| Objective:Hepatocellular carcinoma (HCC) is one of the most common malignancies in China. Heterogeneous phenotypic and genetic traits of affected individuals and a wide range of risk factors have classified it a complex disease. HCC typically has poor prognosis, because it is often diagnosed at an advanced stage. HCC is not amenable to standard chemotherapy and is resistant to radiotherapy. In most cases, surgical resection and liver transplantation remain the only curative treatment options. But after radical resection, both the patient’s5-year recurrence rate and transfer rate are very high. Therefore, development of novel, effective therapies is of prime importance. p100protein is a multifunctional protein. In the study of some malignant tumors, the p100protein plays an important role in tumor development. Therefore, we speculated that the p100may be involved in the regulation of liver cancer formation. This project is designed to study the expression of p100protein in human hepatocellular carcinoma and the relationship with its clinical features. We look forward to explore the feasibility of the p100protein as a novel liver cancer detection marker and therapeutic target protein for HCC.Methods:The experiment is divided into three parts:in part I, the expression of p100in27cases of HCC tissues and paraneoplastic tissues were detected by En Vision immunohistochemistry. In part Ⅱ,a double-stranded shRNA targeting the p100gene was designed, synthesized and cloned into the pLKO.3G vector. Positive clones were verified with double enzyme digestion and sequencing. Then the valid constructions were cotransfected with the other2packaging plasmids into293T cells to package lentiviral particles carrying p100shRNA expression element. The lentivirus was then harvested and used to infect SMMC7721cells. The knock-down efficiency of p100in infected SMMC7721cells was verified with western blot. In part Ⅲ, we observe the influence of silencing p100by lentivirus-mediated RNA interference on the tumor growth of human liver cancer cells in nude mice and to investigate the effects of p100on the oncogenesis and progression of liver cancer. SMMC7721cells steady integrated with lentiviruses deliverred shRNA expression element against p100gene had been constructed. Then the SMMC7721cells were injected subcutaneously into the back of BALB/c nude mice group. Tumorigenicity and the growth of transplanted tumor in nude mice were observed. Meanwhile the negative control group which SMMC7721cells steady infected with lentiviruses delivering a empty vector control was set up as well.Result:①p1000protein is overexpressed in human hepatocellular carcinoma tissues.②A lentiviral vector carrying a shRNA targeting the p100gene was successfully constructed and a p100stable knock-downed SMMC7721cell line was established by using this lentiviral system. After viral particles infection of SMMC7721cells, strong green fluorescence was observed in SMMC7721cells under fluorescent microscope, the infection efficiency exceeded90%.Western blot analyses confirmed that the expression of p100was significantly down-regulated in this infected SMMC7721cell line.③The constructed p100-shRNA lentivirus vector could suppress the expression of p100protein in SMMC7721cells effectively. And the tumor genesis ability of this p100knock-downed SMMC7721cells were significantly reduced in comparison with the control group.Conclusion:①p100protein is overexpressed in human hepatocellular carcinoma.②A lentiviral vector carrying a shRNA targeting the p100gene was successfully constructed and a SMMC7721cell line stably expressing p100shRNA was established with this lentiviral system.③The silencing p100gene can strongly suppress the tumor formation and transplanted tumor growth of hepatocellular carcinoma in nude mice which suggests that p100may play an important role in the oncogenesis and progression of HCC and may constitute a potential therapeutic target in this disease. |
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