MicroRNA Regulates Osteogenesis Of BMSCs By Targeting Id1

[Background]Although Bone morphogenetic protein 2 (BMP2) promote early osteogenic differentiation of BMSCs in vitro, BMP2 gene-modified BMSCs show poor terminal osteogenesis in vitro and insufficiently yield functional and mechanically competent bone in vivo. The alteration of inhibitor of DNA binding or differentiation(Id) family’s expression in BMSCs during differentiation is the key process that guarantees successful differentiation of BMSCs toward an osteogenic lineage.It’s found that there is significant difference in some microRNAs expression level between BMP2 gene-modified BMSCs with BMSCs through microRNA chip screening, which is coincided with the same microRNAs which are likely to regulate the expression of Idl by means of bioinformatics prediction.[Purpose]State the effect on the differentiation of BMSCs osteogenesis in the environment of high expression of BMP2 in vitro and the change of Idl, verify Idl factor plays an important part in the differentiation of BMSCs development in rat, to prove Id expression level is the inhibition of BMP2-modified BMSCs osteogenetic differentiation and explore the BMP2 regulatory mechanism of Idl; to screen and identify microRNAs targeting in Idl, and prove it may be a new molecular mechanism that the change of some specific miRNAs expression promotes BMP2 increases Idl express, which will provides a new research strategy for maxillofacial bone defects repair.[Materials and methods]1. Mononuclear cells from autogenous animal bone marrow had strong proliferative ability when cultured in plastic culture flask in vitro and would amplified in short period, which finally were induced to BMSCs with high purity and quantity. Under some specific conditions,BMSCs could differentiate into osteoblast and adipocyte.2. The prime was designed and synthetised according to Id1 mRNA sequences in Genbank, then PCR amplified genes. BMP2 gene was constructed into recombinant adenovirus expression vector by AdMax vector system(Ad-BMP2). Purified recombinant adenovirus and determined for virus titer, Ad-BMP2 transfected BMSCs, drawn the cell growth curve to test the affection of virus to cell proliferation, MOI=40, Ad-BMP2 and Ad-GFP transfected BMSCs,and collected BMSCs at different time points including Ad-BMP2-BMSCs, the Ad-GFP-BMSCs, BMSCs, and then Real-TimePCR detected the purpose gene(Id1) level.3. Screening microRNAs targeting Id1, with the application of microRNAs online database, to predict the microRNAs that could regulate the expression of Id1. Through the microRNA expression profile chip and related technology selection, the outcome and the prediction was analyzed, and then Real-TimePCR detected the changed microRNA to screening the target microRNA.[Result]1. BMSCs induced by osteogenesis grew slowly relatively, and cell shape was irregular, rendering polygon osteoblast form. A small amount of dotted red mineralized nodule was tested by Alizarin S Red staining after 7 days’ induction while the mineralized nodule was much more obvious after 14 days’.2. BMSCs with adipogenic induce supplements were in relatively flattened shape, and became slow slightly in cell proliferation. Oil Red O staining of the BMSCs after 14 days of culture demonstrated numerousintracellular lipid droplets.3. After Ad-BMP2 transferred into BMSCs 48 hours, cells green fluorescent protein expression was tested by fluorescence microscope. According to the expression of green fluorescent observation to determine the best MOI viral transfection is 40, and the transfection efficency could be 95%.4. Adenoviruses had no effect on cell proliferation after transfection, there was no significant difference between the number of groups of proliferation(P>0.05).5. According to microRNA expression profile chip and the related technology, it’s found that nearly 123 microRNA reduced and 114 microRNAs raised. Analyzing the results of the chip screening and the predicted results, we found miRNA-338、miRNA-195 and miRNA-98 changed, one of which raised and two of reduced.[Conclusion]1. Bone marrow mononuclear cells were separated by density gradient centrifugation which can be induced to BMSCs under certain conditions. This method can obtain the high quality BMSCs.2. Osteogenic and adipogenic differentiation capability of BMSCs can be identified by the cell function test, respectively.3. Ad-BMP2 recombinant adenovirus expression vector, with high drop degree and transfection efficiency, as a stable and effective gene expression vector, could make BMSCs after transfection over express BMP2, and the best MOI values to 40.4. Both the predicted results and the chip screening were consistent:the miRNA-338 increased, the miRNA-195 and the miRNA-195 reduced.