蛋白质组学检测P4HB蛋白在结直肠癌组织中的表达及其意义

目的 分析结直肠癌(CRC)组织和正常癌旁组织之间的差异表达蛋白质,探讨其与CRC疾病特征的关系. 方法 应用二维聚丙烯酰胺凝胶电泳法(2D-PAGE)分析12例配对CRC组织和正常癌旁组织之间的差异表达蛋白,并运用激光解吸电离飞行时间质谱分析(MALDI-TOF-MS)和MS/MS法对差异表达的蛋白质标记点进行鉴定和分析.找出CRC组织中表达上调的蛋白脯氨酰-4-羟化酶(P4HB)β亚单位作为兴趣蛋白后,通过Western-blot验证,并运用免疫组织化学技术检测P4HB在CRC中的表达情况,分析其与CRC的临床病理特征及预后的关系. 结果 2D-PAGE分析和MALDI-TOF-MS鉴定结果显示,CRC组织中 P4HB表达丰度比正常黏膜组织上调2.01倍(P<0.01).Western-blot结果显示,CRC组织中P4HB的表达水平明显高于其配对的正常黏膜组织(1.36 ± 0.54)vs(0.74 ± 0.35)](P<0.05).P4HB蛋白在CRC组织、腺瘤组织和正常黏膜组织中的表达阳性率依次减低,分别为43.85%(82/187),19.35%(12/62)及12.67%(19/150),三者间的差别均有统计学意义(P<0.001).P4HB蛋白表达与CRC的TNM分期(P=0.003)、肿瘤分化程度(P=0.020)、肿瘤大小(P<0.001)有关,而与患者的性别(P=0.880)、年龄(P=0.464)及肿瘤部位(P=0.293)无关.Log-rank检验结果显示,在CRC患者中,P4HB表达阳性组与阴性组的生存时间差别无统计学意义(P=0.110). 结论 CRC组织中P4HB表达上调,这与CRC更晚的分期、低分化及较大的肿瘤相关.

The Expression and Clinical Significance of P4HB in Colorectal Cancer by Proteomic Technology

Objective To investigate the differential expression of protein between colorectal canc-er tissue(CRC)and normal adjacent tissues,and to explore its relationship with CRC characteristics. Methods Two dimensional polyacrylamide gel electrophoresis(2D-PAGE)was used to analyze the differ-ential expression proteins between 12 cases of colorectal cancer tissues and normal adjacent tissues,and Matrix-assisted laser desorption ionization time of flight mass spectrometry(MALDI-TOF-MS)and MS/MS were used to identify and analyze the markers of differential expression proteins. After being found out as a protein of interest,which was expressed at a higher level in the colorectal cancer tissues,the prolyl 4 hydroxylase β subunit(P4HB)was validated using Western-blot. The expression of P4HB in colorectal cancer was detected by immunohistochemistry ,and the relationship between the expression of P4HB and clinicopathologic features and prognosis was analyzed . Results 2D-PAGE analysis and MALDI-TOF-MS showed that the abundance expression of P4HB in CRC tissue was 2 .01 times higher than that in normal mucosa ( P < 0 .01) . The results of Western-blot showed that the expression level of P4HB in CRC tissues was significantly higher than that in normal mucosa tissues ( P < 0 .05 ) (1 .36 ± 0 .54 ) vs (0 .74 ± 0 .35 )] .The positive expression rate of P4HB protein was successively decreased by 43 .85% (82/187)in colorectal cancer tissues ,19 .35% (12/62)in adenoma and 12 .67% (19/150) in normal mucosa ,and difference had statistical significance ( P < 0 .001) .The expression of P4HB protein was significantly correlated with TNM stage (P = 0 .003) ,tumor differentiation (P = 0 .020)) and tumor size ( P < 0 .001) ,however significant associations were not observed between P4HB expression and patient's gender ( P = 0 .880) ,age (P = 0 .464) and tumor location ( P = 0 .293) .The median survival time of patients with positive expression of P4HB protein was 70 months ,and the median survival for patients of postoperative without P4HB expression was 104 months .Log-rank test showed that the survival rate of patients with positive expression of P4HB protein was not statistically significant than that with negative expression of P4HB( P = 0 .11) . Conclusion The expression of P4HB is up-regulated in CRC tissue ,and the positive expression of P4HB is related to later CRC stage ,poorly differentiated and larger tumor of colorectal cancer.