Abstract
To investigate the role of miR-122 in the development and regression of non-alcoholic fatty liver disease (NAFLD) in vitro, we used multicellular 3D human liver organoids developed in our laboratory. These organoids consist of primary human hepatocytes, Kupffer cells, quiescent stellate cells and liver sinusoidal endothelial cells. They remain viable and functional for 4 weeks expressing typical markers of liver function such as synthesis of albumin, urea, and alpha-1 p450 drug metabolism. Before mixing, hepatic cells were transduced with lentivirus to inhibit miR122 expression (ABM, CA). Immediately after the organoids were fully formed (day 4) or after 1 or 2 weeks of additional incubation (days 11 or 18), the organoids were analyzed using fluorescent live/dead staining and ATP production; total RNA was extracted for qPCR gene expression profiling. Our results show that miR-122 inhibition in liver organoids leads to inflammation, necrosis, steatosis and fibrosis. This was associated with increase in inflammatory cytokines (IL6, TNF), chemokines (CCL2, CCL3) and increase in a subset of Matrix Metaloproteinases (MMP8, MMP9). An altered expression of key genes in lipid metabolism (i.e LPL, LDLR) and insulin signaling (i.e GLUT4, IRS1) was also identified. Conclusion: Our results highlight the role of miR-122 inhibition in liver inflammation, steatofibrosis and dysregulation of insulin signaling. Patients with NAFLD are known to have altered levels of miR-122, therefore we suggest that miR-122 mimics could play a useful role in reversing liver steatofibrosis and insulin resistance seen in patients with NAFLD.
Highlights
Primary human hepatocytes (PHH) when maintained in conventional 2D monolayer cultures de-differentiate and rapidly lose hepatocyte-specific functions [1]
Liver fibrosis is characterized by excessive extracellular matrix (ECM) deposition and fibrous scar formation, which is mainly due to the activation and transition of hepatic stellate cells [HSC, known as Ito or fat-storing cells] to myofibroblasts in response to fibrogenic stimuli
Nonalcoholic steatohepatitis (NASH), which is characterized by the presence of hepatic steatosis and inflammation with hepatocyte injury with or without fibrosis is a more advanced stage of fatty liver diseases that has reached epidemic proportions world-wide-with a global prevalence estimated as high as 25% [5,6]
Summary
Primary human hepatocytes (PHH) when maintained in conventional 2D monolayer cultures de-differentiate and rapidly lose hepatocyte-specific functions [1]. Such cultures only partially mimic the microenvironment of the liver because they lack other major hepatic cells and cellular microarchitecture. It is known that liver inflammation plays a major role in development of hepatic fibrosis. Nonalcoholic steatohepatitis (NASH), which is characterized by the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis is a more advanced stage of fatty liver diseases that has reached epidemic proportions world-wide-with a global prevalence estimated as high as 25% [5,6]. Consistent with this notion, it was recently found that pharmacological inhibition of CCL2 can decrease hepatic inflammation and steatohepatitis in animal models of NASH [7]
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