| Background:Metastasis is one of the most important biological characteristics of malignant tumor, at the same time, it is the fundamental cause of high mortality and poor prognosis of the patients who suffer from malignant tumors.Lymphatic metastasis is the early mode in epithelial malignant tumors, but the mechanism of it is unclear. As one of the most common malignant tumor, the primary carcinoma of the liver includes 3 types:hepatocellular carcinoma (HCC), which accounts more than 90%, and the other types are intrahepatic cholangiocarcinoma (IHCC) and the mixed type carcinoma. There are about 626,000 new cases of HCC and more than half in China. Meanwhile, as one of the most fatal malignant tumor, HCC has a high relapse incidence though the mass has been cut off closely. Therefore, to reveal the mechanism and the key steps in metastasis of HCC is helpful for reducing the mortality of it.Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines which have different rates of lymphatic metastasis. Hca-F is the cell line whose metastatic rate is more than 70%, compared with it, Hca-P has a low lymphatic metastasis rate, which is less than 30%. They are the ideal models for the research of mechanism of lymphatic metastasis. Our research team has engaged in the study about the mechanism of lymphatic metastasis. We have picked out the lymphatic metastasis-associated genes in mouse hepatocarcinoma cell lines by using suppressive subtractive hybridization and high throughput screen of genechips assays respectively and obtained the lymphatic metastasis-associated proteins by using quantitative proteomics technique. The expressing level of CLIC1 was much higher in Hca-F than that in Hca-P cell lines in both mRNA and protein levels which showed that CLIC1 maybe play an important role in lymphatic metastasis of mouse hepatocarcinoma. Because there is the same origin of CLIC1 protein in the tissue of both human being and mouse, it is very helpful to study the function of CLIC1 in the lymphatic metastasis of mouse hepatocarcinoma.CLIC1, also known as NCC27, is a member of the family of chloride intracellular ion channels. As a chloride channel, which play an important role in the cells.CLIC1 usually exists in both soluble and memberane-assosiated forms. Studies have shown that the role of CLIC1 is not limited to intra and extra cellular ion transport, but also, and many diseases, especially neoplastic diseases are closely related to. Some scholars have found that CLIC1 markedly enhanced expression in breast cancer, lung cancer, gallbladder cancer, hepatocellular carcinoma, colorectal cancer, gastric cancer, as well as a variety of tumor cells, and found it was significantly correlated with lymph node invasion in stomach and gallbladder carcinoma, suggesting that CLIC1 may be one of the important genes involved in tumor occurrence and lymphatic metastasis. In this study, CLIC1 as the research object to explore in Hca-F cell line. There was not the same reports home and abroad by retrieval.Objective:1. To observe the different expression levels of CLIC1 in mouse hepatocarcinoma ascites cell lines Hca-F and Hca-P by Westen Blottin for further study of its function and mechanism in lymphatic metastasis of hepatocarcinoma.2.To build expression vector of pGPU6/GFP/ Neo-shRNA and get cell line Hca-F which expression of CLIC1 has been markedly decreased after stable transfection.3. To study the influence on proliferation, cell cycle, apoptosis, migration and invasion of mouse hepatocellular carcinoma cell line Hca-F after down regulation of CLIC1 expression by shRNA and discuss the correlation between expressing level of CLIC1 and lymphatic metastasis of mouse hepatocarcinoma.Methods:1. The expression of CLIC1 protein was detected in mouse hepatocarcinoma ascites cell lines of Hca-F and Hca-P by Western Blotting. 2. Three shRNAs (shRNA-1, shRNA-2, shRNA-3) and unrelated sequence were designed according to the gene sequence of CLIC1 (NM0033444) in Gene Bank. pGPU6/GFP/Neo vector was obtained and restriction enzymes were used to digest. The annealing of shRNA compositive sequence was performed and T4 DNA ligase was used to connect the pGPU6/GFP/Neo vector with three shRNAs and unrelated sequence(negative control, NC) after annealing. The connecting products were named pGPU6/GFP/Neo-shRNA-1, pGPU6/GFP/Neo-shRNA-2, pGPU6/GFP/Neo-shRNA-3 and pGPU6/GFP/Neo-shRNA-NC. The expressing vector was obtained by extraction of vectors. The sequence of expressing vectors was identified by sequencing and the result was compared to that in Gene Bank. The three pGPU6/GFP/Neo-shRNA expressing vectors were transfected into Hca-F cells respectively by SofastTM reagent and the most effective pGPU6/ GFP/Neo-shRNA vector was selected according to the results of Real-time RT-PCR and Western Blotting. The unrelated shRNA transfected Hca-F cell line and normal Hca-F cell line were negative control group and blank control group respectively.3. The cell viability was evaluated by CCK-8. The cell cycle and apoptosis were evaluated by flow cytometry. The cell migration and invasion capability were evaluated by transwell assays.Results:1. The expression of CLIC1 in cell line Hca-F was 1.33 times more than that in Hca-P.2. The expression vector of pGPU6/GFP/Neo-shRNA was built and transfected into Hca-F cells successfully. The sequence of three shRNAs was identified by sequencing and the result was identical to that in Gene Bank. At the same time, all expression vectors were verified by restriction enzyme cutting to be built successfully. The expression vector pGPU6/GFP/Neo-shRNA-2 was the most effective one. The expressions of mRNA and protein of CLIC1 in Hca-F cells after transfection with pGPU6/GFP/Neo-shRNA-2 were markedly decreased compared with the other groups. pGPU6/GFP/Neo-shRNA-2 group was chosen as the cell line for further study.3. The results of CCK-8 showed an obvious increase of cell proliferation was detected in pGPU6/GFP/Neo-shRNA-2 group cells after the expression of CLIC1 has been decreased. The results detected by flow cytometer showed more cells were arrested in G2/M phase and the cell percentage of apoptosis significantly decreased after the expression of CLIC1 was decreased compared with the blank group and negative control group cells (P<0.05). Transwell assays showed that both migration and invasion capability were decreased after knock-down of CLIC1 expression in Hca-F cell line (128.67±33.54* vs 214.53±24.96, 197.73±24.48,*P<0.05; 50.73±3.89* vs98.93±5.00,96.27±2.60,*P <0.05).Conclusions:1. The expreesion of CLIC1 in Hca-F was greatly higher than that in Hca-P.2. The expression vectors pGPU6/GFP/Neo-shRNAs were constrcted successfully with plasmid pGPU6/GFP/Neo and shRNAs. After the expression vectors were stable transfected into Hca-F cell line, pGPU6/GFP/Neo-shRNA-2 was proved by Real-time RT-PCR and Western Blotting to be the most effective one, which can decreased the expression of CLIC1 remarkably. And pGPU6/GFP/Neo-shRNA-2 was chosen as the cell line for further research which provided a solid foundation for further studies about the relationship between CLIC1 and lymphatic metastasis of hepatocellular carcinoma.3. As one of the proteins expressed differently in Hca-F and Hca-P cell lines, CLIC1 could inhibit proliferation, promote apoptosis, promote cells to pass G2/M phase, increase migration and invasion capability of mouse hepatocellular carcinoma cells. Results above pointed out that CLIC1 may play an important role in lymphatic metastasis of hepatocellular carcinoma. |
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