桥本甲状腺炎患者Th17细胞变化及相关调控因素的研究
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摘要
目的：
通过研究桥本甲状腺炎(HT)患者外周血中Th17细胞及相关因子表达水平,分析Th17细胞与自身抗体变化的关系,了解Th17细胞及相关因子在HT发病中的作用；研究Th17细胞和Treg细胞在外周血中的比例及其特异性转录因子的表达水平,分析Th17/Treg细胞比率与自身抗体水平的关系,探讨HT患者Th17/Treg细胞免疫偏移与HT发生的相关性；研究HT患者外周血糖皮质激素诱导的肿瘤坏死因子受体相关配体(GITRL)表达水平,及其与Th17细胞和Treg细胞的相关性,探讨GITR/GITRL系统在Th17细胞和Treg细胞平衡中的作用；通过研究调控白介素-23受体(IL-23R)表达的的miRNAs,探讨了miR-125a-3p对HT患者外周血单个核细胞(PBMC)IL-23R的调控作用,为HT的诊治提供新思路。
方法：
1、采用流式细胞术(FCM)检测HT患者与健康对照PBMC中CD3+CD8-IL-17+T细胞(即Th17)比例,ELISA检测HT患者与健康对照外周血血浆中IL-6、IL-23的水平,并进行比较。通过实时荧光定量PCR(qRT-PCR)检测HT患者与健康对照组PBMC中RORyt mRNA表达水平,逆转录PCR(RT-PCR)检测患者甲状腺组织中RORyt和IL-17mRNA的表达情况。
2、应用化学发光免疫分析法(CIA)检测HT患者抗甲状腺球蛋白抗体(TG-Ab)和抗甲状腺过氧化物酶抗体(TPO-Ab)的水平,分析患者PBMC中Th17细胞比例与自身抗体水平的相关性。
3、采用流式细胞术检测HT患者与健康对照PBMC中CD4+CD25+CD127lowT细胞(即Treg)比例,通过qRT-PCR检测HT患者与健康对照PBMC中Foxp3mRNA表达水平。
4、分析HT患者及健康对照PBMC中Th17/Treg细胞比率,并与TG-Ab水平进行相关性分析。
5、采用ELISA检测HT患者和健康对照血浆中GITRL的水平,qRT-PCR检测HT患者甲状腺组织中GITRL mRNA的表达情况。分析血浆中GITRL水平与Th17和Treg细胞比例的相关性。
6、免疫磁珠分选小鼠初始型CD4+T细胞,在Th17细胞诱导条件下进行体外培养,观察GITRL对促进小鼠Th17细胞分化的影响。
7、采用qRT-PCR检测HT组与健康对照组PBMC中IL-23R mRNA的表达情况。以IL-23R为目的基因,利用targetscan/miRanda/Pictar数据库软件,预测可能调控IL-23R的miRNAs。
8、通过qRT-PCR检测预测miRNAs在HT患者以及健康对照PBMC中的表达情况,筛选出符合预期的miRNA。
9、利用荧光素酶报告基因检测实验验证miR-125a-3p与IL-23R的靶向作用,明确miR-125a-3p与IL-23R3'UTR结合位点的作用强度。将表达IL-23R的健康体检者PBMC作为靶细胞,转染miR-125a-3p mimics和NC,流式细胞术检测PBMCIL-23R表达变化。
10、采用qRT-PCR方法检测HT患者和健康对照外周血PBMC miR-125a-3p的表达情况,并与IL-23R mRNA水平进行相关性分析。同时对HT患者PBMC中miR-125a-3p的表达水平与其自身抗体水平进行相关性分析。
结果：
1、与健康对照组相比,HT患者PBMC中的Th17细胞比例明显增高[(0.75+0.79)%vs(0.28+0.23)%,P<0.05]。HT组PBMC中Thl7细胞特异性转录因子RORγt的相对表达量显著高于健康对照组(0.30±0.38vs0.04±0.02,P<0.05)。HT患者血浆中IL-6、IL-23的表达量显著高于健康对照组(3.66±4.70vs0.47±1.11pg/ml, P<0.05)(154.7±75.81vs80.65±61.41pg/ml, P<0.05)。与单纯性甲状腺肿组相比,HT组患者甲状腺组织中表达RORyt和IL-17基因。
2、桥本甲状腺炎患者PBMC中的Thl7细胞比例与其血清TG-Ab水平存在较强的正相关(r=0.8484,P=0.0077),与其血清TPO-Ab水平可能存在一定的正相关(r=0.6224,P=0.0994)。
3、HT患者PBMC中Treg细胞比例明显降低(P<0.01)。Treg细胞特异性转录因子Foxp3mRNA的相对表达量也显著低于健康对照组(P<0.05)。
4、HT患者外周血PBMCs中Th17/Treg细胞比率明显增高(P<0.01),并与TG-Ab呈现明显正相关(r=0.5243,P=0.0176)。
5、HT患者外周血中GITRL的水平明显上调(P<0.05),相关分析显示,GITRL与Th17细胞比例呈明显正相关(r=0.5282,P=0.0167)。在Th17细胞诱导条件下,mGITRL可明显促进Th17细胞的诱导分化。
