| Background and objectiveHepatocellular carcinoma (HCC) is one of the most common malignant tumors which seriously threat the body health of human being.HCC is the most common histological type of liver cancer.70% to 85% of liver cancer is HCC.The highest liver cancer rates are found in East and South-East Asia,Africa and South Europe.China is among the high incidence area of HCC.Liver cancer in men is the fifth most frequently diagnosed cancer worldwide but the second most frequent cause of cancer death. In women, it is the seventh most commonly diagnosed cancer and the sixth leading cause of cancer death. According to the latest global cancer statistics show that 748 300 new liver cancer cases and 695 900 cancer deaths occurred worldwide in 2008. Half of these cases and deaths were estimated to occur in China.Nowadays, liver cancer is the second major cause of cancer deaths in China, with a mortality rate of 26.26 per 100 000 and incidence rate of 37.9 per 100 000 for men and of 14.2 per 100 000 for women.HCC has become a significant problem which threaten people's health in our country. Nowadays, many clinic methods are used for treating HCC including surgical resection, liver transplantation, vascular intervention, ablation technology, radiotherapy, chemotherapy,etc. The new drug,multikinase inhibitor sorafenib made a major breakthroughs in cure sphere of liver cancer. At present,the first line of liver cancer treatment is liver transplantation and surgical resection.But more than 80% of HCC patients are on the progressive stage when they dectected HCC. They have lose the chance to surgery. Even after the radical correction, recurernce rate is high up to 43.5%～62.5% within five years.Metastasis and recurrence have become the crux of further improvement on survival rate of HCC patients. Therefore, to seek and predicate HCC's metastasis and recurrence tendency, judge prognosis of HCC patients is the key and focus of HCC's research, as well as its emphasis and difficulty. EpCAM (Epithelial cell adhesion molecule,also called CD326,CO17-1A, EGP,EGP40,GA733-2,KSA,Ly74,M1S2,M4S1,MIC18,MK-1, TROP1 and hEGP-2) is a glycosylated,39 to 42 kDa typeⅠmembrane protein, which is comprised of an extracellular domain with epidermal growth factor (EGF) and thyroglobulin repeat-like domains, a single transmembrane domain, and a short 26-amino acid intracellular domain called EpICD. It is encoded by the GA733-2 gene located on the long arm of chromosome 4.Initially discovered as a dominant antigen on colon carcinomas, EpCAM was considered a mere cell adhesion molecule and reliable surface-binding site for therapeutic antibodies. EpCAM expressed in a variety of human epithelial tissues,cancers, and progenitor and stem cells.It is also expressed on essentially all human adenocarcinoma, on certain squamous cell carcinoma,on retinoblastoma, and on hepatocellular carcinoma.EpCAM participates in the control of cell proliferation, differentiation, adhesion, migration and so on.It is considered to be a surface marker of hepatic stem cell.So far research shows that EpCAM participates in the control of signal transduction pathway which closely related to tumor development such as Wnt-β-Catenin. Researches also show that silencing EpCAM gene expression with EpCAM short interfering RNA (siRNA) resulted in a decrease in the rate of cell proliferation, cell migration and cell invasion in breast cancer cell and gastric cancer cell in vitro, which suggests that EpCAM is a worth-expecting specific marker of multiple tumors.Double-stranded RNA (dsRNA) can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs, a process known as RNA interference. Small interference RNA(siRNA) or shot hairpin RNA(shRNA) both are the medium of RNAi.There are several characters of RNAi,including specificity of sequence which can discriminate mutation gene sequence with normal gene sequence,high efficiency and amplification effect.shRNA is more stable than siRNA and also can extent the time of target gene silencing.MHCC97 is a cell line with a highly metastatic potential was established from subcutaneous xenograft of a metastatic model of human HCC in nude mice (LCI-D20) by means of alternating cell culture in vitro and growth in nude mice.The subcutaneous and intrahepatic xenografts were formed and metastatic lesions in lungs were found after the cells were inoculated into nude mice. The rate of metastasis to lungs was 100% using orthotopic inoculation. MHCC97H is a monoclone cell line with high metastatic potential which is isolated from the parent cell line.Its rate of metastasis to lungs was 100% spontaneously.The cell line MHCC97 is a best model for studies of HCC metastatic mechanisms.This research intent to test EpCAM's influence on biological behavior of MHCC97H cells by transient transfecting the EpCAM-targeted siRNA into MHCC97H cells and observing the changes of MHCC97H cells on cell proliferation,cell apoptosis and cell migration.The object of the study is to provide the evidence to the feasibility of hepatocellular carcinoma gene therapy.MethodsDesign EpCAM-targeted siRNA,transient transfect MHCC97H cells,and detect the intracellular expression of EpCAM gene.1. Design of siRNA Apply siRNA design software for screening out of nine pairs of siRNA,and then remove the siRNA which are homologous with the other encoding genes,the final select two pairs of EpCAM-targeted siRNA.2. Transient transfection Transfect EpCAM-siRNA into MHCC97H cells by lipofection technique,and detect after 48h.3. Detecting Expression of EpCAM mRNA was detected by real-time PCR,expression of EpCAM protein was identified by Western blot,cell apoptosis rate was evaluated by flow cytometry, cell proliferation was detected by MTT assay,and cell migration was detected by wound healing assay.4. Statistical methods The difference of EpCAM protein and mRNA expression rate was analyzed by Chi-square test and Wilcoxon rank sum test respectively.The difference among groups on cell proliferation,cell apoptosis and cell migration was analyzed by Factorial design analysis of variance.Results1. Expression of EpCAM mRNA The result showed that the expression of EpCAM mRNA of siRNA interfered MHCC97H cells of two groups was further lower than control MHCC97H cells group.2. Expression of EpCAM protein The result displayed that protein level of siRNA transfected MHCC97H cells of two groups was lower than control MHCC97H cells group.3. Apoptosis The result of 48h and 72h after transfection demonstrated that apoptosis rate of two experimental groups was higher than control group.However,there are smaller difference between two experimental groups and control group at the time point of 96h.4. Apoptosis morphological Fluorescence microscopy with Hoechst 33342/PI staining detected typical apoptotic changes (condensed chromatin, shrunken nuclei and high blue fluorescence by Hoechst 33 342 staining) and necrotic cells (high red fluorescence) in the two experimental groups. In contrast, few apoptotic and necrotic cells were observed in the cells of control group.5. Cell proliferation The result of 48hrs and 72h after transfection also show that OD490nm detected by Spectrophotometer of two experimental groups is lower than of control group.Small difference can be seen in 96h among groups.6. Cell migration The result of wound healing assay displayed that the number of migratiing cells of experimental groups were significantly lower than the control group.Conclusion1. Application of RNAi technology,EpCAM-targeted siRNA can induce EpCAM gene silencing to achieve the purpose of closure of EpCAM gene.2. The results showed that the biological behavior of MHCC97H cells interfered by EpCAM-targeted siRNA changed significantly,including decline of cell proliferation and cell migration and increase of cell apoptosis.It suggested that EpCAM gene may play a key role on regulation of biological behavior of hepatoma cells.3. Transient transfection of EpCAM-targeted siRNA can efficiently knock out the EpCAM gene expression of MHCC97H cells,but only last for a short time. |
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