| Background:Hashimoto’s thyroiditis(HT)is a common organ specific immune disease,which is more common in women and is characterized by circulating autoantibodies.HT induces chronic thyroid inflammation and thyroid tissue infiltration through lymphocytes,including CD4+and CD8+T cells.In HT,the antithyroid immune response begins with the activation of thyroid antigen-specific helper T cells.CD4+T cells were activated,proliferated and differentiated after antigen exposure.CD4+T cells differentiate into different T cell subtypes according to different cytokine signals,including effector T cells or helper T cell lineages,such as Th1,Th2 and Th17 cells,or regulatory T cells.Once Th1 and Th17 cells are activated,they induce B cells to secrete thyroid antibodies,thereby promoting the autoimmune response.In addition,Treg cells inhibit immunity and inflammation.Studies have shown that HT patients have increased Th1 and Th17 subsets,and overexpress IFN-G and IL-17 A.Long non-coding Rnas(Lnc RNAs)belong to non-protein coding rnas,and their own length is more than 200 base pairs.These RNAs widely exist in animals and humans,and play a variety of biological roles of antisense transcription in transcriptional regulation,cell function and many diseases,including cancer.Lnc RNA is involved in many cancer-related processes,Including apoptosis,proliferation,stem cell differentiation,metastasis and therapeutic drug resistance.One of the potential mechanisms of lncrna is that they can bind to mi RNA as competitive endogenous RNA(Cerna)to protect target genes.In this study,we aimed to screen lncrnas related to the progression and pathological stage of HT,explore the biological activity of lncrnas in T cells,and further analyze the molecular biological mechanism of lncrna release activity.The results of this study showed that NONHSAT135873.2 had effects on the proliferation,migration,apoptosis and differentiation of T cell lines,as well as the corresponding molecular targets,and play a pro-inflammatory role in HT by regulating the secretion of IL17 A through competitive binding with mi R-1587.Finally,the biological function of mir-1587 in T cell line was studied.NONHSAT135873.2 provides experimental evidence for finding new therapeutic targets of HT.Methods:1.Peripheral blood was collected from 37 HT patients and 50 healthy individuals attending the Department of Endocrinology and Metabolic Diseases of the First Hospital of China Medical University from September 2018 to May 2019,relevant clinicopathological data were recorded,and samples were sorted by CD4+ T-cell magnetic beads for transcriptome sequencing.In addition,peripheral blood of 40 HT patients and 50 healthy people were collected for verification of sequencing results.2.Using QRT-PCR to detect the expression of NONHSAT135873.2 in CD4+T cells of the research subjects,the relationship between the expression level of NONHSAT135873.2 and clinicopathological data was analyzed.3.To localize NONHSAT135873.2 in T cell line using the method of nucleocytoplasmic isolation and extraction of RNA.4.The expression of NONHSAT135873.2 in Jurkat cell line was changed by si RNA transfection,and the interference efficiency was detected by QRT-PCR.CCK8 and Edu experiments were used to detect the effect of NONHSAT135873.2 on T cell proliferation,flow cytometry(FCM)was used to detect the effect of NONHSAT135873.2 on T cell apoptosis,and transwell assay was used to detect the cell migration activity.The expressions of PCNA,MMP9,AKT,MMP2,Ki67,Bcl-XL,Bad and Bcl-2 were detected by Western or QRT-PCR.The related molecules of T cell subtype or differentiation were detected by QRT-PCR.The inflammatory factor IL17 A and IFNG were detected by QRT-PCR and ELISA.5.Use the bioinformatics database to predict the mi RNAs that can simultaneously bind to NONHSAT135873.2 and IL17 A.6.Silencing NONHSAT135873.2,followed by QRT-PCR to verify the mi RNAs obtained by bioinformatics analysis.7.Interfere or overexpress mi R-1587,and detect the transfection efficiency.8.The regulation of IL17 A expression by mi R-1587 was detected by QRT-PCR and ELISA experiments.9.Co-transfect NONHSAT135873.2,mi R-1587,and then detect the expression of IL17 A.10.The dual-luciferase reporter gene experiment verified whether mi R-1587 could directly bind to NONHSAT135873.2 and IL17 A.11.The effect of mi R-1587 on T cell proliferation was measured by CCK8 and Edu experiments,the influence of mi R-1587 on T cell apoptosis was detected by FCM,the cell migration activity was detected by Transwell assay,and the expressions of PCNA,MMP2,Ki67,Bcl-XL,AKT,Bcl-2,Bad and MMP9 were detected by QRT-PCR or Western.QRT-PCR was used to detect the expression of FOXP3,RORγT,STAT3 and T-bet,and QRT-PCR and ELISA were used to detect the expression of IL17 A,IFNG.Result:1.NONHSAT135873.2 was significantly expressed in CD4+T cells of HT patients,and was positively correlated with TPOAb.2.NONHSAT135873.2 exists in the cytoplasm and nucleus,and the content in the cytoplasm is more.3.NONHSAT135873.2can promote T cell proliferation,inhibit apoptosis,promote cell migration and regulate cell differentiation in vitro.4.After silencing NONHSAT135873.2,the expression of PCNA,AKT,Ki67,Bad,Bcl-2,Bcl-xl,MMP2,MMP9 were inhibited,and the expression of FOXP3,RORγT,STAT3,and T-bet were also significantly down-regulated.5.Silencing NONHSAT135873.2 in Jurkat cell line can significantly down-regulate the expression levels of IL17 A and IFNG.6.Silencing the expression of NONHSAT135873.2 in T cells can increase the m RNA level of mi R-1587.7.mi R-1587 can regulate the expression of IL17 A.8.mi R-1587 can regulate T cell proliferation,apoptosis,migration and cell differentiation.9.mi R-1587 affects proliferation,apoptosis,migration by regulating the expression of AKT,Ki67,Bad,Bcl-2,Bcl-xl,MMP2,MMP9,and also affects the expression of T cell subtype molecules FOXP3,RORγT,STAT3,T-bet.10.The double luciferase reporter gene experiment proved that mi R-1587 could directly bind to NONHSAT135873.2 and IL17 A.11.NONHSAT135873.2 can regulate the expression of IL17 A by competitively binding to mi R-1587.Conclusion:NONHSAT135873.2 was significantly highly expressed in CD4+T cells of HT patients,and its expression was positively correlated with TPOAb.NONHSAT135873.2 regulates the expression of IL17 A by competitively binding to mi R-1587 in HT,and regulates T cell proliferation,apoptosis,migration and differentiation,and exerts biological functions.It is a potential biomarker and therapeutic target for HT. |
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