The Association And Molecular Mechanism Between The Infection Of Helicobacter Pylori And The Carcinogenesis Of Primary Hepatocellular Carcinoma

Objectives: To investigate the association between the infection of Helicobacter pylori and the carcinogenesis of primary hepatocellular carcinoma (HCC), and preliminary elucidate the molecular mechanism involved.Methods:First part1. PCR amplification and sequence analysis of Helicobacter pylori from the liver tissues from HCC patients: Liver samples from 80 patients with HCC as well as 30 without HCC were selected. Tissues DNA were extrated by Trizol, and the nested-PCR was introduced to amplify the helicobacter-specific 16SrRNA genes. As for the obtained PCR fragments , homologous analysis was carried out by using Bioedit software based on the standard Helicobacter pylori sequences (GeneBank accession number was AF302106). As for 16SrRNA positive samples, Helicobacter pylori specific 26Kda protein gene and the other 4 related genes, i.e., cagA ,vacA, rps4, and glmM were amppfied to further ascertain the exsit of the bacteria.2. Immunohistochemical staining: IHC was introduced for detection of H. pylori in both carcinoma tissues and adjacent non-tumor tissues.Second part1.Effects of Helicobacter pylori on the proliferation of HepG2 cells: Helicobacter pylori strain NCTC11637 with different MOI were incubated with HepG2 cells, CCK-8 reagent was used to determine appropriate MOI for the HepG2 infection. 2. Effects of Helicobacter pylori on the gene expression profile of HepG2 cells were analyzed by using Affymetrix human oligonucleotide genechip, and the results were further checked by semi-reverse transcription PCR.Third part1. Association between the infection of Helicobacter pylori and the invasiveness of HCC2. Analysis by IHC of the expression differences of transgelin in high and low invasiveness tomor tissues.3. Analysis by IHC of the expression differences of integrin in high and low invasiveness tomor tissues.Results:First part1. Totally 57 liver tissues (either carcinoma tissues or adjacent non-tumor tissue or both)from HCC patients were helicobacter specific gene positive. Among them, 48.75% (39/80) samples of carcinoma and 47.5% (38/80) samples of paracarcinoma tissue were positive, while none was positive in controls (P<0.01) . Sequence analysis of these PCR products obtained from HCC patients indicated that they were related most closely to the 16S rDNA sequence of Helicobacter pylori. Of these 57 positive samples, 37 were positive in the 26-kDa protein gene of H. pylori(77.19%). The cagA was detectable in 12samples(21.05%) and the glmM was detected in 10 samples(17.50%), while the vacA and rps4 genes were never detected. H. pylori was detected by IHC in 39 (48.75%) of the 80 liver samples, among them 32.5% were carcinoma tissues and 30.0% were adjacent non-tumor tissues, while no bacteria was found in benigh liver tissues. IHC also showed that in most of the cases, H. pylori were located in sinus hepaticus, and in some cases, in portal area. In addition, IHC positive tissues were all shown PCR positive. Second partHelicobacter pylori enhance the proliferation of HepG2 cells while the MOI ranging from 0.15:1 to 0.075:1.The gene chip analysis showed that totally 35 genes (19 out of them were up-regulated, and 16 were down-regulated) were changed after the treatment of HepG2 cells with Helicobacter pylori.Most importantly, 6 of them were categorized to celluar skeleton/matrix, 8 of them were DNA binding proteins and transcriptional factors, 2 of them were cytokines. 5 of the changed genes were selcted, and the semi-RT PCR were carried out, the results fit the that of gene chip analysis.Third partPositive rate of Helicobacter pylori 16SrRNA in high invasiveness HCC samples were significantly higher than that in low invasiveness HCC samples (86.44% vs 58.06%). IHC revealed that the expression level of trangelin and integrinα2 were higher in high invasiveness HCC samples that in low invasiveness HCC samples.Conclusions:1. Helicobacter pylori exist in hepatocellular carcinoma tissues , and the infection of Helicobacter pylori may associated with the carcinogenesis of primary hepatocellular carcinoma.2. Helicobacter pylori could enhance the proliferation of HepG2 cells, and had broad influences in cellular function.3. Infection of Helicobacter pylori may closely co-related with the invasiveness of HCC by promoting the expression level of transgelin and integrinα2 .