| Background: The high mortality rates associated with cancers are mainly attributable to their strong tendency to metastasize into surrounding or distant tissues. Lymphatic metastases are usually an early step, with lymph nodes often the first organs to develop metastases. It is believed that the presence of lymph node metastases (LNM) is associated with badly prognosis in head and neck cancer. Despite the importance of this in malignancies, a detailed understanding of the mechanism of lymphatic metastasis is still lacking. Immunocytochemistry (ICC) analysis and Western blot (WB) analysis indicated that the protein levels of Ech1 in Hca-F were 1.5-fold and 1.2-fold of that in Hca-P , which was consistent with our previous genomic (3.0 fold) and proteomic (2.7 fold) results.Hca-F with a high lymphatic metastasis rate of 75%, while Hca-P with a low lymphatic metastasis rate of 30%, which has been proved to be an ideal cell model for studying the lymphatic metastasis.Ech1 is the second enzyme in fatty acid degradation viaβ-oxidation pathway. A variety of studies already demonstrated that Ech1 was associated with tumor progression. The abnormal expressions of Ech1 were associated with putative preneoplasia and neoplastia and hepatocellular carcinoma infected by HCV and the pathogenesis of gastric cancer. And the down-regulation of Ech1 at the gene level was found to be associated with the DNA damage-induced apoptosis resistantance of B cell chronic lymphoid leukemia (B-CLL). However, no study has been addressed on the relevance of Ech1 with tumor lymphatic metastasis. To gain an insight into the role that Ech1 is playing in a high LNM potential tumor cell line, an RNA interference (RNAi) study was performed to downregulate Ech1 expression in the Hca-F cell line with observation of the subsequent effects. Objective: Short hairpin RNA (shRNA)-Ech1 expression plasmids were constructed and got the stably transfected cells. To study the effect to cell proliferation, adhesion, migration, invasion, and cell cycle status when down-regulation of Ech1 gene expression in mouse hepatocarcinoma cell. And to study the expression of Annexin A7, Clic1 and Gelsolin when downregulation of Ech1.Methods: 1 Four shRNAs that targeted different sites of the mouse Ech1 gene (NM-016772)(Ech1-430)5-CTCATCAGCAAGTACCAGA-3,(Ech1-91)5-GCCAGCTGTACTTCAACATCA-3, (Ech1-348)5-GCAGGAAA GATGT TCACTTCA-3,(Ech1 -460)5-GCAAGTACCAGAAGACCTTCA-3 were designed, synthesized and inserted into the pGPU6/GFP/Neo expression vector. pGPU6/GFP/Neo-shRNA-Ech1 were constructed,and all the expression plasmids were confirmed by DNA sequence analysis. Hca-F cells were added into a 24-well plate prior to transfection, and transfected with 2μg of pGPU6/GFP/Neo plasmid using sofastTM transfection reagent (Sunma Corp., China) according to the instructions of the manufacturer. Transfection efficiencies were determined by fluorescence microscopy at 24 h and 48 h. The stably transfected cells were obtained by growing them in neomycin (400μg/ml) for 3 weeks. The levels of expression of Ech1 mRNA and protein were detected by real time quantitative polymerase chain reaction (qRT-PCR) and WB analysis, respectively. The shRNA-Ech1-91- Hca-F cells were chosen for further experiments. 2. Cells were divided into three groups: (a) shRNA-Ech1-91-Hca-F; (b) control shRNA-Hca-F; (c) unmanipulated Hca-F cells were used as control. Cell proliferation was assessed by Cell counting kit-8 (CCK8) assay according to the protocol of the manufacturer. Cell adhesion in vitro was measured using the CytoSelectTM 48-well extracellular matrix (ECM) array, with kits that contained collagen I, collagen IV, fibrinogen, fibronectin and laminin. While Cell adhesion in vivo was measured using cell suspension (1×106 cells/ml) placed onto the surface of frozen slices of lymph nodes and stained with hematoxylin and eosin (HE), then adherent cells were counted. Cell migration assay and invasion assay were measured using the Boyden-transwell assay (8-μm pore size). The cell cycle status was measured by fluorescence activated cell sorting, to obtain the distribution and percentage of cells in the G1, S, and G2/M phases. 