The Study Of Loss Of Heterozygosity On Chromosomes 8p And 16q In Primary Hepatocellular Carcinoma

Background:Primary liver cancer, hepatocellular carcinoma (HCC) is one of the most common cancer on world scale. The recent epidemiological survey showed that there had been about 620 thousand new HCC patients annual globally. And as globalization has further intensified, not only in developing countries, but also in some western countries, the incidence and mortality of HCC has shown a upward trend in the past 25 years. However, the underlying molecular mechanism of tumor initiation, progression and metastasis are still not well fully understood, is a hotspot in the area of cancer research. At present, molecular genetics of cancer showed that the carcinogenesis was a complex event, involving a number of factors involved, where genetic alterations is a key factor, and activation of oncogenes and (or) inactivation of tumor suppressor genes (TSG) play a very crucial role. Among them, the reports of inactivation of TSG are increasing in the process of hepatic carcinogenesis. Recently many research centers found some high-frequency of loss of heterozygosity on chromosomes 1p, 1q, 4q, 6q, 8p, 10q, 13q, 14q, 16q, 17p, and 22q in HCC tissues samples. Theses genetic events, loss of heterozygosity sites (LOH) of some specific loci on chromosome, induced inactivation of TSG. The microsatellite loci on these chromosomes exist regional differences, screened chromosome segment yet to be precise. Thus, these knowledge about the molecular mechanism of these genetic alterations on some chromosomes of HCC have still need to discussed in the follow-up research.Objective:This study analyzed the frequency of loss of heterozygosity of the gene and chromosome segment on chromosomes 8p and 16q in hepatocellular carcinoma (HCC), that screened the specific loci by SNP6.0 chip, evaluated the relationship between the features of LOH and clinicopathologic features and tried to screened some HCC-related tumor suppressor genes in order to provide possible molecular markers for early diagnose, molecular typing and prognosis warning of HCC screening.Methods:1. 45 HCC tissue samples and corresponding non neoplastic tissue samples were collected from Tang-du hospital. The pathological specimens were examined and confirmed by two senior pathologists.2. We selected eight microsatellite loci with high-frequency abnormality on the segment of some specific chromosomes in HCC tissue by SNP6.0 gene chip screened, namely: D8S261,D8S499,D8S1125,D8S1810,D8S1827,D16S402,D16S422,D16S511.These primers of DNA microsatellite loci were designed by the GenBank database.3. The frequency of loss of heterozygosity (LOH) at the specific microsatellite loci on chromosome in tissue samples from these patients with HCC were examined by using PCR-denaturing PAGE-silver staining. And the results were analyzed combined with the frequency of LOH and clinicopathologic features by Chi-square or Fisher's exact probability test. Results:1. Frequency of LOH The frequency of the 8 microsatellite loci on 45 HCC tissues were detected : D8S261(39.02%,16/41),D8S499(34.88%,15/43),D8S1125(15.38%,4/26),D8S1810(19.51%,8/41),D8S1827(9.76%,4/41),D16S402(19.04%,8/42),D16S422(21.21%,7/33),D16S511(53.33%,24/45), respectively. The three most more frequent Loci were D16S511(53.33%,31/45),D8S261(39.02%,16/41)and D8S499(34.88%,15/43). The overall frequency of the LOH was 68.89% (31/45) at least one locus of the 8 loci on the chromosomes. We did not find that the LOH occurred on the all loci.2. The relationship between the LOH and clinicopathologic features Through analysis the frequency of loss of heterozygosity of the 8 highly polymorphic microsatellite loci on chromosomes 8p and 16q in hepatocellular carcinoma (HCC), we found that the frequency have no significant difference with pathogenic and clinical features such as history, HBsAg infection, serum AFP, tumor size, differentiation, cirrhosis, intrahepatic metastasis, and so on. However, The LOH on some specific loci were associated with the clinicopathologic features of HCC. The frequency of LOH on D8S261 in HBSAg-positive HCC samples were higher than that in HBSAg-negative HCC(P<0.028).The frequency of LOH on D8S1810 in HCC patients with cirrhosis were more frequent than that in those without cirrhosis(P<0.045).The LOH frequency on D16S511 and D8S499 had significant difference in the samples with EdmondsonⅢ-ⅣandⅠ-Ⅱ(P<0.003, P<0.033, respectively).Conclusions:1. We found that the frequency of LOH are significantly higher on D16S511,D8S261 and D8S499 than that on other loci. This experiment results showed that there may be a new putative tumor suppressor gene related to the occurrence and development on some specific chromosome region 16q23, 8p22-21.3 or 8p12 with high-frequent LOH. Further, it provides a new clue for us in order to screening some new tumor suppressor gene.2. The high-frequent LOH on some microsatellite specific loci may be related with the clinicopathologic features of HCC such as the positive HBsAg, differentiated degrees, and so on. Thus, this may guide the early clinical diagnosis, molecular typing and prognosis of early warning, which gives us provides a new theoretical basis.3. In this study, we simply discussed that there may be a new putative tumor suppressor gene related to the occurrence and development on some specific chromosome fragment and some gene region. In the foreseeable future, this experimental research basis give us some theories on precise positioning HCC-related tumor suppressor gene mapping, cloning HCC-related TSG and the molecular signaling pathway and conduction mechanism.