Preliminary Study And Molecular Cloning Of CRC-related EST-2

Colorectal cancer (CRC) is one of the most common cancers with low survival rate in human beings .The incidence has an increasing tendency worldwide in recent years. With the development of molecular biology, tumor has been identified as a kind of genic disease. And the role of cell growth regulators in formation and development was considered more and more important. The intrinsically properties of tumor are activation of oncogene and inactivation of antioncogene. With the post genomic era arriving, there have been many researches on colorectal genes by microarray technique with the character of high-throughput, high-flux, miniaturization and automation. As a result, colorectal genic data-bank is becoming more and more abundant. However, there are no systemic researches based on the strategy "data mining of gene profiles→screening candidate genes→investigating function of these genes".This study is the extension of Hong-min Xu's research, "Gene expression profiling of human colorectal cancer with different differentiation grades". Hong-Min Xu et al firstly used Affymetrix GeneChip HG-U133 to monitor gene expression of about 19308 known genes and 12956 ESTs (altogether 32264) on 9 single samples of CRC with different differentiation grades (3 well differentiated, 3 moderately and 3 poorly) paired normal mucosa tissue (9 samples in one pools);a series of CRC-related gene and ESTs were obtained.Important CRC-related EST2 (temporary name whose Affymetrix number is 244669 at) was one of the obtained gene. The different expression level between CRC and normal mucosa tissues was statistically significant (rank sum test, P=0.04,<0.05); The different expression level among well, moderately, poorly differentiated groups was statistically significant (F test, P=0.0240,<0.05). Thesedata shows CRC-EST2 was not only CRC-related gene but also CRC differentiation-related gene.On the basis of previous study, we cloned CRC-related EST2 by silicon clone combined with RACE technique, then blastn the sequence in Genebank; besides, reverse transcription followed by PCR(RT-PCR) and real-time fluorescent quantitative PCR(Q-PCR) was used to detect the existence and different expression level of of target gene respectively. The results were as follows:1. The interested CRC-EST2, that has been registered as a EST by Affymetrix (whose Affymetrix number is 244669_at), was cloned by bioinformatics tools and experiment technique of RACE, then it was confirmed to be U50HG, whose Genebank number is AB017710.2. U50HG's expression was detected on 10 CRC samples, 6 normal mucosa tissues, which were coincident with previous study; and U50HG's expression was also detected on the human colon cancer LoVo cells.3. To detect the relationship of U50HG with CRC, Q-PCR technique was applied; the data verified a relative high expression in tumor compared with the normal tissue by statistics analysis (non-parametric test, p=0.0393), which was coincident with previous study.The expression of U50HG in colorectal cancer, normal mucosa tissues and human colon cancer LoVo cells was not reported yet; Besides, U50HG was CRC-related gene was also not reported.