6、与单纯性甲状腺肿组相比,HT组甲状腺组织中RORγt mRNA、IL-17mRNA和GITRL mRNA相对表达量明显增高(P均<0.05)。
7、HT患者PBMC中IL-23R mRNA的表达水平显著上调(P<0.05)。以IL-23R为目的基因,经过软件分析,取预测结果的交集,结合文献查询结果,筛选出可能调控IL-23R的miRNAs:miR-155、miR-125a-3p。miR-125a-3p的表达显著下调(P<0.05),与上调的IL-23R mRNA表达水平趋势相反。
8、荧光素酶报告基因检测实验证实,miR-125a-3p能够结合IL-23R的种子区,抑制下游IL-23R基因表达,降低荧光强度,结果表明,miR-125a-3p与IL-23R基因存在直接靶向关系。流式细胞术检测发现,与转染NC组相比,转染miR-125a-3p的PBMC表达IL-23R的细胞比例明显减少,提示miR-125a-3p可以抑制IL-23R的表达。
9、qRT-PCR检测表明HT患者PBMC中miR-125a-3p相对表达量下调(P<0.05),相关性分析显示,miR-125a-3p表达水平与IL-23R mRNA表达水平呈负相关(r=-0.5195,P=0.0226)。miR-125a-3p表达水平与TG-Ab水平呈显著负相关(r=-0.4811,P=0.0371)。
结论：
1、HT患者体内Th17细胞及相关因子表达升高,Thl7细胞与自身抗体(TG-Ab)水平呈正相关,提示Th17细胞及相关细胞因子在HT的发生发展中起重要作用。
2、HT患者外周血Th17细胞比例增加、Treg细胞比例减少,其特异性转录因子RORγt和Foxp3变化趋势与细胞比例相同,Th17/Treg细胞比率与TG-Ab水平呈显著相关,提示HT患者存在Th17/Treg失衡,为阐明HT的发病机制提供了新的思路。
3、GITRL能够促进Th17细胞的产生,HT患者外周血血浆中GITRL高表达,与Th17细胞呈明显正相关。HT患者甲状腺组织中GITRL、RORγt、IL-17mRNA水平均高表达。结果表明,GITRL可能参与Th17/Treg细胞平衡的调节。
4、miR-125a-3p可以直接调控IL-23R的表达。HT患者外周血PBMC中miR-125a-3p的表达显著下调,与IL-23R mRNA表达水平呈负相关,且与体内自身抗体水平呈负相关。提示miR-125a-3p与HT的发生及疾病严重程度有关。
Objective:
To investigate the variations of CD4+T-helper17(Th17) cells and the level of Thl7cells related cytokines in patients with Hashimoto's Thyroiditis (HT), analyze the relations between Th17cells and autoantibodies, evaluate the role of Th17cells and related cytokines in the pathogenesis of patients with HT. To investigate the proportion of Th17and regulatory T cells (Treg) and the expression of specific transcription factors, analyze the relations between the ratio of Th17/Treg and autoantibodies, explore the role of immune deviation of Th17/Treg in HT patients.To investigate the level of Glucocorticoid-induced TNF receptor-related protein ligand (GITRL) in peripheral blood of patients with HT and its correlation with Th17cells or Treg cells, explore the role of GITR/GITRL in the balance of Th17/Treg. To study miRNAs of regulating interleukin-23receptor (IL-23R) expression, explore the regulation role of miR-125a-3p on IL-23R in peripheral blood mononuclear cells (PBMC) of patients with HT.
