| Hepatocelluar carcinoma (HCC) is one of the ten most prevalent malignant cancers in the world, ranking the fourth on the occurrence frequency. HCC is epidemic in non-developed or developing countries, and is the one of most common cancers with high frequency in China. Mortality derived from primary HCC ranks the second in the mortalities from the malignancies, and ranks the first in some suburban areas in China. In the newly happened 500-600 thousand HCC per year in the world, 42.5% occurred in China. The development and progression of HCC are a complicated process involving multiple genes and multiple steps in human bodies. Up to now, though some suppressors in HCC were identified such as p53 and p16INK4A, and confirmed their specific inactivation, few specific activated oncogenes in HCC was found and reported. In 2000, a novel gene named gankyrin with repeated sequences coding 'ankyrin' motif was cloned in human HCC by Japanese scientists Fujita and his colleagues using subtractive hybridization method. We found this gankyrin gene was identical to the p28 (Nas6p) gene reported by Hori in 1998 by searching the GenBank data, thus named it as p28GANK in our work.Large amounts of research were previously conducted in our laboratory. Northern blot analysis showed the mRNA of p28GANK was detected in 62 of 64 examined HCCs (96.9%). among which the increased expression in HCC versus para-cancerous tissues was found in 57 cases (91.9%), absent expression was found in normal liver tissues. The high expression of p28GANK mRNA was also observed in hepatoma cell lines, HepG2, Hep3B, HuH7. PLC/PRF/5, SK-Hepl, Chang liver and SMMC7721. The carcinogentic trait of p28GANK may be ascribed to its phosphorylation and inactivation ability to tumor suppressor RB1, leading to the cell transmission from Gl phase to S phase. RBI combined with transcription factor E2F1 regulates the expression of down stream genes which are highly related to the cell proliferation and mitosis. The function of RB1-E2F1 complex is regulated by the signal pathway of CDK4-CyclinDl-pl6INK4A. We and other laboratories confirmed the interaction of p28GANK and CDK4, indicating the involvement of p28GANK in the CDK4-CyclinDl-pl6INK4A-RBl-E2Fl signal transduction pathway.Further investigation regarding to the novel gene biological function is needed. In this project, we applied the RNA interference (RNAi) technology to study p28GANK function enhancing our knowledge on this hepatoma gene. The mechanism of RNAi is through double chains (dsRNA) mediated specific degradation of targeted mRNA sequence, the transcription of targeted gene was inhibited due to the silence effect at the RNA level. Many researchers substantiated the phenomena of RNAi exists in most species from low to high evolution animals, which implies the conservative regulation mode of RNAi in nature and its prevalent application perspective of RNAi technology. Compared with gene targeted technology, RNAi is a kind of effective and simple way to acquire the 'knock down' consequence versus the 'knock out' by gene targeted. Current research has successfully showed the wide application of RNAi in the silence of some specific oncogenes, shedding a light in the gene function study and in the prevention and treatment of human cancers.The planning of targeted sequence is the key step of RNAi experiment. There are five methods in the execution of RNAi in current research. The chemically synthesized small interference RNA (siRNA) can effectively decrease the expression of targeted gene, but the expensive cost restricts its widely application. Vector mediated RNAi is suitable for the research after specific sequences are screened and identified, while the procedure of screening is comparatively complicated. Cock-tail and siRNA expression frame methods can't be applied in the screening of targeted sequence. Considering above factors, we conducted T7 transcription in vitro to acquire siRNA, avoiding shortcomings of other methods. T7 transcription in vitro system was firstly established in our work, appl...
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