Underexpression of hepatocyte growth factor activator inhibitors HAI-1 and HAI-2 was associated with promoter hypermethylation in hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a major malignancy worldwide and is prevalent in Southeast Asia and Africa. The risk factors are well established but the molecular mechanism is unclear. We have carried out a high-throughput methylation study using cDNA microarray approach on HCC cells and investigated the genome-wide expression changes upon pharmacological demethylation using 5-aza-2’-deoxycytidine (5-aza-dC) in three HCC cell lines (Hep3B, SMMC7721, and BEL7402). We found that about 300 transcripts were commonly upregulated in at least two of the three HCC cell lines after 5-aza-dC treatment. Upon selection based on genomic sequence data analysis, a few putative tumor suppressor genes were identified and it is likely that aberrant promoter methylation significantly contributes to the inactivation of these tumor suppressor genes. Among these genes were two closely related genes of the same family, HGF activator inhibitors, HAI-1 and HAI-2. Both HAI-1 and HAI-2 can inhibit the generation of biologically active hepatocyte growth factor (HGF) through their interaction with HGF activator (HGFA). Structurally, these two HAIs belong to Kunitz-type serine protease inhibitor containing two Kunitz domains and a transmembrane domain. They are first synthesized as integral membrane proteins and subsequently released from the cell surface by proteolysis. Functionally, HAI-1 and HAI-2 inhibit not only HGFA but also other serine proteases, such as membrane-type serine protease 1 and plasmin. To further define the roles and importance of HAI-1 and HAI-2 in HCC, we examined the expression of HAI-1 and HAI-2 and their methylation status in HCC cell lines and human HCCs. Using RT-PCR on 14 HCC cell lines, we found that HAI-1 and HAI-2 mRNA expression were either absent or low in most of the cell lines, and 5-Aza-dC treatment significantly upregulated HAI-1 and HAI-2 expression in these cell lines (9/14 and 11/14, respectively). The mRNA expression levels of HAI-1 and HHAI-2 were also found to be frequently under-expressed in human HCC samples as compared with their corresponding nontumorous livers (58%, 11/19; 80%, 16/20, respectively) . Using bisulfite DNA sequencing, we confirmed that the promoter of HAI-1 and HAI-2 gene were frequently hypermethlyated in HCC cell lines and human HCC samples, and HAI-1 and HAI-2 promoter hypermethylation was associated with the low mRNA expression levels of HAI-1 and HAI-2. These results showed that HAI-1 and HAI-2 were frequently hypermethylated and under-expressed in human HCC. Our findings suggest that HAI-1 and HAI-2 inactivation may play a role in the development of HCC. (This study was funded in part by the Michael Kadoorie Cancer Genetic Research Program of the Kadoorie Charitable Foundation)