Methods:
1. The frequency of CD3+CD8-IL-17+T (Thl7) cells in PBMC of patients with HT and healthy controls was analyzed by flow cytometry (FCM), the concentration of plasma IL-6、IL-23were measured by enzyme-linked immunosorbent assay (ELISA). QRT-PCR was used to measure the expression of RORyt mRNA in PBMC, the expression of RORyt and IL-17mRNA in thyroid tissue was measured by RT-PCR.
2. The levels of anti-thyroglobulin antibody (TG-Ab) and anti-thyroperoxidase antibody (TPO-Ab) in patients with HT were detected by chemiluminescence immunoassay(CIA), correlation between Th17and the levels of autoantibodies was analyzed.
3. The frequeney of CD4+CD25+CD127lowT (Treg) cells in PBMC of patients with HT and healthy controls was analyzed by FCM, qRT-PCR was used to measure the expression of Foxp3mRNA in PBMC.
4. The ratio of Th17/Treg and correlation with the levels of TG-Ab was analyzed.
5. The concentration of plasma GITRL in HT patients and healthy controls were measured by ELISA. QRT-PCR was used to measure the expression of GITRL mRNA in thyroid tissue of patients with HT and healthy controls. Correlation between the levels of plasma GITRL and ratio of T17/Treg was analyzed.
6. Naive CD4+T cells were purified by immune magnetic beads from normal murine, and incubated under the Th17-differentiation condition. The role of GITRL on the differentiation of Th17cells was observed.
7. QRT-PCR was used to measure the expression of IL-23R mRNA in PBMC of patients with HT and healthy controls. Targing IL-23R gene, through miRanda and PicTar software analysis, we listed the miRNAs probably target to IL-23R.
8. The expression of the predicted miRNAs in HT patients and healthy controls were detected by qRT-PCR. The miRNA in line with expectations was chosen for further research.
9. The targeting role of miR-125a-3p on IL-23R was validated by luciferase reporter assay, to clear the binding site strength of miR-125a-3p and IL-23R3'UTR. MiR-125a-3p mimics and control transfection was done in PBMC expressing IL-23R from the people conducted health examination as target cells. The expression of IL-23R in PBMC was measured by FCM.
10. QRT-PCR was used to measure the expression of miR-125a-3p in PBMCs of HT patients and healthy controls. Correlation between the levels of miR-125a-3p in PBMCs and the levels of autoantibodies was analyzed.
Results:
1. The proportion of Th17cells were significantly increased in HT patients compared with healthy controls[(0.75±0.79)%versus (0.28±0.23)%, P<0.05]. The level of RORγt mRNA in PBMC was significantly higher in HT patients compared with that from healthy controls (0.30±0.38versus0.04±0.02, P<0.05). The concentrations of plasma IL-6、IL-23were increased in HT patients compared with healthy controls (3.66±4.70versus0.47±1.11pg/ml, P<0.05)(154.7±75.81versus80.65±61.41pg/ml, P<0.05). Compared with the simple goiter group, the expression of RORyt and IL-17mRNA in thyroid tissue was measured in HT patients.
2. A strong positive correlation between the proportion of Th17cells in PBMC and levels of TG-Ab (r=0.8484, P=0.0077) and a borderline correlation that with the levels of TPO-Ab (r=0.6224, P=0.0994) were found in HT patients.
3. The proportion of Treg cells was significantly decreased in HT patients(P<0.01). The expression of Foxp3mRNA was significantly decreased in HT patients compared with healthy controls (P<0.05).
4. The ratio of Thl7/Treg was significantly increased in HT patients (P<0.01) and had significant correlation with the levels of TG-Ab (r=0.5243, P=0.0176)
5. The concentration of plasma GITRL was significantly increased in HT patients (P<0.05). The levels of GITRL had significantly positive correlation with proportion of Th17cells (r=0.5282, P=0.0167). mGITRL can significantly promote the differentiation of Thl7cells under the Th17-differentiation condition.
6. The expression of RORγt、IL-17mRNA and GITRL mRNA in thyroid tissues was significantly increased in HT patients compared with the simple goiter group (P<0.05).
7. The level of IL-23R mRNA in PBMCs was significantly increased in HT patients (P<0.05). Through software analysis, we listed two miRNAs probably target to IL-23R:miR-155and miR-125a-3p. The expression level of miR-125a-3p was significantly decreased in HT patient (P<0.05), in contrast with the upregulation of IL-23R mRNA level.
