| Background and significance: Aging will lead to a steady increase in bone defects,which will lead to osteoporosis,fracture and other diseases.However,its pathogenesis has not been fully elucidated,and the treatment of bone defects related diseases is still not ideal,which further restricts the development of bone regeneration.In recent years,studies on exosomes and microRNAs have developed rapidly,bringing new perspectives to the field of bone defects and bone regeneration.Our group previously found that when mouse Bone marrow macrophage exosomes were co-cultured with Bone marrow-derived mesenchymal stem cells(BM-MSCs),exosomes could be absorbed by BM-MSCs.The effect of bone marrow macrophage-exosomes on BM-MSCs differentiation was different in mice of different ages.In order to explore the molecular mechanism,microRNAs from exosomes derived from bone marrow macrophages of elderly mice and young mice were sequenced on chip,and 21 microRNAs were found to have different expression levels.This study further explored and predicted the role and possible mechanism of21 differentially expressed microRNAs previously discovered,in order to further reveal the way that aging affects bone properties,and did not provide a theoretical basis for the clinical application of bone marrow macrophage exosome-microRNAs in bone regeneration.Research objective: To verify the expression differences of human bone marrow-macrophage exosomal microRNAs before and after osteogenic and lipid differentiation of BM-MSCs,preliminarily clarify the regulatory role of human microRNAs on the differentiation of BM-MSCs,screening microRNAs with further research value and predict their downstream target genes,and provide a basis for subsequent studies.Research methods: The primary BM-MSCs were extracted by modified whole bone marrow adherent method,and the surface antigens of BM-MSCs were identified by Flow cytometry(FCM).The BM-MSCs were induced to osteogenic and lipid differentiation,and Alkaline phosphatase(ALP)was used.ALP staining was used to detect osteoblast formation,Alizarin Red S(ARS)staining was used to detect calcified nodules,and oil Red O(ORO)fat staining was used to detect lipid droplets.Real-time quantities Polymerase chain reaction(RT-q PCR)was used to quantitatively detect osteogenic markers ALP and Runt-related transcription factor 2(RUNX2)of BM-MSCs.Bone Morphogenetic protein-2(BMP-2)and Osteocalcin(OCN),And lipid-differentiation marker peroxisome proliferator-Activated receptor γ(PPARγ),Adipocyte lipid binding protein(ALBP),CCAAT/enhancer binding protein(C/EBP),and microRNAs differentially expressed after osteogenesis and lipid formation were detected by tail-added RT-q PCR.Four databases,Target Scan,miRWalk,DIANA and miRDB,were used to predict downstream target genes of differentially expressed microRNAs.Results:(1)FCM showed that the primary BM-MSCs were positive for CD105 and CD73,but negative for CD34,CD45 and HLA-DR,which met the identification criteria of BM-MSCs;(2)After osteogenic induction of BM-MSCs,ALP staining was positive,ARS staining showed calcium nodules,and the relative expression levels of ALP,RUNX2,BMP2 and OCN were significantly increased.ORO staining showed the formation of lipid droplets,and the relative expression levels of PPARγ,ALBP and C/EBP were significantly increased.(3)The expression levels of miR-767-5p,miR-211-3p,miR-380-3p,miR-193B-3p and miR-25-3p did not change significantly before and after osteogenic differentiation of BM-MSCs.The expression levels of miR-671-5p,miR-488-5p,miR-493-5p,miR-532-3p,miR-489-5p,miR-342-3p and miR-130b-3p increased significantly after differentiation.The expression levels of miR-382-5p and miR-670-5p decreased significantly after differentiation.The expression levels of miR-130b-3p,miR-25-3p,miR-671-5p,miR-382-5p,miR-493-5p,miR-767-5p and miR-211-3p did not change significantly before and after adipogenic differentiation of BM-MSCs.The expression levels of miR-193B-3p,miR-488-5p,miR-532-3p,miR-489-5p,miR-342-3p and miR-670-5p were significantly increased after differentiation.The expression of miR-380-3p decreased significantly after differentiation.The differences were statistically significant(P <0.05).Among them,miR-532-3p,miR-342-3p,miR-488-5p,miR-489-5p,miR-493-5p,miR-193B-3p,miR-382-5p and miR-670-5p were consistent with the results of the chip;(4)Further target gene prediction results showed that miR-382-5p and miR-670-5p may regulate BM-MSCs differentiation by targeting TOP1,MAPRE2,PDLIM5 and BRWD1 genes.Conclusions:(1)The expression levels of exosome microRNAs derived from bone marrow macrophages were significantly different before and after osteogenic and lipid differentiation of BM-MSCs;(2)The expression levels of miR-382-5p and miR-670-5p in bone marrow macrophage exosomes increased with age,which may inhibit the osteogenic differentiation of BM-MSCs and promote the adipogenic differentiation of BM-MSCs,thereby aggravating bone marrow adipose and increasing bone loss;(3)This effect may be realized by targeting TOP1,MAPRE2,PDLIM5 and BRWD1 genes,which is worthy of further exploration in future studies. |
