| Primary hepatocellular carcinoma (Hepatocellularcarcinoma; HCC) is a high incidence of malignant tumors in the world and also the common malignant tumors in China and some Asia era. According to WHO 2009 statistics, the incidence of HCC in male in the world, HCC incidence of living in the first four. Female incidence rate was slightly lower than male, but for the first seven. About 50 million men and women each year die from the disease patients. During the last 20 years, Chinese HCC patients causes of death from cancer No.3 to No.2. The liver cancer patients features in clinical are early non-specific abdominal symptoms, the condition is quite more hidden, difficult to detect and diagnose. About 70% to 80% patients had a clinical diagnosis they were late to do the surgery(III orⅣ).Liver cancer and the invasive growth and early metastasis were leaded to tumor recurrence and poor efficacy of one of the most important reason, which is leading to poor treatment of cancer patients, the death of the most common and most important reason. Although the clinical application in recent years, many new treatments, but the overall survival time of HCC has not been significantly improved. Cancer gene therapy clinical research in recent years one of the hot, Although found in many liver cancer invasion and metastasis-related target molecules, such as p53, PTEN, etc., but research shows that they are only part of the liver plays a role in inhibiting the growth of tumor cells and a certain lack of specificity. Therefore, starting from the biological mechanisms, research, and seeking participation in the development of vast majority of liver cancer and the critical molecular target specific molecular oncology at home and abroad at the present stage of researchPaper is divided into the following four parts.Objective:MDA-7 gene consists of seven exons and six introns, and IL-10, IL-19, IL-20 co-located on the 1st chromosome, also known as IL-24. We constructed the adenovirus vector carrying MDA-7/IL-24 gene. Using denovirus vector transfected the normal liver cells and liver cancer cells which were ectopic expression of MDA-7/IL-24 gene, studied on the biological effects of MDA-7/IL-24 on liver cancer cells. Methods: cultured the 293 cells in 6 well plate, using the PSGCMV MDA-7/pSGCMV-GFP shuttle plasmid and adenovirus backbone plasmid to Polyfect mediated transfection of 293 cells. appear plaque after plaque picked two were transferred to 500ul 5%DMEM culture medium. Results:the Plaque visible CPE in 6 well plate. the cells become large, rounded, floating, grapes were gathered. Clone 1 and clone 2 get a virus DNA in 1 of this as a template, PCR amplification of specific fragments of about 630bp, its size in line with the positive control, while the negative control adenovirus pSGCMV-GFP virus solution without PCR amplification.Objective:Under hypoxia environment, cultured the hepatoma cells.Comparison the ability of cells migration and metastasis changing in the different condition. Methods: established the hypoxia model of hepatoma cell line MHCC-97H in vitro. cultured the hepatoma cell in the normal and hypoxia group, each groups were cultured for 48 h,72 h. MTT and Transwell found the cell growth of MHCC-97H under the hypoxia and the change of tumor cell invasion and metastasis. The expression of E-cadherin, Twist, Vimentin, FAK of mRNA and protein were analysised by quantitative PCR and Western-blot. Results:the Hepatoma cells cultured in the hypoxia that no significant differentance the tumor cell growth was inhibited. The hepatric cells were change from the stromal cells form over to the epithelial cells, finally resulting in EMT. The cellls metastasis and invasion ability were increased. Tumor cells were further reduced the expression of E-cadherin but the Twist, Vimentin, FAK were significantly increased and then it promoted tumor cells to invasion and metastasis.Objective To explore the mechanism of the Melanoma differentiation associated gene-7(MDA-7/IL-24) inhibit hepatocellular carcinoma cell line MHCC-97H invasion and metastasis. Methods Based on the human hepatoma carcinoma cell(MHCC-97L) and hepatic cell line LO2, Using FCM and Transwall to observe the ability of invasion and metastasis after Ad.mda-7 treated MHCC-97H and LO2. Using immunofluorescence staining to indicate the expressing of E-cadherin in the heptaic cell lines. The expression levels of TWIST,Vimentin and E-cadherin was using Real-time PCR. Western blot perform to observe the change of protein twist, Vimentin,E-cadherin and FAK among teams. Result After transfection with 1000VP/cell Ad.mda-7, the growth of MHCC-97H had been arrested but no to LO2. After Ad.mda.7 transfected MHCC-97H, the EMT was reversed in the tumor cells. MHCC-97H marked CFSE and then FCM showed that MDA-7 selectively arrested MHCC-97H cell proliferation (P＜0.05). Then, transwall showed that MDA-7 could inhibited the tumor cell invasion and metastasis (P＜0.05) Finally, Real-timePCR and Western-blot showed that mda-7 could block the expressing of Twist, Vimentin and FAK were decreased.Objective To explore the mechanism of the Melanoma differentiation associated gene-7(HepG2) enhance adriamycin(ADM) kill the hepatoma carcinoma cell and reverses multidrug resistance(MDR). Methods Based on the human hepatoma carcinoma cell(HepG2), Using MTT assay and FCM to determine the difference among the 100VP/cell Ad.mda-7 add ADM, ADM, Ad.mda-7.To interpret the MDA-7/IL-24 reverses multidrug resistance. The expression levels of MDR-1,STAT-3,BCL-2,BAXmRNA was using real-time PCR. Westernblot was perform to observe the change of protein gp-170, stat-3, P-stat3, PKB, bcl-2, bax among those teams. Result After transfection with 100VP/cell Ad.mda-7, the growth suppression rate of HepG2 which was treated by ADM(1.5mg/L) rising from 17.46% to 79.5%.According to the change, killed cell of HepG2 was increase 4.55 time. It is important that real-time PCR showed MDR-1 mRNA was decreased from(16.49±0.11) to (5.48±0.05) and STAT-3 mRNA increased from (13.17±0.08) to (21.57±0.11) (P＜0.05).WB analyses also showed that P-170 and PKB was decreased after and the phosphorylation-stat-3 was increased. Conclusion Ad.mda-7 can reverse the multidrug resistance of ADM to HepG2. It inhibit the expression of MDR-1 mRNA, then arrest PKB protein active the signing of stat-3 to induce hepatoma carcinoma cell apoptosis.After all, MDA-7/IL-24 gene can significantly reverse the EMT, the inhibition of liver cancer cell invasion and metastasis of change. In the multi-drug resistance mechanisms, MDA-7 can also inhibit the endoplasmic reticulum stress the role of proteins and related proteins increase resistance of chemotherapeutic drugs on tumor cells in vitro. It also suggested that not only will we MDA-7 is an effective treatment of liver cancer molecular target, but also to explore the biological mechanisms of liver cancer is an effective tool.
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