MiR-338-5p Promotes Inflammatory Response Of Fibroblast-Like Synoviocytes In Rheumatoid Arthritis Via Targeting SPRY1

Introduction:Known as a common autoimmune disease,rheumatoid arthritis?RA?is distinguished by synovitis.The main manifestations in RA patients include joint swelling,cartilage destruction,joint stiffness,and even deformity.RA affects almost 5-50individuals per 100,000 population in developing countries and 0.5%-1%of adults in developed countries.RA onset is rare under the age of 15,but its incidence shows a steady increase with aging to 80.Women are 3-5 times more susceptible than men.Synovial fibroblasts?SFs?,also widely known as fibroblast-like synoviocytes?FLSs?,are a special cell type located inside joints in the synovium,and they play a crucial part in RA pathogenesis.Although it is known that the interaction of internal and external factors participates in the disease development,the interplay between microRNAs?miRNAs/miRs?and their target mRNAs during RA pathogenesis remained to be completely elucidated.MicroRNAs are endogenous small and non-coding RNAs conserved across from worms to mammals and function as negative regulators of gene expression that fine-tune the processing of mRNA including cleavage,translational repression and destabilization.And the specific mechanism is that after loading into the RNA-induced silencing complex,miRNAs prevent the translation or promote the degradation of target mRNAs by complimentarily binding to the 3?-untranslated region?3'UTR?.In the last few years,it has become explicit that miRNAs are fundamental in managing cell cycle,apoptosis,cell differentiation and immunity.Hence,it is not astonishing that dysregulated expression of miRNA is found in an increasing number of pathologic conditions,such as cardiovascular diseases,cancers,infections and autoimmune diseases.A plethora of up-regulated and down-regulated miRNAs have also been identified in RA,however,there are still no researches exploring the dysregulation of miR-338-5p during RA pathogenesis.SPRY proteins are feedback regulators of receptors of receptors tyrosine kinases?RTKs?that restrain RTK-mediated ERK signaling,and therefore play key roles in the process of cell survival,proliferation and differentiation.Until now,four isoforms of SPRY proteins,designated SPRY 1-4,have been identified.SPRY 1 was the first SPRY isoform to be identified as a valid feedback inhibitor of the FGF receptor signaling during the tracheal development in the Drosophila embryos.Sharing a unique,highly conserved cysteine-rich domain at the COOH terminus,which is believed to be crucial for them to target phosphatidylinositol?4,5-bisphosphate?within the plasma membrane,thus conducting their inhibitory role on the mitogen-activated protein kinase?MAPK?pathway.Due to their functions of regulation in cellular cycle,the contribution of SPRY dysregulation to carcinogenesis has already been studied in various malignancies.But very few studies have been done to explore the dysregulation of SPRY1 in autoimmune diseases especially RA.SPRY1 has been predicted to be a target of miRNA-338-5p.To elucidate the mechanism of how miRNA-338-5p influences synovial fibroblasts activities by targeting SPRY1,we conducted a series of cellular level experiments in the present study.Methods:1.Tissue samples and cell groupingSynovial tissues of rheumatoid arthritis were acquired from 17 patients diagnosed with RA and 17 patients diagnosed with OA according to American College of Rheumatology in the First Hospital of China Medical University?the First Hospital of China Medical University?.These synovial tissues were collected from patients during arthroscopic debridement or knee joint debridement?total knee arthroplasty?.Control synovial tissues were taken from 8 cases of emergency trauma amputation patients(Patients with ligament reconstruction under arthroscopy;patellar fracture patients who were diagnosed with neither RA nor OA.Fibroblast-like synovial cells?FLSs?were obtained from synovial tissues using the tissue-culture method and enzyme-digestion method.The cells were then cultured in DMEM?Dulbecco's Modified Eagle Medium?in 5%CO₂ at 37°C.The cells were passaged to the 3^rd-8^th generation,which were then used to conduct further experiments.These cells were randomly divided into 4 groups,including the control group without transfection,NC group,in which the cells were transfected with miR-338-5p nonsense sequences,miR-338-5p mimics group,in which the cells were transfected with miR-338-5p mimics?GenePharma?and the miR-338-5p inhibitors group,in which the cells were transfected with miR-338-5p inhibitors?GenePharma?.The transfection was conducted using Lipofectamine 2000?Invitrogen?.2.3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide?MTT?assayRASFs cultured in DMEM medium containing 