8. The luciferase reporter assay confirmed that miR-125a-3p can target to the seed region of IL-23R and can inhibit the expression of downstream IL-23R gene, fluorescence intensity was decreased. The results show that miR-125a-3p had direct targeted relationship with IL-23R gene.The percentage of IL-23R+cells was decreased after miR-125a-3p transfection in PBMC, indicated miR-125a-3p can inhibit the expression of IL-23R.
9. The expression levels of miR-125a-3p measured by qRT-PCR were significantly decreased in PBMC of HT (P<0.05). A negative correlation was found between the levels of miR-125a-3p and the levels of IL-23R mRNA in HT (r=-0.5195, P=0.0226). The expression levels of miR-125a-3p had a significantly negative correlation with the levels of TG-Ab(r=-0.4811, P=0.0371).
Conclusions:
1. The increased proportion of Th17cells and related cytokine were observed in patients with HT. The proportion of Th17cells was positively correlated with the levels of autoantibodies. The results show that Th17cells and related cytokines may play an important role in development of HT.
2. The elevated of Th17cells were accompanied with the decreased Treg cells as same as changes of transcription factors RORγt and Foxp3with the significant correlation between the ratio of Th17/Treg and TG-Ab, suggesting that Th17/Treg imbalance may be involved in HT patients. It provided a new insight in elucidating the pathogenesis of HT.
3. GITRL could promote the development of Th17cells in vitro. The increased expression levels of GITRL in HT patients were positively correlated with Th17cells. HT patients had significantly increased levels of GITRL、RORγt、 IL-17mRNA in thyroid tissues. The results show that GITRL may be involved in the regulation the balance of Thl7/Treg cells.
4. MiR-125a-3p can derectly regulate the expression of IL-23R. The decreased expression levels of miR-125a-3p in PBMCs of HT patients were negatively correlated with increased expression levels of IL-23R mRNA. Moreover, the expression levels of miR-125a-3p had negative correlation with the levels of autoantibodies. It is indicated the possible involvement of miR-125a-3p in the pathogenesis of HT, and also in the severity of desease.
通过研究桥本甲状腺炎(HT)患者外周血中Th17细胞及相关因子表达水平,分析Th17细胞与自身抗体变化的关系,了解Th17细胞及相关因子在HT发病中的作用；研究Th17细胞和Treg细胞在外周血中的比例及其特异性转录因子的表达水平,分析Th17/Treg细胞比率与自身抗体水平的关系,探讨HT患者Th17/Treg细胞免疫偏移与HT发生的相关性；研究HT患者外周血糖皮质激素诱导的肿瘤坏死因子受体相关配体(GITRL)表达水平,及其与Th17细胞和Treg细胞的相关性,探讨GITR/GITRL系统在Th17细胞和Treg细胞平衡中的作用；通过研究调控白介素-23受体(IL-23R)表达的的miRNAs,探讨了miR-125a-3p对HT患者外周血单个核细胞(PBMC)IL-23R的调控作用,为HT的诊治提供新思路。
方法：
1、采用流式细胞术(FCM)检测HT患者与健康对照PBMC中CD3+CD8-IL-17+T细胞(即Th17)比例,ELISA检测HT患者与健康对照外周血血浆中IL-6、IL-23的水平,并进行比较。通过实时荧光定量PCR(qRT-PCR)检测HT患者与健康对照组PBMC中RORyt mRNA表达水平,逆转录PCR(RT-PCR)检测患者甲状腺组织中RORyt和IL-17mRNA的表达情况。
2、应用化学发光免疫分析法(CIA)检测HT患者抗甲状腺球蛋白抗体(TG-Ab)和抗甲状腺过氧化物酶抗体(TPO-Ab)的水平,分析患者PBMC中Th17细胞比例与自身抗体水平的相关性。
