Association Of BRAF T1799A Mutation With TGF-β And Its Effect On The Prognosis Of Patients With Papillary Thyroid Cancer

Objective:BRAF T1799 A gene mutation is the most common molecular defect in papillary thyroid carcinoma.It is a risk factor for predicting the prognosis of tumors,but the exact mechanism of affecting prognosis is unknown.TGF-β is a kind of polypeptide growth factor that influences cell growth,differentiation and apoptosis.The expression of TGF-βis enhanced in thyroid cancer cells,whose function is promoting differentiation and invasion.The relationship between BRAF T1799 A gene mutation and the abnormal expression of TGF-β remains controversial.The purpose of this study is to clarify the association between BRAF T1799 A gene mutation and TGF-β expression in papillary thyroid cancer,and to explore the specific mechanism of BRAF T1799 A gene mutation and TGF-β on the prognosis of papillary thyroid cancer.Methods:Samples were collected from 80 postoperative cases with PTC.BRAF T1799 A gene mutation were examined by gene sequencing and polymerase chain reaction(PCR).m RNA levels of TGF-β and NIS expression were detected by Realtime quantitative PCR,and protein expression of TGF-β and NIS were tested by immunohistochemical.ROC analysis was used to construct the model of tumor staging risk factors,and the specificity and sensitivity of these risk factors on prognosis were analysed.We tested the m RNA expression of TβRⅡ,E-cadherin and Vimentin with or without BRAF T1799 A gene mutation by Real-time quantitative PCR.The protein expression of TβRⅡ,E-cadherin and Vimentin were examined by western blot.Treating BHT101 cells with U0126,a ERK/MAPK signaling pathway inhibitor,or DMSO,expressions of v-mintin and E-cadherin were examined by Western blot.Results:1.The incidence of BRAF T1799 A gene mutation was 57.5% in our study.The gene mutation was associated with lymph node metastasis and infiltration outside glands.The tumor staging was not correlated with age,tumor size,nodule number,and thyroid stimulating hormone(TSH)level.2.TGF-β m RNA expression was positively correlated with tumor size.Higher m RNA TGF-β levels were found in tumors larger than 1cm in diameter than in microcarcinoma.The protein expression of TGF-β was associated with lymph node metastasis.In other words,the expression of TGF-βprotein in lymph node metastasis was increased.No connection existed between BRAF T1799 A gene mutation and the expression of m RNA and protein of TGF-β.3.NIS m RNA expression was not associated with BRAF T1799 Amutation,TGF-β m RNA and protein level.However,NIS protein expression was correlated with BRAF T1799 Amutation,that was to say,NIS protein expression was decreased in BRAF T1799 Amutation tumors.4.ROC analysis showed that the specificity and sensitivity of BRAF T1799 A gene mutation combined with high expression of and TGF-β was significantly higher than that of the single index in predicting prognosis of tumor.5.The expression E-cadherin m RNA and protein in the high TGF-b-expressing tissues were lower than in the low-expressing group,while vimentin m RNA and protein expression in the high TGF-b-expressing tissues were greater than in the low-expressing group.6.The expression of Vimentin both in m RNA and protein level was enhanced in tumor with BRAF T1799 A gene mutation.E-cadherin protein expression in tumor with BRAF T1799 A gene mutation was lower than in BRAF wildtype group,while the expression of E-cadherin m RNA had no change neither in BRAF T1799 A geng mutation group nor in BRAF wildtype group.7.The groups were divided by whether or not BRAF T1799 A gene mutation and high expression of TGF-b,group 1 was BRAF T1799 A geng mutation & TGF-b high expression;group 2 was BRAF T1799 A geng mutation & TGF-b low expression;group 3 was BRAF wildtype & TGF-bhigh expression;group 4 was BRAF wildtype & TGF-b low expression.The expression of Vimentin m RNA in group 1 was significantly higher than in group 2,3,4.8.Inhibiting ERK/MAPK signaling pathway in BRAF T1799 Amutation cells,decreased Vimentin protein expression and increased E-acdherin protein expression were found.Conclusion:1.There was no correlation between BRAF T1799 A gene mutation and TGF-β in PTC,neither in transcription level nor in protein level.2.Both BRAF T1799 A gene mutation and TGF-β high expression can affect the prognosis of tumor,and the combined role is far greater than the single factor inpredictive value to the prognosis of tumor.3.The effect of BRAF T1799 A gene mutation and TGF-β on the prognosis of tumorwas not through NIS.4.The activity of epithelial-mesenchymal transition(EMT)was enhanced in tumor with BRAF T1799 A gene mutation.5.BRAF T1799 A gene mutation was involved in the process of TGF-β/no Smad pathway induced EMT,and affected the prognosis of tumor as a catalyst with TGF-β.