The Mutation And Expression Feature In Hepataellular Carcinoma From Qidong And Its Hepatocarcinogenic Mechanism

AKR1C2 (Aldo-Keto Reductase full-length sequence was obtained by bioimformatics homologous screening using EST Sequence (AB02165) as information probe, and its structure and function was predicted by bioimformatics. Simulated bioimformation analysis showed that (l)the AKR1C2 full-length contains 1900 bases pairs and encodes 332 amino acids, and produces polyadenylic acid,poly (A) and AATAAA tail signal. There is 84.3% homology of this gene between human sequence and mice sequence, and 88.6% homology between their encoded domain. (2)Within encoded domain (ORF), there maybe two N-glycosylation sites, three phosphorylated casein kinase n sites, four phosporylated PKC sites, and there is C-terminal nuclear localization signal and endoplasmic reticulum retention motif, such as, RMTM, FKTV, but no N-terminal signal peptide in its expression product. (3)It may be a ER membrane pretein , but may mostly be a plasmic protein. There exists LJM structure riched cysteine which may act as interface of proteins interaction each other when it is integrated with Zn2+. In additon, we found that there are two phosphorylated tyrosine protein active enzyme sites , one phoshorylated tyrosine site within the protein encoded by AKR1C2 . We infer that AKR1C2 is related to phoshorylation of protein. (4)Electronic expression chart (ePCR. eNorthem blot) showed that the expression of AKR1C2 exists in the some tissues, such as,heart,placenta,rectum,liver,skeletal muscles,overy,prostate,thymus,testicle,epididymis,oviduct,hair,large intestine, cecum, nose, breast, ear, stomach, etc, especially higher expression in the liver, prostate, epididymis, oviduct than in others tissue.According to these results above simulated bioimformation analysis ,we analyzedthe AKR1C2 mutation and its expression in Qidong hepatocellular carcinoma patients whose disease was caused by HBV infection,, AFB1 pollution, P53 (249 codon on exon) mutation ,etc and its hepatocarcinogenic effect using QGY7703 liver cancer line (from Qidong) by a series of cellular and molecular methods, such as, RT-PCR, Northern blot, Southern blot, Western blot, 5'-RACE, 3'-RACE, PCR-SSCP, direct sequence analysis, immunohistochemistry , In site hybridization, preparing polyclonal antibody, gene expression chip, gene or antisense gene transfection, gene frameshift mutant,co-immunoprecipitation, yeast two-hybridization,the tumorigencicity assay in vivo or in vitro, gene therapy experiment, etc.Our 5',3'-RECE results showed that the AKR1C2 cDNA full-length is 1865 bp, including 5'-nonencoding sequence (253 bp), 3'-nonencoding sequence (640bp), initiater codon (ATG), terminater codon (TAA), ORF(972bp). Using isotope label probe and mRNA MTN membrane drawed 36 human tissue, Northern blot showed its expression in transcription exists in some tissues(transcript about 1.9kb,or 1.2kb), such as, heart, placenta, rectum, liver,skeleta muscles,ovary,prostate, large intestine, cecum, hair(1.2kb), thymus, testicle , epididymis, oviduct, nose, breast, stomach, and there are very higher expression in liver, prostate, large intestine, cecum, epididymis oviduct than the other tissues.It shows that AKR1C2 expression is different and special among several human tissues. Overlay image of fluorescence of AKR1C2-GFP and anti-mitochondria antibody indicates that the subcellular localization of AKR1C2 is located at mitochondria membrane. After AKR1C2 was expressed by prokaryotic expression, we obtain a 42ku product by degeneration,renaturation .purification and it was confirmed by western blot. The AKR1C2 protein isoelectric point is about 7.5 and its anti- AKR1C2 titer adds up to 1:10000 (ELISA)after the new Zealand rabbits was immunized with purified product by Niaffinity Chromatography. By purification of anti- AKR1C2and then dilution by 1:2000,1:4000, 1:5000, western blot all showed a 42 ku band each lane ,only the expression amount not the same. In order to examine theAKR1C2 expression in different tumor line, we choice the human esophagus carcinoma EC8712, human rectal cancer cell line HR8348, human l...