miR-122 inhibition in a human liver organoid model leads to liver inflammation, necrosis, steatofibrosis and dysregulated insulin signaling

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.

miR-122 promotes hepatic lipogenesis via inhibiting the LKB1/AMPK pathway by targeting Sirt1 in non-alcoholic fatty liver disease

“…Furthermore, several studies also confirmed a positive association between circulating miR-122 and histopathological features in NAFLD patients (Jampoka et al, 2018; Ye et al, 2018; Akuta et al, 2016; Miyaaki et al, 2014). However, some previous studies showed that liver miR-122 levels were decreased while serum miR-122 levels were increased in human non-alcoholic steatohepatitis (Braza-Boils et al, 2016; Latorre et al, 2017; Sendi et al, 2018), which was not consistent with our results. We speculated that the different results might be due to the different experimental models and induction conditions of NAFLD, as well as the different severities of NAFLD patients.…”

Expression of inflammatory interleukins and selected miRNAs in non-small cell lung cancer

“…In addition, miR-122 affects expression level of both IL-1β 17 and IL-6 18 as well as is believed to impact a range of cancer-related processes, such as inhibiting metastatic processes in lung cancer 19 and promoting cell proliferation and invasion in renal cell carcinoma 20 . In addition to its influence on the cell cycle, it also affects glucose metabolism in breast cancer 21 and inflammation in the liver 22 . Very few studies have examined the influence of miR-122 on inflammatory processes in lung cancer.…”

Development of 3D Hepatic Constructs Within Polysaccharide-Based Scaffolds with Tunable Properties

“…Initially, sandwiched cultures promoted survival of functional primary hepatocytes and have been widely used since then [21,22]. Later on, spheroids were produced using different simple techniques such as culture on non-adherent plates, stirred plates or bioreactors [23][24][25][26], that offered spheroid mass production. To improve reproducibility and produce organoids with a homogeneous size, hanging-drop and emulsion-based techniques [19], as well as microwells made of non-adherent materials, were developed [18,[27][28][29].…”