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

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

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。

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 polyaerylamide gel electrophoresis （2D-PAGE） was used to analyze the differential 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 13 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 tis sues 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 coloreetal 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.