The Mechanism Of Osteoblast Differentiation And Dysfunction Involved In MiR-143

BackgroundBone formation is directly regulated by the number and function of osteoblasts and osteoclasts[1-3]Osteogenesis is under the regulation of several signaling pathways,including the transforming growth factor beta（TGF-β）pathway and the receptor activator of the NF-κB ligand（RANKL;also known as TNFSF11）,the receptor activator of the NF-κB（RANK）pathway[4,5].which involves several transcription factors,such as Runt-related transcription factor 2（Runx2）,nuclear factor of activated T cells（NFAT）and Osterix（Osx）[6,7].Runx2 is a vital determinant of osteoblast differentiation,and its inactivation delays osteoblast differentiation,whereas the low-density lipoprotein receptor-related protein 5（LRP5）signaling pathway controls osteoblast proliferation.Therefore,Runx2 is the key to osteoblastic bone formation and is accurately regulated by a set of mechanisms,such as Osterix,which acts downstream of Runx2.However,the regulation of Runx2activation on osteoblast differentiation remains to be determined.MicroRNAs（miRNAs）are single-stranded noncoding²2 nucleotide small RNAs that are involved in the regulation of gene processing and biological processes and regulate mRNA expression by binding 3’-untranslated regions（3’-UTRs）[8-11].A series of miRNAs have been identified to participate in osteogenic differentiation and osteoblastic bone formation.The deletion of Dicer alleles in mice at different stages of bone formation has confirmed that miRNAs control every step of osteogenesis,such as miR-214 inhibition of bone formation by targeting ATF4[12,13].However,most of these miRNAs have only been characterized in vitro,and their roles in physiological and pathophysiological mechanisms of bone loss in skeletal disorders remain to be elucidated.Histone deacetylases（HDACs）are a family of enzymes that are involved in gene transcriptional regulation by removing acetyl groups from lysine residues on histones[14-16].Several HDACs have been reported to contribute to bone development.For example,class II HDACs repress Runx2 function through Smad3[17],HDAC7 and Runx2 have been found to be colocalized in nuclei;and HDAC7 is associated with Runx2 in osseous cells.Furthermore,HDAC7 represses Runx2 transcriptional activity in a deacetylase-independent manner and suppresses osteoblast maturation.¹⁸ However,the interaction between HDACs and miRNA in the regulation of osteoblast differentiation is still under rigorous investigation.In this study,we screened the miRNAs that are important in osteogenic differentiation by miRNA sequencing analysis between pre-osteoblast MC3T3-E1 cells and osteoblast,and found that miR-143 is the most important miRNA in osteoblast differentiation.Furthermore,we found that the expression levels of miR-143 in serum from aged patients with fractures were lower than those of younger patients.Therefore,we generated miR-143 knockout mice using CRISPR/Cas 9 technology and found that bone formation was inhibited in miR-143knockout mice.The administration of agomiR-143 in vivo promoted osteoblastic bone formation and prevented bone loss in age-related osteoporosis.Therefore,we focused on the roles of miR-143 in bone formation in this study.ObjectiveMiRNAs can play an active and effective role in cardiovascular disease,immune system diseases,tumor-related diagnosis and treatment.However,miR-143 has not been reported for osteoblast differentiation and bone formation.We mainly focus on the studies of miR-143 on osteoblast differentiation and bone formation,and its related mechanisms.In this study,we first cultured osteogenic precursor cells MC3T3-E1 and osteoblasts after osteogenic induction medium.High-throughput sequencing was used to screen out keymiRNAs.Secondly,we verified whether osteoblasts were delayed or promoted osteogenic differentiation by miRNA inhibitors and mimics in vitro,and constructed knockout mice in vivo.Finally,it is verified by regular injection of agomiRNA whether it has a therapeutic effect on osteoporosis.Part Ⅰ:Screening and identification of key miRNAsMethods:（1）MC3T3-E1 osteogenic precursor cells and osteoblasts induced byosteogenic induction medium were cultured and perform high-throughput sequencing.（2）Collecting blood serum from patients with femoral fractures,extracting total RNA from blood serum,and detecting key miRNAs in fracture patients by qRT-PCR.Results:High-throughput sequencing was used to screen for the differentiation of MC3T3-E1 into osteoblasts.miR-143 was the most abundant and most variable inosteoblasts;and miR-143 was found decreased with aging.Conclusion:miR-143 plays a key role in the development and differentiation of osteoblasts.Part Ⅱ:Related studies of osteoblasts in vitro and in vivo by miR-143Methods:（1）MC3T3-E1 cell line was cultured,and mimicsand inhibitors of miR-143were transfected into cells.The osteogenic differentiation was observed by toluidine blue staining and alizarin red staining.（2）the expression level of the relevant osteogenic marker was verified by qPCR.（3）The miR-143 knockout mice were constructed,and the mice were sacrificed at 8 weeks and micro-CT scans were performed on the femur.H-E staining and ALP staining was performed on the embedded sections.And then observe the trabecular bone and osteoblast differentiation status.Results:By toluidine blue staining and alizarin red staining of the osteoblast differentiation and qRT-PCR showed that the differentiation of osteoblasts from miR-143mimics increased significantly,and the differentiation of osteoblasts induced by miR-143inhibitors decreased significantly.Construction of miR-143 knockout mice also revealed a markable reduction in the trabecular bone of the femur and cortical bone formation.The reduction of osteoblast was also observed in H-E staining and ALP staining.Conclusion:miR-143 can promote osteoblast differentiation in vitro,while inhibition of miR-143 inhibits osteoblast differentiation.Part Ⅲ:Mechanism and therapeutic effect of miR-143Methods:（1）BMSCs in miR-143 knockout mice were extracted and induced to osteoblasts differentiation by osteogenic induction medium.Morphology was observed by ALP and alizarin red staining,and its possible targeted mRNA was observed by high-throughput sequencing technology.（2）It was verified by Western blotting experiments and luciferase reporter experiments that it may target mRNA.（3）The effect was verified by simultaneous injection of interfering RNA of HDAC7 and agonistic miR-143 in vivo.（4）The therapeutic effect of miR-143 in vivo was verified by injecting agomiR-143.Results:（1）High-throughput sequencing revealed that HDAC7 may be the targets of miR-143;（2）the expression of HDAC7 was increased in western blotting experiment transfected by miR-143 mimics and decreased when transfected by miR-143 inhibitor.It was also comfirmed in the luciferase reporter assay that miR-143 exerts its function by acting directly on HDAC7.（3）By simultaneously injecting HDAC7 interfering RNA and stimulating miR-143 into wild-type mice,micro-CT scan and hard tissue sections showed significantly increased trabecular bone and cortical bone compared with miR-143 knockout mice.（4）By injecting agomiR-143 into wild type mice,it was found that the trabecular bone and cortical bone were significantly more than wild type mice,which further indicated that miR-143 may be a treatment to prevent osteoporosis.Conclusion:（1）miR-143 promotes osteoblast differentiation by targeting HDAC7;（2）miR-143 may be used as a treatment to prevent osteoporosis.