Effect of siRNA targeted against survivin on the malignant behaviors of human hepatoma cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study survivin expression in human hepatoma cells and the effects of survivin siRNA on the malignant phenotypes of human hepatocellular cell line HCCLM6.</p><p><b>METHODS</b>Four hepatocellular carcinoma (HCC) cell lines were used. Semi-quantitative RT-PCR and Western blot were used to measure and compare their survivin expressions. The siRNA expression vector pshRNA-survivin targeting the mRNA of survivin and vector pGPU6/GFP/Neo-NC (as a control) were constructed, and then transfected into HCCLM6 cells. FQ-PCR was used to quantify the mRNA levels of survivin. The malignant phenotypes of transfected HCCLM6 cells, including invasive activities and adhesive capabilities, were analyzed.</p><p><b>RESULTS</b>Survivin expression gradually increased with the increase of the invasion and metastasis behaviors of the four HCC cell lines (P<0.05). The expression of survivin was highest in cell line HCCLM6. Survivin mRNA level was decreased by 93.500%+/-3.117% after the pshRNA-survivin transfection. The cell adhesion rates significantly decreased in the cells transfected with pshRNA-survivin (cell adhesion rates were 11.403%+/-1.256% vs 32.545%+/-1.367%, t=20.732, P<0.01). The migrating number of HCCLM6 cells (13.5+/-0.9) transfected with pshRNA-survivin was also significantly decreased (t=14.5, P<0.01) as compared with the control group (32.6+/-1.4).</p><p><b>CONCLUSION</b>The expression of survivin in HCC might have a close relationship to their invasion and metastasis properties. Sequence-specific shRNA can significantly reduce the survivin expression in the HCCLM6 cell line. Suppression of survivin expression in HCCLM6 cells transfected with pshRNA-survivin can reduce their invasive and adhesive capabilities.</p>

Relationship between epithelial-mesenchymal transition and lung metastasis in hepatocellular carcinoma / 中华外科杂志

<p><b>OBJECTIVE</b>To evaluate the relationship between epithelial mesenchymal transition (EMT) and lung metastasis in hepatocellular carcinoma.</p><p><b>METHODS</b>There were 100 patients who underwent surgical resection of hepatocellular carcinoma between January 2000 and March 2004. They were classified with non-distance metastasis and lung metastasis depend on the close following up till March 2007. Their hepatocellular carcinoma specimens were retrospectively examined for EMT markers (E-cadherin, Vimentin, Fibronectin) with immunochemistry staining in tissue microarray. Univariate and multivariate analysis were used for study the relationship between EMT and lung metastasis.</p><p><b>RESULTS</b>Univariate analysis showed that down regulation of E-cadherin, overexpression of fibronectin, cytosolic expression of vimentin, AFP >or= 400 ng/ml, tumor size more than 10 cm, portal vein involvement, poorly differentiated of tumor had close correlation with lung metastasis. Multivariate analysis indicated that overexpression of fibronectin was independent factor for lung metastasis apart from tumor size more than 10 cm, portal vein involvement and poorly differentiated of tumor.</p><p><b>CONCLUSION</b>The results proposed that EMT has close relation with lung metastasis in hepatocellular carcinoma.</p>

Different expressions of chemokine receptors in human hepatocellular carcinoma cell lines with different metastatic potentials / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To compare different expression profiles of all known chemokine receptors in human hepatocellular carcinoma (HCC) cell lines with different metastasis potentials.</p><p><b>METHODS</b>Eighteen pairs of chemokine receptor primers were designed using Premier software. Expression profiles of the 18 chemokine receptors on four HCC cell lines of lower to higher potentials of metastasis (SMMC-7721, MHCC97-L, MHCC97-H and HCCLM6) were analyzed by RT-PCR. Expression of CXCR4 was detected by RT-PCR.</p><p><b>RESULTS</b>Expression profiles of chemokine receptors on four HCC cell lines with different metastatic potentials had significant differences (P < 0.01), in which CCR10, CXCR4 and CXCR6 expressions decreased gradually as the metastatic potential of the cell lines increased. The expressions of CCR3, CCR4, CCR10, CCR12 and XCR1 on HCCLM6 were significantly reduced compared with SMMC-7721 (P < 0.01), whereas the expressions of CXCR1 (P = 0.006) and CXCR5 (P = 0.003) exceeded that of SMMC-7721. Except for CXCR2, CXCR6 and XCR1, most of chemokine receptors on MHCC97-H were expressed differently compared with MHCC97-L (P < 0.05), in which expressions of CCR1 (P = 0.002), CCR2 (P = 0.004) and CCR5 (P = 0.046) exceeded MHCC97-L. CXCR4 was detected only on the positive controls and SMMC-7721 when the template of total RNA was reduced one-half in RT-PCR.</p><p><b>CONCLUSION</b>Chemokine receptors are expressed very differently at mRNA level on HCC cell lines with different metastatic potentials. The different profiles of chemokine receptors in tumor microenvironment and the function of CXCR4 in HCC should be further studied. Our findings have important implications in understanding the relationship between chemokine receptors and the metastatic potential of HCC.</p>

Osteopontin promotes the malignant phenotypes of human hepatocellular carcinoma cell line SMMC-7721 / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the effects of osteopontin (OPN) on the phenotypes of human hepatocellular carcinoma cell line SMMC-7721.</p><p><b>METHODS</b>Human hepatocellular carcinoma SMMC-7721 cells were transfected with plasmid pcDNA 3.1(-)/OPN and cells transfected with a mock plasmid served as controls. OPN expression was verified by RT-PCR and Western blot, and concentrations of OPN, MMP-2, MMP-9 and uPA were measured by ELISA. A series of functional assays in vitro were used to monitor the changes of SMMC-7721 malignant phenotypes.</p><p><b>RESULTS</b>OPN expression of SMMC-7721 cells was elevated after transfection. Concentrations of OPN, MMP-2 and uPA in the medium of SMMC-7721 cells after transfection were higher than those of the controls [(3.02+/-0.12) ng/ml vs (1.43+/-0.07) ng/ml, (43.04+/-3.06) ng/ml vs (22.15+/-4.34) ng/ml, and (4.78+/-0.70) ng/ml vs (1.61+/-0.34) ng/ml respectively, P less than 0.01], but MMP-9 concentration did not increase [(7.82+/-2.25) ng/ml vs (7.70+/-1.92) ng/ml]. Functional assays in vitro indicated that SMMC-7721 cells after transfection showed higher adhesive, migrant and invasive capabilities than those of the controls (cell adhesion rates were 75.33%+/-10.59% vs 57.34%+/-2.52%; number of outer surface cells in migrant assay was 14.33+/-2.51 vs 6.34+/-1.53; cell number in the invasive assay was 8.23+/-1.53 vs 4.12+/-1.29 respectively, P less than 0.05).</p><p><b>CONCLUSION</b>OPN might enhance the expression of MMP-2 and uPA to promote malignant phenotypes of SMMC-7721 cells.</p>