| Objective: Mesenchymal stem cells(MSCs)are adult stem cells that have the potential for self-renewal and differentiation.They are widely available and can be obtained in various tissues and organs.Toll like receptors(TLRs)are a kind of natural immune receptor,which plays an important role in the process of inherent immunity and acquired immunity.The researches found that TLRs can participate in the regulation of MSCs migration,differentiation and cytokine secretion and other biological functions.Micro RNAs(mi RNAs)are a small RNA of about 19~23 nucleotides,which negatively regulate a target gene by binding to a target gene,resulting in the degradation or translational inhibition of the target gene.Mi RNA involved in regulating a variety of biological characteristics of MSCs,such as proliferation,migration,differentiation,apoptosis and others.In many cell types,TLRs have been shown to regulate the expression of mi RNAs,whereas mi RNAs can serve as important regulators of TLR signaling.Therefore,we believe TLRs related mi RNAs are more likely to regulate the biological characteristics of MSCs.Our group previously confirmed that BM-MSCs express TLR2 and TLR4 and that BM-MSCs stimulated by Pam3CSK4(TLR2 agonist)or LPS(TLR4 agonist)can influence the migration,differentiation,cytokine secretion and hematopoietic support of BM-MSCs.We used Illumine high-throughput sequencing to detect intracellular micro RNA expression profiles of BM-MSCs before and after Pam3CSK4 or LPS stimulation.Based on the results,we selected mi R-99a-5p and mi R-425-5p,which were significantly changed after stimulation,and explored the effect of these two mi RNAs on the biological characteristics of BM-MSCs,such as proliferation,differentiation and cytokine secretion.Methods: The BM-MSCs of healthy volunteers were isolated and cultured.The cells were transfected with mi R-99a-5p and mi R-425-5p mimcs or inhibitor.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect transfection efficiency.The effects of mi R-99a-5p and mi R-425-5p on the proliferation,differentiation and cytokine differentiation of BM-MSCs were detected by proliferation assay,differentiation assay and enzyme-linked immunosorbent assay(ELISA).Results: Compared with the control group,the corresponding mi RNA expression could be significantly increased after transfection of mi R-99a-5p and mi R-425-5p mimcs(P<0.0001).On the other hand,after transfection of mi R-99a-5p and mi R-425-5p inhibitor,the corresponding expression of mi RNA was down-regulated(P<0.0001).After alizarin red stained,we observed in inverted microscope,showing that overexpression of mi R-99a-5p can promote the osteogenic differentiation,while down-regulation of mi R-99a-5p expression can inhibit the osteogenic differentiation.The same results were obtained by microplate Readere detection of spectrophotometer.However,in the adipogenic differentiation assay,transfection of mi R-99a-5p mimcs or inhibitor did not significantly affect adipogenic differentiation of BM-MSCs.In addition,overexpression or low expression of mi R-425-5p had no significant effect on adipogenic and osteogenic differentiation of BM-MSCs.The results of EILSA suggested that the concentration of TGF-?1 secreted by BM-MSCs was significantly higher than that of IL-1? and GM-CSF.Overexpression of mi R-99a-5p promoted the secretion of TGF-?1 by BM-MSCs(P <0.05),but the low expression of mi R-99a-5p did not affect the secretion of TGF-?1.Overexpression of mi R-425-5p inhibited GM-CSF secretion(P <0.05),while low expression of mi R-425-5p had no significant effect on GM-CSF.Conclusion: 1.mi R-99a-5p,mi R-425-5p had no significant effect on BM-MSCs proliferation.2.Overexpression of mi R-99a-5p promoted osteogenic differentiation of BM-MSCs,but had no significant effect on adipogenic differentiation.3.Overexpression of mi R-99a-5p promoted the secretion of TGF-?1 by BM-MSCs,and the over-expression of mi R-425-5p inhibited the secretion of GM-CSF by BM-MSCs,indicating that mi R-99a-5p and mi R-425-5p affect BM-MSCs cytokine secretion.4.TLR2/4 related mi RNA has certain influence on the biological characteristics of BM-MSCs. |
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