3、采用流式细胞术检测HT患者与健康对照PBMC中CD4+CD25+CD127lowT细胞(即Treg)比例,通过qRT-PCR检测HT患者与健康对照PBMC中Foxp3mRNA表达水平。
4、分析HT患者及健康对照PBMC中Th17/Treg细胞比率,并与TG-Ab水平进行相关性分析。
5、采用ELISA检测HT患者和健康对照血浆中GITRL的水平,qRT-PCR检测HT患者甲状腺组织中GITRL mRNA的表达情况。分析血浆中GITRL水平与Th17和Treg细胞比例的相关性。
6、免疫磁珠分选小鼠初始型CD4+T细胞,在Th17细胞诱导条件下进行体外培养,观察GITRL对促进小鼠Th17细胞分化的影响。
7、采用qRT-PCR检测HT组与健康对照组PBMC中IL-23R mRNA的表达情况。以IL-23R为目的基因,利用targetscan/miRanda/Pictar数据库软件,预测可能调控IL-23R的miRNAs。
8、通过qRT-PCR检测预测miRNAs在HT患者以及健康对照PBMC中的表达情况,筛选出符合预期的miRNA。
9、利用荧光素酶报告基因检测实验验证miR-125a-3p与IL-23R的靶向作用,明确miR-125a-3p与IL-23R3'UTR结合位点的作用强度。将表达IL-23R的健康体检者PBMC作为靶细胞,转染miR-125a-3p mimics和NC,流式细胞术检测PBMCIL-23R表达变化。
10、采用qRT-PCR方法检测HT患者和健康对照外周血PBMC miR-125a-3p的表达情况,并与IL-23R mRNA水平进行相关性分析。同时对HT患者PBMC中miR-125a-3p的表达水平与其自身抗体水平进行相关性分析。
结果：
1、与健康对照组相比,HT患者PBMC中的Th17细胞比例明显增高[(0.75+0.79)%vs(0.28+0.23)%,P<0.05]。HT组PBMC中Thl7细胞特异性转录因子RORγt的相对表达量显著高于健康对照组(0.30±0.38vs0.04±0.02,P<0.05)。HT患者血浆中IL-6、IL-23的表达量显著高于健康对照组(3.66±4.70vs0.47±1.11pg/ml, P<0.05)(154.7±75.81vs80.65±61.41pg/ml, P<0.05)。与单纯性甲状腺肿组相比,HT组患者甲状腺组织中表达RORyt和IL-17基因。
2、桥本甲状腺炎患者PBMC中的Thl7细胞比例与其血清TG-Ab水平存在较强的正相关(r=0.8484,P=0.0077),与其血清TPO-Ab水平可能存在一定的正相关(r=0.6224,P=0.0994)。
3、HT患者PBMC中Treg细胞比例明显降低(P<0.01)。Treg细胞特异性转录因子Foxp3mRNA的相对表达量也显著低于健康对照组(P<0.05)。
4、HT患者外周血PBMCs中Th17/Treg细胞比率明显增高(P<0.01),并与TG-Ab呈现明显正相关(r=0.5243,P=0.0176)。
5、HT患者外周血中GITRL的水平明显上调(P<0.05),相关分析显示,GITRL与Th17细胞比例呈明显正相关(r=0.5282,P=0.0167)。在Th17细胞诱导条件下,mGITRL可明显促进Th17细胞的诱导分化。
6、与单纯性甲状腺肿组相比,HT组甲状腺组织中RORγt mRNA、IL-17mRNA和GITRL mRNA相对表达量明显增高(P均<0.05)。
7、HT患者PBMC中IL-23R mRNA的表达水平显著上调(P<0.05)。以IL-23R为目的基因,经过软件分析,取预测结果的交集,结合文献查询结果,筛选出可能调控IL-23R的miRNAs:miR-155、miR-125a-3p。miR-125a-3p的表达显著下调(P<0.05),与上调的IL-23R mRNA表达水平趋势相反。
8、荧光素酶报告基因检测实验证实,miR-125a-3p能够结合IL-23R的种子区,抑制下游IL-23R基因表达,降低荧光强度,结果表明,miR-125a-3p与IL-23R基因存在直接靶向关系。流式细胞术检测发现,与转染NC组相比,转染miR-125a-3p的PBMC表达IL-23R的细胞比例明显减少,提示miR-125a-3p可以抑制IL-23R的表达。
9、qRT-PCR检测表明HT患者PBMC中miR-125a-3p相对表达量下调(P<0.05),相关性分析显示,miR-125a-3p表达水平与IL-23R mRNA表达水平呈负相关(r=-0.5195,P=0.0226)。miR-125a-3p表达水平与TG-Ab水平呈显著负相关(r=-0.4811,P=0.0371)。
结论：
1、HT患者体内Th17细胞及相关因子表达升高,Thl7细胞与自身抗体(TG-Ab)水平呈正相关,提示Th17细胞及相关细胞因子在HT的发生发展中起重要作用。
2、HT患者外周血Th17细胞比例增加、Treg细胞比例减少,其特异性转录因子RORγt和Foxp3变化趋势与细胞比例相同,Th17/Treg细胞比率与TG-Ab水平呈显著相关,提示HT患者存在Th17/Treg失衡,为阐明HT的发病机制提供了新的思路。
3、GITRL能够促进Th17细胞的产生,HT患者外周血血浆中GITRL高表达,与Th17细胞呈明显正相关。HT患者甲状腺组织中GITRL、RORγt、IL-17mRNA水平均高表达。结果表明,GITRL可能参与Th17/Treg细胞平衡的调节。
4、miR-125a-3p可以直接调控IL-23R的表达。HT患者外周血PBMC中miR-125a-3p的表达显著下调,与IL-23R mRNA表达水平呈负相关,且与体内自身抗体水平呈负相关。提示miR-125a-3p与HT的发生及疾病严重程度有关。
Objective:
