Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

Super-Enhancer-Associated Long Non-Coding RNA LINC01485 Promotes Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Regulating MiR-619-5p/RUNX2 Axis

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ObjectiveTo investigate the mechanisms of super-enhancer-associated LINC01485/miR-619-5p/RUNX2 signaling axis involvement in osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs).MethodsOsteogenic differentiation of hBMSCs was induced in vitro.The expression levels of LINC01485 and miR-619-5p during osteogenesis were measured using quantitative real-time polymerase chain reaction (qRT-PCR).Osteogenic differentiation was examined by qRT-PCR, western blot, alkaline smokeaers.shop phosphatase (ALP) staining, ALP activity measurement, and Alizarin Red S (ARS) staining assays.Thereafter, the effects of LINC01485 and miR-619-5p on osteogenic differentiation of hBMSCs were evaluated by performing loss- and gain-of-function experiments.

Subsequently, a fluorescence in situ hybridization (FISH) assay was employed to determine the cellular localization of LINC01485.Bioinformatics analysis, RNA antisense purification (RAP) assay, and dual-luciferase reporter assays were conducted to analyze the interactions of LINC01485, miR-619-5p, and RUNX2.Rescue experiments were performed to further delineate the role of the competitive endogenous RNA (ceRNA) signaling axis consisting of LINC01485/miR-619-5p/RUNX2 in osteogenic differentiation of hBMSCs.ResultsThe expression of LINC01485 was up-regulated during osteogenic differentiation of hBMSCs.The overexpression of LINC01485 promoted osteogenic differentiation of hBMSCs by up-regulating the expression of osteogenesis-related genes [e.

g., runt-related transcription factor 2 (RUNX2), osterix (OSX), collagen type 1 alpha 1 (COL1A1), osteocalcin (OCN), and osteopontin (OPN)], and increasing the activity of ALP.ALP staining and ARS staining were also found to be increased upon overexpression of LINC01485.The opposing results were obtained upon LINC01485 interference in hBMSCs.miR-619-5p was found to inhibit osteogenic differentiation.

FISH assay displayed that LINC01485 was mainly localized in Protection the cytoplasm.RAP assay results showed that LINC01485 bound to miR-619-5p, and dual-luciferase reporter assay verified that LINC01485 bound to miR-619-5p, while miR-619-5p and RUNX2 bound to each other.Rescue experiments illustrated that LINC01485 could promote osteogenesis by increasing RUNX2 expression by sponging miR-619-5p.ConclusionLINC01485 could influence RUNX2 expression by acting as a ceRNA of miR-619-5p, thereby promoting osteogenic differentiation of hBMSCs.The LINC01485/miR-619-5p/RUNX2 axis might comprise a novel target in the bone tissue engineering field.