| The adenoma-carcinoma sequence has been accepted as a main pathway in colorectal carcinogenesis which includes a series of molecular changes such as activation of oncogenes and inactivation of tumor suppressor genes. Combinations of altered expressions of many different genes are involved in multiple steps. However, mechanisms, implicated in the initiation and progression of colorectal cancer, have not been fully clarified. Thus, independent analysis of any single gene would be insufficient to understand all of the aspects of colorectal cancer. There are many ways to search for candidate differentially expressed genes such as DD PCR, RDA and SSH. SSH is a powerful PCR based method that allows us to compare gene expressions between two experimental samples.In this study, suppression subtractive hybridization (SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three libraries were constructed including, the subtracted A-N cDNA library using normal mucosa as tester cDNA and adenoma tissue as driver cDNA; the subtracted T-N cDNA library using adenocarcinoma tissue as tester cDNA and normal mucosa as driver cDNA; the subtracted T-A cDNA library using adenocarcinoma tissue as tester cDNA and adenoma tissue as driver cDNA. We performed a global analysis of the differential gene expression of a colorectal adenoma - normal mucosa library.Contigs were assembled by electronic cloning (in silico cloning) with the ESTdatabase. The ammo acid sequence of a novel gene, isolated in 1999, was determined with ORF prediction software and was found to be 100% homologous to the protein product of Reg IV, a novel gene isolated from a large inflammatory bowel disease library in 2001 . In 109 clones of the A-N library, there were tumor associated genes, 9 hypothetical proteins and 33 candidate novel genes. Reg IV had been selected for 13 times in this library. Reverse Northern blot results showed 3 1/40 clones including Reg IV were up-regulated in adenoma compared with matched normal mucosa. Two fragments of differential expression were associated with Alu sequences.Semi-quantitative RT-PCR was performed to detect mRNA levels to validate the above findings. Twenty nine cases of fresh colorectal tumors were collected from April 2000 to October 2001, including 27 colorectal cancers, 12 colorectal adenomas (16 from rectum and 13 from colon; 17 specimens from men and 12 from women). Reg IV mRNA levels were determined by RT-PCR normalized by G3PDH or p-actin. It was up-regulated in 22/27 carcinomas (81.5%) (P <0.01, paired rank test) and 12/12 adenomas (100%) (P <0.01, paired rank test). Reg IV was up-regulated in 4 poorly differentiated colorectal carcinomas and 12 carcinomas with metastasis. There were no significant difference between two groups with poor and high-moderate histological differentiation (P>0.05, paired X2 test).In addition, Reg IV mRNA level was compared between colon and rectum. Reg IV mRNA level was higher in colon than that in rectum (P =0.05, rank analysis).Northern blot analysis further confirmed the results of SSH, Reverse Northern and RT-PCR. Northern blot showed that Reg IV mRNA expression level was higher in most of adenomas (7/8) and the majority of cancers (11/15) compared with that of normal tissues.In situ hybridization(ISH) was performed to assess the cellular localization of Reg IV and further testify the overexpression of Reg IV in adenomas and carcinomas. Colorectal tumors from 73 patients were collected. RNAs of 11 samples were degraded, which had been determined by B-actin positive control. Among other 62 colorectal tumors detected, there were 29 carcinomas and 33 adenomas with mild dysplasia, moderate dysplasia, and severe dysplasia with/ without carcinomatouschange. Reg IV mRNA was detected in colorectal normal mucosa cells, adenoma cells and carcinoma cells by ISH. It was up-regulated in 74 % (14/19) adenomas with mild or moderate dysplasia and 12/12 adenomas with carcinomatous change (P=0.002, paired rank test). Reg IV... |
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