Depression Of Invasion And Migration Of Mouse Hepatocarcinoma Cell Lines Hca-F With High Metastatic Potential By Gelsolin

Background: Many biological processes, such as embryonic morpho- genesis, immune surveillance, and tissue repair and regeneration, are required for cell migration which drives progression of many diseases, including cancer invasion and metastasis.Cell migration is a highly integrated multistep process which is initiated by the protrusion of cell membrane. In the process of tumor invasion and metastasis, the relationship among tumor cell,host cell and extracellular matrix (ECM) is complicated. The actin cytoskeleton is crucial for cell motility, and it connects with cell disintegration, cell differentiation, apoptosis and canceration. Gelsolin is best known for its involvement in dynamic changes in the actin cytoskeleton during a variety of forms of cell motility.Hca-P and Hca-F is a pair of syngenetic mouse hepatocarcinoma ascites cell lines, presenting a specific potential of lymphatic metastasis when inoculated subcutaneously in 615 mice. Hca-F with a high metastasis potential (>70%) and Hca-P with a low one (<30%) are rare and available cell model as a control to each other in the research on the molecular mechanisms of lymphatic metastasis.Based on the gene chip experiment, we found that the expression of gelsolin in Hca-F cells was higher than that in Hca-P cells (21.9 fold compared with Hca-P cells). In order to obtain lymphatic metastasis associated proteins, the protein expressed profiles of Hca-F cells and Hca-P cells were compared using fluorescent two-dimensional difference gel electrophoresis (2D DIGE).Gelsolin protein was up-regulated in Hca-F cells (1.7 fold compared with Hca-P cells) . It was suggestted that gelsolin was a lymph node metastasis-associated protein.Objective: 1. To observed the expression of gelsolin in Hca-F cells and Hca-P cells. 2. Down-regulating the gelsolin expression in Hca-F cells by using pSilencer 3.1-shRNA expressing vectors. 3. To investigate the effect of gelsolin on the proliferation and invasion of mouse hepatocarcinoma cell lines.Method:1. Immunohistochemistry and western blot were utilized to show the expression of gelsolin in Hca-F cells and Hca-P cells. 2. Three shRNAs and scrambled shRNA sequences were designed and synthesized according to gelsolin gene sequences .Then three shRNAs and scrambled shRNA sequences were inserted into the pSilencer-3.1 vector to silence gelsolin gene ,and named gelsolin1,gelsolin2,gelsolin3 and gelsolin wg. The four pSilencer 3.1-shRNA expressing vectors were transfected into Hca-F cells respectively using Lipofectamine 2000 according to the manufacturer,s interfering. For RT-PCR analysis of gelsolin mRNA levels, total RNA was isolated from cells using Trizol and cDNA was synthesized using RT-PCR Kit .PCR analysis was performed. Total cell lysates were separated by SDS-PAGE and transferred to PVDF membranes, which were incubated with the indicated antibodies. Bound antibodies were visualized by ECL. And the most effective pSilencer 3.1-shRNA vector was selected based on the results of RT-PCR and Western blotting. In the end, the Hca-F cells were transfected stably with the most effective pSilencer 3.1- gelsolin- shRNA expressing vector. 3. Hca-F cells, control cells and shRNAi cells were used for next experiment. About 1×106 cells in 100μl -RPMI1640 were seeded in duplicate into each well of the 96-well culture plates, and 10μl CCK-8 was added at 24,48,72 hours respectively. After 1 h incubation at 37℃in 5% CO2, the absorbency A450 was measured . The inhibitory effect of RNAi on Hca-F cells migration in vitro was demonstrated using 24-well transwell units with 8μm pore size polycarbonate filter. cells were harvested in 100μl serum-free medium containing 0.1% BSA and added to the upper chamber. The lower chamber contained 10μg/μl FN in 500μl RPMI 1640. Cells were incubated for 24 h at 37℃, 5% CO2 incubator. At the end of incubation the cells on the upper surface of filter were completely removed by wiping with a cotton swab. Then the filters were fixed in methanol and were stained with 0.1% gentian violet. 33% acetate acid was used to wash filters. The absorbency A450 of elution solution was measured. ECM was added in upper filter when metastasis in vitro was performed .In vivo tumor metastasis assay, three group cells were inoculated into footpad of 615 mouse subcutaneously. After three weeks, the mice were killed and their lymph nodes were isolated.Result: 1.The result of immunofluorescence showed that gelsolin protein was located at cytoplasm of hepatic cells; western blot showed that the expression of gelsolin was positive in Hca-F and Hca-P. Its expression in Hca-F cells was higher than that in Hca-P cells﹙P<0.05﹚. 2. The recombinant vectors were confirmed by enzyme digestion analysis and DNA sequencing.The shRNA expressing recombinants were transfected into Hca-F cells successfully. Gelsolin transcript (gelsolin1,gelsolin2,gelsolin3) was reduced by 46.8%,48.2%,20.2% respectively, and the protein of GSN was reduced by 73.9%,45.1%,31.2% respectively. shRNA-1 was found to have the best effect against gelsolin protein expression(P<0.01).Hca-F cell line with gelsolin stably down regulated using shRNA technique was successfully established, which laid a favorable foundation on the gene function study of gelsolin. 3. There was no influence of gelsolin to the proliferation of hepatic cells. In vitro transwell migration , the absorbency A450 of elution solution from Hca-F cells was(0.2480±0.0447), control cells (0.2239±0.0274) to shRNAi cells (0.1716±0.0232)(P<0.05) ; In vitro invasion assay the absorbency A450 of elution solution from Hca-F cells (0.2007±0.0129), control cells (0.2236±0.0446)to shRNAi cells (0.1557±0.0051)(P<0.05) ;In vivo tumor metastasis assay, lymph node metastasis rates of Hca-F cells and control cells was 90% and 80%; lymph node metastasis rates of shRNAi cells was 50%.Conclusion: 1.The expression of gelsolin was higher in Hca-F cells than that in Hca-P cells. 2. Hca-F cell lines with gelsolin stably down- regulated using shRNA technique was successfully established. 3. The ability of metastasis and invasion of mouse hepatocarcinoma ascites cell lines Hca-F was reduced by gelsolin after its down-regulation and proved that gelsolin played an important role in process of lymphatic metastasis of hepatocarcinoma.