first study to identify and characterize exosomal miRNAs derived from osteogenically differentiated ADSCs. This study confirms that alterations in the expression of exosomal miRNAs can promote osteogenic differentiation of ADSCs, which also provides the foundation for further research on the regulatory functions of exosomal miRNAs in the context of ADSC osteogenesis.
Project description:MicroRNAs (miRNAs) emerge as important regulators of stem cell lineage commitment and bone development. MiRNA-26a (miR-26a) is one of the important miRNAs regulating osteogenic differentiation of both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived mesenchymal stem cells (ADSCs). However, miR-26a functions oppositely in osteogenic differentiation of BMSCs and ADSCs, suggesting distinct post-transcriptional regulation of tissue-specific MSC differentiation. However, the molecular basis is largely unknown. Here, we report that the function of miR-26a is largely depended on the intrinsic signaling regulation network of MSCs. Using bioinformatics and functional assay, we confirmed that miR-26a potentially targeted on GSK3? and Smad1 to regulate Wnt and BMP signaling pathway. Overall comparative analysis revealed that Wnt signaling was enhanced more potently and played a more important role than BMP signaling in osteogenic differentiation of BMSCs, whereas BMP pathway was more essential for promoting osteogenic differentiation of ADSCs. The distinct activation pattern and role of signaling pathways determined that miR-26a majorly targeted on GSK3? to activate Wnt signaling for promoting osteogenic differentiation of BMSCs, whereas it inhibited Smad1 to suppress BMP signaling for interfering with the osteogenic differentiation of ADSCs. Taken together, our study demonstrated that BMSCs and ADSCs applied different signaling pathway to facilitate their osteogenic differentiation, which determined the inverse function of miR-26a. The distinct transcriptional regulation and post-transcriptional regulation network suggested the intrinsic molecular differences between tissue-specific MSCs and the complexity in MSC research and MSC-based cell therapy.
Project description:BACKGROUND:Many studies have shown that long noncoding RNAs (lncRNAs) are closely related to the stimulation of osteogenic differentiation of adipose-derived stem cells (ADSCs) and the prevention of osteoporosis. Current research aimed to investigate the novel lncRNA and explored the function and molecular mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. METHODS:LncRNA and miRNA sequencing was performed in normal and osteogenic differentiation-induced ADSCs (osteogenic group). Abnormally expressed lncRNAs and miRNAs were obtained by the R software and the relative expression of LINC00314, miR-129-5p, and GRM5 during osteogenic induction was measured by RT-PCR. ADSCs were then transfected with pcDNA3.1-sh-LINC00314 and agomiR-129-5p. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were performed to identify the mechanism of the LINC00314/miR-129-5p/GRM5 axis in regulating osteogenic differentiation of ADSCs. RESULTS:LINC00314 was significantly upregulated in the group of osteogenic-induced ADSCs. LINC00314 and GRM5 mimics increased the early and late markers of osteogenic differentiation, which manifest in not only the markedly increased ALP activity but also higher calcium deposition, while miR-129-5p mimic had the opposite effects. LINC00314 directly targeted miR-129-5p through luciferase reporter assay, and miR-129-5p suppressed GRM5 expression. Moreover, the LINC00314/miR-129-5p/GRM5 regulatory axis activated the Wnt/?-catenin signaling pathway. CONCLUSIONS:LINC00314 confers contributory function in the osteogenic differentiation of ADSCs and thus the LINC00314/miR-129-5p/GRM5 axis may be a novel mechanism for osteogenic-related disease.
Project description:Exosomal microRNAs (miRNAs) are promising candidate biomarkers for diagnosis or prognosis for breast cancer. We investigated the prognostic role of exosomal miRNAs in serum samples derived from patients with breast cancer and compared miRNA expression between serum and tumor tissues.The miRNA profile derived from exosome between breast cancer patients with recurrence (n = 16) and without recurrence (n = 16) were compared by miRNA PCR array. Further, we examined the expression of miRNAs derived from tissues in the patients with breast cancer with (n = 35) and without recurrence (n = 39) by qRT-PCR.Of 384 miRNAs, three miRNAs (miR-338-3p, miR-340-5p, and miR-124-3p) were significantly upregulated and eight (miR-29b-3p, miR-20b-5p, miR-17-5p, miR-130a-3p, miR-18a-5p, miR-195-5p, miR-486-5p, and miR-93-5p) were significantly downregulated in the patients with recurrence. We evaluated the expression of the miRNAs in tumor tissues. The patients with recurrence had higher levels of miR-340 at their primary site as well as in the serum. In contrast, miR-195-5p, miR-17-5p, miR-93-5p, and miR-130a-3p, derived from tumor tissues that were downregulated in the serum from patients with recurrence, were higher in the patients with recurrence than in those with no recurrence. In logistic regression analysis, miR-340-5p, miR-17-5p, miR-130a-3p, and miR-93-5p were significantly associated with recurrence.Several exosomal miRNAs may be useful biomarkers to predict breast cancer recurrence. We show the different expression patterns of miRNAs between tumor tissues and serum. These findings may suggest selective mechanism of release of exosomal miRNAs by cancer cells to regulate their progression.
