miR-134-5p inhibits osteoclastogenesis through a novel miR-134-5p/Itgb1/MAPK pathway

Abstract

Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy. Osteoporosis affects approximately 200 million people and severely affects quality of life, but the exact pathological mechanisms behind this disease remain unclear. Various miRNAs have been shown to play a predominant role in the regulation of osteoclast formation. In this study, we explored the role of miR-134-5p in osteoclastogenesis both in vivo and in vitro. We constructed an ovariectomized (OVX) mouse model and performed microarray analysis using bone tissue from OVX mice and their control counterparts. Quantitative RT-PCR data from bone tissue and bone marrow macrophages (BMMs) confirmed the decreased expression of miR-134-5p in OVX mice observed in microarray analysis. In addition, a decrease in miR-134-5p was also observed during induced osteoclastogenesis of BMMs collected from C57BL/6N mice. Through transfection with miR-134-5p agomirs and antagomirs, we found that miR-134-5p knockdown significantly accelerated osteoclast formation and cell proliferation and inhibited apoptosis. Furthermore, a luciferase reporter assay showed that miR-134-5p directly targets the integrin surface receptor gene Itgb1. Cotransfection with Itgb1 siRNA reversed the effect of the miR-134-5p antagomir in promoting osteoclastogenesis. Moreover, the abundance levels of MAPK pathway proteins phosphorylated-p38 (p-p38) and phosphorylated-ERK (p-ERK) were significantly increased after transfection with the miR-134-5p antagomir but decreased after transfection with the miR-134-5p agomir or Itgb1 siRNA, which indicated a potential relationship between the miR-134-5p/Itgb1 axis and the MAPK pathway. Collectively, these results revealed that miR-134-5p inhibits osteoclast differentiation of BMMs both in vivo and in vitro and that the miR-134-5p/Itgb1/MAPK pathway might be a potential target for osteoporosis therapy. Osteoporosis is a widely concerning disease affecting a population of approximately 200 million that interferes with the patients’ quality of life and subsequently imposes burdens on their families and society (1Pisani P. Renna M.D. Conversano F. Casciaro E. Di Paola M. Quarta E. et al.Major osteoporotic fragility fractures: risk factor updates and societal impact.World J. Orthop. 2016; 7: 171-181Crossref PubMed Scopus (316) Google Scholar). In pathological settings, patients with osteoporosis display an uncontrolled erosion of bone mass, a deteriorated microarchitecture, and bone fracture with an unbalanced microenvironment (2Wu Y.Z. Huang H.T. Cheng T.L. Lu Y.M. Lin S.Y. Ho C.J. et al.Application of microRNA in human osteoporosis and fragility fracture: a systemic review of literatures.Int. J. Mol. Sci. 2021; 22: 5232Crossref PubMed Scopus (5) Google Scholar). The homeostasis of a healthy bone microenvironment is mainly maintained by osteoclastic bone resorption and osteoblastic bone formation (3Datta H.K. Ng W.F. Walker J.A. Tuck S.P. Varanasi S.S. The cell biology of bone metabolism.J. Clin. Pathol. 2008; 61: 577-587Crossref PubMed Scopus (369) Google Scholar, 4Lee K.S. Lee J. Kim H.K. Yeom S.H. Woo C.H. Jung Y.J. et al.Extracellular vesicles from adipose tissue-derived stem cells alleviate osteoporosis through osteoprotegerin and miR-21-5p.J. Extracell. Vesicles. 2021; 10e12152Crossref Scopus (62) Google Scholar, 5Wu K. Feng J. Lyu F. Xing F. Sharma S. Liu Y. et al.Exosomal miR-19a and IBSP cooperate to induce osteolytic bone metastasis of estrogen receptor-positive breast cancer.Nat. 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Osteoclasts are large multinucleated cells from differentiation which factor and receptor of factor are a on the the of osteoclast after receptor 2019; PubMed Scopus (47) Google Scholar). their from the bone macrophages on the bone surface and multinucleated with the of and cells the bone structure by and to the formation of bone resorption J. review of J. Mol. Sci. Scopus Google Scholar). this cytokines and are activated in a pathological that bone which may an effective for miRNAs are with a of approximately to that of gene expression by that of human are microRNA Full Text Full Text PDF PubMed Scopus Google Scholar). of that miRNAs in osteoclastogenesis S. and of the microRNA PubMed Scopus Google Scholar). miRNAs the bone mass by that play a role in osteoclast formation. have shown that inhibits by the factor and osteoclastogenesis P. S. S. M. uncontrolled osteoclastogenesis of signaling PubMed Scopus Google Scholar). et L.J. L. L. Y. factor to Res. 2014; PubMed Scopus Google that osteoclast differentiation through the of and However, bone resorption by factor to the pathway J. L. C. B. Lu C. et targets to to osteolytic bone metastasis