Tissue Factor Pathway Inhibitor-2 Gene Cloning And Expression, And Its Effects On Hepatocarcinoma

Objective: To clone TFPI-2 cDNA and construct a recombinant eukaryotic expression vector of Chinese TFPI-2 gene; to explore the role of TFPI-2 in hepatocellular carcinoma in vitro and elucidate the relationship between TFPI-2 expression and hepatoma migration and invasion. To find a new therapy of hepatocarcinoma.Methods: First stage: RNA from hepatic tissue of human fetor was isolated and full length TFPI-2 cDNA was amplified by RT-PCR. The cloned gene was inserted into plasmid pcDNA2.1 and sequenced from forward and reverse direction, then it was inserted into eukaryotic expression vetor pcDNA3.1, verified by enzyme digestion and sequencing. Second stage: TFPI-2- pcDNA3.1 was transfected into hepatoma cells line HepG2. After the transfected cells were screened by G418, transfected cells were examined for TFPI-2 mRNA and protein expressions by RT-PCR and Westenblot analysis. Third stage: The number of transfected or nontransfected cells passing through membrane of Transwell chamber was counted as the basis assessing tumor cells migratory and invasive behaviors. Used TFPI-2 probe to detect the expression difference of TFPI-2 mRNA among common, cirrhosis and hepatoma hepatic tissue.Results: Chinese TFPI-2 gene is 1222bp. Sequencing results showed that the cloned Chinese TFPI-2 gene has three bases different with that registered in Genbank. Our sequencing results has been transmitted and accepted by Genbank, accession number is TFPI AY691946. TFPI-2 gene was inserted to eukaryotic expression vetor pcDNA3.1 successfully. The result of nucleotide sequencing confirmed that the recombinant vector pcDNA3.1-TFPI-2 was constructed accurately. Expression of mRNA and protein of TFPI-2 were confirmed in transfected cells. In situ hybridization study showed that there are abundant TFPI-2 expressions in common hepatic tissue and greatly decreased in hepatic cirrhosis and hardly any in hepatoma. In invasion assay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obvisously decreased compared with that of nonexpressing cells (49.3±5.9 vs 98.7±6.8, P<0.01); While in migration assay, no significant difference was observed between transfected and nontransfected cells (115.3±6.0 vs 121.7±7.1, P>0.05).Conclutions: There are abundant expressions of TFPI-2 in normal hepatic tissue and significant down-regulation of expression in hepatoma. The expression of TFPI-2 would inhibit the invasive ability of HepG2 cells in vitro, which may provide a target for treating human hepatocellular carcinoma with TFPI-2 protein inhibitors in therapy.