Effect And Mechanism Of LncRNA H19 In BMP9 Induced Osteogenesis And Angiogenesis Differentiation Of MSCS

Objective:To explore the effect and mechanism of lncRNA H19 in BMP9 induced osteogenesis and angiogenesis of MSCs,and provide theoretical and experimental evidence for the construction of BMP9mediated tissue engineering bone.Methods:The recombinant adenoviruses AdGFP,AdBMP9,AdsimH19 were constructed by AdEasy technology.Adenoviruses were amplified in HEK293 cells in vitro.Immortalized mouse embryonic fibroblasts were cultured in vitro and infected with adenoviruses,which were divided into three groups:AdGFP,AdBMP9,AdBMP9+AdsimH19.After iMEFs cells were successfully infected with adenovirus,protein and mRNA was extracted at 3 days,5 days and 7 days respectively and then subjected to Western blot and qPCR analysis.The expressions of molecular markers related to osteogenic and angiogenic differentiation and Notch signal as the key regulatory pathway of angiogenesis were detected.Three and five days after iMEFs cells were successfully infected with adenovirus,the expression of vascular endothelial molecular markers was analyzed by flow cytometry.As for in vivo test,six healthy male nude mice at 2 weeks were selected and divided into three groups of AdGFP,AdBMP9,AdBMP9+AdsimH19.Among them,1 in AdGFP group,3 in AdBMP9group and 2 in AdBMP9+AdsimH19 group.The iMEFs cell suspension successfully infected with adenovirus was injected subcutaneously into nude mice.The number of cells was about 10~6 cells/100ul.The nude mice were sacrificed under anesthesia at 3w,4w and 5w respectively,and the subcutaneous ectopic bony mass was collected.In the 4th week,only the osteogenic mass of AdBMP9 group was collected.All the collected osteogenic masses were analyzed by micro-CT;then all ectopic masses were embedded in paraffin and sliced,paraffin sections were stained with HE and immunohistochemical staining were used for detecting blood vessel formation markers,and the images were recorded and analyzed by microscope.Results:BMP9 could promote the osteogenic differentiation of MSCs,and significantly up-regulated the expressions of VEGFa and CD31 at the mRNA and protein levels;silencing H19 resulted in a decrease in the volume of ectopic bony mass induced by BMP9,and significantly down-regulated the expressions of VEGFa and CD31 at the protein level;silencing H19 significantly down-regulated the expressions of NICD1 and the downstream target genes Hey1,Hey2 and Hes7 at mRNA level.Conclusion:BMP9 hold the potential to induce osteogenic differentiation and angiogenesis of MSCs at the same time,and BMP9induced angiogenesis is combined with BMP9 induced bone formation;lncRNA H19 regulates BMP9 induced osteogenic differentiation of MSCs through angiogenesis signal;lncRNA H19 regulates BMP9 induced osteogenic and angiogenic differentiation of MSCs by influence the expression of Notch signaling pathway.