of breast Scholar). results that miRNAs might be of osteoclastogenesis. miR-134-5p a role in the of and in and disease. Moreover, a the of miR-134-5p in in cells in S. H. J. Kim Y. et microRNA miR-134-5p by in Mol. PubMed Scopus (6) Google Scholar, S. and in an model of PubMed Scopus Google Scholar, cell through of 2016; 7: PubMed Scopus Google which been to in bone formation. However, a role for miR-134-5p in bone resorption is In the study, to the of miR-134-5p on bone we bone marrow macrophages (BMMs) of mice for the of osteoclast formation. We found that miR-134-5p knockdown osteoclast differentiation both in vivo and in vitro. and that miR-134-5p osteoclast differentiation by directly integrin pathway revealed that the miR-134-5p/Itgb1 axis to osteoclastogenesis and bone and might for the mechanism of bone the osteoclast formation process in we constructed a mouse model of The of ovariectomized (OVX) mice was significantly that of that an in adipose tissue for osteoporosis of revealed bone in OVX was by a trabecular trabecular bone was observed in mice Furthermore, an of bone of of the bone bone to tissue trabecular trabecular was shown in revealed that OVX mice a in bone mass with in the analysis with and with a of osteoclast were shown by OVX mice and a of mice The OVX mice also a osteoblast or osteoblast which indicated of the underlying the of and the osteoclast surface the trabecular bone were increased in OVX by In addition, the expression levels of the and in bone tissue were these play a role in osteoclast The levels of and were in OVX mice In the expression of and was the of miRNAs play an role in human osteoporosis K. Ng C. Y. B. of osteoclastogenesis and bone resorption by 2021; Scopus Google we the expression of miRNAs in and OVX mice. miRNAs was and to microarray analysis. The microarray analysis revealed a of and of these miRNAs were and shown in the and the expression of and in mice and expression we to the miRNAs expression between and OVX mice. We constructed with and to the relationship between in expression and In the the significantly the and the miRNAs that are significantly We also to the in miRNAs between The of the and in the the of miRNAs from and OVX mice. The the or the the in the or miRNAs with a of and analysis revealed that the miRNAs were in the regulation of osteoclast differentiation tissue or BMMs from and OVX mice was and decreased expression of miR-134-5p was in OVX mice by RT-PCR the results for miR-134-5p in the microarray analysis and the in vitro study, we the exact that miR-134-5p on BMMs in to and we induced BMMs to by with and for The multinucleated cells were a a for these cells were multinucleated were observed after for in the induced for were observed and formation of was and the was by with and of the of that indicated by was increased and analysis was performed to the expression levels of and The for and in and the expression was on the Moreover, the of miR-134-5p in induced BMMs was by In to gene the expression of miR-134-5p showed after and of results confirmed that the of and osteoclast the role of miR-134-5p during we miR-134-5p antagomir and miR-134-5p antagomir control BMMs and the knockdown by The antagomir and control were The cell after transfection with antagomir and control was by cell and The indicated that the knockdown of miR-134-5p accelerated cell proliferation Moreover, cell was with a and showed that miR-134-5p knockdown inhibited cell with the control and osteoclast differentiation was induced by with and for was performed to identify the formation of The of multinucleated cells in miR-134-5p antagomir was that found for the miR-134-5p antagomir and The regulation of miR-134-5p in bone resorption was in bone and bone resorption were by of bone was observed in the antagomir in the control and In addition, formation in was by to the of revealed the on formation in multinucleated cells observed with which also showed an of and the and expression levels of and were The expression levels of these were significantly in the miR-134-5p antagomir these data indicated that the miR-134-5p antagomir a of osteoclast the exact effect of we also miR-134-5p agomir and miR-134-5p agomir BMMs to determine miR-134-5p agomir the process of osteoclast The transfection was also by which showed an in miR-134-5p after agomir transfection with that after agomir transfection was by after and and the results showed that the cells with agomir a of with with the control agomir Moreover, cell was using a The results were confirmed by in the agomir with the and the of the agomir was also that with the control and of bone resorption and were performed to the and resorption of The of cells and bone resorption formation were significantly in cells with miR-134-5p agomir with with