酪氨酸和苏氨酸蛋白激酶在结直肠癌发生发展中的表达变化及其与预后的关系

Expression of tyrosine and threonine protein kinase in carcinogenic process of colorectal cancer and its relationship with prognosis

摘要目的探讨酪氨酸和苏氨酸蛋白激酶（TTK）在结直肠癌发生发展中的表达变化及与结直肠癌患者预后的关系。方法联合应用氧化偶氮甲烷（AOM）和葡聚糖硫酸钠（DSS）诱导C57BL/6小鼠发生结肠炎相关的结肠癌，得到结肠癌发生发展的4个不同阶段的小鼠模型（分别为炎症恢复期、轻度不典型增生、腺瘤、腺癌），同时设立无任何处理的对照组。应用实时荧光定量PCR和免疫组化法检测不同阶段小鼠结肠和24对结直肠癌及其配对的癌旁组织中TTK mRNA和蛋白的表达水平。从美国国家生物技术信息中心（NCBI）基因表达公共数据库中获取结直肠癌全基因组芯片数据，分析TTK的表达与结直肠癌患者预后的关系。结果AOM-DSS结肠癌小鼠模型的结肠全基因组表达芯片显示，TTK mRNA的表达水平随着结肠癌的发生发展逐渐升高。实时荧光定量PCR检测结果显示，对照组、炎症恢复期、轻度不典型增生、腺瘤和腺癌小鼠结肠组织中TTK mRNA的表达水平分别为1.05±0.42、1.10±0.03、1.38±0.15、1.33±0.17和2.12±0.22，轻度不典型增生、腺瘤和腺癌小鼠结肠组织中TTK mRNA的表达水平均明显高于对照组（均P〈0.05）。免疫组化检测对照组、炎症恢复期、轻度不典型增生、腺瘤和腺癌小鼠结肠组织中TTK蛋白的表达变化与TTK mRNA的检测结果一致。24例结直肠癌患者肿瘤组织中TTK mRNA的表达水平为0.71±0.10，明显高于配对的癌旁组织（0.18±0.04，P〈0.001）。免疫组化检测5例结直肠癌患者肿瘤组织中TTK蛋白的阳性表达率为80.0%，明显高于配对的癌旁组织（30.8%，P＝0.014）。全基因组芯片网络数据（GSE17536）分析显示，TTK的高表达与结直肠癌患者的不良预后有关。结论在结肠癌的小鼠模型中，TTK的表达随着肿瘤的发生发展逐渐升高；在临床结直肠癌标本中，TTK的高表达与患者的不良预后相关。

Objective To investigate the expression of TTK （tyrosine and threonine protein kinase） in the process of colorectal cancer （CRC） development and its relationship to prognosis in CRC patients. Methods Colitis-associated colon cancer model was induced by azoxymethane （AOM） and dextran sulfate sodium （DSS） in C57BL/6 mice. Mice at four different stages of colon cancer development were obtained, named AD1 （inflammation of the recovery） , AD2 （mild dysplasia） , AD3 （adenoma） and AD4 （ adenocarcinoma）, as well as negative control （ no treatment）. The expression of TTK was measured by real time fluorescent quantitative PCR （qPCR） and immunohistoehemical staining in mouse colon tissues and 24 pairs of CRC specimens. The relationship between TTK and prognosis was analyzed in a set of CRC genome- wide gene expression microarray data that was obtained from Gene Expression Omnibus （GEO） of National Center for Biotechnology Information （NCBI）. Results The genome-wide microarray data from mouse AOM-DSS model indicated that the expression of TTK mRNA was gradually elevated during the development of colon cancer. The subsequent qPCR results showed that TTK mRNA levels in negative control, AD1, AD2, AD3 andAD4 groups were 1.05±0.42, 1.10±0.03, 1.38±0.15, 1.33±0.17 and 2.12±0.22, respectively. And TTK expression in AD2, AD3 and AD4 groups were significantly higher than that in negative control （P〈0.05）. The protein expression of TTK showed by immunohistochemical staining had similar tendency as the results of TFK mRNA. Besides that, the TTK mRNA levels in tumor tissues （0.71± 0.10） from 24 CRC patients were significantly higher than those in paired adjacent normal tissues （0.18± 0.04 ; P〈0.001 ）. The positive expression rate of TTK protein in 5 pairs of CRC clinical samples was 80.0%, and it was significantly higher than that in adjacent normal tissues （30.8%, P = 0. 014）. Furthermore, according to a public transcriptomic data （GSE17536） , the high levels of TTK were associated with poor prognosis in CRC patients. Conclusions Elevated expression of TTK is related to colonic carcinogenesis in both of mouse model and human CRC samples. TFK is a poor prognostic factor in CRC.