**3. TFPI-2 plasmid expression vector construct**

#### **3.1 TFPI-2 gene sequencing**

162 Hepatocellular Carcinoma – Basic Research

In Situ Hybridization Detection Kit (Boster, Wuhan, China) according to the manufacturer's instructions. Briefly, the sections were hybridized in prehybridization buffer supplemented with 0.1 ug/ml digoxigenin-labeled, 1.2-kb antisenese TFPI-2 probe overnight at 37C and incubated with biotinylated mouse antidigoxigenin antibody (1:1000 dilution), then incubated with biotinylated peroxidase. Staining was developed with DAB. Slides were counterstained with hematoxylin, dehydrated, and mounted. The number of cells stained brown (indicating the presense of TFPI-2 mRNA) were assessed by light microscopy. The hybridization probe replaced with PBS was used as a negative control. Mature placenta

Tissue sections were prepared in the same manner as above. Then the expression of TFPI-2 was determined by incubation with a mouse polyclonal antibody against human TFPI-2 (Santa Cruz, CA), horseradish peroxidase (HRP)-conjugated sheep anti-mouse lgG secondary antibodies (Chinagen, Shenzhen, China), and final detection using the non Biotin-labeled

Fig. 1. TFPI-2 expression in normal hepatic and hepatocarcinoma tissues. Expression of (A) TFPI-2 mRNA in hepatocarcinoma tissue and (B) tumor-adjacent normal hepatic tissue was examined by *in situ* hybridization with a digoxigenin-labeled TFPI-2 probe. Expression of (C) TFPI-2 protein in hepatocarcinoma tissue and (D) tumor-adjacent normal hepatic tissue was examined by immunohistochemical analyses with TFPI-2 antibody. Magnification, x400.

tissue, known to express large amounts of TFPI-2, was used as a positive control.

**2.3 Immunohistochemistry** 

The RNA from hepatic tissue of human fetor (Shenzhen People's Hospital) was isolated and full length TFPI-2 cDNA was amplified with RT-PCR kit (TaKaRa). The cloned gene was inserted into plasmid pcDNA2.1 (Chinagen, Shenzhen, China), and sequenced from forward and reverse direction at Shanghai Biotechnology (China), then it was inserted into eukaryotic expression vetor pcDNA3.1, a gift from Dr Tiyuan Li (Central Laboratory, Shenzhen People's Hospital) verified by enzyme digestion and sequencing.


Tissue Factor Pathway Inhibitor-2 Inhibits the Growth and Invasion of

vector pcDNA3.1-TFPI-2 was constructed accurately.

**4. Construct HepG2-TFPI-2 stable cell line** 

**4.1 Cell culture and transfection** 

construct (HepG2-TFPI-2).

**4.2 RT-PCR** 

establishing HepG2-TFPI-2 stable cell line.

ATAGGATCCACATGGACCCGCTCGC-3'

**3.2 Plasmid construct** 

sequencing.

Hepatocellular Carcinoma Cells and is Inactivated in Human Hepatocellular Carcinoma 165

A 0.7-kb fragment encoding TFPI-2 cDNA was amplified from normal liver tissue with the primers 5'-GCTTTCTCGGACGCCTTGC-3' and 5'-GAATACGACCCCAAGAAATGAGTGA-3'. PCR product was purified and cloned into the BamHΙ and XhoΙ sites of the pCDNA3.1 expressing vector. The DNA sequence of the recombinant plasmid was confirmed via DNA

The Chinese TFPI-2 gene is 1222bp. Sequencing results showed that the cloned Chinese TFPI-2 gene has three bases different (258, 585 and 884 bp) with that registered in Genbank (Fig. 2). 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

To explore the functional role of TFPI-2 in HCC, we employed HepG2 cells as a model. Based on RT-PCR, we found that the expression of TFPI-2 mRNA in HepG2 cells was undetected (data not shown), therefore we introduced TFPI-2 into HepG2 cells by

Human hepatoma HepG2 cells were obtained from Cancer Institute, Chinese Academy of Medical Sciences, and cultured in 6% CO2 to 94% air and 96% humidity at 37C in DMEM supplemented with 10% bovine calf serum (Hyclone, Logan, UT), 1.0% glutamine, 100 ug/ml strepotomycin, 100 ug/ml penicillin. The recombinant constructs or pCDNA3.1 vector was transfected into HepG2 cells using Lipofectamine 2000 transfection reagent (Invitrogen) according to the manufacturer's instructions. Selection of transfected cells with 0.8 mg/ml G418 sulfate (Invitrogen) was initiated 48 h after transfection. After a 4 week selection, stable transfectants were expanded and used for the study. The HepG2 cells were divided into three groups: HepG2 parental cells (HepG2-P), HepG2 cells transfected by pCDNA3.1 vector (HepG2-V) and HepG2 cells transfected by TFPI-2

Total RNA was isolated from HepG2 cells using TRIZOL reagent (Invitrogen) following a standard protocol. Using the 2-step RT-PCR kit (TaKaRa), cDNA was synthesized with RNA as the template. PCR amplification of human TFPI-2 and ß-actin was performed with Taq Master Mix (Promega, Madison, WI, USA) with synthesized cDNA. The primer were

and 5'-GGCCTCGAGAAATTGCTTCTTCCGAATTTCC-3', amplicion 700 bp. ß-actin 5'-CTGGCACCACACCTTCTACAATG-3' and 5'-AATGTCACGCACGATTTCCCGC-3'. The PCR condition were: denaturing at 95C for 30 sec, annealing at 52C for 30 sec, and extension at 72C for 40 sec for 32 cycles. After electrophoresis of PCR products, the data were analyzed by Image Master Tatal Laboratory ID software. The level of TFPI-2 mRNA

synthesized by Shanghai Biotechnology (China) as follows: TFPI-2 5'-

was calculated by the ratio of density of TFPI-2 to ß-actin.


Fig. 2. Homology analysis of TFPI-2 gene

#### **3.2 Plasmid construct**

164 Hepatocellular Carcinoma – Basic Research

Fig. 2. Homology analysis of TFPI-2 gene

A 0.7-kb fragment encoding TFPI-2 cDNA was amplified from normal liver tissue with the primers 5'-GCTTTCTCGGACGCCTTGC-3' and 5'-GAATACGACCCCAAGAAATGAGTGA-3'. PCR product was purified and cloned into the BamHΙ and XhoΙ sites of the pCDNA3.1 expressing vector. The DNA sequence of the recombinant plasmid was confirmed via DNA sequencing.

The Chinese TFPI-2 gene is 1222bp. Sequencing results showed that the cloned Chinese TFPI-2 gene has three bases different (258, 585 and 884 bp) with that registered in Genbank (Fig. 2). 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.
