ABSTRACT
We established a colonic adenoma-normal mucosa suppressive subtraction hybridization (SSH) library in 1999. In this study, we wanted to explore the expression profile of all candidate genes in this library. We developed an EST pipeline which contained two in-house software packages, nucleic acid analytical software and GetUni. The nucleic acid analytical software, an integrator of the universal bioinformatics tools including phred, phd2fasta, cross_match, repeatmasker and blast2.0, can blast sequences of differential clones with the downloaded non-redundant nucleotide (NR) database. GetUni can cluster these NR sequences into Unigene via matching with the downloaded Homo Sapiens UniGene database. Sixty-two candidate genes in A-N library were obtained via the high throughput automatic gene expression bioinformatics pipeline. Gene Ontology online analysis revealed that ribosome genes and immunity-regulating genes were the two most common categories in the KEGG or Biocarta Pathway. We also detected the expression of 2 genes with highest hits, Reg4 and FAM46A, by semi-quantitative RT-PCR. Both genes were up-regulated in 10 or 9 out of 10 adenomas in comparison with the paired normal mucosa, respectively. The candidate genes in A-N library would be of great significance in disclosing the molecular mechanism underlying in colonic adenoma initiation and progression.
Subject(s)
Adenoma/genetics , Cluster Analysis , Colonic Neoplasms/genetics , Computational Biology , Gene Expression Profiling , Adenoma/metabolism , Colon/pathology , Colonic Neoplasms/pathology , Databases, Nucleic Acid , Expressed Sequence Tags/chemistry , Expressed Sequence Tags/metabolism , Gene Expression , Gene Library , Humans , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
OBJECTIVE: To get a complete cDNA sequence of B2 and evaluate the correlation on structure and expression between B2 and insulin like growth factor binding protein related protein 1 (IGFBP-rP1) in colorectal carcinomas, paired normal tissues, adenomas, tissues adjacent to the tumor, and colorectal carcinoma cell lines. METHODS: 5'RACE (rapid amplification of cDNA end) was applied to get the sequence of the 5' end of B2. Semi-quantitative RT-PCR and immunohistochemistry were used to detect the expression of B2 in colorectal cancer tissues and cell lines (SW480, SW1116, SW620, HCT8, CoLo205 and LoVo). RESULTS: A sequence of 1,125 bp was obtained by combining the sequence from 5'RACE product and the known sequence of B2. It shared 1,122/1,125 identities with IGFBP-rP1. At the level of mRNA, the expression of B2/IGFBP-rP1 was high in colorectal carcinomas, moderate in adenomas and tissues adjacent to tumor, low in normal tissues (P < 0.05). Five cell lines except SW480 showed no expression of B2/IGFBP-rP1. A significant difference was obtained in the immunoreactivity of B2/IGFBP-rP1 between normal tissue and cancer (P < 0.05). In 28.9% (22/76) samples, cancer cells locating at the invasive front of cancer nest had a stronger staining of B2/IGFBP-rP1 than those surrounding the lumen. These samples had also an increased frequency of lymph node metastases, increased depth of invasion and a stronger staining of B2/IGFBP-rP1 than in other samples (P < 0.05). CONCLUSIONS: B2 is the same gene as IGFBP-rP1. Overexpression of B2/IGFBP-rP1 may play an important role in the initiation and promotion of colorectal cancer. Its overexpression in invading tumor cells may be linking with an increased potential of invasion.
Subject(s)
Adenocarcinoma/metabolism , Colonic Neoplasms/metabolism , Insulin-Like Growth Factor Binding Proteins/genetics , Rectal Neoplasms/metabolism , Adenocarcinoma/secondary , Adenoma/metabolism , Adenoma/pathology , Aged , Cell Line, Tumor , Colonic Neoplasms/pathology , Female , Humans , Insulin-Like Growth Factor Binding Proteins/biosynthesis , Lymphatic Metastasis , Male , Middle Aged , Neoplasm Invasiveness , RNA, Messenger/genetics , Rectal Neoplasms/pathology , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
AIM:To detect the presence of HPV DNA and study the alteration of p53 expression in anal cancers in Chinese.METHODS:HPV DNA was amplified by PCR.The amplified HPV DNA was classified by DBH. HPV antigen and p53 expression were respectively detected by immunohistochemistry.RESULTS:HPV DNA was amplified only in one case of squamous cell carcinoma of the 72 Chinese anal cancers and further classified as HPV type 16. Others were all HPV negative. HPV antigen and p53 expression were also detected in this case. Positive stainings with anti-p53 antibody were seen in 61.2% anal cancers.There were no statistically significant differences between anal squamous cell carcinomas and adenocarcinomas and between anal adenocarcinomas and rectal adenocarcinomas.p53 protein expression was observed in the basal cells of squamous epithelium of condyloma acuminatum and morphologically normal squamous epithelium in 2 cases invaded by anal adenocarcinoma.CONCLUSION:HPV infection was not associated with these cases of anal cancer.p53 alteration was a common event. Positive p53 immunostaining can not be regarded as a marker for differentiating benign from malignant lesions.