To investigate the variations of CD4+T-helper17(Th17) cells and the level of Thl7cells related cytokines in patients with Hashimoto's Thyroiditis (HT), analyze the relations between Th17cells and autoantibodies, evaluate the role of Th17cells and related cytokines in the pathogenesis of patients with HT. To investigate the proportion of Th17and regulatory T cells (Treg) and the expression of specific transcription factors, analyze the relations between the ratio of Th17/Treg and autoantibodies, explore the role of immune deviation of Th17/Treg in HT patients.To investigate the level of Glucocorticoid-induced TNF receptor-related protein ligand (GITRL) in peripheral blood of patients with HT and its correlation with Th17cells or Treg cells, explore the role of GITR/GITRL in the balance of Th17/Treg. To study miRNAs of regulating interleukin-23receptor (IL-23R) expression, explore the regulation role of miR-125a-3p on IL-23R in peripheral blood mononuclear cells (PBMC) of patients with HT.
Methods:
1. The frequency of CD3+CD8-IL-17+T (Thl7) cells in PBMC of patients with HT and healthy controls was analyzed by flow cytometry (FCM), the concentration of plasma IL-6、IL-23were measured by enzyme-linked immunosorbent assay (ELISA). QRT-PCR was used to measure the expression of RORyt mRNA in PBMC, the expression of RORyt and IL-17mRNA in thyroid tissue was measured by RT-PCR.
2. The levels of anti-thyroglobulin antibody (TG-Ab) and anti-thyroperoxidase antibody (TPO-Ab) in patients with HT were detected by chemiluminescence immunoassay(CIA), correlation between Th17and the levels of autoantibodies was analyzed.
3. The frequeney of CD4+CD25+CD127lowT (Treg) cells in PBMC of patients with HT and healthy controls was analyzed by FCM, qRT-PCR was used to measure the expression of Foxp3mRNA in PBMC.
4. The ratio of Th17/Treg and correlation with the levels of TG-Ab was analyzed.
5. The concentration of plasma GITRL in HT patients and healthy controls were measured by ELISA. QRT-PCR was used to measure the expression of GITRL mRNA in thyroid tissue of patients with HT and healthy controls. Correlation between the levels of plasma GITRL and ratio of T17/Treg was analyzed.
6. Naive CD4+T cells were purified by immune magnetic beads from normal murine, and incubated under the Th17-differentiation condition. The role of GITRL on the differentiation of Th17cells was observed.
7. QRT-PCR was used to measure the expression of IL-23R mRNA in PBMC of patients with HT and healthy controls. Targing IL-23R gene, through miRanda and PicTar software analysis, we listed the miRNAs probably target to IL-23R.
8. The expression of the predicted miRNAs in HT patients and healthy controls were detected by qRT-PCR. The miRNA in line with expectations was chosen for further research.
9. The targeting role of miR-125a-3p on IL-23R was validated by luciferase reporter assay, to clear the binding site strength of miR-125a-3p and IL-23R3'UTR. MiR-125a-3p mimics and control transfection was done in PBMC expressing IL-23R from the people conducted health examination as target cells. The expression of IL-23R in PBMC was measured by FCM.
