| Background and purposeOsteoporosis is one of the most common bone diseases.It is a systemic bone disease characterized by low bone mass,damage to the microstructure of bone tissue,increased bone fragility,decreased bone strength,and increased risk of fracture.Epidemiological survey shows that the prevalence of osteoporosis in people over 60 years old is significantly increased,especially in women.Osteoporosis has become an important public health problem in our country.The cause of osteoporosis is the weakening of bone development and renewal and the enhancement of bone destruction.The ability of bone marrow mesenchymal stem cells(BMSCs)to differentiate into osteoblasts is particularly important during bone development and renewal.Therefore,the dysfunction of BMSCs is closely related to the occurrence of osteoporosis.Targeting the osteogenic ability of BMSCs may become a new strategy for osteoporosis treatment.The differentiation process of bone marrow mesenchymal stem cells in vivo is precisely regulated by a variety of factors,among which the most studied are tissue-specific transcription factor Runx2,cytokine BMP,etc.In recent years,N6-methyladenosine methylation(m6A),as an epigenetic modification,regulates various cell biological activities.The role it plays in bone development has been gradually paid attention by more and more researchers.m6A is the most common post-transcriptional modification in eukaryotic cells.It is believed that this modification is mainly performed by m6A Methyltransferase(also known as"Writers"),Demethylase(also known as "Erasers"),and methylated reading recognition proteins(also known as "Readers").The main Methyltransferase members include methyltransferase-like protein 3(METTL3),Methyltransferase-like protein 14(METTL14)and Wilms’ tumor-associated protein(WTAP).Previous studies have explored the specific mechanisms of METTL3 and METTL14 in regulating BMSCs.However,as an important member of the m6A functional protein family,WTAP’s role in regulating the differentiation of bone marrow mesenchymal stem cells remains unclear.This paper is divided into two parts:(1)WTAP expression analysis and study on the effect of the m6A modification mechanism on the maturation of pri-miR-181a and pri-miR-181c on the differentiation potential of bone marrow mesenchymal stem cells.(2)YTHDC1 recognizes methylated pri-miR-181a and pri-miR-181c,promotes the maturation of miR-181a and miR-181c,and jointly targets SFRP1 to affect the differentiation potential of bone marrow mesenchymal stem cells.Materials and methods(1)RT-qPCR and Western blot tests were performed on patient-derived and mouse model-derived specimens to determine the expression of related genes.(2)The differentiation potential of bone marrow mesenchymal stem cells was evaluated by RT-qPCR,Western blot assay,ALP activity assay,ALP staining and alizarin red staining.(3)The osteoporosis model of ovariectomized mice was constructed,and WTAP expression was manipulated by virus injection in vivo.The bone development changes were evaluated by Micro-CT examination,histological staining analysis,RT-qPCR and Western blot.(4)The expression of miRNA and pri-miRNA in bone marrow mesenchymal stem cells was detected by RT-qPCR under different conditions(5)The binding protein of WTAP was explored through Co-IP experiment,and RNA-pull-down experiment was used.RIP and MeRIP experiments explored the direct mechanism of WTAP regulation of pri-miR-181a and pri-miR-181c and searched for methylation recognition proteins of pri-miR-181a and pri-miR-181c after methylation.(6)Dual luciferase reporter gene assay was used to investigate the methylation sites of WTAP binding to pri-miR-181a and pri-miR-181c,and the binding sites of miR-181a and miR-181c to SFRP1 mRNA 3’UTR.Result(1)The expression of WTAP in bone tissue of osteoporosis patients and ovariectomized osteoporosis mouse models is decreased,and the expression of WTAP is up-regulated in bone marrow mesenchymal stem cells during osteogenic differentiation,but down-regulated in lipogenic differentiation.In vitro,overexpression of WTAP promoted osteogenic differentiation of bone marrow mesenchymal stem cells and inhibited lipogenic differentiation.In vivo,WTAP supplementation delayed bone mass loss in mice with osteoporosis.That is,WTAP can participate in the pathogenesis of osteoporosis by regulating the differentiation potential of bone marrow mesenchymal stem cells.(2)The expression levels of miR-181a and miR-181c in bone tissue were decreased in osteoporosis patients and ovariectomized osteoporosis mouse models,and the expression levels of miR-181a and miR-181c were increased during osteogenic differentiation of bone marrow mesenchymal stem cells.miR-181a mimics and miR-181c mimics were injected into bone marrow mesenchymal stem cells to inhibit lipogenic differentiation,while miR-181a inhibitor and miR-181c inhibitor had the opposite effect.miR-181a mimics and miR-181c mimics can salvage the decreased osteogenic differentiation potential caused by WTAP knockdown,that is,WTAP affects the differentiation fate of bone marrow mesenchymal stem cells by regulating miR-181a and miR-181c.(3)Overexpression of WTAP increased the activity of methyltransferase complex and thus mediated the increase of m6A methylation modification levels of pri-miR-181a and pri-miR-181c,thus promoting their maturation into miR-181a and miR-181c.Dual luciferase reporter gene assay identified pri-miR-181a and pri-miR-181c methylation sites.(4)YTHDC1 as a "reader" mediates the recognition and maturation of pri-miR-181a and pri-miR-181c after methylation.After YTHDC1 knockout,the osteogenic differentiation ability of bone marrow mesenchymal stem cells was weakened,and the lipogenic differentiation potential was enhanced.The deletion of YTHDC1 restored the enhanced osteogenic potential caused by WTAP overexpression.On the other hand,miR-181a mimics and miR-181c mimics can restore the effect of YTHDC1 knockout on the differentiation ability of bone marrow mesenchymal stem cells.(5)SFRP1 is the common downstream target of miR-181a and miR-181c in bone marrow mesenchymal stem cells.Dual luciferase reporter gene assay identified the direct binding sites of the two,and SFRP1 expression increased in bone tissue of osteoporosis patients and ovaries removed mice.Phenotypic changes induced by miR-181a inhibitor and miR-181c inhibitor were saved by SFRP1 knockdown.ConclusionThe expression of WTAP is decreased during the onset of osteoporosis,and WTAP can delay the occurrence of osteoporosis by promoting the osteogenic differentiation of bone marrow mesenchymal stem cells.Mechanically,WTAP acts as "Writers" to increase the m6A modifications of pri-miR-181a and pri-miR-181c.YTHDC1 then acts as a "reader" to recognize the m6A-modified pri-miR-181a and pri-miR-181c and promote their maturation into miR-181a and miR-181c.Mature miR-181a and miR-181c use SFRP1 as a common target to down-regulate SFRP1 expression during osteogenic differentiation of bone marrow mesenchymal stem cells.In summary,the WTAP/YTHDC1/miR-181a and miR-181c/SFRP1 axes are involved in the pathogenesis of osteoporosis by regulating the differentiation fate of bone marrow mesenchymal stem cells.This discovery not only improves the pathogenesis of osteoporosis,but also provides a possible new target for the prevention and treatment of osteoporosis. |
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