miR-134-5p agomir The formation was decreased in the agomir with the control and The expression of and by and that the of miR-134-5p in a in the expression of data revealed that the miR-134-5p agomir inhibits osteoclastogenesis. Therefore, miR-134-5p osteoclast shown by an in miR-134-5p knockdown and a decrease in miR-134-5p miRNAs are with a large of and in pathways cell differentiation P. Yu M. W. in vesicles and their 2021; Full Text Full Text PDF PubMed Scopus Google Scholar). the potential by we and to identify potential targets of a of potential were that induce osteoclast differentiation were and Itgb1. The expression levels of these after the or knockdown of miR-134-5p in BMMs were Itgb1 was after miR-134-5p but after miR-134-5p The and display the and a of these Itgb1 was a potential target of and a in the of Itgb1 was using Moreover, we a luciferase reporter assay to the between miR-134-5p and Itgb1. We constructed of luciferase reporter and with miR-134-5p and the luciferase by the luciferase of the was significantly by miR-134-5p the showed The expression of Itgb1 was increased and to in osteoclastogenesis the exact between miR-134-5p and we to that the of Itgb1 was decreased by the miR-134-5p agomir and increased by the miR-134-5p antagomir with the control and was to the expression of Itgb1 in BMMs with agomir or The Itgb1 was in BMMs with antagomir in BMMs with agomir and In addition, the results from in vivo were Itgb1 expression levels and Itgb1 expression were in bone from of OVX mice of mice that Itgb1 is a target of the mechanism underlying the miR-134-5p with Itgb1 in osteoclast osteoclast differentiation with Itgb1 knockdown was The transfection of Itgb1 siRNA was by and and The of BMMs with antagomir and Itgb1 was to determine the of Itgb1 in miR-134-5p knockdown Itgb1 reversed the excessive cell proliferation induced by the antagomir and were performed with the with antagomir or antagomir and with or The results showed a in the of multinucleated cells in the miR-134-5p antagomir Itgb1 knockdown reversed the of osteoclast differentiation observed with the miR-134-5p Moreover, Itgb1 the of in with that found with the control and revealed the In the of that was by the of antagomir and Itgb1 transfection with antagomir the of and and data showed that the miR-134-5p antagomir increased the and expression Itgb1 reversed the promoting effect of miR-134-5p antagomir on osteoclastogenesis of these results that miR-134-5p osteoclast differentiation by Itgb1. that miR-134-5p promotes osteoclast we subsequently to the potential pathway in this indicated by the the was to the MAPK pathway Moreover, the MAPK pathway been to be for bone and osteoclast differentiation Y. Z. X. B. J. Y. et surface promotes by osteoclastogenesis integrin PubMed Scopus Google Scholar). and and their and in BMMs with agomir or antagomir or with Itgb1 or control were The of and in BMMs with Itgb1 siRNA or agomir were significantly decreased and that were increased in antagomir shown by but the expression showed in siRNA, and antagomir Collectively, data indicated that the regulation of osteoclast differentiation by miR-134-5p mainly the pathway results revealed a role of the miR-134-5p/Itgb1 axis in osteoclastogenesis. gene we for miRNAs in OVX mice and that miR-134-5p was significantly The expression of miR-134-5p was decreased in both bone and BMMs of OVX mice with a potential role in osteoclastogenesis. The results were confirmed by in vitro which that miR-134-5p significantly osteoclast differentiation by Itgb1 and that Itgb1 is an underlying target in the bone with the of osteoclast during control of the activation of and S. F. H. H. J. and in the regulation of bone PubMed Scopus Google Scholar). In this the to the of and pathways and induce the of factor of activated cells which results in the of osteoclast differentiation M. H. in in and 2019; PubMed Scopus Google Scholar, J. H. S.S. Cao X. et enhances formation and bone in ovariectomized PubMed Scopus Google Scholar). miRNAs are a of that of and of the PubMed Scopus Google Scholar). of process which to the of target to and target of microRNA in Mol. 2019; PubMed Scopus Google Scholar). the of miRNAs have been a for osteoclast formation and bone and in osteoclast in induce expression to decrease the to the osteoclastogenesis M. J. M. Y. Y. et inhibits cell bone and Res. 2014; 12: PubMed Scopus Google Scholar, B. F. inhibits osteoclast formation through Mol. Res. 2016; Scopus Google Scholar, J. H. Zeng X. Feng S. osteoprotegerin expression the of expression in PubMed Scopus Google Scholar). 