**7. Statistical analysis**

All data were presented as mean±SD. Stastical analysis was performed with SPSS statistical software. The Student two-tailed *t* test was used to compare the difference between groups, *p*<0.05 was considered to be statistically significant.

TFPI-2 is a serine proteinase inhibitor which is frequently downregulated in malignant tumors (18). Previous studies have demonstrated that silencing of TFPI-2 by either histone deacetylation (19) or promoter hypermethylation contributes to its inactivation and tumor progression in several cancers including glioma (18), choricarcinoma (20), pancreatic carcinoma (17), lung carcinoma (21), breast cancer (22), melanoma (23) and hepatocarcinoma (24). In addition, the aberrant splicing form of TFPI-2 was detected during cancer progression (25), which represents an untranslated form providing another mechanism by which TFPI-2 is downregulated in tumor cells.

In this study, we investigated the expression and function of TFPI-2 in HCC. We first applied the *in situ* hybridization and immunohistochemistry methods to evaluate the expression of TFPI-2 mRNA and protein in hepatocarcinoma tissues and tumor-adjacent normal hepatic tissues. Consistent with previous studies, our results showed that TFPI-2 expression at both mRNA and protein levels was low in hepatocarcinoma tissues compared to adjacent normal hepatic tissues. These results indicated that a decreased expression of TFPI-2 is implicated in HCC.

To find the mechanism by which TFPI-2 loss contributes to HCC, we employed HepG2 cells as a model. Our results demonstrate that reconstitution of TFPI-2 into HepG2 cells could inhibit the proliferation and invasion of HepG2 cells. Although the details for TFPI-2-mediated growth suppression are unknown, a previous study suggested that TFPI-2 induces apoptosis in glioma cells (26). Further studies are necessary to examine whether TFPI-2 promotes apoptosis of HepG2 cells. In agreement with previous reports that overexpression of TFPI-2 reduced the invasion of cancer cell lines derived from melanoma (27), prostate cancer (28), choriocarcinoma (29), glioblastoma (30) or meningiomas (31), our results showed that restoration of TFPI-2 was associated with a twofold decrease in invasive ability of HepG2 cells. In fact, TFPI-2 is thought to play a pivotal role in the regulation of plasmin-mediated ECM proteolysis during tumor invasion and metastasis (14). TFPI-2 inhibits the release of plasminor trypsin-dependent activation of pro-matrix metalloproteinase (MMP)-1 and pro-MMP-3, which leads to diminished ECM degradation and decreased invasion of HT-1080 fibrosarcoma cell lines (32, 33). In addition, TFPI-2 can inhibit MMP-2 activation in HT-1080 cells (34) and inhibit MMP-1, MMP-13, MMP-2 and MMP-9 in experimental models (35). Thus we assume that TFPI-2 inhibits HCC invasion and metastasis through modulating the activity of MMPs.

In summary, we reported that TFPI-2 expression is lost in HCC. The results of our *in vitro* studies confirm that restoration of TFPI-2 caused decreased proliferative and invasive behaviors of HepG2 cells. Taken together, these data suggest that inactivation of TFPI-2 may contribute to the malignant behavior in hepatocarcinoma. Additional in vivo studies will help determine whether restoration of TFPI-2 in hepatocarcinoma cells may represent a novel therapeutic approach for HCC.
