MiR-140-5p Affects The Biological Characteristics Of Chondrocytes By Targeting Smad3 In The Inflammatory Microenvironment

Temporomandibular joint(TMJ)is a unique bilateral joint with both rotational and sliding movements in the maxillofacial region.It participates in refined and complex functional activities such as chewing,swallowing,speech,expression and breathing.Osteoarthritis(O A)is a typical representative of joint diseases.Due to the high incidence,OA is harmful to the human body and seriously influence the quality of life of patients.Although many scholars have reported the related type of joint diseases,there is still a lack of complete understanding of the complex pathological mechanism of OA,especially for temporomandibular joint osteoarthritis(TMJ-OA).Previous studies reported that miR-140-5p played an important role in chondrogenesis and cartilage homeostasis.MiR-140(-/-)mice showed age-related OA-like changes.However,clinical studies found a significant increase of miR-140-5p in patients with OA.Although more and more studies indicated a relationship between miR-140-5p and OA,the functional role of miR-140-5p is still controversial.The regulatory mechanism of miR-140-5p in OA needs to be elucidated urgently,especially for TMJ-OA.In the first part of this study,TMJ-OA-like lesion changes were simulated by an experimental model in the inflammatory microenvironment in vitro.The second part predicted and identified the target of miR-140-5p in the condylar chondrocytes in the inflammatory microenvironment.The third part further clarified the regulatory network of miR-140-5p by targeting Smad3.The results of this study explored the regulatory mechanism of miR-140-5p in cells proliferation and chondrogenesis in the inflammatory microenvironment,which provided evidence for understanding the development and clinical treatment of TMJ-OA.Part ? Comparison of mandibular condylar chondrocytes and tibia growth plate chondrocytes in the inflammatory microenvironmentObjectives:To establish an inflammatory response model of mandibular condylar chondrocytes and tibia growth plate chondrocytes in vitro,and to explore the biological differences of different types of chondrocytes in the inflammatory microenvironment.Methods:1.The primary mandibular condylar chondrocytes and tibia growth plate chondrocytes were extracted by two-step enzymatic digestion.H-E's staining and type II collagen immunocytochemical staining were used to identify the cell sources.2.The cell proliferation rates of both cells were measured by CCK-8 and the growth curves were depicted.3.To establish an experimental model of inflammatory microenvironment in vitro,IL-1? was used to induce mandibular condylar chondrocytes and tibia growth plate chondrocytes from 0 h to 24 h.To identify the experimental model and explore the optimal experimental conditions,the expression of miR-140-5p,MMP13 and Col2a1 were detected by qRT-PCR or Western blotting.4.To compare the differences between mandibular condylar chondrocytes and tibia growth plate chondrocytes in the inflammatory microenvironment,Western blotting was used to analyze the relative expression of inflammatory response-related protein NF-?B1 and chondrogenesis correlated proteins,such as Smad3/4,p-Aktl,Runx2,p-CREB,Sox9 and TGF-?3.Results:1.Primary cells were stained by H-E's staining and immunocytochemical staining.Positive expression of type II collagen in immunocytochemical staining indicated that the extracted primary cells exhibit specific characteristics of chondrocytes and the primary chondrocytes extraction was reliable.2.The results of CCK-8 indicated that the optical density of the tibia growth plate chondrocytes was significant higher than that of the condylar chondrocytes at 5 d.3.The results of qRT-PCR showed that MMP13 and miR-140-5p gradually increased by IL-1?.The difference was most significant at 24 h of induction.In contrast,the mRNA expression of Col2a1 exhibit in a converse manner.4.Western blotting showed that the protein expression of Col2al was decreased while NF-?B1 increased in IL-1?