BRAF^(V600E)突变对甲状腺乳头状癌HMGB1表达的影响

Effect of BRAF^(V600E) mutations on the expression of HMGB1 in papillary thyroid cancer

目的近年来甲状腺乳头状癌(PTC)的发病率逐渐升高,发生复发和转移的患者也在增多。本文意在探索在PTC中BRAF^(V600E)突变对高迁移率族蛋白(HMGB1)表达的影响,寻求BRAF基因影响PTC发展及预后的机制,指导临床进行精准治疗。方法收集2015年9—12月青岛大学附属医院收治的44例PTC患者的术前血清及术后新鲜病理组织,组织提取DNA进行基因测序。根据有无BRAF^(V600E)突变将患者分为BRAF突变阳性组和BRAF突变阴性组,运用免疫组化和Western blot检测组织中HMGB1蛋白的分布和含量;荧光定量PCR检测组织中HMGB1 mRNA的水平;应用ELISA法检测血清中HMGB1蛋白的水平。Western blot数据应用Image J软件计算灰度值,采用相对定量法计算荧光定量PCR数据,用2-△Ct进行分析,应用ELISA Calc回归/拟合计算程序计算血清中的HMGB1蛋白浓度。所得数据均采用SPSS 20.0进行统计分析。Western blot、荧光定量PCR和ELISA数据分别用χ2检验、Mann-Whitney-Wilcoxon test和独立样本t检验的方法进行统计学处理。淋巴结转移和腺体外浸润与BRAF^(V600E)突变发生的关系采用χ~2检验进行统计分析。结果在PTC组织中,HMGB1蛋白主要定位于胞浆,BRAF^(V600E)突变阳性组HMGB1的转录水平及蛋白水平均低于BRAF^(V600E)突变阴性组(Z=2.117,P<0.01;χ~2=19.989,P<0.05),而这种变化在外周血中并未呈现(t=1.135,P>0.05)。BRAF^(V600E)突变增加淋巴结转移和腺体外浸润的风险(χ~2=6.117,P<0.05;χ2=5.587,P<0.05)。发生淋巴结转移PTC中的HMGB1 mRNA和蛋白的表达量均低于无淋巴结转移PTC(Z=-2.216,P<0.05;t=-2.217,P<0.05),发生腺体外浸润的PTC也呈现此种趋势(Z=-2.267,P<0.05;t=-3.885,P<0.01)。结论在PTC中,BRAF^(V600E)突变可能通过下调HMGB1的表达加速肿瘤的恶性发展。

Objective In recent years, the incidence of papillary thyroid carcinoma （ PTC ） increased gradually and the recurrence and metastasis of PTC patients are also increasing. The aim of this study was to investigate the effect of BRAF^V600E mutation on the expression of HMGB1 in PTC ,to explore the mechanism of BRAF gene affecting the development and prognosis of PTC, and then guiding clinical practice of precise therapy. Methods The preoperative serum and postoperative fresh tissue were collected from 44 cases of PTC patients in our hospital from September,2015 to December, 2015. According to BRAF^V600E mutations divided into positive group and negative group. Western blot,immunohistochemistry and fluorescence quantitative PCR were used to detect the level of HMGB1 in cancer tissues. The level of HMGB1 in serum was tested by ELISA method. Western blot was performed using Image J software. The relative quantitative method was used to calculate the fluorescence quantitative PCR data. The concentration of HMGB1 protein in serum was calculated by ELISA Calc regression/fit calculation program. All the data were analyzed by SPSS 20.0. Western blot, fluorescence quantitative PCR and ELISA data were analyzed by Chi-square test, Mann-Whitney-Wilcoxon test and independent sample t test. The relationship between lymph node metastasis and extrathyroid infiltration and the occurrence of BRAF^V600E mutation was analyzed by chi-square test. Results In PTC cancer tissues,HMGB1 was mainly located in the cytoplasm,the levels of HMGB1 mRNA and protein in positive group were lower than that of the negative group （ Z = 2.117, P 〈 0. 01, Х^2 = 19. 989,P 〈 0.05 ）, and the change did not show in peripheral blood （t = 1. 135, P 〉 0. 05 ）. The expression of HMGB1 mRNA and protein in PTC with lymph node metastasis was lower than those without lymph node metastasis（ Z = -2.216,P〈0. OS;t = -2. 217,P 〈0.05） ,and so as in the extrathyroid infihration （Z = - 2. 267,P 〈0.05;t = - 3. 885,P 〈 0.01 ）. Conclusion BRAF^V600E mutations may accelerate the malignant development of PTC by down regulating the expression of HMGB1