Epigenetic inactivation of LHX6 mediated microcystin-LR induced hepatocarcinogenesis via the Wnt/ß-catenin and P53 signaling pathways.

Microcystins (MCs) have been shown to be carcinogenic by animal and cellular experiments and found to be associated with the development of human hepatocellular carcinoma (HCC) through epidemiological studies. However, the molecular mechanism of microcystin-LR (MC-LR) induced HCC is still unclear. This study is determined to clarify the role and mechanism of LHX6 in MC-LR-induced hepatocarcinogenesis. Using the previously established MC-LR-induced malignant transformation model in L02 cells, we screened out LHX6, homeobox gene that was significantly changed. We found that LHX6 was significantly down-regulated in MC-LR treated L02 cells and the liver tissue of rats treated for 35 weeks with 10 µg/kg body weight of MC-LR. Expression of LHX6 in human tumor tissue was significantly down-regulated in high MC-LR-exposure group. LHX6 was hypermethylated in MC-LR treated L02 cells and up-regulated after treatment with 10 µM of 5-aza-2'-deoxycytidine. Furthermore, overexpression of LHX6 inhibited proliferation, invasion and migration of malignantly transformed L02 cells in vitro and in vivo, while knockdown of LHX6 resulted in an opposite phenotype. In addition, we found that up-regulation of P53 and Bax resulted in apoptosis, and that down-regulation of CTNNB1 and MMP7 led to migration of MC-LR treated L02 cells. Blockade of P53 and CTNNB1 by its inhibitor significantly diminished the effect of LHX6. These genes were working together during the process of MC-LR-induced hepatocarcinogenesis. Our study demonstrated for the first time that LHX6 gene expression is regulated by DNA methylation and can inhibit the proliferation, invasion and migration through Wnt/ß-catenin and P53 signaling pathways during the MC-LR-induced hepatocarcinogenesis. This result may suggest that LHX6 gene can be used as a potential target gene and a biomarker for liver cancer treatment.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway.

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.