| Background: Hepatocellular carcinoma (HCC) is a devastating neoplasm that is usually associated with difficulty of early diagnosis, early hematogenous metastasis, high recurrence, poor prognosis. As one of the most frequent cancers in China, its development, progression and metastasis is a complexed process involved many genes and several transforming steps in liver. Up to now, though several tumor suppressors such as p53 and p16 were identified in HCC and be confirmed their specific inactivation events occur in carcinogenesis, few reports introduced the important activated oncogenes playing vital role in HCC. Gankyrin (Gann ankyrin repeated) gene is an overexpressed gene cloned in HCC recently. Its carcinogenesis activity mey be realized through Gankyrin protein's hyperphosphorylation and degradation on Retinoblastoma (Rb) protein, leading to the transferring from G1 to S in cell cycle regulation. The expression of Gankyrin protein can be found in HCC, which be more evident with HCC's progression. Although many advances obtained in HCC treatment in recent years, surgical curative resection and liver transplantation are still the most effective primary treatments. Despite apparently curative resection or liver transplantation, significant number of patients die of the recurrence and the metastasis of HCC due to development of undetected micrometastasis in peripheral blood. Recent years advances in the detection of both isolated tumor cells and micrometastasis in distant organs or peripheral blood by means of molecular biological techniques have brought a new pathway for the investigation of HCC metastasis biology. The specificity and sensitivity of detection with HCC micrometastasis in peripheral blood maybe increased by highly sensitive technique of nested reverse transcription-polymerase chain reaction (nested RT-PCR). Objective: 1. To detect the expression of Gankyrin protein in the progression of Wistar rat HCC carcinogenesis. 2. To detect the expression of Rb protein in the progression of Wistar rat HCC carcinogenesis. 3. To assess the relationship of Gankyrin protein and Rb protein in the progression of Wistar rat HCC carcinogenesis. 4. To investigate the frequency of peripheral blood micrometastasis and the relationship with the expression of Gankyrin protein and Rb protein in the progression of Wistar rat HCC carcinogenesis, assess specificity and sensitivity of nested RT-PCR assay for detecting circulating tumor cells and investigated the influence in matters of recurrence and metastasis of HCC. Methods: HCC was induced in Wistar rat by N-Nitrosodiethylamine (DEN). After the liver of Wistar rats scaned by MRI dynamically, the rats were killed in the 16th week, 18th week, 20th week, 22th week, 25th week respectively. On the base of the animal model which we established, the pathological changes were observed dynamically, the expression of Gankyrin protein and Rb protein were detected dynamically by immunohistochemistry during the progression of Wistar rat HCC carcinogenesis. Extracting 10 ml total peripheral blood, then AFPmRNA was detected by nested RT-PCR in the peripheral blood of Wistar rat. Results: 1. Wistar rat model of HCC was established successfully, the rate of carcinogenesis of Wistar rat is 71.7%(86/120) by induction with 2‰DEN. The primary pathological type is cirrhosis before the 16th week, HCC between the 17th-22th week and metastasis occur frequently after the 23th week. We found 35 cases of well-differentiated type HCC, 29 cases of moderate-differentiated type HCC and 22 cases of poor-differentiated type HCC in the 86 cases of Wistar rat HCC. And 18 cases of HCC with metastasis, the frequency of metastasis is 20.9%(18/86). 2. Gankyrin protein has such a hepatocellular antigen speciality that negatively stained in bile duct epithelial cells, vascular endothelial cells, Kupffer cells and other interstitial cells. The positive rates of the expression of Gankyrin protein are 94.4%(17/18) in the group of HCC (with metastasis), 67.6%(46/68) in the group of HCC (without metastasis), 14.3%(3/21) in the group of liver cirrhosis, 0.0%(0/20) in the control group. There is a significant difference among four groups (P<0.01). 3. Rb protein stains in normal hepatic epithelial cells completely. The positive rates ofthe expression of Rb protein are 16.7%(3/18) in the group of HCC (with metastasis), 54.4%(37/68) in the group of HCC (without metastasis), 85.7%(18/21) in the group of liver cirrhosis, 100.0%(20/20) in the control group. There is a significant difference among four groups (P<0.01). 4. Pearson's correlation is used to analyze the correlation of the expression of Gankyrin protein and Rb protein among three groups. The rank correlation coefficient is -0.611(P<0.01) in the group of liver cirrhosis, -0.444(P<0.01) in the group of HCC (without metastasis) and -0.542(P<0.05) in the group of HCC (with metastasis), which are all negative correlation respectively. 5. The positive rates of the expression of AFPmRNA are 81.8%(9/11) in the group of HCC (with metastasis), 42.3%(22/52) in the group of HCC (without metastasis), 6.3%(1/16) in the group of liver cirrhosis, 0.0%(0/20) in the control group. There is a significant difference among four groups (P<0.01). 6. Pearson's correlation is used to analyze the correlation of the expression of Gankyrin protein and AFPmRNA among three groups. They are all positive correlation respectively at 0.05 two-tailed level, r=0.683 in the group of liver cirrhosis, r=0.287 in the group of HCC (without metastasis), r=0.671 in the group of HCC (with metastasis). McNemar test is used to analyze the correlation of the two methods and no significant difference is found, p=1.000. 7. Pearson's correlation is used to analyze the correlation of the expression of Rb protein and AFPmRNA among three groups. The rank correlation coefficient is -0.806(P<0.05) in the group of liver cirrhosis, -0.413(P<0.01) in the group of HCC (without metastasis) and -0.671(P<0.05) in the group of HCC (with metastasis), which are all negative correlation respectively. McNemar test is used to analyze the correlation of the two methods and no significant difference is found, p=1.000 (p=0.324 in the group of liver cirrhosis). Conclusions: 1. Gankyrin protein maybe promotes carcinogenesis in HCC through degradating the anti-oncoprotein of Rb, which also plays a major role in the invasion and metastasis of HCC. 2. The overexpression of Gankyrin protein and/or loss expression of Rb protein maybe plays a synergetic role in the pathogenesis, progression and metastasis of HCC.3. Gankyrin protein and Rb protein may be an effective referenced index to early monitoring for metastasis in HCC. 4. Nested reverse transcription-polymerase chain reaction is a feasible method for detecting circulating tumor cells in peripheral blood with a liver-specific AFPmRNA marker, which can increase the sensitivity and specificity of detection to micrometastasis in HCC. Moreover, the early detection of micrometastasis can increase the predictable ability of recurrence and metastasis in HCC. 5. Micrometastasis in peripheral blood of HCC maybe correlated with the overexpression of Gankyrin protein and/or loss expression of Rb protein.
|
PDF Full Text Request