| Research Objectives:Rheumatoid arthritis(RA)is a common autoimmune disease with symmetric multiple arthritis as the main clinical manifestations.The etiology and pathogenesis of RA are complex and still unclear.Recent studies have confirmed that non-coding Rnas are involved in the occurrence and development of rheumatoid arthritis by targeting downstream related genes.Based on this,this paper discussed from the following three aspects: From the perspective of clinical research,the evaluation value of Lnc RNA Lnc RNA Plnc RNA-1?mi R-146 a and TGF-?1 on the severity of rheumatoid arthritis patients was explored.To explore the targeting relationship between Lnc RNA Lnc RNA Plnc RNA-1?mi R-146 a /TGF-?1 axis from the perspective of cytology,and to explore the regulation of the three on the proliferation,migration and invasion of synovial fibroblasts,providing a theoretical basis for clinical exploration of the pathological mechanism of rheumatoid arthritis.To explore the mechanism of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 involved in the occurrence and development of rheumatoid arthritis from in vivo animal models,and to determine the regulatory behavior of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 on inflammatory factors in rats.Materials and Methods:(1)30 female Wistar rats were selected to construct CIA rat Model of type II collagen-induced arthritis,and Control group and Model group were set.Determine the success of model establishment;The expressions of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 in synovial fluid and synovial tissue were detected by RT-PCR.The expression of inflammatory factors in synovial fluid and synovial tissue was detected by ELISA.TUNEL assay was used to detect the apoptosis index of synovial tissue.To determine the correlation between the expression of Lnc RNA Plnc RNA-1?mi R-146 a and TGF-?1 in synovial tissues and inflammatory factors and apoptosis index.(2)Clinical study: 70 patients with rheumatoid arthritis diagnosed in our hospital were selected according to whether they were in.The active period was divided into two groups: active RA(n=34)and non-active RA(n=36).Forty healthy subjects were selected as the control group.The expressions of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 in serum were detected by RT-PCR.The expression levels of CRP,ESR,INF-?,TNF-?,IL-17 and IL-10 in serum were determined by ELISA.The expression of Lnc RNA Plnc RNA-1?mi R-146 a and TGF-?1 were correlated with CRP,ESR,INF-?,TNF-?,IL-17 and IL-10.Serum levels of Lnc RNA Plnc RNA-1?mi R-146 a,mi R-146 a and TGF-?1 and Lnc RNA Plnc RNA-1and TGF-?1 were correlated.The diagnostic value of Lnc RNA Plnc RNA-1 active RA was analyzed by ROC curve.(3)In vitro cell study: The synovial fibroblasts(RA-FLS)were selected as the research object,and primary culture was performed.P3 cells were selected as the research object.After LPS treatment,the cells were transfected with Lnc RNA Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 overexpressed and knocked out plasmids.Control group,pc DNA-Plnc RNA-1,si Plnc RNA-1,pc DNA-mi R-146 a,PCDNA-Plnc RNA-1+mi R-146 a and pc DNA-Plnc RNA-1+ mi R-146 a + TGF-?1 were set.The cells were cultured for 48 hours.The successful transfection of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 in each group was determined by RT-PCR.Dual luciferase gene assay was used to determine the targeted regulation of Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1.Cell proliferation activity was detected by CCK-8.Apoptosis was detected by Annexin V assay.The expressions of factors INF-?,TNF-?,IL-17 and IL-10 were detected by ELISA.The migration and invasion of cells were determined by scratch assay and Transwell assay.The concentration expression of migration,invasion and proliferation related proteins was detected by WB.Results:(1)Verification of the success of model establishment: three weeks later,in the fourth week after model establishment,the skin on the surface of the joints of rats in the model group was hyperemic and pink;The joints of rats in the control group were normal without obvious redness and swelling.The weight gain of rats in control group was faster than that in model group at 3 and 4 weeks.Hind foot volume in model group increased gradually at 2,3 and 4 weeks after model establishment(P<0.05).With the prolongation of time,arthritis index of rats in model group increased(P<0.05);In the control group,the articular surface was smooth,the synovium was thin and intact,and there was no obvious tissue edema or inflammatory cell infiltration.Synovial tissue in CIA model rats was infiltrated by inflammatory cells,the proliferation and arrangement of synovial cells were disorder,and some synovium was missing.After CIA model was established,the expression of Lnc RNA Plnc RNA-1 and TGF-?1 in synovial fluid and synovial tissue of joints of rats was decreased,and the expression of Mir-146 a was increased(P<0.05).After CIA model was established,the apoptosis of synovial tissue cells was increased(P<0.05).After the establishment of CIA model,the expression of INFLAMMATORY factors INF-?,TNF-? and IL-17 in synovial fluid and synovial tissue increased,while the expression of IL-10 decreased(P<0.05).The expression of Lnc RNA Plnc RNA-1 and TGF-?1 was negatively correlated with the expression of INF-?,TNF-? and IL-17,and positively correlated with the expression of IL-10.The expression of mi R-146 a was positively correlated with the expression of INF-?,TNF-? and IL-17,and negatively correlated with the concentration of IL-10(P<0.05).