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PubMed Scopus (6) Google is exact in bone formation and Therefore, in this study, we miR-134-5p expression was decreased in the osteoclast formation process in vitro and in OVX mice in on these the of this were by the or knockdown of The results showed that the knockdown of miR-134-5p accelerated osteoclast formation and that inhibited this The confirmed that miR-134-5p is a of osteoclastogenesis. miRNAs osteoclast differentiation and formation by K. Ng C. Y. B. of osteoclastogenesis and bone resorption by 2021; Scopus Google Scholar). was confirmed to induce osteoclast formation through of a of the factor P. C. He H. et osteoclastogenesis by Res. PubMed Scopus Google Scholar). et C. Cheng P. H. et osteoclastogenesis Res. 2014; PubMed Scopus Google that which is the receptor of in osteoclastogenesis. been shown to osteoclast formation by which is with osteoporosis H. Z. W. 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Y. by promotes osteoclastogenesis and bone 22: Full Text Full Text PDF PubMed Scopus Google that during OVX mice bone mice. integrin in osteoclast during bone resorption H. Lu H. J. et a osteoclast integrin that osteoclast formation and Res. PubMed Scopus Google Scholar). In addition, in attenuates osteoclast formation by and and analysis revealed that cells the cells Y. K. K. Y. J. S. inhibits the of in 2016; PubMed Scopus Google Scholar). We that miR-134-5p to the of Itgb1. We performed luciferase reporter to the relationship between miR-134-5p and Itgb1. The and expression of Itgb1 indicated that Itgb1 was directly by determine the of Itgb1 in we a with miR-134-5p antagomir The results showed that the of Itgb1 in a decrease in the osteoclast and the of BMMs with miR-134-5p and Itgb1 inhibited osteoclast formation with that with transfection with miR-134-5p antagomir the role of MAPK signaling in of and osteoclastogenesis. MAPK signaling is mainly activated by an that is by with X. K. K. Y. et are for osteoclast differentiation but for osteoclast PubMed Scopus Google Scholar). The of MAPK is for the activation of and X. Y. L. S. F. inhibits osteoclastogenesis and promotes through promoting expression the MAPK 2021; PubMed Scopus (9) Google Scholar). have also shown that mice are to osteoporosis and a decrease in bone mass H. P. S. J. Y. et MAPK proliferation and differentiation of osteoclast and bone in an Scopus Google Scholar). In addition, osteoclast are to the from cell proliferation to differentiation by the MAPK pathway K. H. Kim H. J. regulation of MAPK signaling osteoclast J. Sci. 2016; 12: PubMed Scopus Google Scholar). Moreover, through which enhances osteoclast formation. have that Itgb1 a with MAPK pathway signaling in osteoclastogenesis. is by the integrin pathway on a surface Y. Z. X. B. J. Y. et surface promotes by osteoclastogenesis integrin PubMed Scopus Google Scholar). In study, a analysis of OVX mice revealed that miRNAs were mainly to the MAPK underlying a potential miR-134-5p/Itgb1 in osteoclast differentiation through the MAPK pathway. we on the integrin and MAPK relationship to the mechanism through which miR-134-5p osteoclast formation. The expression levels of phosphorylated-p38 phosphorylated-ERK and in miR-134-5p agomir or BMMs and in BMMs with were by and the results indicated that the knockdown of miR-134-5p increased and expression and that these were decreased after miR-134-5p The and were to determine the of activation in and the in BMMs with antagomir a of osteoclast the osteoclast differentiation process was Itgb1 is an of the MAPK the in Itgb1 BMMs showed in the expression of MAPK which the of Itgb1 on MAPK the Itgb1 after miR-134-5p was and were increased with miR-134-5p the of miR-134-5p in osteoclastogenesis through MAPK pathway. results that the MAPK pathway was in miR-134-5p osteoclastogenesis through activation of the miR-134-5p target Itgb1. miRNAs are to pathological and but have been found with to bone H. Z. X. J. K. potential targets for of PubMed Scopus Google Scholar). have been in bone a the a pathological Moreover, and mechanisms be found to target these and these for the role of of miRNAs in miRNAs be a for bone The of miR-134-5p in mice osteoporotic and in cells induced on the of osteoporotic may a target for the of osteoporosis In the indicated that miR-134-5p osteoclast differentiation and inhibited bone resorption by the axis miR-134-5p was both in mice with osteoporosis and in in vitro. The knockdown of this the expression of target Itgb1 and induced an in osteoclast miR-134-5p/Itgb1 may be a potential target in the regulation of bone and osteoporosis therapy. C57BL/6N mice were from the mice were in a and with and were by the of the of of mice were the or OVX The of the OVX were from an on the of the were to X. Liu Z. X. W. et bone resorption by osteoclastogenesis and Pharmacol. 9: PubMed Scopus (33) Google Scholar). 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