Experimental Research Of MicorRNA-146b-5p On The Biological Function

Objective:To explore the effect of micro RNA-146b-5p(mi R-146b-5p)on the ability of murine bone marrow derived monocytes develop into mature of dendritic cells(DC)and murine compact bone derived-mesenchymal stem cells(MSC)differentiate into adipocytes.Methods:(1)We isolated CD11b+ monocytes from the bone marrow of C57BL/6 mouse throughimmunomagnetic beads,after 5 days or 7 days induction,the immature dendritic cells(i DC)or mature dendritic cells(m DC)were obtained and then the cell phenotypes was analyzed by flow cytometry.Through the Chip analysis of monocytes,i DC and m DC,we screened for mi RNAs highly expressed during the DC maturation.QPCR results showed that the expression of mi R-146b-5p is remarkable changed.We transfected mi R-146b-5p mimics or inhibitors into the CD11b+ monocytes and examined cell phenotypes by flow cytometry.Furthermore,the function of mi R-146b-5p during the DC maturation was analyzed by phagocytosis experiment and mixed leukocyte reaction(MLR).(2)Meanwhile,MSCs were isolated from the murine compact bone,and analyzed the surface markers by flow cytometry.We validated the expression levels of mi R-146b-5p by quantitative reverse transcriptase polymerase chain reaction(q-PCR)in adipogenic differentiation.Subsequently,the synthetic mimics and inhibitors of mi R-146b-5p were transfected into murine compact bone derived MSCs and the mi RNAs were PCR-amplified on a real time PCR system using the specific mi R-146b-5p primer.To assess the effect of miR-146b-5p on the adipogenic differentiation for murine MSCs,we transfected mi R-146b-5p mimics or inhibitors into the murine MSCs.After 14 days culture,the transfection MSCs was induced adipogenic differentiation,and induction ability was determined by Oil-red-O staining.The key adipocyte transcription factor of C/EBPα and PPARγ for murine MSCs was detected by q-PCR,and the protein expression of PPARγ was displayed by western blot.Results:(1)We isolated the murine bone marrow CD11b+ monocytes through immunomagnetic beads,and induced i DC and m DC differentiation.By using flow cytometry,we further analyzed DC phenotypes.i DC exhibited increased CD11 b expression level and decreased CD80,CD86,CD11 c,MHCⅡ expression level;m DC exhibited decreased CD11 b expression level and increased CD80,CD86,CD11 c,MHCⅡ expression level.According to Mi RNA Chip Analysis,we found mi R-146b-5p,mi R-21a-5p,mi R-690,mi R-223-3p,mi R-155-5p,mi R-107-3p,mi R-1224-5p and mi R-5112 highly expressed during DC maturation.Furthermore,q PCR results indicated that mi R-146b-5p exhibit remarkable change.The expression of mi R-146b-5p was gradually reduced in the murine bone marrow CD11b+ monocytes derived DC maturation(p<0.01).We transfected CD11b+ monocyteswith mi R-146b-5p mimics or inhibitors and found that the modulation of mi R-146b-5p on DC maturation was dose-dependent.Meanwhile,the phagocytic capacity of the monocytes was reduced when mi R-146b-5p expression was blocked,exhibiting m DC phenotypes.These data suggest that mi R-146b-5p inhibitor is capable of promoting DC maturation.Moreover,MLR experiment of mi R-146b-5p mimics can decrease DC stimulate T cell proliferation,strongly support the conclusion that mi R-146b-5p inhibits DC maturation.(2)Additionally,MSCs were isolated from C57 mice by cultivating the digested compact bone.These cells exhibited the typical fibroblast-like cells morphology;the cell was positive for menchymal phenotypes for CD29 and CD105,stem cell phenotype for Sca-1;negative for endothelial marker for CD31,hematopoietic marker for CD34,CD45,and CD11 b,major histocompatibility complex marker for MHCⅡ and CD140 a.The expression of mi R-146b-5p in adipogenic differentiation of murine compact bone derived MSC was measured by q-PCR.The results showed that mi R-146b-5p expression could significantly increase in a dose-dependent manner,but day 7 was not upregulated(p<0.01).To identify the role of mi R-146b-5p in regulating MSC property,we transfected mi R-146b-5p mimics or inhibitors into murine MSC.The results determined that the expression levels of mi R-146b-5p in murine MSC was dramaticlly increased or decreased(p<0.001),comparing with control.In adipogenic culture system,quantity of adipocytes and lipid droplets were significantly increased in MSC transfected with mi R-146b-5p mimics,the key transcript factor for adipocyte C/EBPα and PPARγ was greatly up-regulated(p<0.01),and the protein expression of PPARγ was also oberviously increased by western blot.Conversely,MSC transfected with mi R-146b-5p inhibitor was observed inverse phenomenon,which the quantity of adipocytes and lipid droplets were significantly decreased,the key transcript factor for adipocyte C/EBPα and PPARγ was significantly down-regulated(p<0.05),and western blot analyzed that PPARγ protein was lower expression.These data suggest that mi R-146b-5p could regulate the adipogenic differentiation of MSC.Conclusion:Mi R-146b-5p inhibits murine bone marrow derived CD11b+ monocytes differetiation and block the process of DC maturation.MSC-transfected mi R-146b-5p mimics promoted adipogenic differentiation.In summary,our findings reveal a novel function for mi R-146b-5p in the DC maturation and adipogenic differentiation of MSC.