A miR-340/SPP1 axis inhibits the activation and proliferation of hepatic stellate cells by inhibiting the TGF-β1/Smads pathway.

Abstract

Hepatic fibrosis (HF) is a common pathological complication of liver cirrhosis which affects human health. It is well established that microRNAs (miRNAs) regulate the proliferation, activation and apoptosis of hepatic stellate cells (HSCs). To determine the function and molecular mechanism of miR-340-5p/secreted phosphoprotein 1 (SPP1) axis in HF and identify potential therapeutic targets. The HF model in cholestatic rats was induced by ligating the common bile duct. The histological sections of the liver tissues were stained with hematoxylin and eosin (H&E), Masson's trichrome or Sirius Red. The differential expression of mRNAs in the liver tissues was examined using the microarray analysis. The expression levels of miR-340-5p, SPP1, alpha-smooth muscle actin (α-SMA), Collagen I, phosphorylated Smad2 (p-Smad2), and p-Smad3 were determined using quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Cell proliferation was quantified using cell counting kit-8 (CCK-8) assays. The regulatory effect of miR-340-5p on SPP1 was determined with fluorescent reporter assay. The bile duct ligation (BDL) rat model was successfully induced, and SPP1 was upregulated in liver tissue from the BDL group compared to that of the sham group. The expression level of miR-340-5p was decreased in activated human primary normal fibroblasts (NFs) and activated LX-2 cells, and the mRNA and protein expression levels of SPP1 were increased in activated LX-2 cells. The SPP1 was the target of miR-340-5p, and the overexpression of SPP1 increased the proliferation of LX-2 cells, the expression of HF markers α-SMA and Collagen I, and key factors p-Smad2 and p-Smad3 (all p < 0.05). However, reverse results were obtained with the overexpression of miR-340-5p in LX-2 cells. Our findings provide evidence that SPP1 targeted by miR-340-5p promotes LX-2 cell proliferation and activation through the TGF-β1/Smads signaling pathway. Therefore, miR-340-5p and SPP1 may be possible therapeutic targets for HF.