| miRNAs are non-coding single-strand RNAs(ssRNAs),of～22nt in length in their mature form that can specially bind to 3’-untranslated region of the target mRNA and negatively regulate gene expression post-transcriptionally, and make biological sense,through repressing or degradating of mRNA translation. It is knew that miRNAs were closely related to bone germination, growth, metabolism. ConnexinCx43 protein as the main gap junction in bone tissue, the gap connection with Cx43 and its half channel make directly communicating between bone tissue cells, play an important part in bone normal development and bone remodeling. Tremendous researches had showed that microRNA-206 regulation of osteoblast through Cx43/miRNA-206 signal path. And osteoblast cell is one of the most important cells in bone development and reconstruction, also significant to SANFH. We hypothesized that the pathologic process of SANFH were connected with osteoblast cell differentiation regulated by Cx43/miRNA-206 signal path. The purpose of this research was to investigate the relationship between miR-206 and SANFH and find the possible pathogenesis of SANFH.Method and Result1. The expressions of miRNA-206 and Cx43 in steroid -induced avascular necrosis of the femoral head(SANFH).Method:(1) Forty-six of whole femoral heads obtained from patients who underwent prosthetic hip replacement because of osteonecrosis due to chronic glucocorticoid treatment (n=26)and fresh femoral neck fracture(n=20).(2) HE staining was used to check the femoral head tissue morphology.(3) Immunohistochemical staining to detect the expression of Cx43 in SANFH.(4) Total RNA were extracted from the femoral head, the expression levels of miR-206 and Cx43mRNA were tested using real-time PCR.(5) Total protein were extracted Cx43 quantify were detected by Western blot.Result:(1)Histological examination suggested the subchondral bone trabecula became thin and sparse with disorder structure; a great quantity of empty osteocyte; hematopoietic tissue in medullary cavity was significantly decreased in SANFH; In contrast, apoptotic bone cells were absent and hematopoietic tissue in medullary cavity was active in control group.(2)Immunohistochemical results showed that, positive expression of Cx43 was seen in osteoblast, osteocyte and marrow cavity:(3) The PCR result showed that compared with femoral neck fracture, the expression levels of miR-206 was significantly up-regulated by 4.3 times in SANFH(P<0.05),while 44% down-regulation was recorded in expression of Cx43mRNA in SANFH as comparable to femoral neck fracture (P<0.05).(4) The protein expression of Cx43 in SANFH group(0.236±0.017)was significantly lower than the femoral neck fracture group(0.496±0.022)(P<0.05).2. miRNA-206 through inhibited connexin43 expressions to regulated osteoblast differetitaion.Method:(1) Mimic, inhibitor and NC of miRNA-206 were transfected into osteoblasts to produce over-expression or declining expression of miRNA-206.(2) Total RNA and protein were extracted, the expression levels of miR-206 and Cx43mRNA were tested using real-time PCR, Cx43 and ALP quantify were detected by Western blot,CCK-8 to detect osteoblast cell proliferation capability.Result:(1)Post-transfection with miRNA-206 mimicCompared with N.C.group number of osteoblast declined in mimic group. Gene expression of miRNA-206 were remarkably increased, (P<0.05);Cx43mRNA remained unchanged (P>0.05),Cx43,ALP activity significantly decreased(P<0.05).(2)Post-transfection with miRNA-206 inhibiters Compared with N.C. group number of osteoblast increased significantly in inhibiter group. Gene expression of miRNA-206 were remarkably decreased(P<0.05), Cx43mRNA remained unchanged (P>0.05),while Cx43,ALP activity significantly increased(P<0.05).Conclusion:(1) The expression levels of miR-206 was up-regulated in SANFH, while the Cx43mRNA and Cx43protein were decreased.(2) miRNA-206 through inhibited connexin43 expressions to regulated osteoblast differetitaion.(3) miRNA-206,at lease in part, through connexin43 signaling pathways in ostoblast, may play a regulatory role in the development of SANFH. |
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