KRAS基因突变与分化型甲状腺癌131I 放疗耐受性及预后的关系

摘要] 目的：探究KRAS基因突变与分化型甲状腺癌（differentiated thyroid carcinoma，DTC）131I 放疗疗效和预后的相关性，并阐明其可能的机制。方法：收集经131I 放射治疗DTC临床组织样本，聚合酶链反应-单链构象分析法（single strand conformation polymorphism analysis of polymerase chain reaction products，PCR-SSCP）检测KRAS的遗传突变；采用qPCR 和Wb检测p21 蛋白的表达水平；亚致死剂量的131I 放射治疗DTC细胞系，采用CCK-8、流式细胞术（FCM）、Transwell 实验检测细胞活力的变化，并通过动物模型验证。结果：131I 放射治疗耐受DTC患者的KRAS基因突变增加（P<0.01），KRAS基因突变导致p21 蛋白表达下调（P<0.05），且与DTC临床分期及预后较差相关（P<0.05，P<0.01）。体内外实验证明，亚致死剂量的131I 放射治疗导致DTC细胞KRAS基因的突变率增加、p21 蛋白的表达水平降低，导致DTC细胞产生131I 放射耐受，而超表达KRAS基因显著提高p21 的表达，抑制肿瘤增长及转移。结论：KRAS基因突变与DTC临床分期及131I 放射耐受相关，亚致死剂量131I 放射治疗DTC促进KRAS基因突变产生放射耐受，而超表达KRAS基因能够提高DTC对131I放射治疗的敏感性。

Correlation between KRAS gene mutation and DTC resistance to 131I radiotherapy and prognosis

Abstract] Objective: To investigate the correlation between KRAS gene mutation and differentiated thyroid carcinoma (DTC) treatment effect and prognosis, and to explore the mechanism. Methods: Clinical tissue samples from DTC patients undergoing 131I Radiotherapy were collected. Then single strand conformation polymorphism analysis of polymerase chain reaction products (PCRC-SSCP)was used to detect KRAS mutation rate in thyroid cancer patients of different TNM stages; p21 protein expression level was detected by real-time quantitative polymerase chain reaction (qPCR) and western blotting. DTC cells were treated by sub-lethal dose of 131I Radiotherapy,and then CCK-8 assay, transwell assay and flow cytometry (FCM) were used to evaluate the changes of cells viability. Animal models were then constructed for verification. Results: The results showed that KRAS gene mutants were increased in 131I-resistant DTC patients; KRAS gene mutation suppressed p21 protein expression and was associated with clinical stage and poor prognosis. In vivo and in vitro experiments proved that sub-lethal dose of 131I increased KRAS gene mutation rate, suppressed p21 expression level, and caused 131I radiotherapy resistance. Reversely, over-expression of KRAS gene could significantly increase p21 expression, and inhibit tumor proliferation and metastasis. Conclusion: KRAS gene mutations were associated with DTC TNM stages and 131I resistance in DTC patients. Sub-lethal dose of 131I treatment could improve 131I resistance in DTC cells line, inversely, over-expressed KRAS gene could increase the sensitivity to 131I radiotherapy in DTC patients.