Overexpression of proteinase inhibitor 9 is associated with poor prognosis in human hepatocellular carcinoma and with proliferation and apoptosis in HepG2 cells

Excessive proteinase inhibitor 9 (PI9) levels predict a poor outcome for patients with several tumor types. We compared the expression of PI9 in HepG2 cells and in 21 pairs of tumor and peritumor tissue samples using western blotting. Immunohistochemical staining was used to detect PI9 expression in 105 cases of hepatocellular carcinoma (HCC) and in 20 adjacent normal liver tissues. Changes in the degree of apoptosis and proliferation were determined before and after transfection with pcDNA3.1-PI9 and small interfering (si)RNA-PI9 using MTT analysis, colony formation assay, and flow cytometry. The correlation between PI9 expression and prognosis was determined in a large HCC patient cohort (n=105). Western blotting showed that PI9 expression was significantly higher in tumor tissues than in adjacent tissues. PI9 immunohistochemical staining was positive in 78.1% of HCC tissues, which was significantly higher than that seen in adjacent normal liver tissue (35%). PI9 expression in HCC correlated closely with the extent of tumor differentiation, tumor-node-metastasis staging, and tumor size. Cox regression analysis demonstrated that PI9 is an independent predictor of prognosis in patients with HCC, and is related to overall survival. The apoptosis of HepG2 cells was significantly increased following PI9 up-regulation and significantly decreased after siRNA interference against PI9 expression. Cell proliferation was significantly decreased following PI9 up-regulation and significantly increased after siRNA interference of PI9 expression. PI9 appears to contribute to the apoptosis of HCC, and could be an independent predictor of recurrence and a suitable pharmaceutical target in patients with HCC.