Identification And Analysis Of Differentially Expressed Proteins In Colorectal Adenocarcinomas Tissue

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. .