| Hepatocellular carcinoma(HCC) is one of the common types of digestive system malignancies in China, which accounts for approximately 50% of incidence worldwide. HCC has ranked second in cancer mortality in China. Because of metastasis, high mortality and the extremely poor prognosis, the relative 5-year survival of HCC is only 5% to 6%. Surgical resection remains a major approach to a curable outcome of HCC,however,recurrence and metastasis rate remains high after curative hepatectomy, which are main obstacles for further improving the survival and prognosis. In addition, a considerable number of HCC patients with multiple metastasis are not candidates for surgery. Target therapy of anti-metastasis would be a promising way to treat patients for HCC with recurrence and metastasis.Hepatocarcinogenesis is believed to be a complicated multistep and multiple genetic alterations processes involving several classes of proteins (such as cytokines, cell adhesion molecules, angiogenesis factors, extracellular proteases and growth factors, etc.). The pathogenesis of HCC is still not clearly understood and no specific therapies for this disease. Hence, it is of great importance to elucidate the molecular mechanism of HCC for exploring effective therapeutic way, reducting the rate of metastasis and recurrence, improving the survival rate and prognosis.A family of small chemoattractant cytokines called chemokines and their respective receptors have been suggested to be responsible for the metastatic potential of cancer cells. Chemokine receptor CCR6 is an extraordinary conservative seven-transmembrane, G protein-coupled receptor of CCL20(MIP-3a, LARC, exodus-1, ST38). Recent studies disclosed that CCR6/CCL20 axis had played a important role in driving, guiding and locating the organic specific metastatic behavior of several malignant tumors such as pancreatic cancer, prostate cancer, colorectal cancer, lung cancer and malignant tumor of hematological system. By now,the systemic researches on the relationship between CCR6/CCL20 axis and invasion/metastasis of HCC have not been reported. And there is also no report on gene therapy against HCC by small interference RNA(siRNA).To investigate the role of CCR6 in the invasion and metastasis of HCC, silence CCR6 gene expression by miRNA-mediated RNA interference(RNAi) using plasmid vector (pGPH1/GFP/Neo) in HCC cell line HCCLM6 with high metastasis potential in this study. We gain successfully effective interference sequence of CCR6 gene, and explore the feasibility and mechanism of CCR6 targeted gene therapy to inhibit the invasion and metastasis of HCC.Part OneThe expression of CCR6/CCL20 and its relationship with human HCCExpression of CCR6/CCL20 gene and protein in 48 paired specimens of HCC/para-carcinoma tissues, eight specimens of normal liver tissues and six cell lines(one normal, five HCC cell lines) were studied by Real-time PCR, immunohistochemisty, Western-blot and immunofluorescence methods. The relationship of CCR6/CCL20 with the clinicopathological factors and prognosis was analyzed.The expression of CCR6/CCL20 in 48 paired HCC/para-carcinoma and 8 normal liver were studied by Real-time PCR and immunohistochemisty method. The relative levels of CCR6mRNA in HCC, para-carcinoma and normal liver tissues were 0.99±0.21, 0.33±0.09, 0.22±0.06 and CCL20mRNA with 0.46±0.11, 0.31±0.07, 0.28±0.05 respectively. The difference of expressive levels of CCR6mRNA between them are significant(P﹤0.05). The expressive levels of CCL20mRNA between HCC and para-carcinoma/normal liver tissues are significant difference(P﹤0.05). The positive rates of CCR6 protein expression in HCC, para-carcinoma, normal liver tissues were 54.17%, 16.67%, 0% and CCL20 with 50.00%, 33.33%, 25.00% respectively. The difference of CCR6 between them are statistical significance(P﹤0.05), and CCL20 with no significant difference(P﹥0.05). The positive rates of CCR6 and CCL20 expression in HCC tissues were significant correlated by Spearman’s correlation analysis(r=0.42, P﹤0.05).The relationship of CCR6/CCL20 expression with clinicopathological factors such as gender, age, cirrhosis, tumor size, differentiation, pathologic grades, vascular invasion, intrahepatic metastasis and lung metastasis were analyzed by chi-square test. The expression of CCR6 protein was significantly correlated to pathologic grades, vascular invasion, intrahepatic metastasis and lung metastasis of HCC(P﹤0.05), but not gender, age, cirrhosis, tumor size and differentiation. The expression of CCL20 was only significantly correlated to tumor size and pathologic grades of HCC(P﹤0.05).Survival time was also observed and Kaplan-Meier method, log-rank test was used to analyze the relationship between CCR6/CCL20 expression and prognosis of HCC. The DFS(disease-free survival) and OS(overall survival) of CCR6-positive group were significantly lower than those in the CCR6-negaitive group. In contrast, the differences of DFS and OS between CCL20-positive and CCL20-negative groups were not statistical significance(P﹥0.05).The expression of CCR6/CCL20 in six cell lines(one normal, five HCC cell lines)detected by Real-time PCR and Western-blot test. CCR6 expression was all detected in five HCC