Downregulation of heparanase by RNA interference inhibits invasion and tumorigenesis of hepatocellular cancer cells in vitro and in vivo

Abstract

Abstract.Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. The expression of heparanase is associated with invasion, as well as the angiogenic and metastatic potential of diverse malignant tumors. We used RNA interference strategies to evaluate the role of human heparanase in a liver cancer cell line and to explore the therapeutic potential of its specific targeting. Using an online siRNA tool, we designed three small interfering RNA sequences to target the heparanase coding region and cloned them into the pGenesil-1 vector. The siRNA vectors were transfected into HepG2 liver cancer cells. Heparanase expression was measured by real-time RT-PCR and Western blotting. Cell proliferation was detected by MTT staining and plate colony formation. Cell cycle analysis was performed by flow cytometry. In vitro invasion was measured by Matrigel invasion assay. We also analyzed tumorigenicity in heparanase-suppressed HepG2 cells in nude mice. We found that siRNA-1 (1214-1232) and siRNA-3 (611-629) targeting heparanase significantly downregulated the expression of heparanase in HepG2 liver cancer cells. Compared with its controls, siRNA-1 or siRNA-3 vectors efficiently inhibited the proliferation and invasion of HepG2 liver cancer cells in vitro and tumorigenesis in vivo. These results suggest that heparanase-specific RNA interference has potential value as a novel therapeutic agent for human liver cancer.

miR-1207-5p and miR-1266 suppress gastric cancer growth and invasion by targeting telomerase reverse transcriptase

“…39 For the cell cycle analyses, cells were plated into six-well plates and flow cytometry was performed 48 h later, as described previously. 40 …”

Versatile role of heparanase in inflammation

“…Remarkably, induction of heparanase prior to the appearance of malignancy was reported in essentially all of the above-mentioned inflammatory conditions, i.e., Barrett's oesophagus (Brun et al, 2009; Sonoda et al, 2010), hepatitis C infection (El-Assal et al, 2001), chronic pancreatitis (Koliopanos et al, 2001), Crohn disease and ulcerative colitis (Lerner et al, 2011; Waterman et al, 2007). Given the causal role the enzyme plays in tumor progression in tissues in which cancer-related inflammation typically occurs [i.e., gastrointestinal tract, pancreas, liver (Brun et al, 2009; El-Assal et al, 2001; Hoffmann et al, 2008; Koliopanos et al, 2001; Naomoto et al, 2005; Nobuhisa et al, 2005; Sonoda et al, 2010; Xiong et al, 2012; Zhang et al, 2007)], it is conceivable that inflammation-induced heparanase may be involved in coupling inflammation and cancer. The findings obtained in a study utilizing mouse model of colitis-associated colon carcinoma support this notion (Lerner et al, 2011).…”

“…Demonstration of enhanced growth/aggressiveness of numerous cancer cell types following over-expression of heparanase (Cohen et al, 2006; Doviner et al, 2006; Lerner et al, 2008; Mahtouk et al, 2007; Vlodavsky et al, 1999; Yang et al, 2005), as well as inhibition of the tumorigenic/metastatic abilities of cancer cells following heparanase gene silencing (Edovitsky et al, 2004; Jiang et al, 2012; Lerner et al, 2008; Roy et al, 2005; Xiong et al, 2012) provided direct evidence for a crucial role of the enzyme in tumor progression. Preferential over-expression of the enzyme in human carcinomas of various origins, and association of augmented levels of heparanase with reduced patients' survival post operation, further highlight the role of heparanase in sustaining the pathology of malignant tumors (Ilan et al, 2006; Vreys and David, 2007).…”

Gene silencing of heparanase results in suppression of invasion and migration of hepatoma cells

“…Recently, RNAi has been successfully used to interfere with the expression of a variety of target genes to inhibit the growth of different tumor cells. In addition to cancer genes, genes that express anti-apoptotic molecules, telomerase and growth factor receptors as well as some signaling molecules have also been interfered with in other anti-tumor studies [17,18]. RNAi has been used to explore the function and interaction of genes, providing a convenient way to screen the target genes of new drugs.…”