Role And Mechanism Of MiR-377 In Wear Particle-induced Osteolysis

As time advances,loosening of prosthesis previously placed in total joint arthroplasty(TJA)may occur,requiring revision arthroplasty to replace a new joint prosthesis,producing more unnecessary suffering as well as constituting a financial burden for patients.Of note,particle-induced osteolysis(PIO)adjacent to the prosthesis is identified to be the main cause of aseptic loosening of prosthesis,meanwhile,the main factor affecting the long-term therapeutic effects on TJA,as elucidated by certain research.Osteoclasts,which are the only cells with the ability responsible for initiating bone resorption in vivo and play a central part in contributing to osteolysis surrounding the entire prosthesis,exert a crucial effect on the pathogenesis of osteolysis surrounding the prosthesis,yet its potential mechanism demands further investigation.Micro RNA(miRNA),known as a class of small single-stranded non-coding RNA that is highly conserved,regulates protein expression by binding to the 3'UTR end of target genes,thus playing a role in pathophysiological processes.The aim of this study is to explore the molecular and biological function of miR-377 in PIO,so as to provide novel insights to the modification of TJA and prevention of PIO,and for evaluating the potential role of miR-377 in PIO,the following experiments were conducted.Part One Correlation Analysis between miR-377 Expression and PIO Clinicopathological ParametersObjective: To preliminarily understand the correlation between miR-377 expression and PIO clinicopathologic parameters.Methods:1.Blood samples were collected from patients undergoing TJA and then q RT-PCR was used to analyse the peripheral venous blood samples extracted from each subject enrolled in,determining miR-377 m RNA expression level and analyzing the correlation between miR-377 expression and PIO clinicopathological parameters.Meanwhile,peripheral blood macrophages were obtained to make a comparison of the expressions between miR-377 and receptor activator of NF-?B ligand(RANKL),the correlation between which was observed as well.2.Data were expressed as the means±standard deviation(SD)and analyzed using Graphpad Prism version 5.0 software(Graph Pad Software,San Diego,CA,USA).Statistical differences between two groups were assessed using two-tailed Student's T-test.All experiments were repeated at least three times.P < 0.05 indicated as significantly different.Results: There was no significant difference in miR-377 m RNA expression in peripheral blood between different gender,age and diagnosis of patients undergoing TJA(P > 0.05).However,significant difference was observed in miR-377 expression between different numbers of particles in the joint prosthesis(P = 0.000),indicating that miR-377 expression may be in association with the clinicopathological characteristics of PIO.Conclusion: Decreased miR-377 expression was associated with PIO pathogenesis;MiR-377 was downregulated while RANKL was upregulated in patients with PIO.Part Two Effect of miR-377 on Osteoclast Differentiation and FunctionObjective: To determine the effect of miR-377 on osteoclast formation and activation in vitro.Methods:1.Bone marrow macrophages(BMMs)treated with titanium(Ti)particles and/or transfected with miR-377 mimics were obtained.The expression of RANKL,pro-inflammatory cytokine interleukin-6(IL-6)and tumor necrosis factor-?(TNF-?),as well as related molecules of osteoclast including tartrate-resistant acid phosphatase(TRAP),were measured,and real-time PCR or Western blot or ELISA or TRAP staining was performed as needed to determine the m RNA expression level of miR-377 and the protein expression level of RANKL,as well as the correlation between IL-6,TNF-? levels and osteoclast viability.2.Data were expressed as the means±standard deviation(SD)and analyzed using Graphpad Prism version 5.0 software(Graph Pad Software,San Diego,CA,USA).Statistical differences between two groups were assessed using two-tailed Student's T-test.All experiments were repeated at least three times.P < 0.05 indicated as significantly different.Results: The protein expression of RANKL in BMMs culture with Ti particles was higher than that in BMMs culture without Ti particles(P < 0.05).ELISA analysis indicated that the concentrations of IL-6 and TNF-? in the culture solution containing Ti particles were significantly higher than those in the culture solution without Ti particles(P < 0.05).In addition,the number of TRAP + cells formed in the culture containing Ti particles was three times greater that of formed in the culture without Ti(P < 0.05).In light of these findings,it supported that the stimulation of Ti particles promoted the secretion of inflammatory cytokines,up-regulated