| 1.ObjectiveAn in vitro assay and clinical association rules were conducted to explore the mechanism of lncRNA MAPKAPK5-AS1(named lncRNA MK5-AS1 or MK5-AS1)/miR-146a-3p/SIRT1 axis mediated in immune inflammation and apoptotic escape in rheumatoid arthritis(RA),and the intervention of Xinfeng capsule(XFC).2.Methods2.1 Experiment I: Mechanisms of Lnc RNA MK5-AS1/miR-146a-3p/SIRT1combination-mediated immune inflammation and apoptotic escape in RA fibroblast-like synoviocytesA co-culture model of peripheral blood mononuclear cells(PBMCs)and fibroblast-like synoviocytes(FLS)of RA was utilized in this experiment.RNA interference and overexpression plasmid were constructed,and were transfected in RA-FLS.The fluorescence in situ hybridization(FISH)method was used to determine the location of lncRNA MK5-AS1.RNA pull down,RNA immunoprecipitation(RIP),dual-luciferase report experiments and series revertive experiments to verified the targeted regulation relationship between lncRNA MK5-AS1,miR-146a-3p,and SIRT1,and its influence on immune inflammation and apoptotic escape.Expression levels of genes were determined by performing real-time quantitative PCR(RT-q PCR).Cell viability was assessed by performing the Cell Counting Kit-8(CCK-8)assay.The contents of interleukin-6,IL-8,IL-4,and IL-10 were detected by enzyme-linked immuno sorbent assay(ELISA).The bcl2-associated X(Bax),B-cell lymphoma-2(Bcl-2),cysteine-containing aspartate-specific proteases 3,caspas8 and nuclear factor-k-gene binding(NF-κB)protein expression levels were determined by Western blot analysis.Cell apoptosis analysis was assessed by flow cytometry(FCM).lncRNA MK5-AS1/miR-146a-3p/SIRT1 combination2.2 Experiment II: Effects of Xinfeng capsule-containing serum on immune inflammation and apoptotic escape of RA fibroblast-like synoviocytes by regulatingWe observed the effects of XFC-containing serum on cell viability,lncRNA MK5-AS1,miR-146a-3p,SIRT1,inflammation,and apoptosis indexes.Then through a series of recovery experiments,we transfected si-MK5-AS1,miR-146a-3p inhibitor and SIRT1 overexpression plasmids on the basis of XFC-containing serum,and observed the changes of inflammation and apoptosis indexes.2.3 Experiment III: Study on the changes of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis,immune inflammation and apoptosis escape indexes in RA patients and the effect of Xinfeng capsule based on association rulesWe detected the expression of lncRNA MK5-AS1,miR-146a-3p,SIRT1,inflammation,and apoptosis indexes by collecting PBMCs of healthy people and RA patients before and after XFC treatment.At the same time,the laboratory indexes and SPP scores of RA patients were collected.We analyzed the correlation between XFC treatment and lncRNA MK5-AS1,miR-146a-3p,SIRT1,inflammation,apoptosis,laboratory indexes and SPP score by association rules.3.Results3.1 Experiment I: Mechanisms of LncRNA MK5-AS1/miR-146a-3p/SIRT1combination-mediated immune inflammation and apoptotic escape in RA fibroblast-like synoviocytes(1)The co-culture model of RA-PBMCs and RA-FLS: The results of CCK-8showed that the optimal number ratio of RA-PBMCs to stimulate RA-FLS was 2.5:1,and the optimal time was 48 h.(2)Expression of lncRNA MK5-AS1,miR-146a-3p,SIRT1 in RA-FLS: The RT-q PCR results demonstrated that expression level of lncRNA MK5-AS1 and SIRT1 were decreased in RA-FLS,while expression of miR-146a-3p was decreased in RA-FLS(3)Transfection efficiency of lncRNA MK5-AS1,miR-146a-3p,SIRT1 in RA-FLS: The RT-q PCR results showed that the expression of lncRNA MK5-AS1 andSIRT1 decreased after transfection of si-MK5-AS1 and si-SIRT1;the expression of lncRNA MK5-AS1 and SIRT1 increased after transfection of OV-MK5-AS1 and OV-SIRT1;The expression of miR-146a-3p increased after transfection of miR-146a-3p mimic;the expression of miR-146a-3p decreased after transfection of miR-146a-3p inhibitor.