(2)Clinical study: general data: average age,sex ratio,course of disease,BMI index,underlying diseases(hypertension,coronary heart disease,diabetes)of subjects in the three groups(P>0.05);Lnc RNA Plnc RNA-1 ? mi R-146 aand TGF-?1 concentrations showed the same expression trend in serum and cell samples.Lnc RNA Plnc RNA-1 and TGF-?1 were low in RA patients,and mi R-146 was high in RA patients.The trend was most obvious in active patients,followed by inactive patients.The control group was last(P<0.05);The expression levels of CRP,ESR,INF-?,TNF-? and IL-17 in RA patients were higher than those in control group,while the expression levels of IL-10 were lower than those in control group.The expression concentration of inflammatory factors was the highest in RA patients at active stage(P<0.05).The expressions of Lnc RNA Plnc RNA-1and TGF-?1 in serum of RA patients were negatively correlated with CRP,ESR,INF-?,TNF-? and IL-17,but positively correlated with IL-10,and mir-146 showed an opposite trend(P<0.05).Plnc RNA-1 and mi R-146 a,Lnc RNA Plnc RNA-1 and TGF-?1 and mi R-146 a and TGF-?1 in serum of active RA showed negative correlation,positive correlation and negative correlation,respectively,with the strongest correlation in active RA patients.There was no correlation among RA patients in the control group(P<0.05).The AUC areas of active patients,non-active patients and healthy control group were0.8611,0.9004 and 0.6361,respectively,and the 95%CI were 0.7730-0.9493,0.8279-0.9729 and 0.5263-0.7460,respectively(P<0.05).(3)Lnc RNA Plnc RNA-1 ? mi R-146 a and TGF-?1 plasmids were successfully transfected in RLS cells.Overexpression of Lnc RNA Plnc RNA-1 increased the expression of TGF-?1 in RLS and inhibited the expression of mi R-146a(P<0.05).The m RNA 3'non-coding region of Lnc RNA Plnc RNA-1 predicted a mir-146 a binding site,and the wild-type and mutant PGL3-Plnc RNA-1-UTR expression vectors were transfected into RLS cells,and the reporter gene activity of the wild-type expression vectors was significantly decreased(P<0.05).A TGF-?1 binding site was predicted in the m RNA 3'non-coding region of mi R-146 a.Different combinations of wild-type and mutant PGL3-TGF-?1-1-UTR expression vectors were transfected into RLS cells by co-transfection technique,and the reporter gene activity of wild-type expression vectors was significantly decreased(P<0.05).Target Scan analysis did not find a certain binding site between Lnc RNA Plnc RNA-1 and TGF-?1.With the extension of time,RLS cells in each group showed an increasing trend.The overexpression of PCDNA-Plnc RNA-1 in RLS cells inhibited the proliferation,migration,invasion and intracellular inflammatory response of RLS cells,and the concentrations of Survivin,PCNA,Bcl-2,MMP-2 and MMP-9 were significantly decreased.The expression concentration of Bax and Caspase-3increased(P<0.05).Conclusion:(1)The expression of Lnc RNA Plnc RNA-1 and TGF-?1 in synovial fluid and synovial tissue was decreased and the expression of mi R-146 a was increased in CIA model of rat rheumatoid arthritis.Lnc RNA Plnc RNA-1 and TGF-?1 inhibit the expression of inflammatory factors and inhibit the progression of the disease,while the expression of mi R-146 a promotes the expression of inflammatory factors and promotes the development of rheumatoid arthritis,which is consistent with the conclusions of the previous two parts and provides some inspiration for clinical research.(2)Lnc RNA Plnc RNA-1?mi R-146 a and TGF-?1 were differentially expressed in the serum of patients with RHEUMAToid arthritis.Low expression of Lnc RNA Plnc RNA-1and TGF-?1 inhibited the expression of inflammatory factors and the development of the disease.The high expression of mi R-146 a promotes the expression of inflammatory factors and promotes the development of the disease Lnc RNA Lnc RNA Plnc RNA-1?mi R-146 aand TGF-?1 were significantly correlated in active patients,and jointly regulated the development of the disease.(3)Lnc RNA Plnc RNA-1 regulates the biological activity of RLS cells by targeting mi R-146 a /TGF-?1 axis.Overexpressed Lnc RNA Plnc RNA-1 inhibits the proliferation activity,migration,invasion ability and inflammatory response of RLS cells by inhibiting the expression of Mir-146 a and promoting the expression of TGF-?1,thus promoting the apoptotic activity of LRS cells and ultimately inhibiting the occurrence and development of rheumatoid arthritis.To provide some inspiration for clinical research. |
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