Abstract
BackgroundActivation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. We previously demonstrated that exosomes derived from natural killer (NK) cells (NK-Exo) attenuated TGF-β1-induced HSC activation. Herein, this study was designed to investigate the mechanism underlying the action of NK-Exo.MethodsNK-Exo was isolated from NK-92MI cells and then administered into TGF-β1-treated LX-2 (human HSC line) cells. MiR-223 expression in NK-Exo was downregulated by transfecting NK-92MI cells with miR-223 inhibitor followed by exosome isolation. The HSC activation was evaluated by determining cell proliferation using CCK-8 assay and measuring the protein levels of α-SMA and CoL1A1 using western blot in LX-2 cells. The expression of miR-223 was detected by qRT-PCR. The interaction between miR-223 and ATG7 was analyzed by a dual-luciferase activity assay. The autophagy was evaluated by measuring the autophagy-related proteins using western blot.ResultsmiR-223 was highly expressed in NK-Exo and inhibition of miR-223 expression in NK-Exo abrogated the inhibitory effect of NK-Exo on TGF-β-induced HSC activation. ATG7 was confirmed as a direct target of miR-223. Furthermore, treatment with the autophagy activator rapamycin and ATG7 overexpression in LX-2 cells abolished the HSC activation-suppressive effect of NK-Exo.ConclusionNK-Exo attenuated TGF-β-induced HSC activation by transferring miR-223 that inhibited autophagy via targeting ATG7.
Highlights
Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis
natural killer (NK)-Exo-mediated transfer of miR-223 attenuated TGF-β1induced HSC activation Results of the Quantitative real-time PCR (qRT-PCR) analysis showed that miR223 expression was markedly higher in NK-Exo than that in parental NK-92MI cells (Fig. 1a)
To assess whether NK-Exo regulates HSC activation by transferring miR-223, we developed miR-223-deficient NKExo by transfecting NK-92MI cells with miR-223 inhibitor followed by exosomes isolation, namely, NKExo-miR-223I. miR-223 expression was confirmed to be sharply downregulated in NK-Exo-miR-223I when compared with the NK-Exo-negative control (NC) group (Fig. 1b)
Summary
Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis. This study was designed to investigate the mechanism underlying the action of NK-Exo. Activation of hepatic stellate cells (HSCs) is a prominent driver of liver fibrosis that can eventually lead to cirrhosis, liver failure, and even liver cancer (Higashi et al 2017; Parola and Pinzani 2019). Emerging evidence has indicated that miRNAs play a regulatory role in the occurrence and progression of liver fibrosis by influencing HSC activation (Zhao et al 2019). MiR-223 plays an essential role in the pathogenesis of various types of liver diseases, such as hepatitis virus infections, alcohol- or drug- induced liver injury, cirrhosis, and liver cancer (Ye et al 2018). A recent study showed that treatment with miR-223-3p significantly mitigated fibrosis development and HSC activation in a murine model of fibrotic nonalcoholic steatohepatitis (NASH) (Jimenez Calvente et al 2020)
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