cell lines(SMMC-7721, MHCC-97L, MHCC-97H, HCCLM3 and HCCLM6) and expressive levels corresponding with elevated metastasis potentials, but it almost nothing in normal liver cell line(L02). Simultaneously,the expression of CCL20 was detected in all six cell lines and the difference between them was not statistical significance(P﹥0.05). The protein of CCR6 in HCCLM6 cells are mainly expressed in cytoplasm and cytomembrane by immunofluorescence method.The aboving findings indicate that CCR6/CCL20 axis, especially CCR6 may play an important role in malignant progression of HCC. The CCR6 inhibited gene targeted therapy may be a effective therapeutic way for invision and metastasis of HCC. The main aim of this part was to silence CCR6 gene expression in human HCC cell line HCCLM6 using miRNA-mediated RNAi by plasmid vector. HCCLM6 cells was transfected by Lipofectamine2000 and the expression of CCR6 was detected by Real-time PCR and Western-blot test. In addition, the stably-transfected cell line was established by G418 selection.Within a certain range(0.4~2.4ug), the more plasmid DNA used, the higher cell transfection rate obtained. There is highest transfection rate over 85% with Lipofectamine2000 2.0ul and palsmid 1.6ug at the 48 hours after transfection.Four double-stranded DNA vectors pGPH1/GFP/Neo-(CCR6-siRNA-431, -1098, -673, -493) targeting the mRNA of human CCR6 constructed then were transfected into HCCLM6 cell line. We find the expression of CCR6mRNA is signifieantly reduced at the 48 hours after transfection and 72～96 hours reach the peak time. It was more marked in cells transfected with CCR6-siRNA-493 than in those transfected with CCR6-siRNA (-431, -1098, -673)(P﹤0.05). The mRNA level in HCCLM6 cells transfected with CCR6-siRNA-493 was reduced by more than 90%, whereas the CCR6mRNA is not marked changed between the control groups. We also find the CCR6 protein is lowest in 96～120 hours after transfection by Western-blot test, and the expression of CCR6 protein is consistent with the Real-time PCR results. In addition, the stably-transfected cell line of silence CCR6 gene was established by G418 selection.Therefore, CCR6-siRNA-493 has more efficient RNAi effects than CCR6-siRNA(-431, -1098, -673) and achieved better suppression of CCR6 expression at both mRNA and protein levels. Therefore CCR6-siRNA-493 was used in the following experiments. To investgate the role of CCR6 gene on the biological behavior of human HCC cells, the effects of CCR6 RNAi on proliferation, invasion and migration of HCC cells in vitro were tested using stably-transfected cells.It was shown that the CCR6-siRNA cells proliferation was obviously inhibited in comparison with HCCLM6-Mock and HCCLM6-Scrambled groups by MTT methods. Using cell adhesion assay, the results showed that the ratio of the cell adhered coated FN at the time point of 30min in CCR6 RNAi treated group(24.51%) obviously less than that in Mock group(68.27%) and Scrambled group(64.96%)(P﹤0.05). Cell migration assay in vitro revealed that the scratch repaired rate of HCCLM6 RNAi cells at the point of 24 hour and 72 hour significantly decreased in comparison with Mock group and Scrambled group(P﹤0.05). The mean number of cells invaded through transwell was significantly decreased in CCR6-siRNA group(P﹤0.05). The effects of chemokine CCL20 on HCC were evaluated by chemotaxis test. Within a certain range, the chemotaxis was correlated with CCL20 concentration in control groups, but not CCR6-siRNA group. In gelatin zymography assays, we found an marked decreased of MMP-2 and MMP-9 activities, especially MMP-2, in CCR6-siRNA cells compared with control groups. In addition, we find the expression of PCDA, ICAM-1 and OPN protein were inhibited significantly in CCR6-siRNA cell line(P﹤0.05), but E-cadherin was not changed markedly.It was suggested that CCR6-siRNA could inhibit the proliferation, adhesion, migration and invasion of HCC cells. CCR6 might be a potential molecular target for HCC therapy. To investgate the role of CCR6 gene in the progress of HCC and the therapeutic effects of CCR6-siRNA, we established human HCC mode with BALB/c nude mice. The effects of CCR6-siRNA on HCC were evaluated by observing growth, invasion and metastasis of subcutaneous implanted tumor.18 nude mice were randomly divided into three groups and the three prepared cells(6×106/0.2ml) were subcutaneously injected on the back of mice. The results showed that CCR6-siRNA group was significently lower than that of HCCLM6-Mock group and HCCLM6-Scrambled group in volume and weight of tumors, the differences were statistical significance(P﹤0.05). In addition, the metastatic nodules of lung and liver in CCR6-siRNA group significantly reduced in comparision with control groups by H&E staining (P﹤0.05). The expression of VEGF, MMP-2, MMP-9 and microvessel density (MVD)were also significantly decreased in CCR6-siRNA group compared with HCCLM6-Mock and HCCLM6-Scrambled groups (P﹤0.05).The above experiments demonstrated that CCR6-RNAi gene target therapy could effectively inhibit growth and metastasis of HCC in vivo. It was suggested that CCR6 will be a new target for siRNA gene therapy of HCC. |
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