The Study Of MALAT1 And Megalin Regulating Osteogenic Differentiation In Bone Marrow Mesenchymal Stem Cells

Backgroud and Objective:The main characteristics of osteoporosis are the destruction of bone microstructure,the decrease of bone mass per unit structure and the increase of bone brittleness.Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells（hBMSCs）is essential for the human bone formation,and at present,it is the hot research direction of the pathogenesis and treatment of osteoporosis.Emerging evidence shows that long non-coding RNAs（lncRNAs）play important roles in hBMSCs osteogenic differentiation.MALAT1 is often regarded as a tumor-related lncRNA,but its function in mesenchymal stem cell differentiation remains to be defined.Osteoblast differentiation of hBMSCs is stimulated by 1α,25（OH）₂D₃ and by 25（OH）D₃;the latter’s effects require intracellular conversion to 1α,25（OH）₂D₃ by 1α-hydroxylase/CYP27B1.Thus,hMSCs are both a source of and target for 1α,25（OH）₂D₃.Megalin is a transmembrane receptor for serum D binding protein（DBP）in renal tubular epithelial cells and is required for their uptake of the 25（OH）D-DBP complex.In this study,we aimed to investigate the function and specific mechanism of MALAT1in regulating osteogenic differentiation of hBMSCs and the expression of megalin and its function in Vitamin D regulating hBMSCs osteogenic differentiation.Methods:1.hBMSCs from patients undergoing total hip arthroplasty were collected and were divided into osteoporosis group and non-osteoporosis group.Real-time PCR was used to detect the expression of MALAT1 and miR-143 in the two groups.The expression of MALAT1 was specifically knocked down by sh-RNA technique and its effect on osteogenic differentiation of hBMSCs was detected.Double luciferase reporter gene assay was used to verify the downstream targeting binding of MALAT1 to microRNAs and the targeting binding of microRNAs to mRNAs.2.hMSCs from surgically discarded marrow were used for gene expression analysis by RT-PCR.hBMSCs were divided into hBMSCs expressing high megalin（hi-meg）and low megalin（lo-meg）.The specific experimental methods include the following:RT-PCR for effects of 25（OH）D₃ and 1α,25（OH）₂D₃,ELISA kit for biosynthesis of1α,25（OH）₂D₃,alkaline phosphatase（ALP）enzyme activity for osteogenic differentiation,and transient transfection of siRNA for megalin（si-Meg）and control（si-Ctr）.Results:1.Firstly,we found that the expression of MALAT1 was much lower in hBMSCs from osteoporosis patients and miR-143 was contrarily higher.In addition,MALAT1expression increased,and miR-143 decreased when hBMSCs were treated with osteogenic induction.Then,we used short hairpin RNAs to knockdown MALAT1,and the results showed that hBMSCs osteogenic differentiation decreased significantly,indicating that MALAT1 is a positive regulator of osteogenic differentiation in hBMSCs.Furthermore,by luciferase assays,we found that MALAT1 could directly bind to miR-143 and negatively regulate its expression.Similarly,miR-143 could directly bind to the target site on the Osx 3’-UTR and then inhibit Osx expression.Knockdown of MALAT1 decreased Osx expression,and co-transfection of miR-143inhibitor could rescue Osx mRNA expression.While Osx expression was increased in MALAT1-overexpressing hBMSCs,it was reversed by the miR-143 mimics.Moreover,Osx silencing decreased ALP,OCN,and OPN expression induced by the miR-143inhibitor.2.Gene expression analysis with hMSCs from 15 subjects showed a wide range of constitutive expression of megalin.In hi-Meg hMSCs,both 1α,25（OH）₂D₃（10 nM）and25（OH）D₃（100 nM）induced osteoblast signature genes Runx2 and ALP,and stimulated ALP activity（p<0.05）,but only 1α,25（OH）₂D₃ did so in lo-Meg hMSCs（p<0.001）.In si-Ctr cells,both 1α,25（OH）₂D₃ and 25（OH）D₃ increased expression of Runx2,ALP,and BSP,and stimulated ALP activity（p<0.05）,but only 1α,25（OH）₂D₃did so in si-Meg cells（p<0.05）.Similarly,25（OH）D₃ induced target gene CYP24A1 in si-Ctr cells,but was blocked in si-Meg cells.In addition,biosynthesis of 1α,25（OH）₂D₃was significantly lower in lo-Meg（46%,p=0.034）and in si-Meg（23%,p<0.001）,compared with each control.Leptin at 100 ng/mL concentration significantly stimulated megalin expression 2.1-fold in lo-Meg cells（p<0.01）.Conclusion:Altogether,our findings suggest that MALAT1 acts to regulate Osx expression through targeting miR-143 to promote hBMSCs osteogenic differentiation.Megalin is expressed in hBMSCs and is required for their biosynthesis of 1α,25（OH）₂D₃,and for the 25（OH）D-DBP complex to stimulate VDR targets and osteoblastogenesis in hBMSCs.