-induced inflammatory microenvironment,which suggesting that IL-1?promoted the inflammatory reaction of chondrocytes.The expression of Smad3/4,p-Akt1,Runx2,p-CREB,Sox9,and TGF-?3 was suppressed,suggesting that chondrocyte proliferation,differentiation,and phenotype were affected by IL-1?.Notably,the expression of TGF-P3 in the mandibular condylar chondrocytes was more markedly decreased than that of the tibial growth plate chondrocytes.While the expression level of p-Aktl protein was down-regulated more significantly in tibia growth plate chondrocytes.Conclusions:1.Two-step enzymatic digestion was a reliable and reproducible way to extract the primary chondrocytes.2.The proliferation of tibia growth plate was more active than that of mandibular condylar chondrocytes.3.24 h-induction of IL-1? was an stable experimental condition in the present study.4.IL-1? efficiently accelerated inflammation but suppressed chondrocyte proliferation and differentiation.5.Although different parts of articular chondrocytes had tiny differences in inflammatory microenvironment,both of them showed similar OA changes,which suggested that IL-1?-induced MCCs exhibited TMJ-OA-like changes.Part ? Target identification of miR-140-5p in TGF-? pathwayObjectives:To identify the potential target of miR-140-5p in TGF-? pathway,and to explore the IL-1?-induced miR-140-5p regulatory mechanism of proliferation,differentiation and cell apoptosis through TGF-? pathway.Methods:1.The potential binding sites between miR-140-5p and the 3'UTR of Smad3/4 were predicted.Based on the wild-type plasmid vector(WT 3'UTR),the potential binding sequence were mutated and cloned into plasmid vector(MUT 3'UTR).Then HEK293T cells were cotransfected with miR-140-5p and WT 3'UTR,or miR-140-5p and MUT 3'UTR for 48 h.The target gene was verified by the dual luciferase assay.2.To explore the optimal condition for mandibular condylar chondrocytes transfection,miR-140-5p mimics or miR-140-5p inhibitors or the corresponding negative control were transfected into mandibular condylar chondrocytes respectively.Smad3 protein was determined by Western blotting.3.qRT-PCR was utilized to verify the reliability of the miR-140-5p mimics transfection model in vitro.4.Immunofluorescence staining analysis was used to verify the downregulation of Smad3 medicated by miR-140-5p at protein level in mandibular condylar chondrocytes.5.The miR-140-5p was over expressed in mandibular condylar chondrocytes via inflammatory microenvironment transfection model in vitro.To elucidate the relationship between miR-140-5p and TGF-? pathways in the inflammatory microenvironment,Western blotting assay was used to detect chondrogenesis correlated proteins,such as Smad3/4,p-Aktl,Runx2,p-CREB,Sox9,TGF-?3,NF-?B1,NF-?B2,and RelA.Results:1.Predicted by TargetScan and miRTarBase assay analysis,it was found that the 3'UTR of Smad3/4 mRNA contained potential sequences binding to miR-140-5p.Dual luciferase assay revealed that miR-140-5p reported against Smad3 wild-type vector when compared to Negative Control(NC).The downregulation of fluorescence indicated that Smad3 was the direct target of miR-140-5p.What's more,miR-140-5p showed no significant effect on the fluorescence of the Smad4 wild-type vector.The predicted site on the 3'UTR of Smad4 did not significantly interact with miR-140-5p.2.Western blotting analysis indicated that miR-140-5p mimics or miR-140-5p inhibitors inhibited or increased the expression of Smad3 protein production,and transfection for 48 h was a optimal condition.3.To establish a reliable miR-140-5p transfection model,qRT-PCR analysis was utilized in the present study.the qRT-PCR results showed that miR-140-5p mimics significantly increased miR-140-5p miRNA expression in mandibular condylar chondrocytes,while miR-140-5p inhibitors acted a converse manner despite of the stimulation of IL-1?.4.The results of immunofluorescence staining showed that miR-140-5p mimics and IL-1?significantly decreased the fluorescence intensity of Smad3 when compared with the control group(NC),while miR-140-5p inhibitors increased the fluorescence intensity of Smad3 and antagonized the effect of IL-1? on suppressing the expression of Smad3.5.The results of Western blotting showed that IL-1? inhibited cell proliferation-related proteins through miR-140-5p,such as Smad3/4 and p-Aktl.IL-1? reduced the expression of Runx2,a condensation and hypertrophy correlated protein.IL-1? also suppressed chondrocytes phenotype correlated proteins,such as Sox9,p-CREB and TGF-?3.miR-140-5p suppressed the protein expression of NF-?B1,NF-?B2,and RelA.Interestingly,when compared to the miR-140-5p mimics group,the IL-1?