Project description:Osteoporosis is a systemic metabolic bone disease during which bone mass decreases and bone quality is reduced. Maintaining the bone formation capacity of osteoblasts is crucial for the treatment of osteoporosis. In the present study, bioinformatics analysis was performed on online microarray expression profiles to identify miRNA(s) related to osteoblast proliferation and bone marrow?derived mesenchymal stem cell (BMSC) osteogenic differentiation. The specific effects of candidate miRNAs on cell proliferation, osteogenic differentiation and Wnt signaling?related factors were examined. As regards the downstream mechanisms, online tools were employed to predict the downstream targets of candidate miRNAs and the predicted miRNA?mRNA binding was verified. Finally, the dynamic effects of miRNAs and mRNAs were examined. The results revealed that miR?483?3p expression was decreased in bone tissue samples from patients with osteoporosis. In miR?483?3p?overexpressing human osteoblasts, cell viability, DNA synthesis capacity and osteogenesis were promoted, and the protein levels of Wnt1, ??catenin and cyclin D1 were increased. However, the protein receptor activator of nuclear factor kappa?? ligand (RANKL)/osteoprotegerin (OPG) ratio and cell apoptotic rate were decreased. The Wnt signaling, antagonist Dikkopf 2 (DKK2), was targeted and negatively regulated by miR?483?3p. DKK2 knockdown exerted similar effects as miR?483?3p overexpression, while DKK2 overexpression inhibited cell viability, DNA synthesis capacity and osteogenesis. DKK2 overexpression also decreased the Wnt1, ??catenin, and cyclin D1 protein levels, whereas it promoted the the RANKL/OPG ratio and the apoptosis of human osteoblasts. DKK2 overexpression reversed the functions of miR?483?3p overexpression. On the whole, the findings of the present study demonstrate that the miR?483?3p/DKK2 axis modulates the bone formation process by affecting osteoblast proliferation, pre?osteoblast differentiation into mature osteoblasts and new bone matrix formation.
Project description:MicroRNAs (MiRNAs) play critical roles in the regulation of pituitary function. MiR-130a-3p has previously been found to be down-regulated in prolactinoma, but its roles in prolactin (PRL) regulation and the underlying mechanisms are still unclear. Heat stress has been shown to induce alteration of endocrine hormones and miRNAs expressions. However, there is limited information regarding the emerging roles of miRNAs in heat stress response. In this study, we transfected miR-130a-3p mimic into the pituitary adenoma cells (GH3 cells) to investigate the function of miR-130a-3p in regulating PRL. Our results showed that miR-130a-3p overexpression significantly decreased the PRL expression at both mRNA and protein levels. Subsequently, estrogen receptor ? (ER?) was identified as a direct target of miR-130a-3p by bioinformatics prediction, luciferase reporter assay and western blotting assay. Furthermore, the inhibition of ER? caused by estrogen receptor antagonist significantly reduced the PRL expression. Overexpression of ER? rescued the suppressed expression of PRL caused by miR-130a-3p mimic. Besides, we also studied the effect of heat stress on PRL and miRNAs expressions. Interestingly, we found that heat stress reduced PRL and ER? expressions while it increased miR-130a-3p expression both in vitro and in vivo. Taken together, our results indicate that miR-130a-3p represses ER? by targeting its 3'UTR leading to a decrease in PRL expression, and miR-130a-3p is correlative with heat stress-induced PRL reduction, which provides a novel mechanism that miRNAs are involved in PRL regulation.
