| Thyroid cancer is one of the most common malignant tumors of the endocrine system.The incidence of papillary thyroid carcinoma(PTC)in the world is increasing year by year,which is the hotpot of endocrine system tumor research.Hashimoto’s thyroiditis(HT)is the most common autoimmune thyroid disease.The incidence HT combined with PTC is on the rise.A large number of researchers believe that HT is a precancerous lesion of PTC,which many experts and scholars have confirmed.But some researchers believe that the relationship between HT and PTC is occasional phenomenon.It is unclear which factors are indeed involved in the evolution of the HT-PTC biologic transforming process and the specific details of its involvement in the pathogenesis.Researchers have found that CD4+T helper cells(Th)are major tumor-infiltrating lymphocytes infiltrating the PTC tumor microenvironment.They are highly heterogeneous and plastic cells subtype,which are the key point for effective immune responses.The naive CD4+T(Th0)cells,after interacting with pathogen-associated molecules or injury-related molecules,receive different signal stimuli that can differentiate into at least four different subgroups of cells,such as Thl,Th2,Th17 and Treg.In this paper,we analyzed the risk factors of PTC in the Yunnan plateau population by retrospectively analyzing the case of PTC in this region.It was found that HT was closely related to the occurrence of PTC,and then we discussed the the role of Th17 and Treg cells infiltrated from the PTC microenvironment in the evolution of HT-PTC,and to explore the role of immune-related factors in the development of PTC and the prognosis of PTC with HT patients.The main results of this paper are as follows:(1)In this study,we collected 578 cases of PTC patients and 620 cases of benign thyroid tumors(BTD)patients from January 2003 to December 2012 in the First People’s Hospital of Yunnan Province.Using case-control study,it is the first time to obtain clinical data and pathological types of PTC patients from Yunnan Plateau in exploring PTC risk factors in the area.At the same time,the representative articles of different altitude regions are reviewed and analyzed.Univariate regression analysis showed that the average age of patients with PTC in the plateau region of Yunnan was lower than that in the benign thyroid disease(BTD)group.The preoperative TSH level in the PTC patients was higher than that in the BTD group;The positive rate of thyroid peroxidase antibody(TPOAb),thyroglobulin antibody(TGAb)and thyroxine receptor antibody(TRAb)was higher than that of the BTD group;The proportion of PTC patients with Hashimoto’s thyroiditis(HT)(12.1%)or with lymphocytic thyroiditis(LT)(5.6%)was higher than that of BTD group;whose age of menarche<13、numbers of breeding babies≤2 or in premenopausal conditions,were much higher than that of BTD group;The multivariate regression analysis showed that age>45 years(OR=0.114,P<0.001),tumor diameter>1 cm(OR=0.371,P=0.003)and elevated levels of TG(OR = 0.481,P = 0.01)were protective factors for PTC.The abnormally elevated TGAb(OR = 4.894,P<0.001)and TRAb levels(OR = 16.047,P<0.001)were independent risk factors for PTC in the Yunnan Plateau.In summary,we found that age of patient,preoperative serum TSH levels,menstrual period of fertility,HT and its autoimmune antibodies TGAb and TRAb may be associated with the occurrence of PTC in the plateau region of Yunnan.(2)To study the risk factors for the development of PTC children and adolescents in the plateau region,we combined the clinical data of children and adolescents from the First People’s Hospital of Yunnan Province and the Tumor Hospital of Yunnan Provincial for the past 11 years,using case-control study and single factor regression analysis,it is found that among the 84 children and adolescents diagnosed with PTC in the Yunnan Plateau,13 were confirmed to be HT(15.5%)by histopathology.Abnormal elevated level of preoperative TSH levels,TPOAb,combined with Hashimoto’s thyroiditis are associated with the development of PTC in children and adolescents in Yunnan Plateau.Multivariate retrospective analysis showed that tumor diameter>1 cm(OR = 0.344,P<0.01)and abnormally elevated TGAb levels(OR = 25.924,P<0.01)were independent protective factor for PTC of children and adolescents in the plateau region.