Study On The Relationship Between P14～(ARF), ARF-BP1, C-myc Genes And Hepatocellular Carcinoma

Background and objective:More than 90% of primary liver cancers are hepatocellular carcinomas (HCC). Liver cancer is the sixth most common cancers in the world, and the third most common cause of death from cancer, and the second incidence and mortality in China. The etiopathogenesis of HCC is not clear and the ratio of survival is low. Elucidating the etiopathogenesis of HCC and searching for more efficiency therapy to the HCC are still focal point.Hepatocellular carcinoma, like other tumors, is polygene, polystages, polysteps occurrence process with complicated mechanism. The proto-oncogene c-myc is involved in cell proliferation, differentiation, and apoptosis, and plays an important role in tumorigenesis in many tumors. P14^ARF, an important cell cycle regulative protein in p53 pathway, binds with c-Myc and dramatically blocks c-Myc's ability to activate transcription and induce hyperproliferation and transformation, and inhibit expression of the c-Myc induced genes. ARF-BP1, a HECT domain-containing E3 ubiquitin ligase, interacts with ARF and c-Myc. RNA interference (RNAi) is a post-transcriptional regulatory manner of gene expression, is a conservative mode in organism evolution, and has the effect of counteract invading of virus and maintenance stablity of genome. RNAi could knockdown the expression of gene in mammalians, which provide a new approach for gene therapy of tumor. It indicated that specifically inhibiting over-expression of oncogene, cancer related genes or mutant gene by RNAi and retained them in silence status could achieve the result of anticancer effect. But, whether RNAi can be used to reduce the expression of ARF-BP1 gene in HCC is an unknown field. After ARF-BP1 gene was knockdowned by RNAi, whether the expressions of other genes can be affected is also a worthy topic to investigate.According to above metioned consider, in this study, Reverse transcription polymerase chain reaction (RT-PCR) technique and immunohistochemicalmethod were used to detect the expression of p14^ARF,ARF-BP1 and c-myc in HCC. The ARF-BP1 short interfering RNA (siRNA) was transfected into HepG2 cells, silencing the expression of ARF-BP1 gene, the proliferation, cell cycle, apoptosis, and the expressions of other genes were observed in the HepG2 cells. So that to provide experimental evidence for elucidating the etiopathogenesis, treatment and prevention in HCC.Methods:1. Reverse transcription polymerase chain reaction (RT-PCR) technique was used to examine the expressions of p14^ARF,ARF-BP1 and c-myc mRNA in the 52 HCC tissues and their 45 non-tumorous liver tissues.2. Supervision^TM immunohistochemical method was used to detect the expression of P14^ARF, c-Myc proteins in 37 cases of HCC tissues and their non-tumorous liver tissues.3. Lipofectamine 2000 was used to package the ARF-BP1 siRNA and was transfected into HepG2 cells.4. The efficiency transfected of ARF-BP1 siRNA was detected by the fluorescence microscope and the flow cytometry.5. Supervision^TM immunohistochemical method was performed to analyze the influence of RNAi on the ARF-BP1 protein level. RT-PCR method was used to examine the mRNA expression of the ARF-BP1 gene in the HepG2 cells which was treated with ARF-BP1 siRNA. At the same time, the mRNA expression of p14^ARF, c-myc, ODC-1, p53, mcl-1 gene were detected.6. MTT method was adapted to investigate the proliferation of the HepG2 cells after the transfection of ARF-BP1 siRNA.7. Cell cycle was detected by the flow cytometry in control group and treatment groups.8. After 72hs of the transfection of ARF-BP1 siRNA, the HepG2 cells were stained by Annexin V-FIFC and PI, and the flow cytometry was used to examine the early stage apoptosis cells in control group and treatment groups.9. SPSS10.0 statistical software was applied to statistics the date. To categorical variable the chi-square test and sperman correlation analysis were used to analyze. To numerical variable the date was expressed with (?)±s and t test was used to compare these date. The significant level wasα=0.05.Results:1. The expression of ARF-BP1, p14^ARF and c-Myc mRNAs were 76.90%, 76.90%, 75.00% in HCC tissues, and were 20.00%, 11.10%, 53.30% in non-tumorous liver tissue, respectively. (P<0.01) .2. The positive expression rates of P14^ARF, c-Myc proteins were 78.38%, 75.68% in HCC tissues, and were 13.51%, 51.35% in non-tumorous liver tissue, respectively.3. The efficiency transfected of ARF-BP1 siRNA was 84.80%, After 48hs of the transfection of ARF-BP1 siRNA, the ARF-BP1 protein level decline.4. RT-PCR: After 24hs ARF-BP1 siRNA was transfected to the HepG2 cells, the mRNA expression of the ARF-BP1 gene decreased, and the expression of p14^ARF, c-myc, ODC-1, p53, mcl-1 gene mRNAs were changed.5. MTT: After ARF-BP1 siRNA was transfected to the HepG2 cells, thegrowth rate of HepG2 cells was suppressed markedly, and the suppression rates were 38.00%, 56.00% and 58.00% respectively (P<0.01 or P<0.05).6. After 72hs the cells were treated with ARF-BP1 siRNA, the percentage of G₂ stage cells were 33.70±2.12, significantly higher than the control group (P <0.01).7. After 72hs the cells were treated with ARF-BP1 siRNA, the percentage of apoptosis cells were 27.90±1.40%, significantly higher than the control group.Conclusions:1. The higher expressions of p14^ARF, ARF-BP1 and c-Myc in HCC had the close relation to carcinogenesis of HCC.2. ARF-BP1 was close relation with early HCC;p14^ARF was close relation with advanced HCC.3. There, maybe, was a negative feedback loop in HCC: the induction of p14^ARF by c-Myc and subsequent inhibition of ARF-BP1 and then inhibiting c-Myc.4. Lipofectamine^TM2000 is a suitable carrier for transfected ARF-BP1 siRNA to the HepG2 cells.5. ARF-BP1 siRNA was transfected to the HepG2 cells could decline the expression of ARF-BP1 gene, inhibit the proliferation, block the cell cycle, and induce the apoptosis in HepG2 cells.7. ARF-BP1 could regulate the expression of p14^ARF, c-myc, ODC-1, p53, mcl-1 gene mRNAs in HepG2 cells.8. ARF-BP1 maybe a sticking point in carcinogenesis of HCC, and as a potential therapeutic target in HCC.