The Expression Of Foxp3in Papillary Thyroid Carcinoma And Its Regulation By TLR4

The increase of the incidence rate of thyroid carcinoma is one of the fastestamong other cancers in the world, which has the highest incidence rate in manycountries. According to its pathology classification, thyroid carcinoma is divided intofour major different categories, papillary thyroid carcinoma (PTC), follicular thyroidcarcinoma (FTC), medullary thyroid carcinoma (MTC) and anaplastic thyroidcarcinoma (ATC). Among them, PTC accounts for about80%. Because of thedevelopment of examination equipments, patients got their diagnosis in their earlystage. However, there is still lymph node metastasis among many patients, whichinfluences the prognosis. It is important to study how PTC grows and develops, studyits molecular mechanism of invasion and metastasis, and to effectively prevent orreverse its invasion. Those are vital targets for improving the prognosis of patients,and are crucial in the pathogenesis of tumor.Forkhead box protein3(Foxp3) is one of the fork/wing screw transcriptionfactors, which plays a vital role in immune system, especially in theimmunosuppressive process of CD4+CD25+regulatory T cells. Foxp3also plays animportant role in the process of self immune. Foxp3+Tregs participated in tumorimmune escape. some studies found that Tregs had enhanced their immunesuppression, and is closely related to the PTC clinical progress. So it could be clearlyseen that Foxp3+Tregs plays an important role in the development of PTC by itsimmunosuppressive function, and influence the progress of the tumor and theprognosis of patients. In recent years, some researches showed that Foxp3could beexpressed in human’s tumors including pancreatic cancer, prostate cancer, breastcancer, lung cancer and other malignant tumors. However, the related studies werelimited, some even showed opposite roles of Foxp3expression in tumors. Moreover,there is no study of the mechanism of Foxp3expression in PTC.TLR4is a member of Toll like receptors family, and it was originally found inimmune cells. TLR4participates in innate immunity and acquired immunity. Itsexogenous ligand is lipopolysaccharide (LPS) which is the composition of cytoderm in gram-negative bacillus. Recent studies have found that it was also expressed intumor cells, primarily through the release of inflammatory factors andimmunosuppressive factors, to induce immune to the tumor, and to promote theimmune escape of tumor, all of which are closely related to the progress andprognosis of the tumor. It also can be used as an independent risk factor to predictpoor prognosis of some tumor. Studies have confirmed that TLR4signaling pathwayscan promote the activation of Tregs, improve the ability of Tregs proliferation abilityand immune suppression function.Based on the literature review above, we put forward the following questions:whether Foxp3express in thyroid carcinoma? If so, what is its possible role andclinical significance? What is its regulation mechanism? In this study, in order tolooking for Foxp3expression and its mechanism, we used PTC tissue and human’sthyroid carcinoma K1cells as the research object. The study includes three aspects asfollow：1. The expression of Foxp3and TLR4in PTC issue, and their clinicalsignificanceTo explore Foxp3and TLR4expression in PTC issue and the links between thetwo, the study employed immunohistochemical method to look at the expression ofFoxp3and TLR4in78tumor patients and20normal thyroid tissues. The resultsshowed that:(1) High expression of Foxp3in PTC cells, the positive rate is35.90%, and itexpress in cytoplasm, nuclear/cytoplasm and in the nuclear. It has a low expression innormal tissue with a10.00%positive rate. Most of the expression in normal tissue isin the cytoplasm of epithelial cells of the thyroid, and the comparison between tumorgroup and normal group is statistically significant (P <0.05). TLR4has a highexpression in the PTC, with positive rate of61.54%, and expresses in cytoplasm andcell membrane. It has a lower expression in normal thyroid tissue, with positive rateof35.00%, and expresses in the cytoplasm and cell membrane of thyroid epithelialcells. The comparison between tumor group and normal group is statisticallysignificant (P <0.05).(2) The expression of Foxp3in PTC is closely associated with lymph nodemetastasis and TNM stages. The group with lymph node metastasis has a higherexpression rate (56.67%) of Foxp3than the group without lymph node metastasis(22.92%). The comparison between the two groups is statisticallysignificant (P <0.05); The expression rate in TNMⅡ＋Ⅲ stage(61.11%) is higherthan the group in TNMⅠstage (14.29%). The comparison between the two groups isstatistically significant (P <0.05). The expression of Foxp3is not related to patients’gender, age and tumor size (P>0.05). The expression of TLR4in PTC tissue isclosely associated with lymph node metastasis. The group with lymph node metastasishas a higher expression rate (80.00%) of TLR4than the group without lymph nodemetastasis(50.00%). The comparison between the two groups is statisticallysignificant (P <0.05). The expression of TLR4is not related to patients’ gender, age,tumor size and TNM stages (P>0.05).