Silencing of microRNA-122 is an early event during hepatocarcinogenesis from non-alcoholic steatohepatitis

Abstract

Non-alcoholic steatohepatitis (NASH) has emerged as a common cause of chronic liver disease and virus-independent hepatocellular carcinoma (HCC) in patients with obesity, diabetes, and metabolic syndrome. To reveal the molecular mechanism underlying hepatocarcinogenesis from NASH, microRNA (miRNA) expression profiles were analyzed in STAM mice, a NASH-HCC animal model. MicroRNA expression was also examined in 42 clinical samples of HCC tissue. Histopathological images of the liver of STAM mice at the ages of 6, 8, 12, and 18 weeks showed findings compatible with fatty liver, NASH, liver cirrhosis (LC), and HCC, respectively. Expression of miR-122 in non-tumor LC at the age of 18 weeks was significantly lower than that in LC at the age of 12 weeks. Expression of miR-122 was further decreased in HCCs relative to non-tumor LC at the age of 18 weeks. Expression of miR-122 was also decreased in clinical samples of liver tissue showing macrovesicular steatosis and HCC, being consistent with the findings in the NASH model mice. DNA methylation analysis revealed that silencing of miR-122 was not mediated by DNA hypermethylation of the promoter region. These results suggest that silencing of miR-122 is an early event during hepatocarcinogenesis from NASH, and that miR-122 could be a novel molecular marker for evaluating the risk of HCC in patients with NASH.

Keywords: Fatty liver; hepatocellular carcinoma; miR-122; microRNA; non-alcoholic steatohepatitis.

Figure 1

Body and liver weight and biochemical examination of STAM mice. (a) Body and liver weight of control and STAM mice at the ages of 6, 8, 12, and 18 weeks. Blank and filled bars represent the average +SD of control and STAM mice, respectively. (b) Biochemical examination of serum AST, ALT, total cholesterol (T-Cho), and triglyceride (TG) in STAM mice at the ages of 6, 8, 12, and 18 weeks. Blank and filled bars represent the average ±SD for control and STAM mice, respectively.

Figure 2

Histopathological images of the liver of STAM mice. (a) Histopathological images in the liver of control (12 weeks) and STAM mice (8 and 12 weeks). Hematoxylin–eosin (HE), Azan, and Sirius red staining showed fatty liver with moderate inflammatory infiltrate include neutrophils, lymphocytes, and monocytes, and ballooning degeneration of hepatocytes in STAM mice at the age of 8 weeks. Azan and Sirius red staining showed liver fibrosis in STAM mice at the age of 12 weeks. (b) Macroscopic appearance of the liver in STAM mice at the age of 18 weeks. The liver of STAM mice showed a granular surface and tumor protrusion. (c) HE staining of the liver in STAM mice at the age of 18 weeks. Tumors are pathologically compatible with hepatocellular carcinoma (HCC) (arrows). NASH, non-alcoholic steatohepatitis.

Figure 3

Expression profiles of microRNAs (miRNAs) in the liver of STAM mice. (a) Microarray analyses of miRNA expression profile in hepatocellular carcinoma (HCC) tissues (T) compared with non-tumor liver tissues (N) in two STAM mice at the age of 18 weeks (HCC1 and HCC2). (b) MicroRNA-122 (miR-122) expression in the liver of control (6 and 29 weeks) and STAM mice (6, 8, 12, and 18 weeks). miR-122 expression normalized with U6 is represented as average +SD. Downregulation of miR-122 in the liver of STAM mice from the age of 12–18 weeks for non-tumor (N) and HCC (T) was significant (*P < 0.05).

Figure 4

Levels of microRNA-122 (miR-122) expression in clinical samples obtained from hepatocellular carcinoma (HCC) patients. (a) Histopathological images (HE staining, ×100) of non-tumor liver tissue from a hepatitis B virus-negative/hepatitis C virus-negative (NBNC) HCC patient, showing steatosis. This case was considered to be grade 3 (macrovesicular steatosis >66%), with chronic portal inflammation and pericellular fibrosis. (b) The average levels of miR-122 expression in HCC tissues (T, clear bars) and non-tumor liver tissues (N, filled bars). Tissue specimens of HCC and the surrounding non-tumor liver were obtained from patients with NBNC HCC with or without liver steatosis, as well as patients with hepatitis C virus-positive and hepatitis B virus-positive HCC. miR-122 expression is normalized with U6. *P < 0.05.

Figure 5

Promoter assay of microRNA-122 (miR-122) expression. (a) The promoter region of miR-122, which contains a TATA-box, a CCAAT-box, and DR-1 and DR-2 elements. DNA methylation status was determined by bisulfite pyrosequencing at the CpG sites indicated by asterisks. Arrow indicates the transcription start site (TSS), as described previously. (b) Promoter assay of miR-122 expression using a Dual Luciferase Reporter Assay System. Fragments of the human miR-122 promoter with or without the DR-1 and DR-2 elements were inserted between the SacI and HindIII sites within pGL4.10. Plasmids with or without Sss I CpG methylase treatment were cotransfected with Renilla luciferase expression vector into HepG2 cells. Forty-eight hours after transfection, luciferase activities were measured. *P < 0.01; **P < 0.005.

Figure 6

DNA methylation status of the promoter region of microRNA-122 (miR-122) in liver cancer cell lines and hepatocellular carcinoma (HCC) tissues. (a) DNA methylation levels of the miR-122 promoter region in HepG2 and HuH7 cells treated with 1 or 3 μM 5-aza-2′-deoxycytidine (5AZA). Methylated (MC) and unmethylated (UC) control DNAs were used as controls. (b) Average levels of miR-122 expression in HepG2 and HuH7 cells treated with 5AZA. miR-122 expression is normalized with U6. *P < 0.05. (c) Levels of DNA methylation in the miR-122 promoter region in HCC tissues (T, white columns) and non-tumor liver tissues (N, filled columns). Tissue specimens of HCC and the surrounding non-tumor liver were obtained from patients with hepatitis B virus-negative/hepatitis C virus-negative (NBNC) HCC with or without liver steatosis, as well as from patients with hepatitis C virus-positive and hepatitis B virus-positive HCC. Methylated (MC) and unmethylated (UC) control DNAs were used as controls. *P < 0.005. (d) Levels of DNA methylation in the miR-122 promoter region in non-tumor liver tissues (NT) and HCCs of STAM mice. Normal liver tissues obtained from C57BL/6J mice were used as a control (Cont). DNA from normal mouse liver treated with Sss I methylase (in vitro methylated DNA [IVD]) was used as a control for methylated DNA.