Studies On Osteogenesis Mechanisms Of Hedgehog Signaling Pathway Regulated By MiRNA-342-3p

Objectives: The Hedgehog(Hh)signaling pathway is not only an important regulator of bone formation and development during embryonic development,but also a bi-derectional regulator of bone tissue after birth to maintain the stability of the internal environment.Sonic Hedgehog(Shh)is the key ligand for the Hh signaling pathway.There is increasing evidence showing that the Shh pathway plays an important role during osteogenesis.Suppressor of fused protein(Sufu)is an inhibitor of Shh signaling pathway and negatively regulates the Hh transcription factor Gli.Human umbilical cord mesenchymal stem cells(hUCMSCs)are one of stem cells which have been widely used recently and can be used for cell regeneration and stem cell treatment.miRNA-342-3p is expressed in most tissues of human body.The expression level of miRNA-342-3p is significantly down-regulated in primary myelofibrosis and is highly expressed in multiple myeloma.However,little was known about miRNA-342-3p regulating bone formation so far.In this study,we try to investigate whether miRNA-342-3p plays a regulatory role during the osteogenic differentiation via the Hh signaling pathway through the Sufu/Gli signal axis.Methods: We performed a miRNA-seq in hUCMSCs treated with osteogenic induction medium and exogenous Shh,detected the expression of miRNAs during osteogenesis,and identified several miRNAs that affect osteogenic differentiation in vivo.We predicted that miRNA-342-3p may act as a key factor regulating the osteogenesis process through the Hh signaling pathway.After that,we constructed lentiviral vectors overexpressing miRNA-342-3p and inhibiting miRNA-342-3p respectively.Then these two viruses were transduced into hUCMSCs.The expression levels of Hh signaling pathway and osteogenic related factors were observed when miRNA-342-3p was overexpressed or silenced.Subsequently,we used luciferase assay to analyze whether miRNA-342-3p could target Sufu.At the same time,up and down miRNA-342-3p viruses were added combined with Sufu overexpressing virus infected hUCMSCs,the expression level of Hh signaling pathway and osteogenic related factors were observed to further confirm the target effect of miRNA-342-3p on Sufu.Finally,?-TCP was seeded with hUCMSCs which were transduced the recombinant virus expressing miRNA-342-3p or Sufu.These complex materials were filled into the skull defect of rats.In vivo,we observed whether miRNA-342-3p regulated the Hh signaling pathway through Sufu to promote bone regeneration.Results: The results of miRNA-seq and RT-PCR predicted that miRNA-342-3p may act as a key factor to regulate osteogenesis through the Hh signaling pathway(P<0.02).The software predicts that miRNA-342-3p may target Sufu.Subsequently,overexpression and suppression of miRNA-342-3p lentivirus were constructed and transfected into hUCMSCs,our result demonstrates that miRNA-342-3p can activate Hh signaling pathway like up-regulate Smo(P<0.01),Gli1(P<0.01),Gli2(P<0.01)and Gli3(P<0.05),also it can modulate the expression of osteogenic related factors,such as ALP(P<0.01),Cbfa1(P<0.05),OPG(P<0.01)and TGF-?(P<0.01).We further constructed a Sufu overexpression plasmid and verified the targeting relationship between miRNA-342-3p and Sufu by luciferase assay(P<0.01).Then our RT-qPCR and Western Blot results further comfirmed this potential relationship(P<0.01).There was also an interesting phenomenon in our results.The expression level of TGF-? also changed during osteogenesis(P<0.01),which indicated that Hh signaling pathway and TGF-? signaling pathway might have potential interaction while regulating osteogenesis.The bone tissue proliferation was observed in the group filled with overexpression miRNA-342-3p-hUCMSC material complex in vivo(BV/TV,P<0.01),which further supported our results in vitro.Conclusions: These studies demonstrate that miRNA-342-3p may be a key factor modulating Hh signaling pathway by down-regulating Sufu and induce osteogenic differentiation.Our findings provide new mechanistic evidence that miRNA-342-3p might be a valuable therapeutic target in bone regeneration and bone tissue engineering.