(3) The Foxp3expression in the PTC tissue is related to the expression of TLR4in PTC, there is a significant positive correlation (r=0.508, P <0.05). The resultsshowed that Foxp3and TLR4expression in PTC cells is closely related to themalignant biological behavior of PTC, there may be some kind of inner link betweenthem, and promote the development of PTC together.2. The function of Foxp3expression in human’s K1cells and related mechanismAfter initial test of Foxp3expression in PTC tissue, we take human’s K1cell lineas the research object for further discussion of the possible mechanisms of thedevelopment of PTC.(1) Foxp3expression in human’s K1cells, build the cell model of Foxp3expression and detect over expression of Foxp3in rebuilt K1cellsFirst, using RT-PCR and immunocytochemistry staining method to detect Foxp3expression in K1cells line, and then build pEGFP-C1-Foxp3recombinant plasmid,and then transiently transfect K1cells. After that, employing RT-PCR, fluorescencemicroscope and Western blot method to detect the over expression of Foxp3from thelevel of gene and protein. It shows that Foxp3expresses in K1cells, and wesuccessfully build pEGFP-C1-Foxp3recombinant plasmid, after transient transfection,K1cell in the group of pEGFP-C1-Foxp3has a higher expression of Foxp3mRNAand protein than the group of pEGFP-C1-K1and K1group (P <0.05). Foxp3canexpress higher in K1cell after transient transfection, which can be used for thefollowing research.(2) The influence of over expression of Foxp3in K1cell on immune function.For the study of Foxp3expression in K1cells of PTC, we tested the effect of over expression of Foxp3in K1cell and its supernatant on the proliferation of T cells bylymphocyte transformation test. Test the influence of K1cell with over expression ofFoxp3on TGF-β1and IL-10by using the RT-PCR and ELISA. It showed thepEGFP-C1-Foxp3-K1group cell and the culture supernatant have a highersignificance on inhibition to T lymphocyte proliferation than the pEGFP-C1-K1andK1group (P <0.05). The results show that Foxp3expression in K1cells can simulateTregs to inhibit proliferation of T lymphocyte, and it may be regulated by improvingthe expression of TGF-β1and IL-10.(3) The influence of Foxp3over expression on proliferation and migration abilityof K1cellTo explore the influence of Foxp3expression on K1cell, we used MTT test tolook at the proliferation of the K1cells, and used Tranwell test to explore theinfluence of Foxp3over expression on the migration ability of K1cell. It showed that,compared with the K1group and pEGFP-C1-K1group, the cell proliferation andmigration ability of pEGFP-C1-Foxp3-K1group is not promoted, there was nostatistically significant difference (P>0.05). The result showed the over expression ofFoxp3in K1cell did not affect the proliferation and migration ability of K1cell, andit may affect the development of the tumor by other extracellular factors.3. The study of the regulation of TLR4to Foxp3expression in K1cellIn order to further explore the link between TLR4and Foxp3in PTC cell, K1cellis selected as the research object. Choose exogenous ligand LPS to activate TLR4signaling pathway, and test the change of Foxp3expression after activation andblocking of TLR4signaling pathways by the method of RT-PCR and flow cytometry.The result showed that: the Foxp3expression is higher on gene and protein level inthe LPS activation group than the control group(P <0.05), and the Foxp3expressionin the blocking of TLR4group is lower than the activation group(P <0.05). It isclearly shown that the TLR4signaling pathway participated in the regulation ofFoxp3expression, and TLR4may be the upstream signal molecule of the Foxp3expression in K1cell.All in all, we have the conclusions as follows,1. The higher expression of Foxp3and TLR4in human’s PTC tissue is closelyrelated to the malignant biological behavior of thyroid papillary carcinoma. TLR4participates in the regulation of Foxp3expression in K1cell, and TLR4may stimulate the expression of Foxp3. Foxp3and TLR4promote the development ofPTC together.2. The expression of Foxp3in K1cells can simulate Tregs, and inhibit T lymphocyteproliferation by raising the expression of TGF-β1and IL-10, which promotetumor immune escape.3. The expression of Foxp3in K1cells has no influence to K1cell itself, there is nosignificant effect on the proliferation and the migration ability of K1cell. Itshowes that the pathogenesis may be related to extracellular factors.4. The activation of TLR4signaling pathway promotes the expression of Foxp3inthe K1cells, and also raises the expression of TLR4itself.In conclusion, this study discussed the role of Foxp3in the development ofthyroid papillary carcinoma, and also described how Foxp3expression in PTCparticipated in tumor development. The study found that TLR4regulates theexpression of Foxp3in PTC, offering a new insight in the mechanism of tumorinvasion and metastasis. It also provided a new immunotherapy target for thyroidpapillary carcinoma.