Study On The Expression Of Tissue Factor Pathway Inhibitor-2 Gene And Hepatocarcinoma Growth, Invasion And Migration

OBJECTIVETo detect the expressive difference of tissue factor pathway inhibitor-2 (TFPI-2) gene in man live cancer tissue and tumor-adjacent normal tissue. Transfecting the eukaryotic expression vector, pcDNA3.1-TFPI-2, into human live cancer cells line HepG2 and screening TFPI-2 positive clones, explore the effect and mechanism of TFPI-2 gene on cell growth, invasion and migration of hepatocarcinoma cell.METHODSFirst stage: The expressive differences of TFPI-2 mRNA and protein were determined by in situ hybridization (ISH), immunohistochemical staining (IHC) and Western blot in live cancer tissue and hepatic tissue respectively. Second stage: The recombinant expression vector pcDNA3.1-TFPI-2 was transfected into human live tumor cells strain HepG2 with liposome, and TFPI-2 positive clones were screened by G418. Then transfected and nontransfected HepG2 cells were examined for the expressions of TFPI-2 mRNA and protein by reverse transcription- polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Third stage: The growth curve for transfected and nontransfected cells was drawn with MTT assay and the change of cell growth cycle was evaluated by flow cytometry. The number of transfected or nontransfected cells passing through membrane of Transwell chamber was counted as basis assessing tumor cells invasive and migrative behaviors.RESULTSThe expression of TFPI-2 mRNA was obviously lower in carcinoma than normal tissues (P < 0.05); the positive indexes were 30.66±2.47 and 88.23±1.43. The expression of TFPI-2 protein in carcinoma was lower than that of normal tissues (P < 0.05), the positive indexes were 22.54±1.22 and 46.60±1.80. The gray scale of protein bands was analyzed with software Quantity One and the ratios of TFPI-2 toβ-actin were 0.89±0.15 and 1.14±0.13 individually. There was a significant difference between the cancerous tissue and tumor-adjacent tissue (P < 0.05). Expressions of TFPI-2 mRNA and protein were confirmed in transfected cells. Compared with nontransfected cells, the growth of HepG2 cells transfected with TFPI-2 was inhibited significantly and the transfected cells showed a significant increase in G1/G0 phase. In invasion assay, the number of TFPI-2-expressing cells was obviously decreased compare with that of nonexpressing cells(45.2±5.6 vs 87.8±4.5, P < 0.05); while in migration assay, no significant difference was observed between transfected cells and nontransfected cells (140.4±7.4 vs 152.8±9.6, P > 0.05).CONCLUSIONSThe expression of TFPI-2 gene is obviously lower in hepatocarcinoma than normal hepatic tissue. The transfection of exogenous TFPI-2 gene can inhibit the growth and cell proliferation of tumor cells. Expression of TFPI-2 gene may strongly inhibit the invasive ability of liver cancer cells in vitro, but has no effect on the migratory ability, which provides an experimental basis for further study on the new genetic treatment approach of hepatocellular carcinoma.