MicroRNA-206 Regulates Osteogenic Differentiation In Steroid-induced Femoral Head Necrosis By ConneXin43-ERK1/2 Signaling Pathway

Background and objectiveThe pathogenesis of steroid-induced avascular necrosis of the femoral head(SANFH)has not been clarified,and bone marrow mesenchymal stem cells(BMSCs)are closely related to osteogenic differentiation.Studies have shown that microRNA-206(microRNA-206,miR-206)down-regulates its target protein connexin43(Cx43),which in turn inhibits osteoblast differentiation.High expression of Cx43 activates the ERK1/2 classical signaling pathway and promotes Osteoblast differentiation.Extracellular signal regulated kinase 1/2(ERK1/2)is the first and most representative member of the mitogen-activated protein kinase(MAPK)family.The activated ERK1/2 can further affect the expression and activation of its downstream transcription factors.The core binding factor al(Cbfal),(Runx2),is a kind of transcription factors.The Runx2 binds to elements on many osteogenic gene promoters and promotes transcriptional expression of osteogenic target genes.Studies have shown that Runx2 is one of many downstream target genes of ERK1/2,but whether the Cx43-ERK1/2-Runx2 pathway is activated in SANFH is unknown;if it does participate in the osteogenic differentiation of BMSCs in SANFH development,its specificity The molecular mechanism needs to be further studied.In our previous studies,it was found that the expression of Cx43 in the femoral head tissue of patients with femoral neck fracture was significantly higher than that in patients with SANFH,and it was initially confirmed that Cx43 may play an important regulatory role in the process of SANFH;the current study found that Cx43 can be regulated by ERK.Osteogenesis,chondrocyte metabolism,and Src/MEK/ERK pathway are closely osteogenic target genes.Studies have shown that Runx2 is one of many downstream target can regulate SANFH through MAPK-related pathway.We hypothesized that miR-206/Cx43 plays an important role in SANFH by regulating osteoblast differentiation via the downstream ERK1/2-Runx2 classical signaling pathway.Through the establishment of SANFH animal model,the expression changes of Runx2,miR-206,Cx43 and ERK1/2 classical signaling pathways in the femoral head were dynamically detected.To observe the changes of osteogenic differentiation ability of rabbit femoral head,and to elucidate the role and potential mechanism of miR-206 targeting Cx43 in regulating osteogenic differentiation in SANFH,and provide a new target for SANFH diagnosis and treatment.Method1.72 healthy New Zealand white rabbits were randomly divided,which were control group,model group(group B)and inhibitor group(group C).The model group and the inhibitor group were treated with lipopolysaccharide combined with methylprednisolone to establish a model of steroid-induced femoral head necrosis;the inhibitor group was injected with the ERK1/2 pathway inhibitor U0126 at the same time as the first injection of methylprednisolone,and the control group was injected at the same time.The same amount of normal saline.Three groups of animals were examined by magnetic resonance imaging(MRI)at 2nd,4th,and 8th week after modeling,and the model was successfully established by hematoxylin and eosin(HE)staining.2.In situ hybridization(ISH)and real-time-polymerase chain reaction(RT-PCR)were used to detect the expression of miR-206 in rabbit femoral head;RT-PCR and Westernblot were used to detect Cx43.,Runx2,ERK1/2;alkaline phosphatase(ALP)staining method to detect ALP expression;immunohistochemistry(IHC)to detect Cx43 expression.Result1.MRI showed that the signal intensity of T1WI in the bilateral femoral heads of group B and group C decreased,which was linear or spotted,and T2WI showed uneven high signal.There was no abnormal signal in T1WI and T2WI of bilateral femoral head in group A.Light microscopy showed that the trabecular bones in the femoral head of the B group and the C group were thinner and ruptured,the fat cells increased,and the empty bone filling socket increased.The trabecular bone structure of group A was normal,no disorder or broken,no empty space was found.At the time of bone lacunae,the rate of empty bone sag and fat cell diameter in group C were higher than those in group A and B.The difference between group B and group A was statistically significant(P<0.05).2.ALP staining showed that alkaline phosphatase was mainly expressed in osteoblasts at the edge of trabecular bone,and the staining intensity of group C was weaker than that of group A and B,and that of group B was weaker than that of group A(P<0.05).RT-PCR results showed The expression of Runx2 mRNA in group C was lower than that in group A and group B,and that in group B was lower than that in group A(p<0.05).The results of Western blot showed that the expression of Runx2 protein in femoral head of group C was lower than that of group A and group B,B Groups were less than group A(p<0.05).The ISH results showed that miR-206 was positively expressed in the medullary cavity,osteoblasts and a small number of bone cells,and the coloration of the femoral head in the B group and the C group was stronger than that in the A group(p<0.05),and the C group had no comparison with the B group.Significant difference(p>0.05).The results of RT-PCR showed that the expression of miR-206 in the femoral head of rabbits in group B and group C increased compared with group A(p<0.05).There was no significant difference between group C and group B(p>0.05).IHC results showed that Cx43 was positively expressed in medullary cavity cells and osteoblasts,and the intensity of Cx43 staining in femoral head of group C was weaker than that of group A and group B,and group B was weaker than group A(P<0.05).The results of RT-PCR and Western blot showed that the expression of Cx43 mRNA,Cx43 and phosphorylated ERK1/2 protein in group C was lower than that in group A and B,and that in group B was less than that in group A(p<0.05).Conclusion1.The Cx43-ERK1/2-Runx2 signaling pathway was inhibited to a certain extent in the animal model of steroid-induced avascular necrosis of the femoral head.2.Glucocorticoid may down-regulate its target protein Cx43 by miR-206,inhibit the transduction of ERKl/2-Runx2 signaling pathway,inhibit osteogenic differentiation and promote adipocyte formation,and participate in the development of steroid-induced femoral head necrosis.