Abstract
Simple SummaryThe PTS signaling (PI3K/AKT, TGF-β, and STAT3 Signaling) networks, regulated by microRNA-21, play roles in lipogenic factor regulation, tumor suppressor modulation and oncogenic activation. Our zebrafish model recreates the development of hepatocellular carcinoma (HCC) due to nonalcoholic fatty liver disease (NAFLD) concerning the physiological, metabolic, and histological aspects similar to that of human NAFLD-related HCC (NAHCC). Thus, microRNA-21 is critical in the pathogenesis of NAHCC, and serves as a novel therapeutic target in NAHCC progression.MicroRNA-21 (miR-21) is one of the most frequently upregulated miRNAs in liver diseases such as nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). However, mechanistic pathways that connect NAFLD and HCC remain elusive. We developed a doxycycline (Dox)-inducible transgenic zebrafish model (LmiR21) which exhibited an upregulation of miR-21 in the liver, which in turn induced the full spectrum of NAFLD, including steatosis, inflammation, fibrosis, and HCC, in the LmiR21 fish. Diethylnitrosamine (DEN) treatment led to accelerated liver tumor formation and exacerbated their aggressiveness. Moreover, prolonged miR-21 expression for up to ten months induced nonalcoholic steatohepatitis (NASH)-related HCC (NAHCC). Immunoblotting and immunostaining confirmed the presence of miR-21 regulatory proteins (i.e., PTEN, SMAD7, p-AKT, p-SMAD3, and p-STAT3) in human nonviral HCC tissues and LmiR21 models. Thus, we demonstrated that miR-21 can induce NAHCC via at least three mechanisms: First, the occurrence of hepatic steatosis increases with the decrease of ptenb, pparaa, and activation of the PI3K/AKT pathway; second, miR-21 induces hepatic inflammation (or NASH) through an increase in inflammatory gene expression via STAT3 signaling pathways, and induces liver fibrosis through hepatic stellate cell (HSC) activation and collagen deposition via TGF-β/Smad3/Smad7 signaling pathways; finally, oncogenic activation of Smad3/Stat3 signaling pathways induces HCC. Our LmiR21 models showed similar molecular pathology to the human cancer samples in terms of initiation of lipid metabolism disorder, inflammation, fibrosis and activation of the PI3K/AKT, TGF-β/SMADs and STAT3 (PTS) oncogenic signaling pathways. Our findings indicate that miR-21 plays critical roles in the mechanistic perspectives of NAHCC development via the PTS signaling networks.
Highlights
Nonalcoholic fatty liver disease (NAFLD) manifests as a multistep disorder, beginning from a simple liver steatosis to severe forms of nonalcoholic steatohepatitis (NASH) and cirrhosis, with associated high risk of hepatocellular carcinoma (HCC) [1]
Several transgenic zebrafish were identified by the presence of strong red fluorescence signals in the liver (Figure 1C) or whole body (Figure S1B) of transgenic larvae, and the miR-21 expression was confirmed by RT-qPCR (Figure 1B and Figure S1C)
We investigated the effect of miR-21 expression in nonalcoholic fatty liver disease (NAFLD)-related HCC in a zebrafish model-LmiR21, which mimics hepatocellular carcinoma development caused by NAFLD/NASH
Summary
Nonalcoholic fatty liver disease (NAFLD) manifests as a multistep disorder, beginning from a simple liver steatosis to severe forms of nonalcoholic steatohepatitis (NASH) and cirrhosis, with associated high risk of hepatocellular carcinoma (HCC) [1]. The progression of NAFLD-related HCC (NAHCC) is a more complex process, caused by several risk factors, such as genomic instability, obesity, or diabetes [2]. MiR-21 contributes to cell injury, inflammation and fibrosis, through the inhibition of peroxisome proliferator activated receptor alpha (PPAR-α) signaling pathway [13]. These studies suggest that miR-21 has a central role in liver inflammation induced by steatosis
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