Study Of LINC00707 Regulating WNT2B Via MiR-370-3p On Osteogenesis Of Human Bone Marrow Mesenchymal Stem Cells

Research BackgroundStem cell technology is a technology that takes tissue regeneration as the core content and repairs tissue defects through the multi-directional differentiation ability of stem cells.Human marrow-derived mesenchymal stem cells are a type of stem cells that can be induced to differentiate into cartilage tissue,bone tissue and adipose tissue under certain inducing conditions.Our previous study showed LINC00707 was dramatically increased in the process of osteoblastic differentiation,based on the bioinformatics analysis and previous research miR-370-3p might be regulated by LINC00707.LINC00707 and miR-370-3p have not been reported in the literature to interact with each other.Research purposesBy combining literature review,bioinformatics,online database analysis and a variety of molecular biology techniques,the expression of LINC00707 and miR-370-3p in the human marrow-derived mesenchymal stem cells and downstream gene WNT2B expression levels were analyzed experimentally,combined with endogenous regulate the mechanism of competition mechanism to form the theory that LINC00707/miR-370-3p/WNT2B axis regulates osteogenic differentiation of human marrow-derived mesenchymal stem cells and provide a scientific basis for clinical periodontal tissue repair of human marrow-derived mesenchymal stem cells.Research methods1.Effect of LINC00707 on osteogenic differentiation of human marrow-derived mesenchymal stem cells2.LINC00707 and miR-370-3p endogenous competitive regulation of human marrow-derived mesenchymal stem cells osteogenesis mechanism3.The molecular mechanism of LINC00707/miR-370-3p/WNT2B signal axis in promoting osteogenic differentiation of human marrow-derived mesenchymal stem cellsResearch result1.Effect of LINC00707 on osteogenic differentiation of human marrow-derived mesenchymal stem cells(1)Osteogenic induction and differentiation of hBMSCs were induced by osteoinductive medium.The results showed that the content of LINC00707 gradually increased during osteogenic differentiation of hBMSCs.the expression of LINC00707 were positively correlated.with the levels of osteoblast differentiation-related genes.(2)LINC00707 was transfected by overexpression of lentivirus and interference plasmid.The results showed that the expression of RUNX2,OCN and ALP in the hBMSCs transfected with LV-LINC00707 were significantly up-regulated,indicating that the level of LINC00707 expression directly affects the osteogenic differentiation of hBMSCs.2.LINC00707 and endogenous miR-370-3p competitive regulation of human marrow-derived mesenchymal stem cells osteogenic differentiation molecular mechanism(1)Bioinformatics and online database analysis predicted that LINC00707 could combine with the binding site of miR-370-3p and then directly generate the interaction.(2)Luciferase reporter assay and AG02-RIP assay demonstrated that LINC00707 could combine with the binding site of miR-370-3p to form a complex,which in turn affected the expression of downstream target genes.(3)Transfection of LINC00707 with overexpression lentiviral vector and interference plasmid showed that,the level of miR-370-3p in LINC00707 and miR-370-3p was negatively correlated in the process of osteogenic differentiation of hBMSCs,indicating a competing role between the two.(4)The plasmid transfected miR-370-3p,further demonstrating that miR-370-3p can negatively regulate the expression of RUNX2,OCN and ALP in osteogenic differentiation-related genes.(5)The co-transfection of LINC00707 with miR-370-3p resulted in that LINC00707 competitive inhibition of miR-370-3p hBMSCs osteogenic differentiation.(6)The results of co-transfection of sh-LINC00707 with miR-370-3p inhibitor matched the results of co-transfection of LINC00707 with miR-370-3p.3.The molecular mechanism of LINC00707/miR-370-3p/WNT2B signaling axis promoting osteogenic differentiation of human marrow-derived mesenchymal stem cells(1)miR-370-3p,which is predicted by bioinformatics and online database analysis,can target the expression of WNT2B.Targeted regulation of miR-370-3p and WNT2B was verified by luciferase reporter assay.(2)WNT2B was significantly decreased(P<0.05)after transfected with miR-370-3p in hBMSCs Correlation analysis showed that there was a positive correlation between the expression level of LINC00707 and target gene WNT2B in osteogenic differentiation of hBMSCs,while the expression of miR-370-3P and target gene WNT2B were negatively correlated.(3)Transfection of WNT2B with small interfering RNA respectively showed that the expression of RUNX2,a key osteogenic differentiation protein,was down-regulated after WNT2B was successfully disrupted in hBMSCs.However,ALP staining and ARS staining showed that the ability of hBMSCs to differentiate into osteoblasts was significantly inhibited by interfering with the expression of WNT2B.ConclusionThrough a series of experiments,we successfully validate the hypothesis proposed by this research.LINC00707 can competitively reverse the inhibitory effect of miR-370-3p on WNT2B and promote the expression of WNT2B in osteogenic differentiation of hBMSCs,thereby promoting the expression of RUNX2,OCN and ALP expression eventually promote the osteogenic differentiation of hBMSCs.