10%fetal bovine serum?FBS?were seeded into 96-well plates.The cells were allowed to grow for 48 h,subsequently they were treated with MTT solution?MTT solution was added to a final concentration of 0.5mg/ml?.The cells were incubated with MTT solution for another 4 h at 37°.The optical density?OD?was read at 490 nm with a microplate reader?EL405,Bio-Tek Instruments,Winooski,VT,USA?.Each experiment was performed in triplicate and individual measurements were repeated three times.3.Transwell assayCells was seeded onto the upper chambers of Transwell chamber with 8-?m pore size?Corning,USA?at approximately 1×10⁵ cells/mL.The upper chambers were pre-paved with 50?L Matrigel?1:4,BD Biosciences?.500?L of DMEM containing 10%FBS was added into the lower chambers.After 24's incubation,cells that had not penetrated across the membranes were erased,those penetrated across the membranes were stained with0.1%crystal violet.Finally,we randomly selected 5 different fields to count the cell numbers under a microscope.Independent experiments were done in triplicate.4.Western blottingThe cytoplasmic protein and nuclear protein was extracted following the operation instructions of the ReadyPrep protein extraction kit?Bio-Rad?.BCA protein quantitative method was used to assess the protein concentration.Proteins were transferred to a PVDF membrane after the SDS-PAGE.Subsequently,the membrane was blocked for 2 h using 5%skim milk.Thereafter,the membrane was incubated overnight at 4°C with primary antibodies of SPRY1 and GAPDH?ABcam,Cambridge,UK?.Then the membrane was incubated for 2 h with HPR-conjugated secondary antibodies?ABcam,Cambridge,UK?.Finally,the membrane/film was developed using WesternBright^TM ECL luminous liquid?Advansta,USA?.5.Quantitative RT-PCRTrizol agent?Invitrogen?was used to isolate total RNA according to the manufacturer's instructions.The stem-loop RT-qPCR kit?GenePharma?was used to detect miR-338-5p and SPRY1 mRNA expression level.The content of miRNA and mRNA were analyzed using themethod.U6 and GAPDH respectively served as internal reference for miRNA and mRNA.6.Dual luciferase reporter gene assayWild type SPRY1 mRNA 3'UTR?SPRY1 wt 3'UTR?and mutated SPRY1 mRNA 3'UTR were amplified and inserted into the pmirGlo luciferase vectors?Promega?by RT-PCR.Site mutation method was used to delete the first 4 bases of the target sequence of miR-338-5p on the SPRY1 mRNA 3'UTR?SPRY1 mut 3'UTR?.Recombinant luciferase vectors were used to co-transfect RAFLSs with miR-338-5p mimics or NC.DLR dual luciferase reporter system?Promega?was used to assess the relative luciferase activity in the cells.7.Enzyme-linked Immunosorbent Assay?ELISA?After 48 h's transfection,RAFLSs were centrifuged at 6000 rpm for 10 min at 4.IL-1?,IL-6 and COX2 levels in the supernatant were assessed using the ELISA kit?Thermo Fisher Scientific,USA?.The optical density?OD?was measured using R&D Systems?Minneapolis MD?.8.A collagen-induced RA mouse modelNine male DBA/1 collagen-induced RA mice models?aged 13–15weeks?and three normal male DBA/a mice were purchased from Slac laboratory animal Co.Ltd?Shanghai,China?.The mice were kept in the Laboratory Animal Center of First Affiliated Hospital of China Medical University.According to the Slac laboratory animal Co.Ltd,the mice were intradermally injected at the tail base with 0.1 m L of an emulsion containing bovine type II collagen?100 mg in 50 m L 0.1 M acetic acid;CII,Sigma–Aldrich,St.Louis,MO?and Freund's complete adjuvant?50 m L,4 mg/m L,Santa Cruz Biotechnology,Santa Cruz,CA?.Two weeks after the primary injection,a booster injection of CII was administered again.Three of the mice models were injected with anti-inflammatory drug corticosterone?10 mg s.c.,Sigma–Aldrich,St.Louis,MO?,three were injected with mi R-338-5p inhibitors?2x10⁷ transduction units,Gene Pharma,Shanghai,China?and three were injected with saline.Three hours later,the synovial tissues of the nine mouse models and three normal mice were obtained,and the expression of miR-338-5p and SPRY1 were measured.All animal experiments were performed following the Laboratory Animal Care and Use Committee of First Affiliated Hospital of China Medical University guidelines.Results:1.MiR-338-5p was remarkably up-regulated in rheumatoid arthritis tissues and cellsThe miR-338-5p level in the synovial tissues of patients were detected by RT-qPCR,and the results displayed that miR-338-5p expression in RA tissues was remarkably up-regulated compared with that in OA and healthy control tissues?P<0.05?,and there was no notable difference of the miR-338-5p expression between the OA and control tissues?P>0.05,Figure 1?.2.SPRY1 was notably down-regulated in rheumatoid arthritis tissues and cellsThe expression of SPRY1 protein in the synovial tissues of patients were examined using western blot.The results