Role And Mechanism Of Hsa-miR-3202 In Primary Sj(?)gren’s Syndrome

Background and Aim: Primary Sj(?)gren’s syndrome(p SS)is a chronic autoimmune disease characterized by lymphocyte infiltration and exocrine gland dysfunction.Dry mouth and dry eyes are the main clinical manifestations,positive anti-SSA antibody,anti-SSB antibody and anti-nuclear antibody are the main laboratory examination features,and focal lymphocyte infiltration is the pathological feature.The persistent presence of lymphocytes in glands is an important cause of apoptosis of glandular epithelial cells.The pathogenesis of p SS is unclear,but it is believed that mRNA and miRNA play an important role in it.At present,a large number of studies have confirmed that there are a large number of differential expression profiles of mRNA and miRNA in p SS patients.But they are mainly concentrated on virus-related pathway and IFN pathway.Therefore,this study used RNA-seq method to explore mRNA and miRNA related to lymphocyte infiltration pathway.And then we explored its mechanism and tried to provide new evidence for p SS to reduce lymphocyte infiltration leading to glandular destruction,and provide new diagnostic and therapeutic targets for p SS.Methods: 1.mRNA-seq and miRNA-seq: a)The study included 25 untreated p SS patients in the Department of Rheumatology and Immunology of the First Hospital of Jilin University from November 2017 to May 2018.All patients met the 2016 ACR/EULAR revised p SS classification criteria.At the same time,25 healthy individuals with age and gender matched were included as a HC group.The HC group came from the physical examination center of the First Hospital of Jilin University.b)5 cases of p SS and 5 cases of HC were grouped as the training group for RNAseq,and the remaining 20 cases of p SS and 20 cases of HC were grouped as the verification group for differential expression of RNA verification.We collected PBMCs of the above-mentioned individuals for RNA extraction,sequencing analysis,and RTq PCR validation.2.hsa-miR-3202 regulated MMP2 to mediate T cell infiltration: a)Twenty undiagnosed and suspicious p SS patients with dry mouth symptoms were recruited as another group who were treated at the First Hospital of Jilin University and all of them underwent labial gland biopsy.Among them,14 people met the 2016 ACR/EULAR revised p SS criteria(labial gland biopsy needs to meet focal index ≥ 1/4mm2),as the p SS group;6 people did not meet the p SS criteria(labial gland biopsy focal index(27)1/4mm2),as the Control group.b)Labial gland tissues were taken for immunohistochemical staining to verify the expression of MMP2 in labial gland tissue.The dual luciferase experiment was used to verify the target regulation relationship of hsa-miR-3202 and MMP2.c)After transfected Jurkat cells with hsa-miR-3202 mimics,mimics-NC,inhibitor,and inhibitor-NC,RT-q PCR and Western Blot were used to verify the MMP2 expression level.d)Transwell verified the invasion ability of Jurkat cells after transfection pretreatment.3.hsa-miR-3202 regulated FAIM2 to mediate apoptosis: a)After Jurkat cells were transfected with hsa-miR-3202 mimics,mimics-NC,inhibitor,inhibitor-NC,apoptosis was tested.Meanwhile,IFN-γ and TNF-α were detected by ELISA.b)The target gene FAIM2 of hsa-miR-3202 was identified by Target Scan Human(www.targetscan.org).c)After Jurkat cells were transfected with hsa-miR-3202 mimics,mimics-NC,inhibitor,inhibitor-NC,RT-q PCR and Western Blot were used to verify FAIM2 expression level.d)The expression of FAIM2 was verified in 20 cases of p SS and 20 cases of HC.e)After knockdown FAIM2 in Jurkat cells,apoptosis was tested and ELISA were used to detect the secretion level of IFN-γ and TNF-α.f)The dual luciferase experiment verified the targeting relationship between hsamiR-3202 and FAIM2.g)Transwell co-culture system was established.The upper chamber was plated in pretreated Jurkat cells,and the lower chamber was plated in HSG cells.The interaction between infiltrated T cells and glandular epithelial cells was simulated.Apoptosis experiment was used to detect HSG cell apoptosis,Ed U was used to detect HSG cell proliferation after co-culture.Results: 1.Transcriptome-seq and miRNA-seq: a)Compared with the HC group,1671 mRNAs in the PBMC of p SS patients was significantly different expression,of which 1019 were up-regulated and 