Effects Of FOSL1 In Thymic Hyperplasia In Patients With Myasthenia Gravis Patients

ObjectiveMyasthenia gravis(MG)is an autoimmune disease that is mediated by an auto-antibodies which is dominated by IgG targeting the acetyicholine receptor(AChR)on the neuromuscular postsynaptic membrane.The production of AChR antibodies requires the assistance of T cells,and the most MG patients have thymoma and/or thymic hyperplasia,their symptom can be alleviated after thymectomy.Therefore,it is generally believed that dysthymus is closely related to the occurrence and development of MG,especially the germinal center can be seen in MG thymus and the autoantibody against AChR can be isolated from it.How dose the thymus affect the differentiation and maturation of T cells and its effects in occurrence of MG disease?There are no satisfactory answers to these questions.To investigate the mechanism of abnormal thymus hyperplasia in MG patients,we selected thymic tissue of MG patients with hyperplasia for 'Human Signal Transduction Pathway Finder PCR Chip' analysis.This array includes target genes for 10 commonly studied signal transduction pathways.Through the analysis of the results of the microarray,it was found that a variety of signaling molecules in MG thymus tissue were significant differentially expressed compared with the control group.Combined with relevant literature and our research foundation,we chose Fos related antigen 1(FOSL1)as the core content of the study.According to our previous results that FOSL1 is mainly expressed in thymic epithelial cells(TECs),to further clarify the effect of high FOSL1 expression on TECs function and thymocytes proliferation,TECs and thymocytes were isolated and transfected with TECs by constructing lentiviral FOSL1 gene expression and interference vector.The changes of TECs-related signaling molecule SOCS3,STAT3,BCL2 and BAX gene expression and the effect of TECs on thymocytes proliferation were observed.Materials and Methods1.Research ObjectExperimental group:There were 15 clinically confirmed MG patients,including 7 males and 8 females,with the age of 21.5±6.17.According to the following five inclusion criteria:?It has typical symptoms of muscle weakness,eyelid droop or limb weakness,light in the morning and heavy in the evening,and aggravation of fatigue test symptoms;?Positive test for neostigmine;?Blood autoantibodies to the acetylcholine receptor(AChR)were positive;?Thymectomy was performed and the symptoms were relieved;?Pathologic examination of thymus showed no typical thymoma,which was thymic hyperplasia.Control group:15 patients with non-autoimmune heart disease who underwent open-heart surgery,including 9 males and 6 females,aged 18.3±4.23 years,showed no significant difference from the experimental group.Negative autoantibodies,no infectious disease.The sample collection does not increase the patient injury and informed consent of the patient or family members,in line with medical ethics.2.Samples Collection,Storage and transportationThe fresh thymus tissue removed by surgery was placed in sterile normal saline,and then refrigerated and transported to the laboratory within 1h.In the laboratory,the samples were cleaned and the necrotic tissue and fascia were removed with normal saline,and relevant experiments such as RNA extraction,cell isolation,culture and tissue slice were completed within 4h.RNA extraction samples were added with 1 mL Trizol reagent every 50mg to make homogenate.-80 refrigerator or liquid nitrogen was stored on dry ice for transportation.3.RNA extraction,purification and synthesis of cDNAThe sterile thymic tissue by thymectomy from MG patients and control group.The 1mL TRIZOL reagent was added to 50mg thymic tissue,and to make homogemste.The total RNA was extracted acording to instructions.The first strand cDNA synthesis was carried follow the instructions,total RNA,oligo(dT)18 and dNTP Mix were added to a EP tube,fill to volume 13?l.After It is heated to 65? for 5 min,put it on ice for 1 min.Then,add in order SuperScript ? RT,RNase Inhibitor,DTT and First-Strand Buffer for cDNA synthesis.The cDNA was store it at-20?.4.PCR Chip AssayThree RNA samples from thymus tissues of 15 MG patients and 15 control group were respectively selected for PCR chip Assay.Human signal transduction pathway finder PCR chip was selected in this study.5.Fluorescence Quantitative PCR Validation TestBased on the results of the microarray,the thymus tissues of the 15 MG patients and control group were tested for GENE expression validation.GENE sequences of FOSL1,CCL5 and SOCS3 were searched from the GENE database,Primer5.0 was used to design the primers,and the product specifications of SYBR Premix Ex Taq TM(TaKaRa)were followed.Three duplicates were made for each sample,and a reaction system of 20?L per well was prepared on the ice,and the reaction was added to ABI 7500 Fast quantitative fluorescence PCR in the eight-row hole to export the original data for statistical analysis6.Thymic epithelial cells(TEC)isolation,identification and FOSL1 gene transfectionFresh thymus tissue was taken,fascia and blood stains removed,cut into pieces,and inoculated into 6-well plates.Dulbecco's modified Eagle's culture solution containing 10%fetal bovine serum(HyClone)and penicillin streptomycin was added.The culture was conducted in 37? 5%CO2 incubator,and replacement of half the liquid every other day.The formation of single cell colony was observed after culture for 7 day.The colony were selected randomly,moved to the another new plates continues to incubation,observe the growth situation,CK8/18 staining was identified by immunohistochemistry.CK8/18+ cells cloney was selected to next phase of the experiment.The company was entrusted to construct FOSL1 lentivirus expression vector(pcdh-cmv-mcs-ef1-copgfp-fosl1)and interference vector(pmagic7.1-fosllshrna)..Take 2×105 TECs,add 2×107 TU/mL lentiviral vector,and the infection efficiency is greater than 85%.Divided into four groups:FOSLl gene transfection expression group(FOSL1 group),FOSL1 expression interference group(fosll-sh group)overexpression empty vector group(control-sh group)and control group(control group).Lentivirus vectors were added and incubated in a 37? 