Role Of TLR2/TLR4 Activation Related MicroRNA In Migration And Differentiation Of Human Bone Marrow Mesenchymal Stem Cells

Background and objective:Mesenchymal stem cells (MSCs) is a kind of stem cells display self-renewal and a multipotent differentiation potential. Toll-like receptors (TLRs) is an important class of protein molecules involved in innate immunity and acquired immunity. Our previous work demonstrated TLR2 and TLR4 expression in the surface of bone marrow mesenchymal stem cells (BM-MSCs) and can; adjust their migration, differentiation and other biological functions. microRNA (miRNA) are a class of 20-22 nucleotide long RNA molecules that can cause degradation of the target mRNA or translational inhibition, thus inhibiting protein synthesis. In recent years, studies have shown that miRNA can participate in differentiation, proliferation, migration, survival and paracrine series of important biological processes of MSCs. TLR signaling has been demonstrated to modulate the expression of miRNA, whereas miRNA also as an important regulator of TLR signaling in many cell types. Thus, TLR activation related miRNA more likely involved in the regulation of migration and differentiation of MSCs. Our previous work confirmed miR-27b, miR-146a, miR-155 and miR-154 were TLR activation related, suggesting that miR-27b, miR-146a, miR-155 and miR-154 may participate in migration and differentiation of MSCs. In view of this, we study the role of miR-27b, miR-146a, miR-155 and miR-154 in migration and differentiation of BM-MSCs.Methods:BM-MSCs was isolated and cultured from healthy volunteers, after transfected with mimics or inhibitor of miR-27b, miR-146a, miR-155 and miR-154, qRT-PCR (quantitative real-time polymerase chain reaction) were detected expression of miR-27b, miR-146a, miR-155 and miR-154 respectively. Cell migration assay observing the role of miR-27b, miR-146a, miR-155 and miR-154 were in cell migration. Targetscan version 6.2, Miranda, Microcosm Targets Version 5 and gene ontology were used to analysis target genes of miR-155 and dual luciferase reporter gene experiments was used to confirm. Then use Western blotting detection the expression of target gene corresponding protein to further confirm. Study on differentiation of BM-MSCs into adipocytes and osteoblasts cells after transfection were done in vitro. Fuction of miR-27b, miR-146a, miR-155 and miR-154 in the adipogenic and osteogenic differentiation was measured.Results:Expression of miR-27b, miR-146a, miR-155 and miR-154 were significantly up-regulated after transfection with their mimics and down-regulated after transfection with their inhibitors. Transfection with miR-155 mimics significantly suppressed the migration of BM-MSCs. While cell migration was enhanced after transfection with miR-155 inhibitors, indicating that miR-155 inhibits cell migration. Target gene prediction software prediction and gene ontology analysis obtained MYLK may be the target genes miR-155. Dual luciferase reporter showed that miR-155 inhibited the fluorescent activity of MYLK dual luciferase reporter vector, thus confirming the MYLK was miR-155 target genes. Furthermore, western blot analysis showed MYLK protein expression was significantly down-regulated after transfection with miR-155 mimics and up-regulated after transfection with miR-155 inhibitors, indicating that miR-155 inhibits cell migration by targeting MYLK. Cell migration was no significant difference after transfected with miR-27b, miR-146a and miR-154 mimics or inhibitor, indicating that miR-27b, miR-146a, miR-154 had no significant effect on cell migration. In differentiation experiments, transfected miR-146a mimics can promote adipogenic differentiation of BM-MSCs while transfected miR-146a inhibitor inhibits adipogenic differentiation, suggesting that miR-146a promote adipogenic differentiation. Adipogenic differentiation was no significant difference after transfected with miR-27b, miR-155, miR-154 mimics or inhibitor, indicating that miR-27b, miR-155, miR-154 had no significant effect on adipogenic differentiation. In addition, osteogenic differentiation was no significant difference after transfected with miR-27b, miR-146a, miR-155, miR-154 mimics or inhibitor, indicating that miR-27b, miR-146a, miR-155 and miR-154 had no significant effect on osteogenic differentiation.Conclusion:1. miR-155 inhibit migration of BM-MSCs by targeting MYLK; miR-27b, miR-146a, miR-154 had no significant effect on cell migration.2. miR-146a can promote adipogenic differentiation of BM-MSCs; miR-27b, miR-155, miR-154 had no significant effect on adipogenic differentiation.3. miR-27b, miR-146a, miR-155 and miR-154 had no significant effect on osteogenic differentiation of BM-MSCs.