TNF-? Inhibits Osteogenic Differentiation In BMP-2 Induced HBMSCs By Upregulating MiR-33a-5p

Purposes:TNF-? is a major cytokine responsible for bone loss in bone-related inflammatory diseases.Recently,The use of BMP-2 was proved to reduce the inflammatory response and successful in poor bone formation,but exaggerated inflammatory environment can lead to low osteoinductive efficacy of BMP-2,and several clinical cases reported an exaggerated inflammmatory response occurred after administration of BMP-2,The mechanism is not clear.MicroRNAs(miRNAs)are a class of small,evolutionarily conserved non-coding RNA molecules,involved in posttranscriptional gene silencing.miRNAs play critical roles in inflammatory process mediated by TNF-?.Some other miRNAs were also observed play important part in BMP-related bone formation process.Those miRNAs also can be novel therapeutic method to inflammatory treatment.Methods:1.hBMSCs were identified and characterized after isolated from llium boold and cultured for three weeks;To exclude the possibility of negative effect TNF-? did to hBMSCs proliferation,we used CCK-8 toevaluate the role of TNF-? in hBMSCs survival,then performed the double-staining annexin V and PI assay;2.The reliability of the miRNA array analysis with quantified the 7miRNAs levels using RT-PCR in hBMSCs;the target gene of miR-33a-5p was predicted by bioinformatic tools;3.The relationship between miR-33a-5p and SATB2 was testfied by the Luiciferase assay,and the regulation pattern of miR-33a-5p to SATB2 was showed by PCR and Western Blot;To further investigated the role of miR-33a-5p on osteogenic differentiation of hBMSCs,We monitored the expression patterns of key osteogenic differentiation markers in RT-PCR and Western Blot after transfected with miR-33a-5p inhibitor or miR-33a-5p mimic;4.To determine the relationship between TNF-?,BMP-2 and SATB2,hBMSCs were harvested at different time points or different concentration of BMP-2 or TNF-? and tested by RT-PCR and Western Blot;5.To determine what effects SATB2 may have on osteogenic differentiation in hBMSCs,the expression patterns of key osteogenic differentiation markers were tested after transfected the si-SATB2 and miRNA inhibiotr into hBMSCs;At last,We transfected SATB2 overexpression plasmid,termed p-SATB2,into hBMSCs,to test the relationship between up-regulation of SATB2 and the protein expression of Runx2;Results:1.The isolated hBMSCs positively expressed typical MSC associated markers:CD44,CD29,CD90 and CD105,but negatively expressed hematopoietic markers CD34 and CD45.The isolated hBMSCs can differentiate to osteoblasts,adipocytes and chondroblasts after induced in certaincondition;ALP staining and quantitative results showed that TNF-?inhibited osteogenesis of hBMSCs in a concentration-dependent manner,CCK-8 and double-staining annexin V and PI assay showed TNF-? has no influence on the survival and apoptosis of hBMSCs;2.QT-PCR showed only miR-33a-5p significantly induced by TNF-? and BMP-2 both in 48 h and 72 h group,SATB2 was predicted by bioinformatic tools to be potential target gene for miR-33a-5p.3.miR-33a-5p inhibited the luciferase activity of wild type SATB23'UTR reporter while has no effect on mutant type SATB2 3'UTR reporter in which the miR-33a-5p binding site was mutated.WB and RT-PCR results also showed miR-33a-5p regulate SATB2 expression by mRNA translational repression.After blocking miR-33a-5p by miR-33a-5p inhibitor,the expression levels of ALP,OCN,OPN and COL1a1 were increased both in RTPCR and WB.In contrast,hBMSCs transfected with miR-33a-5p mimic showed decreased expression of osteogenic differentiation markers.The Alizarin Red and ALP staining showed a similar tendency.4.The expression of SATB2 increased significantly in a time and dose-dependent manner with BMP-2;After added different concentration of TNF-? to BMP-2,SATB2 mRNA expression decreased in a dose-dependent manner,but SATB2 protein expression didn't change in 10 ng,20ng and 30 ng group;5.RUNX2,OPN,OCN and COL1a1 expression levels in miR-33a-5p-knockdown hBMSCs increased significantly,but after co-transfected with miR-33a-5p inhibitor and siSATB2,their expression didn ' t change;At last,we transfected SATB2 overexpression plasmid,termed p-SATB2,into hBMSCs,results indicated that up-regulation of SATB2 increased the protein expression of Runx2;Conclusion:TNF-? acts as a negative regulator of osteogenic differentiation in BMP-2 induced hBMSCs by inhancing miR-33a-5p expression,miR-33a-5p plays a central role in the regulatory network by targeting SATB2,partly though RUNX2 pathway;...