The Effects And Mechanisms Of Mmu-circHmbox1 On Osteoclast Differentiation In Osteoporosis After Spinal Cord Injury

Objective: Osteoporosis(OP)secondary to spinal cord injury(SCI)is a well known complication characterized by declined bone mineral density,deteriorated bone architecture,decreased bone strength and increased risk of fracture.Currently,enhanced bone resorption after spinal cord injury is considered to be the main pathophysiological mechanism of bone loss,However,the molecular mechanisms underlying SCI-OP have not been adequately understood.circRNA is a class of noncoding RNAs,of which characteristics are high stability,a bundance and evolutionary conservation among species.Accumulated evidences indicate that circRNAs play critical role in process(osteogenic differentiation and osteoclastogenesis)of OP-induced bone dysfunction.Cicr RNAs are known to regulate biological process via multiple mechanism,of which serving as mi RNA sponges is the most frequently reported mechanism.Besides,other functions such as interacting with RNA binding proteins(RBPs),regulating m RNA translation and encoding peptides are emerging.Little is known whether circRNAs can regulate bone-related mechanism via serving as protein decoy molecules.Therefore,the present study aims to explore the functional role of circRNA playing in the pathophysiological process of osteoporosis after spinal cord injury.Methods:1.The contutive spinal cord injury(SCI)models were constructed by modified Allen method using C57BL/6J male mice aged 8-10 weeks.Mice were randomly divided into spinal cord injury group(SCI)and Sham operation group(Sham)according to indicated time: 7-day SCI group,7-day Sham group,14-day SCI group,14-day Sham group,28-day SCI group,and 28-day SHAM group.Bone architectecture was examined by micro CT to evaluate the changes in cancellous and cortical bone of distal femur.HE staining was used to illustrate the pathological changes of femur in SCI group and Sham group.TRAP staining was used to evaluate bone resorption in each group.2.Bone marrow derived macrophages(BMMs)were isolated from the bone marrow cavity of mice in each group and cultured in vitro.Osteoclast related circRNAs were screened by by q RT-PCR in BMMs and bone tissue.In order to further explore the regulatory mechanism of circRNA in osteoclast differentiation,we chose mmu-circHmbox1 for further study.RT-PCR,agarose gel electrophoresis,Sanger sequencing,RNase R digestion assay,FISH in situ hybridization and nuclear-cytoplasmic fractionation assay were used to identify the characteristics of mmu-circHmbox1.3.After the targeted intervention(knockdown or overexpression)of mmu-cirHmbox1 in Raw264.7 cells,in vitro gain and loss of function assays,such as CCK8,flow cytomgraphy and TRAP staining were carried out to investigate the role of mmu-circHmbox1 in osteoclast development.Subsequently,we performed in vivo salvage experiment.mmucircHmbox1-overexpressed BMMs were injected into the femoral marrow cavity of SCI mice,and the rear femur of mice were extracted 28 days after SCI/Sham for micro CT analysis to evaluate the extent of bone loss.4.We performed a circRNA-pulldown assay and mass spectrometry to find the interacting proteins of mmu-circHmbox1 and YBX1,Fus and Hn RNP K were identified.RNAbinding protein immunoprecipitation(RIP)assay was used to further validate that endogenous Hnrnpk,Ybx1 and Fus could directly bind to mmu-circHmbox1.Subsequently,q PCR,immunohistochemistry,Western blot,immunofluorescence and other experimental techniques were used to perform the detection of subcellular localization and protein expression of YBX1,Fus and Hn RNP K.Finally,we selected YBX1 for following research.5.In vitro,YBX1 plasmid was overexpressed in mmu-circHmbox1 knockdown Raw264.7 cells.Subsequently,TRAP staining was used to illustrate the role of mmucircHmbox1/YBX1 in regulating osteoclast differentiation.6.Since YBX1 has been reported as a transcription factor,we found that Fam20 c may be a downstream target gene of YBX1 based on transcriptome high-throughput sequencing(RNA-seq)and literature review.Chromatin Immunoprecipitation assay(Ch IP)and dual luciferase assay were then used to detect the regulatory effect of transcription factor YBX1 on Fam20 c gene.Western Blot analysis confirmed that Fam20 c expression was regulated by the mmu-circHmbox1/YBX1 axis.Results:Part Ⅰ:SCI could significantly change the microstructure of cancellous bone in mice.Cancellous bone loss of femur was observed early after surgery and gradually deteriorated according to the time after SCI.There was no significant difference in cortical bone parameters.TRAP staining showed enhanced bone resorption in SCI mice.The expression of mmu-circHmbox1 in SCI mice BMMs was significantly decreased.Part Ⅱ: mmu-circHmbox1 existed a stable circular structure and is resistant to RNase R digestion.It distributed in both cytoplasm and nucleus,and was mainly locolized in the nucleus.Mmu-circHmbox1 could inhibit the proliferation of osteoclast precursors,promote the apoptosis of mature osteoclasts,and inhibit osteoclast differentiation.Overexpression of mmu-circHmbox1 in vivo could partially alleviate bone loss caused by SCI.Part Ⅲ: Using circRNA-pulldown assay and mass spectrometry,YBX1 was identified as the binding protein of mmu-circHmbox1,of which expression decreased in the femur of SCI mice.Its expression is positively regulated by mmu-circHmbox1.YBX1 can inhibit osteoclast differentiation.Rescue experiment results showed that the overexpression of YBX1 partially alleviated the enhanced osteoclast differentiation induced by mmucircHmbox1 knockdown.RNA-seq data showed that YBX1 up-regulated 96 genes and down-regulated 31 genes.Differentially expressed genes were enriched in toll-like receptor signaling pathway and NF-kappa B signaling pathway,suggesting that YBX1 played a regulatory role in osteoclast differentiation.Subsequently,through biogenic analysis and literature review,Fam20 c was predicted to be the target gene of YBX1.Ch IP and dual luciferase assays confirmed that mmu-circHmbox1 could recruit YBX1 to the Fam20 c promoter and initiate Fam20 c transcription.Conclusion:1.The present study showed that the changes of bone metabolism after SCI were mainly manifested as enhanced bone resorption,decreased bone mass and deteriorated cancellous bone structure.mmu-circHmbox1 was significantly downregulated in the BMMs of SCI mice and showed a down-regulated trend during osteoclast differentiation,which inhibited the proliferation of osteoclast precursor cells and promoted the apoptosis of mature osteoclasts,thus playing a protective role in the process of osteoclast differentiation.2.YBX1 interacted with mmu-circHmbox1 to negatively regulate osteoclast differentiation.3.Fam20 c gene was the target gene of YBX1.mmucircHmbox1 could recruit YBX1 to the Fam20 c promoter and initiate Fam20 c transcription,thus inhibiting osteoclast differentiation through mmucircHmbox1/YBX1/Fam20 c axis.