Project description:Reduced osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs) has been causally linked to the development of aplastic anemia. In this work, we aimed to identify novel microRNAs (miRNAs) that participate in the regulation of differentiation of BMSCs from patients with aplastic anemia. We show that miR-144-3p is significantly upregulated in BMSCs from patients with aplastic anemia relative to control equivalents. Depletion of miR-144-3p significantly enhances osteogenic differentiation of BMSCs from patients with aplastic anemia after culturing in osteogenesis-inducing medium. Conversely, overexpression of miR-144-3p blocks osteogenic differentiation of BMSCs. Mechanistically, miR-144-3p negatively regulates the expression of ten-eleven translocation 2 (TET2) in BMSCs. Reduced TET2 expression is associated with a significant decrease in global 5-hydroxymethyl-cytosine (5hmC) levels and osteogenic gene expression. Knockdown of miR-144-3p elevates the expression of TET2 and total 5hmC levels in BMSCs. Silencing of TET2 inhibits the osteogenic differentiation of BMSCs. Overexpression of TET2 reverses miR-144-3p-mediated inhibition of osteogenesis. In addition, there is a significant negative correlation between the expression of miR-144-3p and TET2 in BMSCs from patients with aplastic anemia. Overall, miR-144-3p impairs the osteogenic capacity of BMSCs from patients with aplastic anemia through repression of TET2. Therefore, the targeting of miR-144-3p may be a therapeutic strategy against aplastic anemia.
Project description:BACKGROUND:The incidence of inflammatory bowel disease, a chronic intestinal inflammatory disorder that includes Crohn's disease (CD) and ulcerative colitis, is rising. Circular RNAs are considered valuable diagnostic biomarkers for CD. Current evidence supports the views that epithelial-mesenchymal transition (EMT) plays an important role in CD pathogenesis, and that hsa-miR-130a-3p can inhibit transforming growth factor-?1 (TGF-?1)-induced EMT. Our previous study revealed that hsa_circRNA_102610 was upregulated in CD patients. Moreover, we predicted an interaction between hsa_circRNA_102610 and hsa-miR-130a-3p. Thus, we hypothesized that hsa_circRNA_102610 may play roles in the proliferation and EMT of intestinal epithelial cells by sponging hsa-miR-130a-3p to participate in the pathogenesis of CD. AIM:To explore the mechanism of hsa_circRNA_102610 in the pathogenesis of CD. METHODS:The relative expression levels of hsa_circRNA_102610 and hsa-miR-130a-3p in patients were detected by quantitative reverse transcription-polymerase chain reaction. The proliferation of human intestinal epithelial cells (HIECs) and normal-derived colon mucosa cell line 460 (NCM460) cells was detected by cell counting kit-8, 5-ethynyl-2'-deoxyuridine staining and cell cycle assays following overexpression or downregulation of hsa_circRNA_102610. Cell proliferation assays were performed as described above in a rescue experiment with hsa-miR-130a-3p mimics. The interaction of hsa_circRNA_102610 and hsa-miR-130a-3p was verified by fluorescence in situ hybridization and dual luciferase reporter assays. The relative expression levels of CyclinD1, mothers against decapentaplegic homolog 4 (SMAD4), E-cadherin, N-cadherin and Vimentin were detected by western blotting following hsa_circRNA_102610 overexpression, TGF-?1-induced EMT or hsa-miR-130a-3p mimic transfection (in rescue experiments). RESULTS:Upregulation of hsa_circRNA_102610 was determined to be positively correlated with elevated fecal calprotectin levels in CD (r = 0.359, P = 0.007) by Pearson correlation analysis. Hsa_circRNA_102610 promoted the proliferation of HIECs and NCM460 cells, while hsa-miR-130a-3p reversed the cell proliferation-promoting effects of hsa_circRNA_102610. Fluorescence in situ hybridization and dual luciferase reporter assays showed that hsa_circRNA_102610 directly bound hsa-miR-130a-3p in NCM460 and 293T cells. An inverse correlation between downregulation of hsa-miR-130a-3p and upregulation of hsa_circRNA_102610 in CD patients was observed (r = -0.290, P = 0.024) by Pearson correlation analysis. Moreover, overexpression of hsa_circRNA_102610 promoted SMAD4 and CyclinD1 protein expression validated by western-blotting. Furthermore, over-expression of hsa_circRNA_102610 promoted TGF-?1 induced EMT in HIECs and NCM460 cells via targeting of hsa-miR-130a-3p, with increased expression of Vimentin and N-cadherin and decreased expression of E-cadherin. CONCLUSION:Hsa_circRNA_102610 upregulation in CD patients could promote the proliferation and EMT of intestinal epithelial cells via sponging of hsa-miR-130a-3p.