10. QRT-PCR was used to measure the expression of miR-125a-3p in PBMCs of HT patients and healthy controls. Correlation between the levels of miR-125a-3p in PBMCs and the levels of autoantibodies was analyzed.
Results:
1. The proportion of Th17cells were significantly increased in HT patients compared with healthy controls[(0.75±0.79)%versus (0.28±0.23)%, P<0.05]. The level of RORγt mRNA in PBMC was significantly higher in HT patients compared with that from healthy controls (0.30±0.38versus0.04±0.02, P<0.05). The concentrations of plasma IL-6、IL-23were increased in HT patients compared with healthy controls (3.66±4.70versus0.47±1.11pg/ml, P<0.05)(154.7±75.81versus80.65±61.41pg/ml, P<0.05). Compared with the simple goiter group, the expression of RORyt and IL-17mRNA in thyroid tissue was measured in HT patients.
2. A strong positive correlation between the proportion of Th17cells in PBMC and levels of TG-Ab (r=0.8484, P=0.0077) and a borderline correlation that with the levels of TPO-Ab (r=0.6224, P=0.0994) were found in HT patients.
3. The proportion of Treg cells was significantly decreased in HT patients(P<0.01). The expression of Foxp3mRNA was significantly decreased in HT patients compared with healthy controls (P<0.05).
4. The ratio of Thl7/Treg was significantly increased in HT patients (P<0.01) and had significant correlation with the levels of TG-Ab (r=0.5243, P=0.0176)
5. The concentration of plasma GITRL was significantly increased in HT patients (P<0.05). The levels of GITRL had significantly positive correlation with proportion of Th17cells (r=0.5282, P=0.0167). mGITRL can significantly promote the differentiation of Thl7cells under the Th17-differentiation condition.
6. The expression of RORγt、IL-17mRNA and GITRL mRNA in thyroid tissues was significantly increased in HT patients compared with the simple goiter group (P<0.05).
7. The level of IL-23R mRNA in PBMCs was significantly increased in HT patients (P<0.05). Through software analysis, we listed two miRNAs probably target to IL-23R:miR-155and miR-125a-3p. The expression level of miR-125a-3p was significantly decreased in HT patient (P<0.05), in contrast with the upregulation of IL-23R mRNA level.
8. The luciferase reporter assay confirmed that miR-125a-3p can target to the seed region of IL-23R and can inhibit the expression of downstream IL-23R gene, fluorescence intensity was decreased. The results show that miR-125a-3p had direct targeted relationship with IL-23R gene.The percentage of IL-23R+cells was decreased after miR-125a-3p transfection in PBMC, indicated miR-125a-3p can inhibit the expression of IL-23R.
9. The expression levels of miR-125a-3p measured by qRT-PCR were significantly decreased in PBMC of HT (P<0.05). A negative correlation was found between the levels of miR-125a-3p and the levels of IL-23R mRNA in HT (r=-0.5195, P=0.0226). The expression levels of miR-125a-3p had a significantly negative correlation with the levels of TG-Ab(r=-0.4811, P=0.0371).
Conclusions:
1. The increased proportion of Th17cells and related cytokine were observed in patients with HT. The proportion of Th17cells was positively correlated with the levels of autoantibodies. The results show that Th17cells and related cytokines may play an important role in development of HT.
2. The elevated of Th17cells were accompanied with the decreased Treg cells as same as changes of transcription factors RORγt and Foxp3with the significant correlation between the ratio of Th17/Treg and TG-Ab, suggesting that Th17/Treg imbalance may be involved in HT patients. It provided a new insight in elucidating the pathogenesis of HT.
3. GITRL could promote the development of Th17cells in vitro. The increased expression levels of GITRL in HT patients were positively correlated with Th17cells. HT patients had significantly increased levels of GITRL、RORγt、 IL-17mRNA in thyroid tissues. The results show that GITRL may be involved in the regulation the balance of Thl7/Treg cells.
4. MiR-125a-3p can derectly regulate the expression of IL-23R. The decreased expression levels of miR-125a-3p in PBMCs of HT patients were negatively correlated with increased expression levels of IL-23R mRNA. Moreover, the expression levels of miR-125a-3p had negative correlation with the levels of autoantibodies. It is indicated the possible involvement of miR-125a-3p in the pathogenesis of HT, and also in the severity of desease.
引文
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