| Background and Purpose Hepatocellular carcinoma (HCC) is one of the severe diseases harmful to human health. As a research reported, the cancer caused the most death during 1991~2000 all over the world were pulmonary carcinoma, gastric carcinoma, HCC and colorectal carcinoma, et al. In the past 30 years, the spectrum of high incidence cancers varied greatly in China. In 1970s, gastric carcinoma, esophageal carcinoma, HCC, pulmonary carcinoma and cervical carcinoma toped the list of death rate of cancer. In 2000, the order changed as pulmonary carcinoma, HCC, gastric carcinoma, esophageal carcinoma and colorectal carcinoma. One million and a half people died from cancer in 2000. Pulmonary carcinoma, HCC, gastric carcinoma caused the most death in cities, however in countries, the order was HCC, pulmonary carcinoma and gastric carcinoma. HCC happened in Chinese mainland annually account for 45% of the total HCC cases around the world. The death rate has an ascending potential.Surgically, less than 25% of HCC can be resected. The recurrent rate is up to 60% while the survival rate for 5 years is between 30.6% and 38.1%.People have found that aflatoxin, and some viruses such as HBV and HCV wereenvironmental risk factors for HCC. In cytogenetics, loss of heterozygosity (LOH) on chromosome 1p, 2p, 4p and amplification of 1q, 5p and 6q happened frequently. Structure and function abnormalities of some genes like p53, P-catenin and AXINI were also found. But the genes related closely with HCC were rarely reported. For example, the mutation rate in HCC is only 10%~20% for p53, less than 30% for P-catenin and AXINI. For the understanding of the mechanism of HCC and its early diagnosis and treatment, new genes associated with HCC must be found.Our laboratory constructed a subtractive cDNA pool of HCC using the method of suppression subtractive hybridization (SSH) in 2000 in order to find the genes that participate in the happening and developing of HCC. Among the 93 clones obtained, 2 EST (expression sequence tag) named P15 and P65 were picked out for further research.Although P15 and P65 had been cloned from HCC, no direct evidence was found that P15 and P65 involved in the biological mechanism of HCC. To prove the hypothesis that P15 and P65 are related to HCC, the experiment was designed as follows: 1, detect mRNAs of P15 and P65 in 21 pairs of HCC and liver cirrhosis tissues with semi-quantitative reverse transcriptase-PCR (RT-PCR) in order to exclude the possibility of false positivity from SSH. 2, Clone the full length cDNAs of P15 and P65 by Bioinformatics and rapid amplification of cDNA ends (RACE). 3, Clone the open reading frame (ORF) of the full-length cDNAs into eukaryotic expression vector pEGFP-Cl to reconstruct fusion expression vector. Transfect the vector into hepatocarcinoma cell line HepG2 and liver cell line L02 to observe the effect of P15 and P65 over-expression on the two cell lines. 4, Construct prokaryotic expression vector of P15 and P65, and get the protein by transforming E.coli TB1 ex vivo, in order to lay the foundation for making special reagents for early diagnosis and bettertreatment of HCC.Materials and Methods 1, Identification of differential expression of the EST. Detect the expression of PI5 and P65 mRNA in HCC and liver cirrhosis tissues with semi-quantitative RT-PCR. The results were analyzed by /-test statistically. 2, Bioinformatics analysis of the EST. Prediction the full length cDNA, ORF, electronic expression pattern, localization on chromosome, characteristics of the protein they code, their subcellular distribution and function on internet with net or localized software such as Blast, DNASIS, ContigExpress, ORF Finder, electronic in situ hybridization and electronic PCR, et al. 3, Verification of the predicted nucleotide sequence. Designed the primers for P15 and P65 according to the predicted full length sequence. PCR product was cloned into T vector, transformed competent cells, and positive clones were selected for sequencing. Analyed the...
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