(4)Localization of lncRNA MK5-AS1 in RA-FLS: The FISH analysis results showed that lncRNA MK5-AS1 was mainly distributed in the cytoplasm(5)Effects of lncRNA MK5-AS1 interference and overexpression inflammation and apoptosis indicators in RA-FLS: The functional phenotype experiment of lncRNA MK5-AS1 showed that The levels of pro-inflammatory factors IL-6,IL-8,anti-apoptotic protein Bcl-2 were decreased,by contrast,the levels of anti-inflammatory factors IL-4,IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 were increased after overexpression of MK5-AS1.While lncRNA MK5-AS1 si RNA silencing played the opposite role.(6)Validation of the regulatory relationship between Lnc RNA MK5-AS1,miR-146a-3p and SIRT1: The dual luciferase assay results demonstrated that miR ‐146a‐3p mimics could significantly reduce the luciferase activity of MK5-AS1-WT and SIRT1-WT,but not MK5-AS1-MUT and SIRT1-MUT.The results of RIP showed that AGO2 is capable of forming compound with miR-146a-3p,the fact that lncRNA MK5-AS1 combined with the AGO2-miR-146a-3p compound further identified the binding relationship between lncRNA MK5-AS1 and miR-146a-3p.Besides,RNA pull-down assay revealed that enrichment of candidate miR-146a-3p pulled down by biotin-labeled lncRNA MK5-AS1.(7)Rescue experiment 1: The effect of miR-146a-3p mimic reversed overexpression of lncRNA MK5-AS1 on RA-FLS inflammation and apoptosis indicators: The results of rescue experiment I showed that,the levels of pro-inflammatory factors IL-6,IL-8,anti-apoptotic protein Bcl-2 were increased,by contrast,the levels of anti-inflammatory factors IL-4,IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 were decreased after miR-146a-3p mimic was co-transfected in RA-FLS with OV-MK5-AS1.(8)Rescue experiment 2: The effect of si-SIRT1 reversed overexpression of lncRNA MK5-AS1 on RA-FLS inflammation and apoptosis indicators: The results of rescue experiment II showed that,the levels of pro-inflammatory factors IL-6,IL-8,anti-apoptotic protein Bcl-2 were increased,the NF-κB pathway was actived,by contrast,the levels of anti-inflammatory factors IL-4,IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 were decreased after si-SIRT1 was co-transfected in RA-FLS with OV-MK5-AS1.3.2 Experiment II: Effects of Xinfeng capsule-containing serum on immune inflammation and apoptotic escape of RA fibroblast-like synoviocytes by regulating lncRNA MK5-AS1/miR-146a-3p/SIRT1 combination(1)Effects of XFC-containing serum on the viability of RA-FLS cells: The results of CCK-8 experiment showed that 10% XFC had the best inhibitory effect on RA-FLS at 48 h,so this was chosen as the optimal intervention concentration and time.(2)Effects of XFC-containing serum on lncRNA MK5-AS1/miR-146a-3p/SIRT1 combination,inflammation and apoptosis indexes in RA-FLS: XFC-containing serum could increase the expression of lncRNA MK5-AS1,SIRT1,IL-4,IL-10,Bax,caspase3,and caspase8,and decrease the expression of miR-146a-3p,IL6,IL-8,Bcl-2,IKKa,and p-p65.(3)Rescue experiment 1: The effect of XFC-containing serum on RA-FLS inflammation and apoptosis indicators through lncRNA MK5-AS1: The results of rescue experiment 1 showed that,the levels of pro-inflammatory factors IL-6,IL-8,anti-apoptotic protein Bcl-2 were increased,while the levels of anti-inflammatory factors IL-4,IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 decreased,after si-MK5-AS1 was transfected with the intervention of XFC-containing serum in RA-FLS.(4)Rescue experiment 2: The effect of XFC-containing serum on RA-FLS inflammation and apoptosis indicators through lncRNA MK5-AS1/miR-146a-3p: The results of rescue experiment 2 showed that based on the intervention of XFC-containing serum and si-MK5-AS1,the levels of pro-inflammatory factors IL-6 and IL-8,and anti-apoptotic protein Bcl-2 were decreased,while anti-inflammatory factors IL-4 and IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 were increased,after miR-146a-3p inhibitor and si-MK5-AS1 were transfected with the intervention of XFC-containing serum in RA-FLS.