The Research Of Mechanism In Transcription Factor ETS2 Promoting PI3K-AKT Signal Transduction And Aggravating Osteoarthritis

Objective:Osteoarthritis,as is known to us,starts slowly,with progressive degeneration of weight-bearing joints,and tends to occur in people over 50 years old,accompanying with subchondral osteogenesis and articular cartilage degradation as the main manifestations frequently,joint pain and limited mobility have become the major complaints of people seeking medical treatment.In today’s society,there is no specific treatment for this disease,which is mainly using conservative treatment,such as acupuncture,physiotherapy and symptomatic treatment of non-steroidal antiinflammatory drugs using to relieve their discomfort.The purpose is mainly to relieve symptoms and promote the resistance of muscles around the joint,but can’t fundamentally figure out the matter.With the procedures of osteoarthritis,finally joint replacement becomes an another treatment option,but the effectiveness varies from person to person.Therefore,to find an appropriate and effective treatment method has become a top priority.The targeted treatment of osteoarthritis can provide reference for it.ETS2,which is a transcription factor associated with cell metabolism,cell aging and death,has been widely studied in some clinically common diseases such as RA,ARDS and IBD,but no research in osteoarthritis.Therefore,understanding whether ETS2 plays a role in osteoarthritis can provide an innovative approach to the treatment of osteoarthritis.JUNB belongs to AP-1(activator protein-1)dimer transcription factor family,a protein-coding gene that is also a cell proliferation inhibitor,an aging inducer,and a tumor inhibitor.JUNB has been shown to promote articular cartilage degeneration by inhibiting autophagy.JUNB was found to be the downstream target of ETS2 by bioinformatic analysis.PI3K-AKT signaling channel is an access often associated with the progression of orthopedic diseases,which is closely related to apoptosis and autophagy.This pathway is powerful and involves a wide range,and has been widely studied in clinical practice.Therefore,our study aims to explore whether transcription factor ETS2 can affect the expression level of downstream target JUNB and promote PI3K-AKT pathway to aggravate the progression of osteoarthritis.Methods:This study first explored whether ETS2 was correlated with osteoarthritis and its expression level,and predicted the genes of its downstream targets.The expression of ETS2 and JUNB in osteoarthritis was preliminarily predicted by TRRUST database,TFDB database,GTEX database and GEO database.In order to further verify the results of the above biogenic analysis,our research group collected clinical osteoarthritis and normal articular cartilage samples,and detected the degree of ETS2 in human tissue with osteoarthritis by using Safranin O-Fast Green Staining and IHC.Then,two kinds of cells,SW1353 cell line and primary chondrocytes,were stimulated by 20ng/ml interleukin-1β for 24 hours to establish a cellular osteoarthritis model.The expressions of ETS2 and JUNB in the model were verified by western blot,RT-PCR and IF.And lentivirus infection was used to lower ETS2 to see if changes in the index caused by the addition of interleukin-1β could be reversed.Then,western blot and RT-PCR were used to detect common phenotypic inflammatory and apoptotic indicators of osteoarthritis.At the same time,possible signaling pathways involved in the pro-inflammatory and pro-apoptotic effects were detected to further study the mechanism.Finally,the DMM osteoarthritis model was constructed by animal modeling,and the expressions of ETS2 and JUNB at animal level were verified by western blot and RT-PCR.Results:The results of biological information analysis of four databases showed that ETS2 may be related to osteoarthritis and its related phenotypes,and PI3K-AKT pathway is the most closely related signaling pathway,and JUNB is the downstream target of ETS2,which is up-regulated in osteoarthritis.At the human tissue level,the results of Safranin O-Fast Green Staining revealed that the articular cartilage layer of the experimental group was thinner and has more fissures appeared,also we can see the OARSI count of the osteoarthritis group was overwhelmingly higher than the control group.IHC showed increased expression of ETS2 and MMP13 with decreased expression of COL2A1 in human tissue.In vitro experiments,at the cellular level,the results of western blot,RT-PCR and IHC tests in SW1353 cell line and primary chondrocytes both suggested that the expressions of ETS2 and JUNB were increased in osteoarthritis,the pro-inflammatory and pro-apoptotic indexes were increased,and the anti-inflammatory and anti-apoptotic indexes were decreased.After lentivirus knockdown of ETS2,the expression of JUNB,a potential downstream target,was decreased,and the inflammatory and apoptotic indicators were also relieved.In terms of signal routes that may be involved in the above phenomena,the results of western blot told us that the PI3K-AKT pathway had different changes before and after modeling.In the mouse DMM model,WB and RT-PCR also revealed that the convey of ETS2 and JUNB in the osteoarthritis group was higher compared with the control group.Conclusion:In the course of osteoarthritis,ETS2 can promote the progression of osteoarthritis and up-regulate the expression levels of inflammation-related indicators and apoptosis-related indicators.In this process,ETS2 may affect the expression of PI3K-AKT pathway and increase the expression level of JUNB,its downstream target.In total,these outcomes prove that ETS2 leads the importance in the pathogenesis of osteoarthritis,and is expected to provide a new therapeutic strategy for osteoarthritis,a disease with no specific treatment.