摘要
目的:蛋白质组学可动态、整体、定量地观察肿瘤发生、进展中蛋白质种类、数量的改变,结合功能蛋白质组的研究,依托大规模双向电泳和质谱分析的体系和生物信息学分析技术,有希望找到控制肿瘤进程关键作用的蛋白分子。本研究拟采用双向电泳分离结直肠癌与正常组织的总蛋白质,建立双向电泳图谱,寻找表达差异点,通过质谱分析鉴定,寻找与结直肠癌发生发展相关的蛋白质。应用Realtime-PCR、Western blot和免疫组化等方法在转录和蛋白质水平对其表达差异进行验证,并分析其与结直肠癌组织学分级、临床分期和预后等的关联性。以期通过此研究,为进一步揭示结直肠癌发病机制,寻找有意义的肿瘤标记物及药物治疗的靶点,提供新的线索和思路。
方法: 1.收集外科手术切除的12例结直肠腺癌和配对的12例正常结直肠粘膜新鲜组织标本,通过双向电泳技术分离总蛋白,得到12组双向电泳凝胶图谱。采用Imaging Master 2D分析软件进行合成、比对和差异分析,寻找结直肠癌组织和正常组织之间的差异表达蛋白斑点。选取差异蛋白质点,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)及蛋白质数据库检索进行差异蛋白质鉴定。
2.应用Realtime PCR和Western blot方法从mRNA水平和蛋白水平对候选分子Prohibitin(PHB)和14-3-3ζ在结直肠腺癌和配对正常肠粘膜组织的表达差异进行验证。
3.组织芯片结合免疫组化方法检测PHB和14-3-3ζ在187例结直肠腺癌、150例正常肠粘膜组织和62例腺瘤石蜡标本中的表达,应用统计学方法对这两种蛋白在不同组织间的表达差异性和组织学分级、病理分期和预后等关联性进行分析。
结果:1.12组结直肠癌与正常组织的双向电泳图谱分辨率高、重复性好,每块凝胶检测平均蛋白质斑点数分别为985±45和1012±53。选择表达差异2倍以上的40个点,经质谱分析和蛋白质数据库检索成功鉴定出35个蛋白,其中17个在癌组织中上调,18个下调。对鉴定出的差异蛋白进行了功能和细胞定位的分析和归类,选择确定结直肠癌相关蛋白候选分子PHB和14-3-3ζ进行深入研究。
2.Western blot结果显示,与正常结直肠粘膜组织相比, PHB蛋白和14-3-3ζ蛋白在结直肠癌癌组织中表达明显增强(P<0.01),与蛋白质组学结果一致。
3.免疫组化结果显示PHB蛋白在结直肠癌癌组织中表达显著高于正常组织和腺瘤组织(P<0.01);但PHB蛋白在正常粘膜组织和腺瘤中表达未见差异(P>0.05)。14-3-3ζ蛋白在正常粘膜组织、腺瘤组织和癌组织的表达呈现逐渐升高的趋势(P <0.001)。
在癌组织中,PHB和14-3-3ζ蛋白表达与病理分级呈负相关(P<0.01);但与患者性别、年龄、肿瘤大小、发病部位、临床分期及预后无关联性(P>0.05)。
4.Realtime PCR结果显示PHB mRNA和14-3-3ζmRNA在结直肠癌癌组织中与正常组织中无显著差异(P>0.05)。
结论:1.运用双向电泳技术结合质谱分析及生物信息学分析方法筛选并鉴定出的35个差异蛋白质可能与结直肠癌的发生、发展有关。
2.应用WesternBlot,组织芯片结合免疫组织化学技术验证筛选差异表达蛋白,发现PHB在结直肠癌中表达显著上调,而腺瘤与正常粘膜组织相比表达未见差异。从正常粘膜组织、腺瘤组织到癌组织,14-3-3ζ蛋白的表达呈现逐渐升高的趋势。PHB、14-3-3ζ表达与结直肠癌癌组织的分化程度相关,随着分化程度的降低其表达逐渐增强。
3. PHB和14-3-3ζ可能在结直肠癌的发生发展过程中发挥作用,其机制与PHB和14-3-3ζ基因的转录无关,而与PHB和14-3-3ζ蛋白翻译后的修饰有关。PHB和14-3-3ζ有望成为结直肠癌潜在的肿瘤标记物和药物治疗的新靶点。
Objective: Proteomics can be used to detect the changes of protein type and quantity in the genesis and development of tumor dynamically, completely and quantitatively. Combined with functional proteome study and relied on large scale of two-dimensional electrophoresis (2-DE) , mass spectrometry (MS) analysis system, and bioinformatics analytical technique, it is promised to find key molecules which control the process of the progression of carcinoma. In present study 2-DE technique was employed to separate total proteins and to identify the differentials proteins in the groups of colorectal carcinoma (CRC) and corresponding normal tissues. Subsequently MS analysis was used to identify proteins in related to the genesis and development of CRC. Realtime PCR, Western blot, and immunohistochemistry etc were used to verify the different expression of mRNAs and proteins in two groups. Furthermore, the correlations between the differential proteins and histological grading, pathological staging, prognosis of CRC were also analysed. In view of this, it is expected to give new clues and thoughts for further revealing the pathogenesis of CRC and searching for significant tumor marker and target for gene interference.
