甲状腺乳头状癌BRAF基因突变及BRAF、MUC15、RKIP蛋白表达的临床意义

Clinical significance of BRAF gene mutation and BRAF,MUC15 and RKIP proteins expressions in papillary thyroid carcinoma

目的探究甲状腺乳头状癌(PTC)鼠类肉瘤滤过性毒菌致癌同源体B1(BRAF)基因突变及BRAF、粘蛋白15(MUC15)及Raf激酶抑制蛋白(RKIP)表达的临床意义。方法 PTC组织标本56例作为研究组(A组),另选滤泡状癌组织标本18例(B组)、甲状腺腺瘤组织标本13例(C组)、结节性甲状腺肿组织标本7例(D组)作为对照。应用逆转录聚合酶链式反应(RT-PCR)PCR产物测序检测上述组织中BRAF基因突变情况,应用免疫组化法分析组织中BRAF、MUC15、RKIP蛋白表达情况。采用四格表χ2检验(或校正χ2检验)和R×C表有序资料的线性趋势χ2检验进行关联性分析。结果 A组56例PTC组织中检测到24例BRAF 15外显子有T1799A杂合突变(BRAFV600E突变),突变率42.86%;在BRAF 11外显子上未检测到突变病例;B、C、D组患者组织中均未检测到BRAF基因突变。随着临床分期的增高(Ⅰ期→Ⅱ期→Ⅲ期→Ⅳ期),PTC组织中BRAF基因突变率逐步升高(7.14%→8.33%→61.11%→91.67%,χ2=27.255,P=0.000)。有淋巴结转移PTC患者BRAF 15外显子基因突变率(57.58%)较无淋巴结转移患者(21.73%)显著增加(χ2=7.108,P=0.008)。A组患者组织中BRAF、MUC15、RKIP蛋白表达阳性率分别为71.43%、76.79%、12.50%。BRAF、MUC15、RKIP蛋白阳性表达率在A、B、C、D组间比较,差异均有统计学意义(P均<0.01)。与无淋巴结转移患者比较,有淋巴结转移患者BRAF和MUC15蛋白阳性表达率显著增高(P均<0.01),RKIP蛋白阳性表达率显著降低(P<0.05)。PTC组织中BRAF和MUC15蛋白阳性表达率随临床分期的增高(Ⅰ期→Ⅱ期→Ⅲ期→Ⅳ期)逐步升高(χ2=12.658,P=0.005;χ2=20.047,P=0.000);RKIP蛋白阳性表达率的降低则与临床分期的增高无相关性(χ2=6.005,P=0.111)。BRAF蛋白表达与BRAF基因突变有关(χ2=14.439,P=0.000);而MUC15和RKIP蛋白表达与BRAF基因突变无相关性(P均>0.05)。结论 BRAF基因在PTC中易发生V600E型突变,其突变可能通过上调BRAF蛋白表达,参与PTC发生进展和淋巴结转移过程。PTC组织中MUC15蛋白表达的上调及RKIP蛋白表达的下调也参与PTC淋巴结转移过程。

Objective To investigate the clinical significance of murine sarcoma filtration pathogens carcinogenic homolog B1 （BRAF） gene mutation and the expressions of BRAF, mucin 15 （ MUC15 ） and Raf kinase inhibitor protein （RKIP） in thyroid papillary carcinoma（ PTC ）. Methods The tissue samples of 56 cases PTC were used as the study group （ group A）. The tissue specimens of 18 cases follicular carcinoma （group B） ,13 cases of thyroid adenoma（ group C） and 7 cases of nodular goiter （ Group D ） were used as controls. Reverse transcription polymerase chain reaction （ RT-PCR ） PCR products sequencing was used to detect BRAF gene mutation in tissues. Immunohistochemistry method was used to detect the expressions of BRAF, MUC15 and RKIP proteins. Fourfold table X2 test （or its correcting method ） and R x C table X2 test to linear trend of ordinal data were used to make the correlation analysis. Results Out of 56 cases in group A,24 had BRAF 15 exon T1799A beterozygous mutation（ V6OOE mutation） with 42.86% mutation rate. The mutation case in BRAFexon 11 was not found. BRAF gene mutation cases were not found in groups B, C and D. With the increase of clinical stage （ I → II→III→IV ） ,the mutation rate of BRAF gene in PTC tissues increased gradually（7.14%→8.33%→61.11% 91.67% ,X2 = 27. 255, P = 0. 000）. The mutation rate of RAF 15 exon in PTC patients with lymph node metastasis was significantly higher than that in PTC patients without lymph node metastasis （57.58% vs 21.73% ,X2 = 7. 108, P = 0. 008）. The positive expression rates of BRAF, MUC15 and RKIP proteins in groups A were 71.43%, 76.79% and 12. 50% respectively in which there were significant differences among four groups（ all P 〈 0.01 ）. Compared with patients without lymph node metastasis,the positive expression rate of BRAF and MUC15 proteins significantly increased （all P 〈 0. 01 ）, and RKIP protein significantly decreased （P 〈 0. 05 ） in patients with lymph node metastasis. With the increase of clinical stage（ Ⅰ → Ⅱ→Ⅲ→Ⅳ） ,the positive expression rates of BRAF and MUC15 in PTC tissues increased gradually 0（2= 12. 658 ,P = 0. 005 ;X2 = 20. 047, P = O. 000）, while the decrease of RKIP protein positive expression rate was not significantly correlated with the increase of clinical stage （X2 = 6. 005, P = 0. 111 ）. The expression of BRAF protein was correlated with BRAF gene mutation（x2 = 14.439 ,P = O. 000） ,while the expressions of MUC15and RKIP proteins were not significantly correlated with BRAF gene mutation （ all P 〉 0. 05 ）. Conclusions BRAF gene is prone to V600E type mutation in PTC which participates in the occurrence and progress of PTC and lymph node metastasis possible through up- regulating BRAF protein expression. The up-regulation of MUC15 protein expression and the down-regulation of RKIP protein expression in PTC tissues also participate in process of lymph node metastasis of PTC.