Regulation And Molecular Mechanisms Of MiRNAs In Atrophic Bone Nonunion

ObjectivemiRNA is a group of single-strand non-coding small RNA involved extensively andclosely in regulating gene expression, but its functions in fracture healing and bonenonunion are still poorly understood. This research was to screen the different expressionof miRNAs in the repairing tissues of atrophic bone nonunion and callus tissues aroundinternal fixation plates in patients whose fractures had healed, and to predict the targetgenes with osteogenic functions of upregulated expressed miRNAs in atrophic bonenonunion through bioinformatics approach. Regulating functions and molecularmechanisms of upregulated miRNAs in atrophic bone nonunion were validated byexploring the relationship of miRNAs and related potential target genes in cell levels. Thisresearch might provide theoretical and practical basis for orthopedic and clinical researchof atrophic bone nonunion.Methods(1) The repairing tissues in atrophic bone nonunion patients (group A,3patients)and callus tissues around internal fixation plates in patients whose fractures had healed(group B,3patients) were observed. We used Exiqon miRCURY LNA microRNAmicroarrays(version11.0) to analysis and select any abnormal expressed miRNAs ingroup A. After abnormal expressed miRNAs in tissues of atrophic bone nonunion werechoosed, qRT-PCR was performed to verify the difference of upregulated miRNAexpression between two kinds of tissues,and then bioinformatic methods such ascomputational biology were used to forecast their potential target genes. By this way, wemay explore the potential pathophysiological processes of fracture healing and atrophicbone nonunion in which miRNAs might play important roles.(2) Validation of relationship between upregulated miRNAs and predicted targetgenes: Human bone marrow blood was collected, hBMSCs were separated by centrifugalization and subcultured in DMEM (dulbecco's minimum essential medium)containing10%fetal bovine serum and1/1000double antibiotics. Four kinds of syntheticdouble-stranded small RNA which imitated4upregulated miRNAs were transfected intothe fourth generation of hBMSCs, total RNA in hBMSCs was extracted after48h, andqRT-PCR, WB, dual-luciferase reporter gene assay were used to verify the relationshipbetween miRNAs and predicted target genes. By this way, we could confirm the specificroles and molecular regulating mechanisms of upregulated miRNAs in atrophic bonenonunion.(3) Expression and function research of miRNAs and related target genes inosteogenic differentiation of hBMSCs: Osteogenic culture medium was used forosteogenic differentiation. We observed the changes of corresponding miRNAs, mRNAand protein expression levels of confimed target genes in the process of osteogenicdifferentiation at different time points (0,12h,1d,2d,4d,7d,14d). By this way, we couldfurther know about the functions of upregulated expressed miRNAs in atrophic nonunion,and explore the potential of osteogenic compositions in osteogenic culture medium forclinical application of early osteogenic differentiation.Results(1) Expression of9miRNAs (hsa-miR-149*, hsa-miR-221, hsa-miR-628-3p,hsa-miR-654-5p, and5kinds of hsa-miRPluses) in group A was1.5-times-above morethan that in group B, while the expression of other9miRNAs (hsa-let-7b*, hsa-miR-220b,hsa-miR-513a-3p, hsa-miR-551a, hsa-miR-576-5p, hsa-miR-1236, kshv-miR-K12-6-5p,and2kinds of hsa-miRPluses) was1.5-times-above less. qRT-PCR showed that thechanges of4upregulated expressed miRNAs which had been verified in miRBase wereconsistent with the results of miRNA microarray. Bioinformatics analysis showed thatthere were many target spots in many osteogenic genes for these abnormally expressedmiRNAs.(2) The hBMSCs we got by subculturing were of typical character of stem cells,which could adhere the flask walls quickly, proliferated and integrated easily,anddifferentiate to osteogenic cells by induction. Our observation showed that hBMSCs were suitable to be used as seed cells for follow-up experimental studies. After syntheticdouble-stranded small RNAs which imitated the sequences of4upregulated expressedmiRNAs were transfected into hBMSCs, qRT-PCR analysis indicated that thecorresponding miRNAs expression levels increased significantly, while the mRNAexpression levels of ALPL(predicted target gene of hsa-miR-149*), PDGFA (predictedtarget gene of hsa-miR-221), BMP2(predicated target gene of hsa-miR-654-5p) decreasedsignificantly. However, the other predicted genes showed no significant increase. WBshowed that their protein expression levels were also repressed significantly. Thereafter,dual-luciferase reporter gene assay proved that ALPL, PDGFA and BMP2are directlytargeted by hsa-miR-149*, hsa-miR-221and hsa-miR-654-5p resepectively.(3) In the process of hBMSCs osteogenic differentiation, the mRNA and proteinexpression levels of osteoblastic target genes at most time points increased significantly,and the changes from2to7days were particularly significant (1～7d for ALPL protein).miRNA expression levels, mRNA and protein expression levels changed significantly atdifferent time points of osteogenic differentiation, while the trends of hsa-miR-149*andhsa-miR-654-5p changes were reverse with the trends of ALPL and BMP2mRNA andprotein expression changes respectively. The trends of hsa-miR-221change didn'tcorrelate negatively with that of PDGFA, and this still need more research.Conclusions(1) There might be a different miRNA expression profile in the reparing tissues ofatrophic bone nonunion；Four kinds of upregulated expressed miRNAs (hsa-miR-149*,hsa-miR-221, hsa-miR-628-3p and hsa-miR-654-5p) might play important roles in theoccurance and development of atrophic bone nonunion by repressing the expression levelsof many osteogenic genes.(2)Results in cell level indicated that hsa-miR-149*, hsa-miR-221andhsa-miR-654-5p directly regulate their predicted target genes ALPL, PDGFA and BMP2respectively. Upregulated miRNAs play biological roles by repressing osteogenic targetgenes ALPL, PDGFA and BMP2, which might cause the occurance and development ofatrophic bone nonunion. (3) Most osteogenic genes were upregulated at most time points in the process ofosteogenic differentiation of hBMSCs, and hsa-miR-149*and hsa-miR-654-5p expressionlevels were negatively correlated with the mRNA and protein expression levels of ALPLand BMP2respectively,indicating that the corresponding miRNA expression levels areclosely related with the regulation of mRNA and protein expression levels in the processof osteogenic differentiation.In summary, miRNAs which play key regulatory roles in the repairing tissues ofatrophic bone nonunion were found. Three abnormally upregulated miRNAs causedatrophic bone nonunion by repressing several osteogenic target genes and the expressionlevels of two miRNAs in them are closely related with the mRNA and protein expressionlevels of their target genes. This would probably provide a new idea for diagnosis andtreatment for atrophic bone nonunion.