| Background:Hashimoto’s thyroiditis(HT),also known as chronic lymphocytic thyroiditis,is a common endocrine system disease and the most common clinical type of autoimmune thyroiditis.The characteristics of HT are:high level of the thyroid peroxidase antibody(TPOAb)and thyroglobulin antibody(TgAb)in patients’serum;low echo in thyroid tissue on ultrasonography;patients’goiters may have a large amount of lymphocyte infiltration or even lymphoid follicles.Patients in the early stages of HT may have no obvious clinical symptoms,but as the thyroid tissue is continuously infiltrated and destroyed by lymphocytes,thyroid tissue and thyroid follicular cells may atrophy in the later stages of the disease and eventually patients may become hypothyroidism.Genetic factors and environmental factors are involved in the pathogenesis of Hashimoto’s thyroiditis,but the specific pathogenesis is not yet clear.Long non-coding RNA(lncRNA)is a group of RNA with a length of more than 200nucleotides.This type of RNA does not encode protein.The mechanism by lncRNA functions without changing the DNA sequence of the gene has become a research hotspot recent years.In previous studies,lncRNA has been shown to be involved in the pathogenesis of some autoimmune diseases,such as systemic lupus erythematosus,rheumatoid arthritis,and inflammatory bowel disease,but we still know little about its function in HT.The purpose of this study is to screen differentially expressed lncRNAs in peripheral blood CD4~+T cells from HT patients and to find lncRNA related to the pathogenesis of HT,so as to elucidate the underlying molecular mechanisms and represent potential biomarkers for HT.Methods:PartⅠ37 HT patients and 50 age-matched healthy volunteers were recruited.Their peripheral blood was collected and the peripheral blood mononuclear cells(PBMCs)were extracted by density centrifugation,then the CD4~+T cells were sorted by a positive selection magnetic separation system.The total RNA of each CD4~+T cell sample was extracted and the qualified RNA samples were used for high throughput sequencing.The sequencing data were analyzed by bioinformatics analysis.In addition,43 HT patients and 50 age-matched healthy volunteers were recruited.Their peripheral blood was collected and the PBMCs were extracted by density centrifugation,then the CD4~+T cells were sorted.The total RNA of CD4~+T cells was extracted and the relative expression of NONHSAT079547.2 was detected using real-time PCR.The concentrations of free T3(FT3),free T4(FT4),thyroid stimulatory hormone(TSH),TPOAb and TgAb were measured using electrochemiluminescent immunoassays.The correlations between the relative expression of NONHSAT079547.2 and the concentrations of serum FT3,FT4,TSH,TPOAb and TgAb were analyzed.PartⅡTo explore the function of NONHSAT079547.2,shRNA-NONHSAT079547.2plasmids were designed and transfected into Jurkat cells.The knockdown efficiency in Jurkat cells is measured using real-time PCR.The CCK8 assay and flow cytometry were used to detect the cell growth and apoptosis level after the downregulation of NONHSAT079547.2.Western blot was used to detect the expression level of proliferation and apoptosis related proteins after the downregulation of NONHSAT079547.2.The expression of T-bet,IFN-γ,IL-17(IL-17A),Ro Rγt,Foxp3,and TGF-βmRNA were detected by real-time PCR.The concentrations of IL-17 in serum and cell culture supernatant were detected using enzyme-linked immunosorbent assay(ELISA).PartⅢAs for lncRNA subcellular distribution,NONHSAT079547.2 expression was detected using real-time PCR in the nucleus and cytoplasm,respectively.MiRNAs that bound to both NONHSAT079547.2 and IL-17 3’UTR were predicted by bioinformatics analysis and target prediction websites(LNCediting and TargetScan).Real-time PCR was used to verify the expression of the predicted miRNAs and ELISA was used to detect the concentration of IL-17 in cell culture supernatant.The direct binding relationship between miR-4716-5p and NONHSAT079547.2 or IL-17 was verified using the dual-luciferase reporter assay.The RNA binding protein immunoprecipitation(RIP)assay was used to verify whether NONHSAT079547.2and miR-4716-5p interacted in an Ago2-dependent manner.Results PartⅠAfter analyzing the data of high-throughput sequencing,7564 significantly differentially expressed lncRNAs between CD4~+T cell samples of HT group and healthy control group were found,of which 3913 lncRNAs were upregulated and3651 lncRNAs were downregulated.Bioinformatics analysis showed that these differentially expressed lncRNAs may be involved in the pathogenesis of HT.It was confirmed that lncRNA NONHSAT079547.2 was highly expressed in peripheral blood CD4~+T cell samples of HT patients and its expression level was positively correlated with TPOAb concentration in patients’serum.PartⅡCompared with the control group,downregulation of NONHSAT079547.2 decreased the level of cell growth but did not affect the level of apoptosis.Similarly,downregulation of NONHSAT079547.2 decreased the expression of PCNA but did not affect the expression level of Bcl-xl.After downregulation of NONHSAT079547.2,the expression of T-bet,IFN-γ,RoRγt and Foxp3 did not change significantly;the expression of TGF-βincreased slightly;while the mRNA expression of IL-17 decreased significantly.In addition,we detected the expression of IL-17 in CD4~+T cells.Compared with the control group,the expression of IL-17 was significantly higher in CD4~+T cells of HT patients.At the same time,there was a positive correlation between IL-17 mRNA expression and NONHSAT079547.2expression.The concentration of IL-17 in serum of HT patients was also significantly higher than that of healthy controls.PartⅢNONHSAT079547.2 was found to be distributed in both the nucleus and cytoplasm,and more NONHSAT079547.2 was found in the cytoplasm.Eight mi RNAs that might bind to both NONHSAT079547.2 and IL-17 3’UTR were predicted by LNCediting and TargetScan.It was shown that miR-30a-3p and miR-4716-5p were upregulated after the downregulation of NONHSAT079547.2.However,only overexpression or downregulation of mi R-4716-5p could decrease or increase the expression and secretion of IL-17.Therefore,it was confirmed that miR-4716-5p could not only bind to NONHSAT079547.2 but also regulate IL-17 expression and secretion.The dual-luciferase reporter assay further showed that miR-4716-5p could directly combine with NONHSAT079547.2 and IL-17 3’UTR.The RIP assay showed that miR4716-5p could bind to NONHSAT079547.2 in an Ago2-dependent manner.ConclusionsThe expression of lncRNAs in peripheral blood CD4~+T cells is significantly different between the HT group and the healthy control group.NONHSAT079547.2 is significantly highly expressed in peripheral blood CD4~+T cells of HT patients.NONHSAT079547.2 can promote cell growth and regulate IL-17 expression and secretion through competitive binding of mi R-4716-5p.Therefore,our research describes the lncRNAs profile in CD4~+T cells from HT patients for the first time,which may prove useful for further studies.The exploration of the function and potential mechanism of NONHSAT079547.2 may lay a foundation for finding new therapeutic targets of HT. |
PDF Full Text Request