-induced miR-140-5p mimics group showed no significant reduction of Smad3/4,p-CREB and Sox9,which suggested that IL-1? may regulate Smad3/4,p-CREB and Sox9 via miR-140-5p.On the contrary,the IL-1?-induced miR-140-5p mimics group showed a lower expression of p-Aktl,Runx2 and TGF-?3 when compared to miR-140-p mimics group,which suggesting that p-Aktl,Runx2,and TGF-?3 might be down-regulated by IL-1? via multiple ways,including miR-140-5p pathway.Conclusions:1.Smad3 was identified as one of the direct targets of miR-140-5p in the TGF-? pathway.2.The IL-1?-induced inflammatory microenvironment regulated the expression of Smad3 via miR-140-5p.3.The signaling mechanism of miR-140-5p regulated chondrogenesis-related proteins in the inflammatory microenvironment and may be closely related to pathological mechanism of TMJ-OA.Part ? miR-140-5p regulated chondrogenesis via TGF-?/Akt/NF-?B signaling pathwaysObjectives:To elucidate the signaling mechanism of miR-140-5p targeting Smad3,and to explore the signal regulatory network that may be involved in miR-140-5p regulation of chondrocytes proliferation,differentiation and apoptosis.Methods:1.To establish a model of chondrocytes with silencing of Smad3 expression in vitro,mandibular condylar chondrocytes were cotransfected with or without small interfering RNA of Smad3(siRNA Smad3)in the presence or absence of miR-140-5p mimics.2.To evaluate the effect of miR-140-5p on the chondrocytes proliferation by suppressing the expression of Smad3,CCK-8 was used to detect the optical density of chondrocytes at 24h,48h,72h after transfection.3.The apoptosis of mandibular condylar chondrocytes in each group was observed by Flow Cytometry.4.Western blotting was used to detect the proteins that related to cell proliferation,differentiation,and apoptosis,including Smad3/4,p-Aktl,Runx2,p-CREB,Sox9,TGF-?3,NF-?B1,NF-?B2,and RelA.Results:1.Western blotting showed that the transfection of siRNA Smad3 or miR-140-5p mimics suppressed the expression of Smad3 and p-Smad3 proteins significantly in mandibular condylar chondrocytes,which provided evidence that the transfection model in vitro was reliable.2.The results of CCK-8 indicated that the optical density of siRNA Smad3 group and miR-140-5p mimics group were lower than that of control group.It suggested that the proliferation of chondrocytes was significantly suppressed by miR-140-5p-regulated Smad3.3.Flow cytometry analysis showed that over expression of miR-140-5p or silencing of Smad3 promoted the apoptosis in mandibular condylar chondrocytes,and the most conspicuous apoptosis rate was found in co-transfection group.4.Silencing Smad3 or over expression of miR-140-5p reduced the expression of chondrogenesis correlated proteins,such as Smad3/4,Sox9,Runx2,TGF-?3,and p-Aktl.Notably,siRNA Smad3 had no effect on the expression of p-CREB compared with the negative control,but p-CREB protein decreased significantly only if miR-140-5p over expressed.It indicated that p-CREB might be a direct target of miR-140-5p.Moreover,co-transfection of siRNA Smad3 miR-140-5p showed a lower expression of p-Akt1,Runx2,Sox9,and TGF-?3,suggesting miR-140-5p might regulated p-Aktl,Sox9,Runx2 and TGF-?3 negatively except from Smad3 pathway.In the present study,over expression of miR-140-5p lead to a reduction of apoptosis correlated proteins,like NF-?B1,NF-?B2,and RelA.However,the silencing Smad3 did not show a significant effect on NF-?B.It suggested that miR-140-5p may affected the NF-?B pathway by some other manners.Conclusions:1.MiR-140-5p affected chondrocytes proliferation,differentiation and apoptosis correlated proteins by targeting Smad3.2.The regulatory mechanism of miR-140-5p was complicated,which involved in TGF-?/Akt/NF-?B signaling pathway.3.The present study provided evidence that miR-140-5p might be a potential predictive factor for TMJ-OA at early stage.