The Genetic Study Of Tumor-suppressor Genes Related To Hepatocellular Carcinoma And Precancerous Lesions

Background:Hepatocellular carcinoma (HCC) is one of the most common malignancies,ranking the fifth, and the third in the cause of death of cancer. Recently,epidemiological investigation shows that its incidence has significant regionaldifferences:80%of the world’s cases are in Asia, Africa and other developingcountries and areas; while in northern Europe, western Europe and northAmerica, its incidence is lower, but the incidence of HCC also showed a risingtrend in these areas. The incidence of HCC is related to not only environmentalrisk factors, such as hepatitis B virus (HBV) infection, hepatitis C virus (HCV)infection, aflatoxin, alcohol and tobacco, but also other genetic factors. In recentyears, studies of the mechanism of HCC have shown that the activation ofoncogene and the inactivation of tumor suppressor gene is the key link thatcauses tumors. Many studies have found that there are high frequency loss ofheterozygosity (LOH) in the specific regions of chromosome1p,4q,6q,8p,13q, 16q and17p in HCC tissues. HCC related to tumor suppressor gene may existnearby these sites. More over, studies have found that precancerous lesion suchas dysplastic nodule (DNs) and hepatocellular adenoma (HA), also occured thesimilar genetic variation such an gene deletion to HCC, which in chromosome8p,17p,5p,13q,14q and16q in the early phase of HCC. The results suggestthat the genetic change appear in HCC may also happen in the precancerouslesion of HCC. So, we need to lucubrate the genetic change involved in thewhole processes of HCC and its precancerous lesion to provide more accuratetheory basis for the diagnosis and treatment of HCC.Objectives:This study is going to detect the rate of LOH of nine high polymorphismsof the microsatellite loci in chromosome4q and8p in HCC and nodules ofaltered hepatocytes (NAH), and further test the protein expression encoding bythe target gene at protein level. Then analyze and investigate the tumorsuppressor genes related to the carcinogenesis of HCC and NAH, and provideprecise molecular markers for the prevention, early diagnosis and treatment ofHCC.Methods:1. The samples of HCC and NAH were collected, and then disstected bymicrodissection. Extract DNA from cells of NAH and HCC specimens byisopyknic phenol chloroform;2. We select9microsatellite sites, including D4S2954、 D4S3331、D4S3030、D4S415、D8S1725、D8S1754、D8S1810、D8S1827and D8S552for polymerase chain reaction(PCR). The primers were got from the GeneBank database, and then using PCR-denaturing polyacrylamide gel electrophoresis-silver staining; respectively according to the results analyse the incidence oftheir LOH, elect the higher microsatellite loci in HCC and NAH, and then locatethe corresponding gene of the selected loci;3. Immunohistochemical SP method was used to test the expression ofprotein coded by the target gene in HCC, NAH and their respective normalcontrol specimens. Then process statistical analysis.Results:1.45cases informative HCC patients were tested and the total ratio ofLOH at the9microsatellite sites was66.7%. In these microsatellite sites,D8S552had a highest rate of LOH, which was41.9%, and there weresignificant differences in both gender (P=0.023), serum α-fetoprotein(AFP)(P=0.004), intrahepatic metastasis conditions (P=0.023),analysed byChi-square test or Fisher exact test.The ratio of LOH at D4S415was22.5%,while D4S3331was22.3%, D8S1810was18.8%, D4S2954and D4S3030were15.4%, D8S1725was12.5%, D8S1827was9.8%, D8S1754was6.3%;2. The ratio of LOH was7.89%at D8S552in38cases nodules of NAHspecimens;3. The protein expression of DLC-1were positive in12cases of45total inHCC, and the ratio was26.7%; while36cases were positive in theircorresponding adjacent tissues, and the ratio was80%.Chi-square test wasperformed on this two group’s protein expression rate (χ2=25.71, P <0.05), therewere statistical differences between the expression of DLC-1in HCC andadjacent tissues, while there were no differences in gender, tumor size, whether with intrahepatic metastasis, whether with liver cirrhosis and the hepatitis B surfaceantigen (HBsAg) level.4. The protein expression of DLC-1was positive in26cases of the38casesof NAH specimens, and the ratio was68.42%.30cases were positive in normalcontrol’s specimens and the ratio was78.9%. Chi-square test was performed onthis two group’s protein expression rate (P=0.442，P>0.05) and there were nostatistical differences between the expression of DLC-1in NAH and normalcontrol tissues.Conclusion:1. The loss of heterozygosity at D8S552, D4S3331and D8S552locus at4qand8p is important events in the development of HCC. It suggests that theremay be potential tumor suppressor genes nearby the position. And it alsoprovides a theoretical basis for the study of other tumor suppressor genes relatedto liver cancer.2. Microsatellite sites D8S552occurred LOH in tissues of NAH, suggestedthat NAH may be a precancerous lesion of HCC.3. The protein expression level of DLC-1in the tissue of HCC wassignificantly lower than that in the normal liver tissues, this verified that theDLC-1gene may be the related tumor suppressor gene of HCC.