RANKL and irritated the viability of osteoclasts in BMMs.At the same time,Ti particles stimulated the production of IL-6 and TNF-? in BMMs(P < 0.05),and enhanced the expression of TRAP staining positive cells,TRAP m RNA(P < 0.05).On the contrary,overexpression of miR-377 resulted in significant reduction of IL-6 and TNF-? levels(P < 0.05)as well as osteoclast viability(P < 0.05),indicating that overexpression of miR-377 minimized PIO-induced inflammation and inhibited osteoclast viability.Conclusion: Ti particles stimulated pro-inflammatory cytokine secretion,upregulated RANKL and promoted osteoclast activity in BMMs;MiR-377 overexpression decreased PIO-induced inflammation and inhibited osteoclast activity.Part Three Mechanism of miR-377 Regulating Osteoclasts via RANKL SystemObjective: To preliminarily explore the regulatory effect of miR-377 on RANKL expression and its molecular mechanism.Methods:1.The potential binding sites of miR-377 and RANKL were predicted by using bioinformatics prediction software Target Scan.Dual-luciferase reporter gene assay was used to verify whether there existed direct binding between miR-377 and RANKL or not.Further,in order to examine the regulatory effect of miR-377 on RANKL expression,HEK-293 cells were transfected with miR-377 mimics or inhibitor and negative control to observe the expression of RANKL at the protein level.2.Data were expressed as the means±standard deviation(SD)and analyzed using Graphpad Prism version 5.0 software(Graph Pad Software,San Diego,CA,USA).Statistical differences between two groups were assessed using two-tailed Student's T-test.All experiments were repeated at least three times.P < 0.05 indicated as significantly different.Results: Co-transfected miR-377 RNA mimics inhibited the expression of firefly luciferase(P < 0.05),while miR-377 inhibitor enhanced the activity of luciferase(P < 0.05).To confirm that this inhibitory or promotive effect was dependent on the predicted miR-377 site or not,we tested p ISoRANKL-MUT,whose recognition site contained mutations.The mutant was no longer in response to miR-377 mimics(P < 0.05)or inhibitor(P < 0.05).In addition,in miR-377 mimics transfected cells,RANKL expression at protein level was significantly down-regulated,in contrast,knocked down miR-377 resulted in an increase in RANKL protein expression level(P < 0.05),suggesting that RANKL was the downstream target of miR-377.Ti particles enhanced the expression of RANKL protein(P < 0.05),stimulated the production of pro-inflammatory cytokines(P < 0.05)and the viability of osteoclasts(P < 0.05).On the contrary,overexpression of miR-377 resulted in a reduction in RANKL expression level(P < 0.05),a decrease in the number of pro-inflammatory cytokines(P < 0.05)and TRAP staining positive cells,as well as a reduction in the expression of TRAP m RNA and CTSK m RNA(P < 0.05).Moreover,the co-overexpression of miR-377 and RANKL would lead to the antagonistic effect.Conclusion: RANKL was a downstream target of miR-377;MiR-377 overexpression led to a decrease in RANKL expression,pro-inflammatory cytokines and osteoclast activity.Part Four Effect of miR-377 on Ti PIO of skull in vivoObjective: To evaluate the in vivo effect of miR-377 in PIO using a mouse skull osteolysis model.Methods:1.Twelve C57BL/J6 mice aged 6-8 weeks were anesthetized by intraperitoneal injection.When the mice were deeply anesthetized,a midline incision was made in the skull,which was then separated from the skull membrane,and 3 mg Ti particles were directly placed on the surface of the calvarium and sutured in full layer.BMMs isolated from mice were induced by M-CSF and RANKL,differentiated after 96 h,and then transfected with pre-NC or miR-377 mimics after exposure to 0.1 mg/m L Ti particles.These BMMs were then surgically transferred to the mouse model.Ten days after operation,the mice were euthanized,and the percentage of bone tissue including trabecular area in the measured bone area was observed.Western blot was used to determine the expression level of RANKL protein.2.Data were expressed as the means±standard deviation(SD)and analyzed using Graphpad Prism version 5.0 software(Graph Pad Software,San Diego,CA,USA).Statistical differences between two groups were assessed using two-tailed Student's T-test.All experiments were repeated at least three times.P < 0.05 indicated as significantly different.Results: In the mouse skull osteolysis model,injection of miR-377 overexpressed BMMs significantly augmented bone area(P < 0.05)and reduced RANKL protein expression(P < 0.05).In general,miR-377 exerted PIO inhibitory effects by binding to RANKL in vivo,demonstrating that miR-377 improved PIO symptoms by targeting RANKL in vivo.Conclusion: miR-377 improved PIO symptoms by binding to RANKL in vivo.