Project description:BACKGROUND:The Wnt/?-catenin pathway is involved in the osteogenic differentiation of human adipose-derived stem cells (hASCs) under cyclic strain. Very little is known about the role of microRNAs in these events. METHODS:Cells were obtained using enzyme digestion methods, and proliferation was detected using Cell Counting Kit 8. Cell cycles and immunophenotypes were detected by flow cytometry. The multilineage potential of hASCs was induced by induction media. Cyclic strain was applied to hASCs (0.5?Hz, 2?h/day, 6?days) to induce osteogenic differentiation and miRNA changes. Bioinformatic and dual-luciferase analyses confirmed lymphoid enhancer factor 1 (LEF1) as a potential target of let-7i-3p. The effect of let-7i-3p on LEF1 in hASCs transfected with a let-7i-3p mimic and inhibitor was analyzed by immunofluorescence. hASCs were transfected with a let-7i-3p mimic, inhibitor, or small interfering RNA (siRNA) against LEF1 and ?-catenin. Quantitative real-time PCR (qPCR) and western blotting were performed to examine the osteogenic markers and Wnt/?-catenin pathway at the mRNA and protein levels, respectively. Immunofluorescence and western blotting were performed to confirm the activation of the Wnt/?-catenin pathway. RESULTS:Flow cytometry showed that 82.12%?±?5.83% of the cells were in G1 phase and 17.88%?±?2.59% of the cells were in S/G2 phase; hASCs were positive for CD29, CD90, and CD105. hASCs could have the potential for osteogenic, chondrogenic, and adipogenic differentiation. MicroRNA screening via microarray showed that let-7i-3p expression was decreased under cyclic strain. Bioinformatic and dual-luciferase analyses confirmed that LEF1 in the Wnt/?-catenin pathway was the target of let-7i-3p. Under cyclic strain, the osteogenic differentiation of hASCs was promoted by overexpression of LEF1and ?-catenin and inhibited by overexpression of let-7i-3p. hASCs were transfected with let-7i-3p mimics and inhibitor. Gain- or loss-of-function analyses of let-7i-3p showed that the osteogenic differentiation of hASCs was promoted by decreased let-7i-3p expression and inhibited by increased let-7i-3p expression. Furthermore, high LEF1 expression inactivated the Wnt/?-catenin pathway in let-7i-3p-enhanced hASCs. In contrast, let-7i-3p inhibition activated the Wnt/?-catenin pathway. CONCLUSIONS:Let-7i-3p, acting as a negative regulator of the Wnt/?-catenin pathway by targeting LEF1, inhibits the osteogenic differentiation of hASCs under cyclic strain in vitro.
Project description:Nonalcoholic fibrosing steatohepatitis is a uniform process that occurs throughout nonalcoholic fatty liver disease (NAFLD). MicroRNAs (miRNAs) have been shown to be involved in the biological processes, but the role and molecular mechanism of miRNAs in NAFLD are not entirely clear. In this study, we observed a significant reduction in the expression of miR-130a-3p in livers of a mouse model with fibrosis induced by a methionine-choline-deficient diet, of NAFLD patients, and in activated hepatic stellate cells (HSCs). A dual-luciferase activity assay confirmed that transforming growth factor-beta receptors (TGFBRs) 1 and 2 were both the target genes of miR-130a-3p. The hepatic expression of TGFBR1 and TGFBR2 was significantly increased. Moreover, the overexpression of miR-130a-3p in HSCs inhibited HSC activation and proliferation, concomitant with the decreased expression of TGFBR1, TGFBR2, Smad2, Smad3, matrix metalloproteinase-2 (MMP-2), MMP-9, type I collagen (Col-1), and Col-4. In addition, the overexpression of miR-130a-3p promoted HSC apoptosis by inducing the expression of caspase-dependent apoptosis genes. Transfection with si-TGFBR1 and si-TGFBR2 revealed effects on HSC function that were consistent with those of miR-130a-3p. TGFBR1 and TGFBR2 rescued the miR-130a-3p-mediated reductions in the mRNA and protein expression levels of Smad2, Smad3, Col-1, and Col-4. In conclusion, our findings suggest that miR-130a-3p might play a critical role in negatively regulating HSC activation and proliferation in the progression of nonalcoholic fibrosing steatohepatitis by directly targeting TGFBR1 and TGFBR2 via the TGF-?/SMAD signaling pathway.