**4.1 Cell culture and transfection**

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 establishing HepG2-TFPI-2 stable cell line.

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 construct (HepG2-TFPI-2).

### **4.2 RT-PCR**

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

ATAGGATCCACATGGACCCGCTCGC-3'

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

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

**6.** *In vitro* **cell migration and invasion assay** 

\* p< 0.05 vs HepG2-V or HepG2-P

TFPI-2).

Hepatocellular Carcinoma Cells and is Inactivated in Human Hepatocellular Carcinoma 167

Fig. 4. The growth curve of different groups of HepG2 cells (HepG2-P, HepG2-V and HepG2-

Invasion and migration of the hepatocarcinoma cells in vitro was measured by the invasion of cells through Matrigel-coated or -uncoated transwell inserts according to a procedure described previously (17). Briefly, transwell inserts (Corning) with 8-um pore size were coated (for invasion assay) or uncoated (for migration assay) with 50 ug of Matrigel matrix (BD Biosciences). Cells suspended in serum-free DMEM medium were seeded into upper chambers (100 ul/well) at a density of 1×10^6 cells/ml (for migration assay) or 3×10^5 cells/ml (for invasion assay). The lower chambers were filled with DMEM supplemented with 10% FBS. After 24 h of incubation, cells attached to the upper side of the filter were removed, and the filters were fixed and stained with hematoxylin and eosin. At this point, there was no difference in the total number of cells (proliferation rate) among the groups in the serum-free medium. The number of cells that had migrated to the undersurface of the

membrane was counted in five randomly-selected microscopic fields in each sample.

HepG2-TFPI-2 45 53 39 48 41 49.3 ± 5.9 **\*** HepG2-V 91 93 80 79 88 86.2 ± 6.4 HepG2-P 90 86 93 81 89 87.8 ± 4.5

HepG2-TFPI-2 142 132 139 152 137 140.4 ± 7.4 HepG2-V 134 148 160 140 147 145.8 ± 9.8 HepG2-P 150 166 159 142 147 152.8 ± 9.6

12345 *x* **±s** 

12345 *x* **±s** 

Groups Membranes in **matrigel invasion assay**

Groups Membranes **in migration assay**

Table 1. In vitro invasion of different groups of HepG2 cells

#### **4.3 Western blot**

HepG2-P HepG2-TFPI-2 and HepG2-V cells were grown to 80-90% confluence in six-well plates, after which the medium was replaced with serum-free medium and incubated for 24 h. Then the cultures were washed several times with PBS and the ECM was prepared as described by Rao *et al* (15). The ECM protein were supplemented with PMSF (1 mmol/L) to inhibit the proteases. The samples was mixed with equal volume of 2×SDS sample buffer and boiled for 5 min. Equal amounts of protein were resolved on 12% sodium dodecyl sulfate (SDS)-polyacrylamid gels, and then transfected onto polyvinylidene difluoride (PVDF) membrane (Millopore). After blocking with 5% non-fat milk, the membranes were incubated, first with primary antibody at 4C overnight and then with horseradish peroxidase (HRP)-conjugated sheep anti-mouse lgG secondary antibody. After washing, the blots were developed with a super-Enhanced Chemiluminescence Detection Kit (Applygen Technologies, Beijing, China).

By Western blot we found that a high level of TFPI-2 protein was detected in conditioned media of HepG2-TFPI-2 cells but not in that of HepG2-V or the HepG2-P cells (Fig. 3). These results proved that we successfully introduced TFPI-2 into HepG2 cells.

Fig. 3. Secretion of TFPI-2 in the conditioned media from HepG2-TFPI-2 cells. The conditioned media were collected from lanes 1, HepG2-P cells; 2, HepG2-TFPI-2 cells and 3, HepG2-V cells, and analyzed by Western blotting.

#### **5. Cell proliferation assay**

Cell proliferation was evaluated by MTT assay (sigma) according to a procedure described previously (16). In brief, every 24 h, for a total of 7 days, the cells from the three groups were harvested and 200 ul of cell suspension was added to each well in 96-well plates. A onetenth volume of MTT solution (5 mg MTT/ml PBS) was added to each well and incubated for 2-4 h at 37C until a purple precipitate was visible. The medium was then carefully removed, and precipitates were dissolved in 150 ul DMSO. Growth rate was plotted as the percentage of viable cells in HepG2-P control (a value arbitrarily set at 100%). Each experiment was repeated at least three times with each treatment given in duplicate or triplicate. Data were presented as an average of the results from individual experiments.

