Antibody Preparation And Expression Of BRD7 Protein In Colorectal Carcinomas And The Clinical Significance

[Study Background of BRD7]BRD7 is a novel gene associated with nasopharyngeal carcinoma (NPC) and it's expression product is a bromodomain-containing protein which functions as a nuclear transcription regulator. Over-expression of the BRD7 gene in NPC cells could effectively inhibit cell growth and cell cycle progression from G1 to S phase by transcriptionally regulating some important molecules involved in MAPK and Wnt signal pathways. Our previous studies have proved that BRD7 was down-expressed in NPC biopsies, and the RT-PCR results showed the mRNA expression of BRD7 was also evidently down-regulated in colorectal carcinomas (CRC) cells. To further investigate the protein expression of BRD7 in CRC and understand the association between the levels of BRD7 protein expression and clinicopathological features of CRC and it's Clinical Significance, in this study we first prepared anti-BRD7 polyclonal antibody and then detected the expression of BRD7 protein in CRC by tissue microarrays technology and immunohistochemistry assay.[Expression and Purification of the GST-BRD7N Fusion Protein]The sequence (spanning from 54 to 1112 nt) encoding the N-terminal 353 amino acids of the BRD7 protein was amplified by PCR from a cDNA library of human fetal brain and cloned into BamHI/XhoI-digested pGEX-4T-1 vector. The positive clones with recombinant construct were identified by PCR and confirmed by DNA sequencing. Then the Escherichia coli BL21 transformed with the recombinant plasmid pGEX-4T-BRD7N was induced by IPTG at a concentration of 0.5 mM for 3.5 hr and the anticipated 64 kDa GST-BRD7N fusion protein was expressed efficiently in BL21. The induced targeting protein was confirmed by Western blot analysis. After purifying by affinity chromatography, the total amount of purified protein was>2 mg in 600μl. The concentration of purified protein was 1.5 mg/ml, and the protein remained intact with no proteolytic degradation during the purification procedure.[Preparation of anti-BRD7 polyclonal antibody]Five immunizations with the purified GST-BRD7N protein were given to two rabbits. The antisera were harvested at 100 days after primary immunization and subjected to affinity purification with a GST-Sepharose 4B affinity chromatography column. Western blot results showed that these antisera were able to recognize recombinant GST-BRD7N protein with a molecular mass of 64 kDa as that recognized by the anti-GST monoclonal antibody. ELISA results showed that the final titer of the purified rabbit anti-BRD7 sera was 1:1,000,000. SDS-PAGE results showed that the estimated concentration of BRD7 antibody was 300μg/ml for rabbit 1 and 150μg/ml for rabbit 2. The specificity of the polyclonal anti-BRD7 antibody was determined by Western blot and immunoprecipitation assays. It showed in Western blot that the serum antibody recognized the full-length recombinant fusion protein Myc-BRD7 with a molecular mass of 75 kDa in COS7 cells transfected with pCMV-Myc-BRD7. In the immunoprecipitation assays, the ectopically expressed BRD7-Myc protein was detected by c-Myc antibody in the immuno-complex precipitated with anti-BRD7 antibody. Similarly, BRD7-Myc protein was detected by BRD7 antibody in the immuno-complex precipitated with anti-c-Myc antibody.[Tissue distribution of BRD7 in human fetus detected with prepared BRD7 antibody]Western blot and immunohistochemistry assays were employed to detect the tissue distribution of BRD7 in human fetus. In the Western blot assay, a high expression level of the BRD7 protein was seen in the cerebellum, cardiocyte, and pancreas. A moderate expression level of the BRD7 protein was seen in the intestine, liver, and kidney. A very low expression level of the BRD7 protein was shown in the human cerebrum (ectocinerea and alba), lung, spleen, and stomach. Immunohistochemistry assays showed similar results. Intensive nuclear staining of BRD7 protein was seen in the human cerebellum, epithelium of pancreas, intestines, liver, and kidney. Cardiomyocyte epithelium showed intensive cytoplasm expression of the BRD7 protein. Very weak nuclear staining of the BRD7 protein was found in the human cerebrum, lung, spleen, and stomach.[Construction of the colorectal carcinomas-specific Tissue Microarrays]The tissue microarrays that contained 103 cases of various differentiated colorectal carcinoma and 103 cases of normal colorectal epithelium samples that paired with carcinoma were successfully constructed using the manual tissue arrayer. Each case was represented by a mean of 3 tissue spots using a 0.5mm punch. H&E-stained slides of TMA were used for the histological validity of the arrayed specimens under the light microscope. It was observed that the slides of TMAs had an appropriate thickness with symmetrically histological morphology of all cel1s and that configuration of tissue was not damaged by punching.[Protein expression of BRD7 in colorectal carcinomas and the clinical significance] Protein expression of BRD7 in colorectal carcinomas was detected by high throughput tissue microarrays technology combined with immunohistochemistry. 75% of the samples contained BRD7 protein localized mainly in the nucleus; some samples (20%) had positive expression of BRD7 in both the cytoplasm and nucleus, and rarely only in cytoplasm. BRD7 positive expression was found in 73 out of 102 cases of colorectal carcinomas (71.6%) and in 84 out of 99 cases in the matched normal colorectal epithelium (84.9%). This indicates a significant decrease of BRD7 expression protein in colorectal cancer tissue compared to the matched normal colorectal epithelium (p=0.023).The association between the levels of BRD7 protein expression and clinicopathological features of colorectal cancer was analyzed. Expression of BRD7 protein was significantly decreased in the samples from patients with tumors larger than 4.0 cm. BRD7 is positive in 64.8% of tumors that are larger than 4 cm, whereas in 87.1% of tumors that are smaller than 4 cm (p=0.031). Also, we found that the expression of BRD7 protein was significantly lower in the colorectal cancer with the Dukes C (62.0%) and D (64.0%) than that in the colorectal cancer with the Dukes A (92.9%) and B (76.5%) (p=0.041). The positive expression of BRD7 protein in the clinical stageⅢ(64.2%) was significantly lower than that it in the clinical stageⅠ(100%) and stageⅡ(80.0%) of colorectal cancer (p=0.041). BRD7 protein expression was significantly low in colorectal cancer with lymph node metastasis (62.0%) compared to cancer without lymph node metastasis (80.8%) (p=0.036). However, no significant association was found between the BRD7 expression and differentiation of colorectal cancer, tumor site, cancer with remote metastasis, patient gender, patient ages (p>0.05).In conclusion, we studied the expression of BRD7 gene in colorectal carcinomas and its clinical significance in the protein level for the first time. We succeeded in constructing the prokaryotic expression vector pGEX-4T-BRD7N and acquiring the purified GST-BRD7N fusion protein expressed in BL21 which was induced by IPTG. We also successfully prepared the anti-BRD7 polyclonal antibody with high specificity and titer. With this polyclonal antibody, we detected the tissue distribution of BRD7 protein in human fetus and analyzed the protein expression of BRD7 gene in colorectal carcinomas with high throughput colorectal carcinomas-specific tissue microarrays. We found that BRD7 protein expression in colorectal cancer tissue was significantly decreased compared to the matched normal colorectal epithelium and was significantly associated with tumor size, Dukes and TMN stage and lymph node metastasis. These data indicate that decreased expression of BRD7 protein may play important roles in the invasion, lymph node metastasis and clinical progress of colorectal carcinomas.