3. Equal amounts of protein from each group were separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). These were incubated with a primary antibody against Annexin A7 (Sigma; 1:1500), Clic1 (Santa Cruz; 1:200), Gelsolin (BD Biosciences; 1:1000) and a monoclonal antibody against GAPDH (Kangcheng; 1:7500),β-actin (Santa Cruz; 1:300) in 5% dried milk for 1 h, then incubated with horseradish peroxidase-linked second antibodies (1:5000 dilution) for 1 h at room temperature. Enhanced chemiluminescence (ECL; GE Healthcare, USA) was performed to visualize the protein bands.Results: 1: Endonuclease activity assays and DNA sequence analysis confirmed the recombinant plasmids of shRNA-Ech1. The levels of mRNA expression of Ech1 in shRNA-Ech1-91-Hca-F and shRNA-Ech1-430-Hca-F were decreased by 78% and 35%, respectively, quantified by qRT-PCR analysis compared to Hca-F cells. WB results indicated shRNA-Ech1-91-Hca-F and shRNA-Ech1-430-Hca-F reduced the Ech1 protein levels by 46% and 35% compared to Hca-F cells, but nonspecific-sequence control shRNA transfected Hca-F cells showed no difference compared to Hca-F cells. shRNA-Ech1-91-Hca-F was more efficient than others, so this was chosen as the shRNA-Ech1-Hca-F for further experiments. 2: Cell proliferation: After 72 h, the number of shRNA-Ech1-Hca-F cells was 84% of the number of unmanipulated Hca-F cells and 87% of the control shRNA-Hca-F cells. The cell number of shRNA-Ech1-Hca-F was 71% that of the Hca-F and 86% of the control shRNA-Hca-F at 96 h. The downregulation of Ech1 may inhibit to some extent the proliferative capacity of the Hca-F cells. Cell adhesion assay in vitro: The absorbance of shRNA-Ech1-Hca-F cells adhered to fibronectin was 1.0±0.3, which was 71% that of the Hca-F cells and 69% of the control shRNA-Hca-F cells. The data for shRNA-Ech1-Hca-F adherence to collagenⅠwas 0.9±0.1, which was 79% of the results for the Hca-F cells and 71% of the control shRNA-Hca-F cells. The adherence of the transfected Hca-F cells to the other tested components of the ECM showed no differences. Cell adhesion assay in vivo: The number of Hca-F and control shRNA-Hca-F cells adhered to lymph nodes were 107±23 and 118±16, respectively. However, the number of shRNA-Ech1-Hca-F cells adhered to lymph nodes was only 58±19, which was only 54% of the number for Hca-F cells and 49% of the control shRNA-Hca-F cells (p<0.05). Cell migration assay: showed that Hca-F and control shRNA had a similar ability to pass through the filter, the numbers of shRNA-Ech1 cell (27±18) passing through the filter were markedly lower than the numbers of Hca-F (59±30) and control shRNA (72±19), and there was no significant difference between control shRNA and Hca-F cells. Cell invasion assay: No differences were observed in invasion ability between shRNA-Ech1-Hca-F and Hca-F cells. Flow cytometric analysis of cell cycle status: The S phase cells were 86.06% in shRNA-Ech1 and 75.75% in negative control and 66.15% in Hca-F cell; The G1 phase cells were 9.42% in shRNA-Ech1 and 24.21% in negative control and 30.29% in Hca-F cell, respectively, the result showed that the ratio of cells was significantly increased at S phase and decreased at G1 phase after Ech1 downregulated when compared with Hca-F cells, and there was no significant difference between control shRNA and Hca-F cells. 3: The expression of Annexin A7 were upregulated 11%, the expression of Clic1 were upregulated 69%, the expression of Gelsolin were upregulated 13%, especially the expression of Clic1 were upregulated obviously.Conclusion: The stably transfected pGPU6/GFP/Neo-shRNA-Ech1- Hca-F cell line was obtained; The down-regulation of Ech1 was proved to inhibit the adhesion ability of Hca-F cell, inhibit the cell proliferation of Hca-F cells, decrease the migration capacities of Hca-F cells, increased the ratio of Hca-F cells in S phase and decreased the ratio of G1 phase. when the expression of Ech1 was downregulated, the expression of Annexin A7, Clic1, Gelsolin were upregulation, especially the expression of Clic1. |
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