displayed that SPRY1 expression in the RA tissues was notably down-regulated compared with OA and control tissues?P<0.05,Figure 2?.3.The miR-338-5p level appeared a negative correlation with SPRY1 level in the synovial tissues of RA patientsThe expression levels of miR-338-5p and SPRY1 in transfected RAFLSs were detected by RT-qPCR,and the results manifested that miR-338-5p expression in miR-338-5p mimics group was remarkably increased,while that in miR-338-5p inhibitors group was notably decreased?P<0.05,Figure 3?.The expression of SPRY1 mRNA and protein in mimics group were significantly decreased,while that in inhibitors group were significantly increased,compared with the corresponding control groups?P<0.05,Figure4?.4.SPRY1 is the target of miR-338-5pMiR-338-5p might directly bind with the possible sequence UAUUGUA in SPRY13'UTR,which was predicted by the online database TargetScanHuman 7.0.Figure 2A demonstrated the sequences of wild-type SPRY 3'UTR,mutated SPRY 3'UTR and miR-338-5p.The luciferase activity of cells transfected with SPRY1-wt 3'UTR and miR-338-5p mimics markedly decreased compared with the SPRY1-wt3'UTR+miR-338-5p control group?P<0.05?.On the contrary,the luciferase activity of those transfected with SPRY1-mut 3'UTR did not notably differ from each other?P>0.05,Figure 5?,suggesting that miR-338-5p can directly silence SPRY1.5.MiR-338-5p promoted the proliferation of RAFLSsMTT assay results demonstrated that the proliferation of cells that were transfected with miR-338-5p mimics notably enhanced compared with the control and NC groups,while the proliferation of cells transfected with miR-338-5p inhibitors significantly declined compared with cells in control and NC groups?P<0.05,Figure 3A?.In addition,there has no remarkable difference between the NC group and the control group?P>0.05,Figure6?.6.MiR-338-5p promoted the invasiveness of RAFLSsThe results of Transwell assay proclaimed that the invasiveness of cells that were transfected with miR-338-5p mimics significantly increased compared with that of cells in control and NC groups,while the invasiveness of cells transfected with miR-338-5p inhibitors significantly decreased compared with the cells in control and NC groups?P<0.05,Figure 7?.7.MiR-338-5p promoted the expression of IL-1?,IL-6 and COX2Results of RT-qPCR proclaimed that the levels of IL-1?,IL-6 and COX2 in cells that were transfected with miR-338-5p mimics significantly increased compared with control and NC groups,while the expression of IL-1?,IL-6 and COX2 in cells transfected with miR-338-5p inhibitors significantly decreased compared with control and NC groups?P<0.05,Figure 8?.And there has no significant difference between the NC group and the control group?P>0.05,Figure 8?.The secreted cytokine levels in miR-338-5p mimics group was substantially higher than those in the mimics control group,whereas the levels in miR-338-5p inhibitors group was dramatically lower than those in the inhibitors control group?P<0.05?.Again,no difference of the cytokines expression was seen between the mimics control group and the inhibitors control group.8.Mir-338-5p inhibitors and corticosterone affected the expression of Mir-338-5p and SPRY1To further investigate whether miR-338-5p inhibitors could mimic anti-inflammatory drug corticosterone to affect the expression of miR-338-5p and SPRY1,in vivo experiments were conducted in collagen-induced RA mice models.The results showed that miR-338-5p inhibitors shared a similar effect with corticosterone in regulating the expression of miR-338-5p and SPRY1.?P<0.05,Figure 10,11?Briefly,compared with the saline and normal groups,miR-338-5p expression was suppressed in miR-338-5p and corticosterone groups but SPRY1 expression was enhanced in both groups.However,mi R-338-5p inhibitors and corticosterone could only bring mi R-338-5p and SPRY1expression back to approximately the normal level,suggesting the m R-338-5p/SPRY1is closely associated with RA and mi R-338-5p could also work as an anti-inflammatory drug.9.Statistical analysesStatistical analyses were performed using GraphPad Prism 6?La Jolla,CA?.The comparison between any two groups was done using t-test,whereas the comparison among more than two groups was done using one-way ANOVA.P<0.05 was considered to be statistically signifificant.10.Informed consent and ethicsThe experiment was approved by the ethical committee of the First Affifiliated Hospital of China Medical University for the use of synovial tissues of patients and informed consent was obtained from all patients.This animal experiment was approved by Animal Ethics Committee of China Medical University.Conclusion:Our results suggested that miR-338-5p promoted the proliferation,invasion and inflammatory reaction in FLSs of rheumatoid arthritis by directly down-regulating SPRY1 expression.