The Mechanism Of MiR-15b Regulating Proliferation And Apoptosis Of Condylar Hypertrophic Chondrocytes

Part 1.The expression of insulin-like growth factor-1,insulin-like growth factor-1 receptor and B-cell lymphoma-2 in condylar hyperplasia cartilageBackground:Insulin-like growth factor-1(IGF1)is a key proliferation inducer that enhances cell proliferation through the insulin-like growth factor-1 receptor(IGF1R).B-cell CLL/lymphoma 2(BCL2)is a major anti-apoptotic factor in its family and can be expressed in many cells,including osteoblasts,acute myeloid leukemia cells and human umbilical vein endothelial cells.The purpose of this part of the study was to investigate the expression of IGF 1,IGF1R and BCL2 in the condylar hyperplasia(CH)cartilage of the human temporomandibular joint.Methods:The condylar tissue specimens of patients with condylar hypertrophy were collected during the temporomandibular joint condyle hypertrophy as the experimental group and the condylar tissue specimens of the patients with condylar fracture were used as the control group.Immunohistochemistry,immunofluorescence,Real-time PCR and Western blot were used to detect the expression of IGF 1,IGF1R and BCL2.Results:Compared with the control group,immunofluorescence and immunohistochemistry showed that IGF1,IGF 1R and BCL2 were significantly increased in CH cartilage,and IGF1,IGF1R and BCL2 were abundantly expressed in the proliferative layer and hypertrophy of the condyle.The results of Real-time PCR and Western blot showed that the expression of IGF 1,IGF1R and BCL2 was significantly enhanced in CH condylar chondrocytes.Conclusion:The expression of IGF I,IGF1R and BCL2 was significantly increased in the condylar hypertrophic condylar cartilage cells,which may be related to the excessive proliferation of condylar hypertrophic condylar chondrocytes.Part 2.Prediction and identification of microRNAs and mechanism studies of related microRNAsObjective:The aim of this study was to predict and identify microRNAs associated with IGF1,IGF1R and BCL2 in condylar hypertrophic chondrocytes and to validate the effects of the corresponding microRNAs on IGF 1,IGF 1R and BCL2.Methods:The MiRanda and TargetScanS algorithms were used to predict microRNAs associated with IGF1,IGF1R and BCL2 in CH chondrocytes.MicroRNA Real-time PCR detects the expression of related microRNAs in CH.Dual luciferase assays was used to verify whether IGF 1,IGF 1R,and BCL2 are direct targets of related microRNAs.Real-time PCR and Western blot were used to detect the expression of IGF1,IGF1R and BCL2 and their related pathways after enhancing or inhibiting the corresponding microRNA.Results:Seven related microRNAs by MiRanda and TargetScanS algorithms were found out,which are miR-15a,miR-15b,mR-16,miR-195,miR-424,miR-497 and miR-6838,respectively,and miR-15b is the only one microRNA significantly changed in CH chondrocytes.IGF1,IGF1R and BCL2 are the direct target genes of miR-15b.Inhibition of miR-15b significantly enhanced the expression of IGF 1,IGF1R and BCL2,and activated IGF 1 and IGF 1R related pathways of PI3K/AKT and BCL2 related pathway of JAK/STAT.Enhanced miR-15b inhibits IGF1 and IGF1R related pathways of PI3K/AKT and BCL2-related pathways of JAK/STAT,and decreased expression of IGF1,IGF1R,and BCL2Conclusion:MiR-15b is involved in the progression of CH in temporomandibular j oint,and miR-15b negatively regulates the expression of IGF 1,IGF1R and BCL2 and their related pathways in CH chondrocytes.Part 3.The Effects of miR-15b on proliferation and apoptosis of condylar chondrocytesObjective:This study was to investigate whether miR-15b is involved in the regulation of proliferation and apoptosis of temporomandibular joint condylar chondrocytes.Methods:The effects of enhancing and inhibiting miR-15b on the proliferation of condylar chondrocytes were verified by cell proliferation assay CCK8 assay and flow cytometry.Hoechst 33342 cell assay and flow cytometry assay were used to verify the effects of enhancing and inhibiting miR-15b on the apoptosis of condylar chondrocytes.Western blot was used to detect the expression of apoptosis-related factors caspase 9 and Fas after enhancing or inhibiting miR-15b.Results:The inhibition of miR-15b in condylar chondrocytes promotes an increase in cell proliferation activity and an increase in the proportion of cells in the S phase.Enhancing miR-15b reduced cell proliferation activity and decreased the proportion of S phase cells.Inhibition of miR-15b in condylar chondrocytes reduced the proportion of cell apoptosis and decreased the expression of apoptosis-related factors caspase 9 and Fas.Enhancement of miR-15b increased the apoptotic rate of chondrocytes and increased the expression of apoptosis-related factors caspase 9 and Fas.Conclusion:In CH condylar chondrocytes,the inhibited miR-15b enhances cell proliferation activity and reduces cell apoptosis,while increased expression of miR15b leads to increased apoptosis of condylar chondrocytes.Part ?.Establishment of an animal model of temporomandibular joint disc displacement in ratsObjective:This study was designed to investigate whether the rat model of the anterior joint displacement caused abnormal proliferation of the condyle.Methods:The rat model of temporomandibular j oint disc displacement was established by operation.Hematoxylin and eosin(HE)was used to detect the overall morphology of the condyle,articular disc and synovium.Micro CT was used to detect changes in subchondral bone.The cartilage matrix glycoprotein changes were detected with saffron green,and osteoprotegerin(OPG)was detected to determine bone destruction.The degree of apoptosis was tested by the TUNEL assay.Immunohistochemistry was used to detect the expression of the stress-related protein pizeol.Results:HE and micro CT showed significant bone destruction,subchondral bone destruction and alteration in the experimental group.Safranin solid staining confirmed that the cartilage matrix glycoprotein was continuously reduced in the experimental group,and the expression of OPG was also decreased in the experimental group.This degenerative process of the temporomandibular joint is accompanied by increased apoptosis and increased expression of pizeol protein.However,this model does not promote the abnormal proliferation of condylar and the development of condylar hypertrophy.Conclusion:The establishment of the rat temporomandibular joint disc displacement animal model did not lead to abnormal proliferation of the condyle,but it can lead to the degeneration of the temporomandibular joint.