| Background:Super-enhancer-associated lncRNAs(SE-lncRNAs)are typically transcribed from super-enhancers(SEs)genomic regions or their adjacent region or interacting with SEs,which harbor specific chromatin states of activation enhancers,H3K4mel,H3K27ac,and related cofactors(i.e.,P300,etc.).SE-lncRNAs can regulate the expression of interconnected signaling pathways and target genes with the SEs by binding to the SEs.With the further study of SE-lncRNAs,scholars have found that SE-lncRNAs play an essential role in cell development,differentiation,and tumor development through a complex signal network.However,the role of SE-lncRNAs in osteogenic differentiation and its regulatory mechanism remains largely unknown.Therefore,based on the post-osteogenic specific SEs of human bone marrow mesenchymal stem cells(hBMSCs)identified by Chromatin Immunoprecipitation-sequencing(ChIP-seq),this study explored the role and molecular mechanism of the post-osteogenic specific SEs related lncRNA LINC01485 in the osteogenic differentiation of hBMSCs.Methods:hBMSCs were purchased for amplification and subculture,osteogenic induce differentiation.Quantitative real-time polymerase chain reaction(qRT-PCR),western blot,alkaline phosphatase(ALP)staining,ALP activity determination,Alizarin Red S staining were used to identify osteogenic differentiation.qRT-PCR was used to detect the expression of LINC01485 and miR-619-5p during osteogenic induction.The expression correlation between LINC01485 and osteogenic differentiation factor and miR-619-5p was analyzed.LINC01485 overexpression and LINC01485 interference lentivirus vector were constructed and transfected into hBMSCs.qRT-PCR,western blot,ALP staining,ALP activity determination,Alizarin Red S staining were employed to examine the effect of LINC01485 on osteogenic differentiation.Subsequently,fluorescence in situ hybridization(FISH)was used to clarify the cellular location of LINC01485.The miRNAs bound to LINC01485 were predicted by the bioinformatics database,and the miRNAs whose target genes were osteogenesis-related genes with high predicted scores were selected from the predicted results for RNA antisense purification(RAP)experiment,and miR-619-5p bound to LINC01485 was selected.Dual-luciferase reporter assay confirmed the combination of LINC01485 and miR-619-5p.Then,the bioinformatics database was used to predict the target gene RUNX2 bound to miR-619-5p for experimental verification.MiR-619-5p mimic and miR-619-5p inhibitor were synthesized and transfected into hBMSCs.The expression level of RUNX2 was detected by qRT-PCR and western blot assay.The effect of miR-619-5p on osteogenic differentiation of hBMSCs was examined by western blot and ALP staining.The binding of miR-619-5p and RUNX2 was verified by a dual-luciferase reporter assay.Finally,rescues assay was performed;that is,miR-619-5p inhibitor and miRNA inhibitor NC group were designed and synthesized to transfect stable cells of LINC01485 interference group and control group,and the osteogenic differentiation of hBMSCs was detected to identify the competitive endogenous RNA(ceRNA)regulatory network between LINC01485/miR-619-5p/RUNX2.Results:Compared with before osteogenic differentiation,the expression of osteogenic related factors RUNX2,COL1A1,Osterix(OSX),and OPN were up-regulated after osteogenic differentiation,and the ALP staining and Alizarin Red S staining were positive after osteogenic induction,indicating that hBMSCs were differentiated into osteoblast.LINC01485 and RUNX2 were gradually upregulated during this process,and miR-619-5p gradually decreased.The the mRNA level of miR-619-5p was negatively correlated with the expression of LINC01485 and RUNX2.In addition,the expression trend of LINC01485 was consistent with that of RUNX2,OCN,and OPN,suggesting that LINC01485 may be closely related to the functions of these genes.Loss-and gain-of-function experiments show that overexpression of LINC01485 promoted osteogenic differentiation of hBMSCs,up-regulating the expression of osteogenic factors RUNX2,COL1A1,OSX,OPN,and OCN and increasing ALP activity.ALP staining was increased,and Alizarin Red S staining showed increased calcium salt deposition.Conversely,interference with LINC01485 produces the opposite result.To explore the mechanism of LINC01485,FISH was performed and FISH results showed that LINC01485 is mainly expressed in the cytoplasm.Combined with bioinformatics prediction results,RAP experiment,and dual-luciferase reporter assay,LINC01485 could effectively bound to miR-619-5p,which confirmed that miR-619-5p is the target of LINC01485.MiR-619-5p loss-and gain-of-function assay results showed that miRNA mimics inhibited the expression of RUNX2,thereby inhibiting osteogenic differentiation,while miR-619-5p inhibitor promoted the expression of RUNX2 and positively regulated osteogenesis,Dual-luciferase reporter assay confirmed binding between miR-619-5p and RUNX2,Rescue assays demonstrated that miR-619-5p inhibitor restored the inhibitory effect of LINC01485 interference on osteogenic differentiation,suggesting that LINC01485 may promote osteogenesis by increasing RUNX2 expression through sponge adsorption of miR-619-5p.Conclusion:In summary,this study found that SE-lncRNA LINC01485 was up-regulated during osteogenic differentiation of hBMSCs.LINC01485 overexpression promoted osteogenic differentiation of hBMSCs,while LINC01485 interference had the opposite effect.LINC01485 is localized in the cytoplasm and acts as a sponge by binding to miR-619-5p,that is,inhibiting the function of miR-619-5p through competitive binding.miR-619-5p inhibits osteogenic differentiation of hBMSCs and binds to and inhibits RUNX2 expression.LINC01485 regulates the LINC01485/miR-619-5p/RUNX2 signaling pathway to promote osteogenic differentiation of hBMSCs by up-regulating RUNX2 expression in combination with miR-619-5p,which provides new ideas and potential targets for bone tissue engineering construction. |
PDF Full Text Request