(3)Through the immunohistochemical study of paraffin-embedded tissue in patients PTC with and without HT,we observed that the proportion of patients with lymph node metastasis in RORγt low expression group was significantly higher than that in overexpression group in PTC patients(71.4%vs.33.3%,P=0.017).The proportion of patients with lymph node metastasis in Foxp3 overexpressing group was significantly higher than that in PTC/HT patients(68.2%vs.37.5%,P=0.037),indicating that the expression of RORyt and Foxp3 in PTC with or without HT microenvironment was correlated with lymph node metastasis.After Spearson correlation analysis,we found that the above two groups of patients with RORyt and lymph node metastasis were negatively correlated(P=0.024,P=0.046).There was a negative correlation between RORγt and TPOAb in PTC group(P=0.027).The expression of RORyt mRNA in patients with PTC with or without HT was significantly higher than that in PTC(t= 2.62,P= 0.011),suggesting that RORγt may have anti-tumor effect of tumor metastasis in the development of HT combined with PTC.(4)In order to evaluate the role of thyroid papillary carcinoma cell lines in the induction of T lymphocytes,follicular epithelial cell line(nthy ori 3-1)as a control,the naive T lymphocytes of the umbilical cord blood were co-cultured with the thyroid papillary carcinoma cell line(TPC-1 or Kl)by transwell chamber.We found that ①PTC cells can induce umbilical cord blood naive T cells to differentiate into Foxp3-expressing Treg cells and RORyt-expressing Th17 cells.Umbilical cord blood naive T cells are highly response to TPC-1 and Kl in this process,whereas umbilical cord blood naive T cells show a low response to normal control thyroid follicular epithelial cells.②It is showed a fluctuating growth that PTC cells and umbilical cord blood initial T cells co-cultured,inducing differentiation of Th17 cells and Treg cells.The proportion of CD3+CD8 IL-17+Th17 cells and CD4+CD25+CD127-Treg cells was the highest when the two groups were treated for 36 hours.The formation ratio of Th17/Treg cells is larger than 1 and significantly higher than the normal control group,suggesting Th17/Treg balance was significantly shifted to Th17 direction,which may be the reason for the better prognosis of PTC patients with HT.③ K1 is a PTC cell line with lymph node metastasis,and the ratio of Th17/Treg cells formed by K1-induced naive T lymphocytes is also higher than that of TPC-1 cancer cell lines without lymph node metastasis,suggesting that changes in Th17 and Treg are associated with PTC lymph node metastases.The above results indicate that PTC can induce the differentiation of naive T lymphocytes into Treg and Th17 CD4+T helper lymphocytes,and the proportion of Th17 cells is higher than that of Treg cells,suggesting that the prognosis of patients with PTC is better.(5)To investigate the expression and significance of cytokines and chemokines during HT-PTC transformation,using of flow cytometry cytometric bead array(CBA),we examined the expression levels of serum cytokines and chemokines,including Th1(TNF-α、IL-2、IFN-γ),Th2(IL-4、IL-6、IL-10)and Thl7(IL-17)in peripheral blood of patients with HT alone,PTC with and without HT.The results are as follows:① The ratio of Th1/Th2 in patients with PTC was 0.84±0.19,which was significantly lower than that of BTD group.The cellular immunity of PTC patients drifts toward Th2 direction,suggesting that anti-tumor immunity is inhibited.Thl/Th2 ratio of PTC/HT patients was 0.88±0.13,which was significantly lower than that of the BTD group.The immunity was also drifted in the Th2 direction and the Thl/Th2 ratio in the PTC/HT group slightly higher than the PTC group.Although the results were not statistically significant,the results suggest that the level of anti-tumor immunity in PTC with HT patients is better than that of patients with PTC alone.②IP-10 and MIG may synergize TIL from regional lymph nodes to tumor tissue in PTC with HT patients and play a role in anti-tumor immunity.(6)We used Illumina gene expression microarray and real-time quantitative PCR(RT-PCR)to explore the differential expression genes in the biologic transforming process of HT-PTC.PCA principal component analysis showed that cancer tissues of 6 cases of PTC patients,4 cases of PTC/HT patients and 4 cases of pathologically confirmed HT tissue belong to three different regions,the distance between the sample space is far,which are completely different three types of tissue;MultiClassDif differential gene screening showed that the three groups were different,the differential expression of a total of 1257 genes,of which function of 945genes is clear and 312 genes is unclear.The significance analysis of signal pathway and gene function showed that there were 45 significant pathways and 290 significant functions(Go).A total of 120 differential genes was associated with immunization were included in the study.We selected 10 immunological-related genes for subsequent bioinformatics analysis and qPCR validation.It was found that all the seven genes ADRBK1,PTPN6,CD247,TNFSF13B,CXCL9,TNFRSF14 and CAV1,which were consistent with the trend,were mainly focused on the regulation of T cells and B cells immunoregulation. |
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