C6orf37, A Novel Gene Overexpressed In Colorectal Tumor

Colorectal Cancer (CRC) is a common gastrointestinal malignancy which threatens the health of human. The adenoma-carcinoma sequence is now widely accepted as the major pathway for the development of colorectal cancer in the general population. Colorectal adenomas are recognized as the precursor lesions for most cases of colorectal cancer. Removal of these adenomas resulted in a markedly lower colorectal cancer occurence than without polypectomy. The progression from adenoma to cancer takes various time, range from 5 years to more than 20 years. A 30% -46% recurrence rate of adenomas has been reported in studies with a follow-up period of 2.5-7 years. There is no effective criterion in evaluating whether adenoma will develop cancer or recur. A better understanding of the mechanism of adenoma development is important for prevention and diagnosis of colorectal cancer. Although many genes have been reported to be closely related to the progression of colorectal carcinogenesis, they are far from enough to explain the transformation. A lot of other genes, known or unknown, remain to be discovered. The identification and characterization of humangenes expressed exclusively or preferentially in tumor tissue will, hopefully, shed light on the mechanisms of colorectal carcinogenesis and provide useful genetic markers for early diagnosis and therapy.Three subtracted cDNA libraries were constructed by SSH in our lab in 1999. They were colonic adenocarcinoma-normal mucosa cDNA subtracted library (subtracted T-N library), colonic adenoma-normal mucosa cDNA subtracted library (subtracted A-N library), colonic adenocarcinoma-adenoma cDNA subtracted library (subtracted T-A library). Four differentially expressed sequences located in the 3'UTR of C6orf37 gene were found in A-N library. C6orf37 had been shown a novel candidate gene associated with human retinal disease, but the function of C6orf37 is still unknown. In this research we planed to confirm the relationship between the 4 differentially expressed sequences and C6orf37, and then study its expression pattern in colorectal tumor and explore the possible functional role of this gene in colorectal carcinogenesis.To obtain the full length of differentially expressed sequences and confirm their identity, 3'RACE was used to amplify 3' end sequence of differentially expressed cDNA and long PCR was used to amplify the upstream sequence 5'RACE was used to amplify the 5' end. The full length of cDNA was obtained by assembling the results of RACE and long PCR. The full length of differentially cDNA was 5547 nucleotides. It contains an open reading frame of 1314bp that encodes a 437-amino acid protein. The similarity between the full length and C6orf37 sequence (AF₃50451) is 98%. The differentially expressed gene that we screened by SSH is C6orf37 gene. Manybioinformatics softwares and internet programmes were used to analyze the characters of C6orf37 mRNA and protein to find hints indicating its function. Results of bioinformatics analysis indicate C6orf37 protein is a globule cytoplasm protein, whose sequence is highly conservative. A number of conserved potential phosphorylation sites exist in C6orf37 protein. C6orf37 gene is expressed in a wide range of human tissues. Differential expression is observed among fetal, juvenile and adult tissues. C6orf37 gene is also screened in many cancer SAGE libraries.A novel VNTR sequence was found in C6orf37 second exon. We tried to find the association of the VNTR polymorphism with colorectal cancer risk. DHPLC was applied in detecting the VNTR genetypes in 166 Chinese healthy people and 122 colorectal cancer patients. The VNTR was composed of 15 base pairs which consensus sequence encoding 5-amino-acid (G-G-D-F-G). The repeat times ranged from 3 to 5. There were three common alleles containing three repeats (a), four repeats (b) and five repeats (c) respectively, which produced three homozygotes (a/a, b/b and c/c) and three heterozygotes (a/b, a/c and b/c). a, b, c alleles frequency were 0.143, 0.332,0.548 respectively in Chinese population. Heterozygosity (H) was 0.583. Polymorphism information content (PIC) was 0.510. The distribution of genotypes and allele frequencies of the VNTR reached no significant difference between patients and healthy controls. There was no correlation between VNTR polymorphism and colorectal cancer clinicopathological features.The expression of C6orf37 gene at mRNA level in colorectal mucosa, adenoma and carcinoma tissues was detected to confirm the result of SSH subtracted A-N library.Semi-quantitative RT-PCR was performed to study the expression of C6orf37 gene at mRNA level in 13 matched normal colorectal mucosa, adenoma and carcinoma tissues. Results of RT-PCR showed that the mRNA expression level of C6orf37 gene in colorectal adenoma was significantly higher than those in paired normal mucosa. Although the expression level of carcinoma was also higher, the difference between carcinoma and normal mucosa did not reach statistic significance. There was no difference between adenoma and carcinoma.ISH was used to further testify the overexpression of C6orf37 mRNA in 24 adenomas and carcinoma tissues. Positive stain of ISH was located in cytoplasm of epithelial tissue. mRNA expression level of C6orf37 in colorectal adenoma and carcinoma was higher than in normal mucosa. There was no difference between adenoma and carcinoma. No relationship was observed between C6orf37 mRNA expression and clinicopathological characters of colorectal tumor.We generated two rabbit anti-C6orf37 polyclonal antibodies by synthesized immunogenic peptides combined with carrier proteins. The titer and speciality of immune serum were confirmed. These polyclonal antibodies were successfully used in immunohistochemistry to detect the expression of C6orf37 protein in 81 colorectal carcinoma and adenoma tissue. C6orf37 protein was highly expressed in adenoma than in normal mucosa. No difference was detected between normal mucosa and carcinoma as well as between adenoma and carcinoma. The cancer cells located in the invasive front of cancer showed stronger staining of C6orf37 protein than those in the center. There was no relationship between C6orO7 protein expression andclincopathological parameters. The distribution of C6orf37 protein stained by IHC was consistent with the results of ISH. C6orf37 expressed in a wide rage of human tissue such as stomach, pancreatic island, lymphoid node and nerve tissue.In conclusion, the differential expressed gene of SSH subtracted A-N library is C6orf37 gene. C6orf37 is a globule cytoplasm protein and may play an important role in human growth;A novel VNTR polymorphism in C6orf37 exists in Chinese population and is not associated with colorectal cancer risk;C6orf37 is a novel gene up-regulated in colorectal adenoma and is associated with colorectal tumorgenesis.