(5)Rescue experiment 3: The effect of XFC-containing serum on RA-FLS inflammation and apoptosis indicators through lncRNA MK5-AS1/SIRT1: The results of rescue experiment 3 showed that based on the intervention of XFC-containing serum and si-MK5-AS1,the levels of pro-inflammatory factors IL-6 and IL-8,and anti-apoptotic protein Bcl-2 were decreased,while anti-inflammatory factors IL-4 and IL-10,pro-apoptotic proteins Bax,caspase3 and caspase8 were increased,after OV-SIRT1 and si-MK5-AS1 were transfected with the intervention of XFC-containing serum in RA-FLS.3.3 Experiment III: Study on the changes of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis,immune inflammation and apoptosis escape indexes in RA patients and the effect of Xinfeng capsule based on association rules(1)Expression of lncRNA MK5-AS1/miR-146a-3p/SIRT1 combination in RA patients: Compared with the healthy control group,the expression of lncRNA MK5-AS1 and SIRT1 were decreased(P<0.01),while the expression of miR-146a-3p was increased in RA patients(P<0.01).We drew receiver operating characteristic(ROC)curve,and the results suggested that the area under the curve(AUC)were greater than70%,which indicated that these genes can be used as molecular biomarkers to assist in the diagnosis of RA.(2)Correlation analysis of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis with clinical indicators,SPP scores,inflammation and apoptosis indicators in RA patients:The results of indicated that a strong correlation between lncRNA MK5-AS1,miR-146a-3p,SIRT1 and clinical laboratory indicators(ESR,hs-CRP,RF,IGG,etc.),SPP(DAS28 score,spleen deficiency and dampness syndrome,SF-36 score),inflammatory and apoptosis indicators in RA patients(P<0.05 or P<0.01 or P<0.001).(3)Association rule analysis of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis with clinical indicators,SPP scores,inflammation and apoptosis indicators in RA patients:The results of association rules showed that the support,confidence,and lift values between lncRNA MK5-AS1,miR-146a-3p,SIRT1 and clinical laboratory indicators,SPP scores,inflammatory and apoptosis indicators in RA patients were greater than40%,60%,and 1,respectively(P<0.01).(4)Changes of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis,clinical indicators,SPP scores,inflammation and apoptosis indicators in RA patients before and after treatment: Compared with before treatment,miR-146a-3p,IL-8,Bcl-2,ESR,hs-CRP,RA symptom scores and spleen deficiency and dampness syndrome scores were decreased in RA patients,while lncRNA MK5-AS1,SIRT1,IL-10,Bax,VT,SF and MH scores increased(P<0.05 or P<0.01).(5)Association rule analysis between XFC and the improvement of lncRNA MK5-AS1/miR-146a-3p/SIRT1 axis,clinical indicators,SPP score,inflammation and apoptosis indicators in RA patients: The results of the association rule between XFC treatment and index improvement showed that the support,confidence,and lift values between XFC treatment and lncRNA MK5-AS1/miR-146a-3p/SIRT1 combination,clinical laboratory indicators,SPP scores,inflammatory and apoptosis indicators were greater than 55%,75%,and 1,respectively(P<0.01).4.Conclusion(1)Lnc RNA MAK5-AS1 acts as a ce RNA to competitively adsorb miR-146a-3p and downregulate the expression of SIRT1;lncRNA MAK5-AS1/miR-146a-3p/SIRT1 forms a ce RNA combination that mediates the pathogenesis of RA immune inflammation and apoptotic escape.(2)XFC-containing serum can up-regulate the expression of lncRNA MK5-AS1 and SIRT1,down-regulate the expression of miR-146a-3p,and regulate the combination of lncRNA MK5-AS1/miR-146a-3p/SIRT1,ameliorated immune inflammation and apoptotic escape in RA.(3)Lnc RNA MK5-AS1/miR-146a-3p/SIRT1 axis in RA patients was significantly correlated with clinical indicators,SPP scores,inflammation and apoptosis indicators;XFC improved the expression of lncRNA MK5-AS1,miR-146a-3p and SIRT1 in RA patients,which can improve clinical indicators,SPP scores,inflammation and apoptosis indicators. |
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