microRNA-34a modulates the senescence of activated hepatic stellate cells in alcohol-associated liver injury

Background: Reversion of activated hepatic stellate cells (HSCs) to quiescence is likely to be a promising pathway in liver fibrosis regression. The development of senescence in HSCs during alcoholic liver injury may lead to liver compensation against fibrosis. The current study aims to characterize the functional role of miR-34a regulated cellular senescence during alcohol-associated hepatitis. Methods: Senescence related gene and miR-34a expression was assessed using a PCR Array and/or real-time PCR analysis. Cellular senescence in cultured HSCs were measured by SA-β-gal staining. Senescence mediators were defined in LPS treated hepatic stellate cells in vitro, and in TLR-4 knockout mice or morpholino antisense oligomer against miR-34a (miR-34a Morpho/AS) treated mice with chronic ethanol feeding in vivo. Results: We identified that 5 weeks of ethanol feeding significantly increased the total liver histopathology score and miR-34a expression, along with the activation of hepatic stellate cells marked by enhanced α-SMA staining. Treatment of HSCs with LPS (20 ng/ml) for 24 hr significantly increased miR-34a expression, along with the enhanced SA-β-gal activity, up-regulated α-SMA/Collagen A1 expression and increased cellular viability. Silencing of miR-34a decreased LPS-induced activation in HSCs by downregulation of fibrosis markers α-SMA, Col1a1 and TIMP-1, and increased SA-β-gal activity as well as the cellular senescence marker CCl2, whereas silencing of the LPS receptor, TLR4, decreased this marker in the same group of HSCs, suggesting anti-miR-34a reversed LPS-mediated HSC activation through inducing cellular senescence. Furthermore, the expression of miR-34a and verified miR-34a related senescence marker CCl2 were significantly altered in hepatic stellate cells from ethanol-fed mouse liver specimens compared to controls. TLR4 knockout mice and miR-34a Morpho/AS treated mice displayed less sensitivity to alcoholic injury, along with enhanced SA-β-gal activity and CCl2 level in HSCs, and recovered expressions of α-SMA, Col1a1 and TIMP-1. Summary and Conclusion: Our results show that miR-34a mediated cellular senescence is essential for the potential reversion from activated to quiescent hepatic stellate cells during alcohol-associated hepatitis. These findings provide new insight into the function of microRNA regulated cellular senescence in human HSCs and increase opportunities for the development of novel treatment paradigms for the management of alcohol-associated liver diseases.