As previously stated, we created a hepatocyte-specific HDAC3-ablation mouse (
HDAC3LCKO) [
16,
18]. HDAC3 was entirely deleted in the hepatocytes of
HDAC3LCKO mice, according to Western blot analysis and immunohistochemistry (Fig.
1A and B). At 2 months of age,
HDAC3LCKO mice had significantly larger livers (Fig.
1 C and
1D) and higher serum ALT and AST levels (Fig.
1E and F). ALT levels in
HDAC3LCKO females were substantially higher than those in
HDAC3LCKO males. Meanwhile, staining for Ki67, a proliferative cell marker, showed a large abundance of proliferating cells in the mutant liver (Fig.
1G), indicating compensatory regeneration upon spontaneous liver injury.
HDAC3LCKO mice had substantial expansion and steatosis of hepatocytes in histology, as revealed by H&E staining (Fig.
1G). Glutamine synthetase (GS) is specifically expressed in the central venous region of the hepatic lobule. However, GS
+ cells were irregularly scattered in the lobules of
HDAC3LCKO livers, showing disarrangement of the lobules. Cytokeratin 19 (CK19) is a marker for bile duct cells and a subset of hepatic progenitor cells (HPCs). Active HPC expansion, known as a ductular reaction, is often considered to repair the chronically injured liver by differentiating into hepatocytes or bile duct cells. The livers of
HDAC3LCKO mice showed substantial proliferation of CK19
+ cells (Fig.
1G), indicating persistent damage to the mutant liver. Notably, at 9 months of age, liver nodules were observed in more than half of the
HDAC3LCKO female mice (64%, 21/33), but not in the
HDAC3LCKO male mice (0%, 0/27) (Fig.
2A and B). The liver-to-body weight ratio in females was not significantly different from that in males (Fig.
2C). The average number of liver nodules per female mouse was 5, and almost all tumours were smaller than 5 mm (Fig.
2D and E). When stained with H&E, tumour cells had basophilic cytoplasm and large, irregular nuclei (Fig.
2F). Hepatocyte nuclear factor 4α (HNF4α) is a marker for the unique recognition of hepatocytes. HNF4α
+ cells were found throughout the tumour, while CK19
+ cells were found outside the tumour (Fig.
2F), indicating that the tumour originated from hepatocytes. In addition, Ki67 immunohistochemistry staining indicated the active growth of the tumour cells (Fig.
2F). All
HDAC3LCKO female mice developed large liver tumours (> 5 mm) at 12 months. Although tumour nodules were seen in the livers of
HDAC3LCKO male mice, the number of total tumours and nodules larger than 5 mm were much lower in males (Fig.
2A, B and D, and E). As a result, the liver-to-body weight ratio in females was much higher than that in males (Fig.
2C). Taken together,
HDAC3LCKO female mice developed spontaneous HCC much earlier than male mice.