652 were down-regulated.290 differentially expressed miRNAs were screened,of which 143 were up-regulated and 147 were down-regulated.b)Among the above-mentioned differentially expressed genes,a total of 84 mRNAs and 49 miRNAs had a mutual regulatory relationship.Among 84 types of mRNAs,39 were up-regulated and 45 were down-regulated.Of the 49 miRNAs,22 were up-regulated and 27 were down-regulated.c)GO term is enriched in secretion,myeloid cell activation involved in immune response and myeloid leukocyte mediated immunity,and alpha-Nacetylgalactosaminide alpha-2,6-sialyltransferase activity.d)The KEGG pathway is enriched in Leukocyte transendothelial migration and Complement and coagulation cascades.In particular,the Leukocyte transendothelial migration is consistent with the pathological characteristics of lymphocyte infiltration in p SS.MMP2 and CLDN5 were enriched in the Leukocyte transendothelial migration.e)It was verified that MMP2,CLDN5,and ANO2 in the p SS group were significantly higher than those in the HC group(P<0.05),which was consistent with the sequencing results.f)Two miRNAs,hsa-miR-3202 and hsa-miR-1291,were found to have regulatory effects with MMP2 and CLDN5 respectively through interaction analysis.hsa-miR-3202 and hsa-miR-1291 in p SS patients were significantly lower than those in the HC group(P <0.05),consistent with the sequencing results.g)Correlation analysis results with clinical characteristics showed that CRP was positively correlated with the relative expression of MMP2(r=0.617,P=0.004);ESSDAI score was positively correlated with the relative expression of MMP2(r=0.710,P=0.001).The relative expression of CLDN5 was positively correlated with ESSDAI score(r=0.644,P=0.002).h)Sequencing results showed that log2 FC of MMP2 was 3.393 and log2 FC of CLDN5 was 2.1719,so Hsa-miR-3202 and MMP2 were selected for mechanism study.2.hsa-miR-3202 regulated MMP2 to mediate T cell infiltration: a)The results of immunohistochemistry showed that compared with the Control group,MMP2 was highly expressed in the labial gland tissues and was highly expressed in the location of lymphocytes infiltrated.b)The results of dual luciferase experiments suggest that hsa-miR-3202 has no direct targeting relationship with MMP2,but the expression of MMP2 was significantly reduced after transfection of hsa-miR-3202 mimics;the expression of MMP2 was significantly increased after transfection of hsa-miR-3202 inhibitor.c)The results of cell invasion experiment were as followed.As for the hsa-miR-3202 mimics transfected group,the proportion of Jurkat cells invasion was lower than that of the control group,which was statistically significant(P=0.047).Compared with the control group,the proportion of Jurkat cells invasion of the hsa-miR-3202 inhibitor transfected group were increased(P=0.032).3.hsa-miR-3202 regulated FAIM2 to mediate apoptosis: a)Overexpression of hsa-miR-3202 can increase the apoptosis of Jurkat cells and decrease the secretion of IFN-γ and TNF-α;low expression of hsa-miR-3202 can decrease the apoptosis of Jurkat cells and increase the secretion of IFN-γ and TNF-α.b)Overexpression of hsa-miR-3202 can reduce the expression of FAIM2 in Jurkat cells,and lowerexpression of hsa-miR-3202 can increase the expression of FAIM2;c)The expression of FAIM2 was increased in p SS group(P=0.002),and FAIM2 level was positively correlated with CRP(r =0.568,P=0.009).d)Knockdown of FAIM2 can increase the apoptosis of Jurkat cells and decrease the secretion of IFN-γ and TNF-α;e)The results of Transwell co-culture indicated that HSG cell apoptosis were decreased and proliferation were increased in the overexpressing hsa-miR-3202 group and the FAIM2 knockdown group;low expression of hsa-miR-3202 increased HSG cell apoptosis and weakened proliferation.Conclusions: 1.A large number of differential expressions of miRNA and mRNA were screened in PBMC of p SS patients,especially MMP2 and its regulatory hsa-miR-3202,which may participate in the pathogenesis of p SS through the Leukocyte transendothelial migration.2.hsa-miR-3202 can reduce the ability of lymphocytes to infiltrate the labial glands in p SS by MMP2 and play a protective role.3.hsa-miR-3202 can promote T cell apoptosis and inhibit secretion of inflammatory factors through FAIM2,thereby reducing the damage to HSG cells,inhibiting apoptosis and protecting HSG.