| Part one:Screen genome wide single nucleotide array to search for the HCC carcinogenesis related gene.Objective:Screen single nucleotide polymorphism(SNP) by Genome-wide association study(GWAS), and search for the HCC related gene for further study.Methods:1. We compared50HbsAg(+) cases with HCC with50HbsAg(+) healthy controls who were similar in age and gender distribution by Affymetrix500K SNP chips, in order to search the genome wide SNPs.2. For all the SNPs, we perfomed statistical analysis of allele frequency, genotype frequency and Cochran-Armitage Trend to get three p value, and the minimal one is called minP.3. Accoding to P<0.001, we selected the most dominant SNP, together with advantage of cluster, so as to pick up the HCC related gene.Results:1. There were totally21154SNPs whose minP were less than0.05, including357with minP<1×10-3,26with minP<10"5and one with minP<10-8.2. Search the357SNPs by PubMed for their haplotype block, and it showed nearly one-third of them have been reported related with carcinoma.3. Among the357SNPs, we statistically analysed their located allele frequency between HCC group and control group. According to P<0.001,we selected325SNPs with most significance, which were located on chromosome1、2、5、6、7、8、14、15、17and19.4. Considering advantage of cluster, we chose Alpha-Amylase1(AMY1) located on chromosome1p21for further, among which rs1334125SNP with minP of10-5, and there were7SNPs with P<0.05in the adjacent area. So far, there haven’t been reports of the relation between AMY1and HCC.Conclusions:Searching genome wide SNPs by GWAS to select HCC related gene for further study is an effective way, which can help to find new genes not ever reported. Part two:study of the relation between AMY1and the clinical or pathological characteristics and prognosis of HCC.Objective:To analyse the relation between AMY1and the clinical or pathological characteristics and prognosis of HCC.Methods:1. We made tissue chips of91HCC patients’tissue and their paracarcinoma tissue, and compared AMY1expression by immunohistochemistry(IHC) method.2. Detected AMY1expression by IHC on96HCC tissue and their paracarcinoma tissue from the patients having surgery during2006-2009and used software--Image Plus and Pro(IPP) to indicate AMY1expression level by IOD value, statistically analysed it’s relation with the clinical or pathological characteristics and prognosis of HCC.3. Detected AMY1expression in8HCC cell strains and1normal liver cell strain by Western Blot.Results:1. Among91patients with79male,12female and median age of55, the AMY1positive rate in HCC tissue is76.92%(70/91),much lower than that in paracarcinoma of94.50%(86/91), P<0.05;2. Among96patients with85male,11female and median age of51.5, AMY1expression average level(IOD) is1.88±2.21×105, much lower than that in paracarcinoma tissue of4.03±3.35×105, P<0.001;3. with enlargement of cancer, AMY1expression in HCC tissue decreased, P<0.05; AMY1expression in HCC tissue of those without cancer embolus is much higher than with cancer embolus, P<0.05. AMY1expression in paracarcinoma tissue has no relation with all the clinical or pathological characteristics.4. Those with high AMY1expression in HCC tissue seemed to survive longer with median overall survival of34.5months and median carcinoma free survival of25.0months, comparing with low AMY1expression of25.8months (P<0.05) and11.9months (P=0.052), respectively.5. Western Blot showed that AMY1only expressed in HepG2HCC cell strain (with good differentiation) and Hep3B HCC cell strain (without invasion and migration potential). No AMY1expression was found in other HCC cell strains and normal liver cell strain (L02).Conclusions:AMY1seems to be related with HCC. It may inhibit proliferation and invasion of cancer to achieve better prognosis. |
PDF Full Text Request