5%CO2 incubator for 7 days.The experimental results were observed and recorded every day.7.Effects of FOSL1 gene transfection or interference on the expression of SOCS3 and related signaling molecules in TECsTECs were collected before and after FOSL1 gene transfection,washed with PBS,RNA was extracted and purified,FOSL1,SOCS3 and related signaling molecules mRNA level of TECs were analyzed by reverse transcription and fluorescence quantitative PCR amplification.TEC was collected by centrifugation,cells were lysed with RIPA containing protease and phosphatase inhibitors,and protein concentration was determined with Thermo Fisher Scientific.Heat the protein sample 100? 10 min,the sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),the protein were transferred to the PVDF membrane by semi-wet method,5%skimmed milk block 2 h,according to the conventional method to add the primary antibody and the horseradish peroxidase-labeled secondary antibody in turn,incubation.The ImageQuant LAS 4000 mini Gel imaging system(GE Healthcare)was used for imaging,and protein expression was quantified by ImageJ.?-actin as reference.8.thymocytes proliferation assayThymus tissues were cut into pieces according to the above method,and was pressed mechanically to obtain a single cell suspension through 100 mesh stainless steel mesh,which was left at room temperature for 2h.Adherent cells were removed,and the suspended cells were washed twice with PBS and counted the cells.The cell concentration was adjusted to 1×105/mL.The TECs of FOSL1 group,FOSL1-sh group and the two control groups were inoculated respectively into 96-well plates at 5.0×103/well,wait until TECs were fully attached(approx 8h),the thymocytes were added at a ratio of 1:10.The thymocytes was co-cultured with TECs for 24h,48h and 72h in 1640 medium(containing 10%fetal bovine serum)and 37? 5%CO2.The 10?L per well MTT solution(5 mg/mL)were added 4 hour before the end of culture.150 ?L DMSO per well was added end of culture and oscillated for 10min to dissolve cells and release Formazan.Absorbance value(value A)was measured by 450nm wavelength automatic microplate reader,with 3 repores per group.9.Data analysis and statistical treatment:Using two independent samples t test,one-way analysis of variance for statistical significance.The test level was mean=0.05,P<0.05 was considered significant difference.The data processing by GraphPad Prism version5 software.Results1.Differentially expressed genes in thymus tissues of MG patientsThe mRNA of signal transduction molecules in the MG thymus tissues was compared with the control group,and it.was found that there were 22 genes with significant differences(the difference multiple was greater than 2),among which 18 genes had significantly increased expression in MG patients,and 4 genes had significantly decreased expression.The differentially expressed genes were ADM?BAX?BCL2A?CDKN1A?CEBDP?CSF1?EGFR?EMP1?FOSL1?GADD45A?GADD45B?ID1?IRF1?ICAM1?HES1?SERPINE1?SOCS3?WISP1 and WNT2B ect.These signaling molecules were involved in multiple signaling pathways including TGF?,Notch,JAK/STAT,NF-?B and WNT signal pathway.Among them,the expression difference of BCL2A,FOSL1 and SOCS3 was the most significant.2.Fluorescence quantitative PCR validationTo verify the correctness of mRNA expression in microarray,FOSL1,SOCS3 and CCL5 was selected for fluorescence quantitativeRT-PCR from thymus RNA of 15 MG patients and control group.The expression of SOCS3 and FOSL1 in thymus tissues of MG patients was significantly different from that of the control group(P<0.05),which was consistent with the microarray results.3.FOSL1 gene transfection affects the expression of TECs related signal moleculesTECs were isolated from thymus tissue for clone culture and immuno-histochemization identification.TECs with positive CK8/18 staining were selected for further culture.The results showed that the expression of FOSL1 mRNA was significantly increased in the FOSL1 gene transfection group(39.01 ± 0.72)compared with the empty vector control group(1.85±0.21)and the blank control group(P<0.01).The expressions of SOCS3(3.30±0.58),STAT3(8.56±0.61),BAX(10.11±0.67)and BCL2(4.31±0.97)were also increased,which was significantly different from the negative control group(1.01±0.66,1.39±0.55,2.18±0.89,1.63±0.11),P<0.05._FOSL1 mRNA expression was significantly decreased in the FOSL1-sh group(0.25±0.11)compared with the empty carrier group(1.10±0.08)and the blank control group(P<0.01).SOCS3 expression was also decreased(0.18±0.04),which was significantly different from that of the empty carrier group(0.89±0.14),P<0.01.However,the expression levels of STAT3(6.33±0.96),BAX(2.69±0.23)and BCL2(5.72±0.88)were significantly higher than those of the negative control group(0.92±0.21,1.33±0.53,0.90±0.75),P<0.01.TECs of FOSL1 transfection group and interference group were collected,protein was extracted from lysis cells,and western-blot results showed that SOCS3 and BCL2 protein levels in FOSL1 transfection group were significantly higher than that in FOSL1 interference group,P<0.05.4.TECs with high FOSL1 expression promotes thymocytes proliferationTECs was co-cultured with thymocytes,and it was found that TECs with high FOSL1 expression could promote the proliferation of thymocytes.-Compared with the control group(0.27±0.02,0.44±0.03,0.37±0.02),there was a significant difference in the proliferation of thymocytes after co-culture for 24h(0.34±0.01),48h(0.58±0.04)and 72h(0.73±0.03),P<0.05.TECs with low FOSL1 expression had no significant effect on thymocytes proliferation,compared with control group,P>0.05.ConclusionsFOSL1 is highly expressed in hyperplastic thymus tissues of MG patients,which affects the proliferation of thymocytes and participates in abnormal MG thymic hyperplasia by regulating TECs function.