Dataset Information

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.

SUBMITTER: Yang S

PROVIDER: S-EPMC7574877 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/β-catenin axis.

Yang Shude S Guo Shu S Tong Shuang S Sun Xu X

Cell proliferation 20200817 10

<h4>Objectives</h4>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.<h4>Materials and methods</h4>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 ...[more]

PMID: 32808361

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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.

Dataset Information

Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/?-catenin axis.

Publications

Exosomal miR-130a-3p regulates osteogenic differentiation of Human Adipose-Derived stem cells through mediating SIRT7/Wnt/β-catenin axis.

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