MiR-130a-3p In Bone Marrow Mesenchymal Stem Cells Inhibits The Proliferation And Differentiation Of Mouse Lung Fibroblast By Regulating The TGF-β/Smad Signaling Pathway

Objective: Pulmonary fibrosis(PF)is a pulmonary disease with high mortality and extremely difficult to treat.The repair of lung tissue injury involves four different stages,including coagulation period,the inflammation stage,the stage of fibroblast proliferation,and the final healing stage of the normal tissue structure.Among which the proliferation and activation of fibroblasts are the key stages of PF development and occurrence.When the lung is damaged,epithelial cells/endothelial cells release large amounts of cytokines,which amplify the inflammatory response and trigger fibroblast proliferation and activation to express α-smooth muscle actin and extracellular matrix components.Mesenchymal stem cells(MSCs)tissue repair/regeneration and immunomodulatory properties make it a cell therapy and tissue regeneration promising candidates,mainly through a paracrine active molecules secreted to activate,MSCs conditioned medium(CM)contains the secretion of various factors,such as exosome,and micro RNAs(miRNAs).And the latter has emerged as effective regulators of various cellular processes,such as proliferation,migration,and differentiation,and are being explored for use as biomarkers of pulmonary fibrosis.We speculated that bone marrow mesenchymal stem cells(BMSCs)and CM might affect the proliferation and differentiation of mouse lung fibroblasts(MLg cells)through miRNAs,so as to play a therapeutic role in PF.The purpose of this study was to explore the relationship between miRNAs and PF,to clarify its role in MLg cell proliferation and differentiation,and to explore its possible related mechanisms.Methods: 1.The influence of BMSCs-CM and transforming growth factor-β1(TGF-β1)on the viability of MLg cells were tested by CCK-8 cell proliferation assay.2.Western Blot assay was used to detect the effect of BMSCs-CM on the levels of fibrosis marker proteins(α-smooth muscle actin,fibronectin)and TGF-β1/Smad signaling pathway proteins in MLg cells at the protein level.q RT-PCR assay was used to detect the m RNA levels.3.q RT-PCR was used to test the expression of miR-130a-3p in BMSCS-CM,BMSCs and MLg cells,and observe the effects of BMSCs-CM and BMSCs co-culture with MLg cells on the expression of miR-130a-3p.4.The effects of miR-130a-3p mimic transfection on the proliferation and differentiation of MLg cells were determined by CCK-8 cell proliferation assay,immunofluorescence assay,Western Blot and q RT-PCR.5.Small interfering RNA was used to specifically knock down type II TGF-β receptor(TGF-βRII)in MLg cells,and CCK-8 cell proliferation assay,immunofluorescence assay,Western Blot and q RT-PCR were used to detect the proliferation and differentiation of MLg cells.6.Dual-Luciferase Reporter Assay detects whether miRNAs act directly on TGF-βRII.7.Western Blot and q RT-PCR experiments were used to determine whether miR-130a-3p participates in the regulation of PF by BMSCs through the TGF-β/Smad signaling pathway.Results: 1.The proliferation of MLg cells reached the maximum after treatment with 10ng/m L TGF-β1 for 24 h.2.BMSCs-CM can inhibit the proliferation of MLg cells.3.BMSCs-CM and BMSCs can reduce the levels of α-smooth muscle actin,fibronectin and TGF-β/Smad signaling pathway proteins in MLg cells at the protein and transcription levels.4.q RT-PCR results showed that BMSCs contained more miR-130a-3p and the expression of miR-130a-3p in MLg cells could be improved after co-culture of BMSCs with MLg cells.5.The proliferation and differentiation of MLg cells transfected with miR-130a-3p mimics were inhibited,and the protein and m RNA levels of α-smooth muscle actin,fibronectin and TGF-β/Smad signaling pathways were decreased.6.TGF-βRII knockdown also inhibited the expression of the above indicators.7.miR-130a-3p can act directly on TGF-βRII,thereby inhibiting its expression.8.miR-130a-3p participates in the regulation of PF by BMSCs through the TGF-β/Smad signaling pathway.Conclusion: BMSCs may affect the proliferation and differentiation of MLg cells by targeting TGF-βRII through miR-130a-3p,which have a certain therapeutic effect on PF.