Methods 1. 12 cases of CRC and 12 intestinal normal mucosal tissues were collected and matched into 12 groups. Total proteins were extracted and separated by 2-DE technique. Then 12 sets of 2-DE gel maps were obtained. Synthesis, comparison and variance analysis were performed by Imaging Master 2D analysis software to identify the differentially expressed protein spots between CRC and normal tissues. The differential proteins in the gel were selected and dug in situ to be identified by matrix-assisted laser desorption ionization time of flight Time mass spectrometry (MALDI-TOF-MS) and protein database searching.
2. PHB and 14-3-3ζwere selected as candidates of CRC-associated molecules, Real-time PCR and Western blot were used to detect the expression of mRNA and protein of these two candidates in the CRC and normal intestinal mucosa tissues.
3. Tissue microarray combined with immunohistochemistry was applied to detect the expressions of PHB and 14-3-3ζproteins in 187 of CRC , 62 of low-grade intraepithelial neoplasia-adenoma, and 150 normal intestinal mucosa specimens. And statistics were used to analyse the the different expressions of the two proteins in three groups and the histological grading, pathological stage, prognosis in CRC.
Results 1. 12 pairs of high-resolution, reproducible 2-DE maps for CRC and normal tissues were prepared. The average number of protein spots that can be detected in each gel were 985±45 and 1012±53 respectively, and 40 points were chosen for more than two-fold expression differences between the two groups. 35 differential proteins, including 17 up-regulated proteins and 18 down-regulated proteins were successfully identified by MS analysis and database searching. PHB and 14-3-3ζwas chosen as candidates of CRC-associated molecule for the further study after analysis and classification of function and cellular localization of these differential proteins.
2. Western blot results showed that the expressions PHB and 14-3-3ζin CRC tissue were markedly enhanced, compared with the corresponding normal intestinal mucosa tissue (P <0.01). And these results were consistent with those of proteomics.
3. Immunohistochemistry revealed that PHB protein expression was significantly higher in CRC than that in normal tissue and adenomas tissues (P <0.01).However, there was no significant difference between normal intestinal mucosa tissues and adenoma tissue(P> 0.05). Immunohistochemistry of 14-3-3ζdemonstrated that the expressions of 14-3-3ζin CRC and adenoma tissues were remarkably higher than that in the normal mucosa , and the expression was gradually increased in the development of CRC from normal intestinal mucosa, adenoma to CRC(P <0.001). In addition, the expression of PHB and 14-3-3ζwere histopathologically dependent. The higher of expression of PHB and 14-3-3ζprotein,the weaker of differentiation (P <0.01); However, we also could not observe the correlation of these two protein expression with gender,age,tumor size, disease location, clinical staging and prognosis (P > 0.05).
Conclusion (ⅰ) 35 differentially expressed proteins which might be associated with the genesis and development of CRC were successfully screened from 12 pairs of CRC and corresponding normal intestinal mucosa tissues after identification by 2-DE combinding with MS and bioinformatics analysis of the total protein expression profile.
(ⅱ) Western blot, tissue microarray combining with immunohistochemistry techniques were employed to further investigate and validate the clinical pathological significance of the differentially expressed proteins PHB and 14-3-3ζin CRC. We found the up-regulated expression of PHB in CRC tissue, whereas the expression was not enhanced in adenoma and normal intestinal mucosa tissue. The expression of 14-3-3ζin carcinoma and adenoma tissues was significantly higher than that in the normal mucosa tissue (P <0.01). The expression of 14-3-3ζdisplayed a gradually increased tendency from normal mucosa, adenoma to carcinoma. The expressions of PHB and 14-3-3ζproteins were differentiation degree dependent. The stronger the expressions of PHB and 14-3-3ζproteins , the weaker the differentiation degree of colorectal tissues. .
(ⅲ) PHB and 14-3-3ζmight be involved in the development and progression of colorectal cacrinoma. And this action may be associated with its post-translational regulation.
They might be involved in the process of malignant transformation from adenoma to carcinoma in CRC, and they are promised to become potential tumor biomarkers and potential therapeutic targets. .
引文
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