| Objective: Autoimmune thyroiditis(AIT)is a common autoimmune disease,Hashimoto thyroiditis(HT)being its main type.HT patients have genetical immune deficiency that makes them susceptible to environmental factors.They are characterized by lymphocyte infiltration and follicular tissue damage in the thyroid gland.In the serum of HT patients,circulating thyroid antibodies mainly anti-thyroid peroxidase antibodies(TPOAb)and thyroglobulin antibodies(TgAb)can lead to thyroid fibrosis or atrophy,accompanied by hypothyroidism.Th17 cells,as members of the family of T helper(Th)cells,mainly secrete cytokines such as IL-17 a.Recent studies have found that Th17 involved in the occurrence and development of many autoimmune diseases,including multiple sclerosis(MS),rheumatoid arthritis(RA)and type 1 diabetes mellitus(T1DM).The level of Th17 cells in AIT is abnormal,but its specific mechanism needs further study.MicroRNAs(miRNAs)are a class of highly conserved endogenous single-stranded non-coding small RNA.They mainly bind to the 3'UTR of mRNA,leading to mRNA degradation and/or translational inhibition of target genes,and play an important role in the development and regulation of the immune system.Nowadays,the pathogenesis of AIT is still not clear,and there are few studies on the role of miRNAs in AIT.In our previous studies,the levels of miR-326 were significantly increased in thyroid and spleen of NOD.H-2h4 mice,as well as PBMC and thyroid tissues of HT patients.In present study,we investigated whether miR-326 participates in Th17 differentiation by targeting Ets-1 protein and ADAM17 in AIT after specifically inhibiting the expression of miR-326,to provide a new theoretical basis for the study of autoimmune thyroiditis.Methods: 1.Human experiment: this part of the study evaluated Ets-1 expression of Hashimoto's disease group(HT group,n=24)and non-Hashimoto's disease control group(n=29)according to their thyroid pathological results and thyroid function.PBMC,serum and plasma of HT patients(n=31)and healthy volunteers(n=23)were collected.The level of Ets-1 mRNA and the expression of Ets-1 protein of PBMC and thyroid tissue were measured,and the correlations between Ets-1 protein and TPOAb,TgAb,miR-326 and Th17 mRNA were assessed.2.AIT animal experiment: firstly,in vitro,spleenmononuclear cells were cultured toward Th17 polarization for 3 days,and then Th17%and IL-17 levels were detected after using the specific siRNA inhibitor(miR-326inhibitor)to knock-down the level of miR-326.Secondly,in vivo,5-week-old NOD.H-2h4 mice were divided into two groups: lentivirus tail vein injection group and lentivirus thyroid local injection group.Tail vein injection was divided into five smaller groups: High iodine,High iodine + therapeutic(the)LV-ctrl,High iodine + therapeutic(the)LV-sponge,High iodine + prophylactic(pro)LV-ctrl and High iodine +prophylactic(pro)LV-sponge group.The prophylactic and prophylactic control group was administration with iodine water at 5 weeks old and lentivirus intervention was given at the same time.The therapeutic and therapeutic control group was given lentivirus intervention at 6 weeks after iodine water was given.The five groups of mice were killed after 12 weeks of iodine water intake,and plasma and tissues were collected.Lentivirus thyroid local injection group was divided into High iodine+ LV-ctrl and High iodine+ LV-sponge group.The mice in both groups were killed after 10 weeks of iodine water feeding.In vivo,the levels of TgAb of mice plasma were measured to evaluate the inflammatory state.Fresh spleens were stained by flow cytometry to detect the proportion of T cell subsets,including T cells,B cells,helper T cells(Th),cytotoxic T cells(Tc),Th1,Th2,Th17 and Treg cells.Total RNA was extracted from spleen.The expression of miR-326 was detected to evaluate the inhibitory effect.Ets-1,Th17-related cytokines,transcription factors and surface receptors were detected to assess changes in downstream indicators.The spleen protein was extracted to evaluate the expression of Ets-1 protein.Results: 1.Human experiment: Compared with control group,the level of Ets-1 mRNA in thyroid tissue and PBMC of HT patients had no difference,but the expressions of Ets-1 protein in thyroid tissue and PBMC were significantly decreased(P=0.034 and P=0.009,respectively).The correlation analysis showed that the relative expression of Ets-1 protein was negatively correlated with the level of miR-326 and Th17 mRNA.2.Animal experiments in vitro: Compared with negative control group,the Th17% in miR-326 inhibitor group was significantly decreased,and the concentration of IL-17 in culture supernatant was also significantly lower(P < 0.05).3.Animal experiment in vivo:tail vein injection group 1)compared with High iodine and High iodine+ LV-ctrl group,the level of miR-326 in High iodine + LV-sponge group was significantly decreased,which indicated that lentivirus inhibiting in tail vein of mice was effective.2)There was no difference in the proportion of T cells,B cells,Th cells and Tc cells in the five groups of NOD.H-2h4 mice fed with high iodine for 12 weeks.3)The areas of the inflammatory lymphocyte cells and the plasma TgAb titers in the LV-sponge group were significantly lower than those in control group.4)The expression of Ets-1 protein in the mice spleen increased in LV-sponge group,particularly in the High iodine + the LV-sponge group(P=0.039),while there were no significant difference in the level of Ets-1 mRNA among the five groups.5)The expression of ADAM17 protein in the mice spleen in LV-sponge group was higher than that in control group,while the expression of IL-23 R was significantly decreased(P < 0.05).6)There was no significant difference in the proportion of Th1 and Th2 cells in the 5 groups;the proportion of Treg cells increased in High iodine + pro LV-sponge group(P=0.047)while the proportion of Th17 cells were decreased(P=0.042).And the ratio of Th17/Treg decreased significantly in the High iodine + pro LV-sponge group(P=0.005).Th17-related cytokines IL-17 a,IL-22,transcription factors Rorgt,Rora and surface receptors CCR6,IL-23 r were significantly decreased in LV-sponge group(P < 0.05).4.Animal experiment in vivo: In the local thyroid injection group,the areas of the inflammatory lymphocyte cells and the serum TgAb titer decreased significantly in LV-sponge group compared with LV-control group(P < 0.05),otherwise,the level of miR-326 in spleen was no significant difference(P=0.239).Conclusion: The expression of Ets-1 protein in PBMC and thyroid tissue of HT patients was significantly lower than that of healthy controls,while Ets-1 mRNA showed no significant difference.The expression of Ets-1 protein was negatively correlated with the level of miR-326 and Th17 mRNA.The results suggested that miR-326 may have a negative regulatory effect on Ets-1 protein.2.By inhibiting the levels of miR-326 in vitro and vivo in AIT model mice,we found that the degree of inflammation decreased in mice.What's more,the expression of Ets-1 protein increased accompanied with the decrease of Th17%,Th17 related cytokines,transcription factors and surface receptor.Based on previous and present studies,we proved that miR-326 regulated Th17 cells in autoimmune thyroiditis through targeting Ets-1. |
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