We examined the effect of TFPI-2 expression on the proliferation of HepG2 cells. The viability of cells was determined by MTT assay for 7 days and cell proliferation was obviously inhibited on the fourth day in HepG2-TFPI-2 cells but not in the other two groups of cells (Fig. 4). These results suggested that TFPI-2 could suppress the growth of hepatocarcinoma cells.

Fig. 4. The growth curve of different groups of HepG2 cells (HepG2-P, HepG2-V and HepG2- TFPI-2).

### **6.** *In vitro* **cell migration and invasion assay**

166 Hepatocellular Carcinoma – Basic Research

HepG2-P HepG2-TFPI-2 and HepG2-V cells were grown to 80-90% confluence in six-well plates, after which the medium was replaced with serum-free medium and incubated for 24 h. Then the cultures were washed several times with PBS and the ECM was prepared as described by Rao *et al* (15). The ECM protein were supplemented with PMSF (1 mmol/L) to inhibit the proteases. The samples was mixed with equal volume of 2×SDS sample buffer and boiled for 5 min. Equal amounts of protein were resolved on 12% sodium dodecyl sulfate (SDS)-polyacrylamid gels, and then transfected onto polyvinylidene difluoride (PVDF) membrane (Millopore). After blocking with 5% non-fat milk, the membranes were incubated, first with primary antibody at 4C overnight and then with horseradish peroxidase (HRP)-conjugated sheep anti-mouse lgG secondary antibody. After washing, the blots were developed with a super-Enhanced Chemiluminescence Detection Kit (Applygen

By Western blot we found that a high level of TFPI-2 protein was detected in conditioned media of HepG2-TFPI-2 cells but not in that of HepG2-V or the HepG2-P cells (Fig. 3). These

results proved that we successfully introduced TFPI-2 into HepG2 cells.

Fig. 3. Secretion of TFPI-2 in the conditioned media from HepG2-TFPI-2 cells. The

HepG2-V cells, and analyzed by Western blotting.

**5. Cell proliferation assay** 

conditioned media were collected from lanes 1, HepG2-P cells; 2, HepG2-TFPI-2 cells and 3,

Cell proliferation was evaluated by MTT assay (sigma) according to a procedure described previously (16). In brief, every 24 h, for a total of 7 days, the cells from the three groups were harvested and 200 ul of cell suspension was added to each well in 96-well plates. A onetenth volume of MTT solution (5 mg MTT/ml PBS) was added to each well and incubated for 2-4 h at 37C until a purple precipitate was visible. The medium was then carefully removed, and precipitates were dissolved in 150 ul DMSO. Growth rate was plotted as the percentage of viable cells in HepG2-P control (a value arbitrarily set at 100%). Each experiment was repeated at least three times with each treatment given in duplicate or triplicate. Data were presented as an average of the results from individual experiments.

We examined the effect of TFPI-2 expression on the proliferation of HepG2 cells. The viability of cells was determined by MTT assay for 7 days and cell proliferation was obviously inhibited on the fourth day in HepG2-TFPI-2 cells but not in the other two groups of cells (Fig. 4). These

results suggested that TFPI-2 could suppress the growth of hepatocarcinoma cells.

**4.3 Western blot** 

Technologies, Beijing, China).

Invasion and migration of the hepatocarcinoma cells in vitro was measured by the invasion of cells through Matrigel-coated or -uncoated transwell inserts according to a procedure described previously (17). Briefly, transwell inserts (Corning) with 8-um pore size were coated (for invasion assay) or uncoated (for migration assay) with 50 ug of Matrigel matrix (BD Biosciences). Cells suspended in serum-free DMEM medium were seeded into upper chambers (100 ul/well) at a density of 1×10^6 cells/ml (for migration assay) or 3×10^5 cells/ml (for invasion assay). The lower chambers were filled with DMEM supplemented with 10% FBS. After 24 h of incubation, cells attached to the upper side of the filter were removed, and the filters were fixed and stained with hematoxylin and eosin. At this point, there was no difference in the total number of cells (proliferation rate) among the groups in the serum-free medium. The number of cells that had migrated to the undersurface of the membrane was counted in five randomly-selected microscopic fields in each sample.


\* p< 0.05 vs HepG2-V or HepG2-P

Table 1. In vitro invasion of different groups of HepG2 cells

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

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We went further to examine the effect of TFPI-2 expression on the invasion of HepG2 cells. Based on invasion and migration assays, we counted the cells that passed through the membranes (Table 1). The results show that the number of cells passing through the membranes was significantly lower in the HepG2-TFPI-2 group than the other two groups ( P<0.05), indicating that TFPI-2 suppresses the invasive potential of hepatocarcinoma cells. While no significant difference in migration ability was observed in the three groups (Table 1).
