早期黏膜下浸润与非黏膜下浸润结直肠癌的差异蛋白鉴定

Identification of differential proteins between early submucosal invasive and non-invasive colorectal cancer

目的构建大鼠早期黏膜下浸润结直肠癌（SICRC）与非黏膜下浸润结直肠癌（SNICRC）模型并鉴定两者的差异蛋白，以筛选早期SICRC的生物学标记。方法应用N一甲基一N一亚硝基脲（MNU）诱导建立大鼠SICRC与SNICRC模型。二维荧光差异定量双向电泳技术（2D-DIGE）和基质辅助激光解吸／电离．飞行时间质谱鉴定出SICRC与SNICRC的差异蛋白。荧光定量Real．timePCR和Westernblot实验验证所选蛋白的鉴定结果。结果成功建立大鼠SICRC与SNICRC模型。2D-DIGE发现SICRC和SNICRC之间有5个差异表达蛋白（P值均〈0．01）。质谱分析鉴定这5个蛋白发现，与SNICRC和正常对照（NC）比较，转凝蛋白（Transgelin）、肽基脯氨酰异构酶A和原肌球蛋白1在SICRC表达上调，而碳酸酐酶lI（CAII）与一种未命名蛋白表达下凋。Real-timePCR和Westernblot验证结果显示，TransgelinmRNA和蛋白水平在SICRC组（33．05±0．75、86．63±1．83）高于SNICRC（22．68-4-0．89、67．93±2．39）和NC组（18．07±0．55、44．25±1．55）（P值均〈0．05），而CAlImRNA和蛋白水平在SICRC组（18．014-0．53、41．55±1．89）低于SNICRC组（26．284-1．08、61．114-1．57）和NC组（33．08±0．76、83．434-1．61）（P值均〈0．05），变化趋势与蛋白质组学一致。

Objective To construct tumor models of early submucosal invasive colorectal cancer （SICRC） and submucosal non-invasive colorectal cancer （SNICRC） in rats, identify the differential proteins between SICRC, SNICRC and normal control （NC） and screen the biomarkers for early detection and diagnosis of SICRC. Methods N-methyl-N-nitrosourea （MNU） was used to induce tumor models of SICRC and SNICRC in rats. 2D-DIGE and MOLDI-TOF-MS/MS were applied to identify the differential pro- teins between SICRC, SNICRC and NC. Quantitative real-time polymerase chain reaction （PCR） and Western blotting assays confirmed the validation of 2D-DIGE analysis. Results MNU-induced tumor mod- els of SICRC and SNICRC in rats were successfully constructed and five differential proteins were found be- tween SICRC, SNICRC and NC by 2D-DIGE （ each P 〈 0. 01 ）. The identified results using mass spec- trometry showed that Transgelin, Peptidylprolyl isomerase A and Tropomyosin 1 were up-regulated, while carbonic anhydrase 2 （ CA I1 ） and an unnamed protein were down-regulated in SICRC compared with SNI- CRC and NC. Furthermore, consistent with the proteomics, real-time PCR and Western blottoing assays demonstrated that the relative mRNA and protein levels of Transgelin were highly expressed in SICRC （33.05 ± O. 75, 86. 63 ± 1.83 ） as compared with SNICRC （ 22. 68 ±O. 89, 67. 93 ±2. 39 ） and NC （ 18. 07± O. 55, 44. 25 ± 1.55 ） （ each P 〈 0. 05 ）, while those of CA II were weakly expressed in SICRC （18.01±0.53, 41.55 ±1.89） as compared with SNICRC （26.28±1.08, 61.11±1.57） and NC （33. 08 ± O. 76, 83.43 ±1.61 ） （ each P 〈 0.05）. Conclusion 2D-DIGE technique is an effective way to screen the differential proteins between early SICRC and SNICRC and identified proteins such as Transgelin and CA II may be the potential biomarkers for early detection and diagnosis of SICRC.