Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGFβ). microRNAs (miRs), small non-coding RNAs modulating mRNA and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGFβ signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Since miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGFβ signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGFβ signaling components demonstrated by decreased TGFβ receptor II (TGFβRII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3’UTR of TGFβRII was validated by luciferase reporter assay. Inhibition of TGFβ signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGFβ-induced expression of α1(I) and α2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased αSMA expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared to normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared to normal human livers. CONCLUSIONS miR 19b is a novel regulator of TGFβ signaling in HSCs suggesting a potential therapeutic approach for hepatic fibrosis.

Background Chronic ethanol consumption increases the risk of hepatic cirrhosis and hepatocellular carcinoma (HCC). While sex differences exist in susceptibility to ethanol-induced liver damage-HCC development, little is known about the effects of ethanol on tumor progression. Methods Neonatal male and female mice were initiated with a single dose of diethylnitrosamine (DEN). 16 or 40 weeks later animals were placed on a 10/20% (v/v) ethanol-drinking water (EtOH-DW; alternate days) regime for 8 weeks. At study end liver tissue and serum were analyzed for liver pathology/function and cytokine expression. Results DEN reproducibly induced hepatic foci/tumors in male and female mice. Ethanol diminished hepatic function and increased liver damage, but ethanol alone did not induce hepatic foci-HCC formation. In DEN-initiated EtOH-DW animals, ethanol significantly increased tumor incidence and burden, but only in male mice. Male and female mice (±DEN) demonstrated comparable blood-alcohol content at necropsy, yet increased hepatic damage and diminished hepatic function/anti-oxidant capacity was significantly greater in males. Analysis of liver mRNA for Th1, Th2 or T-regulatory factors demonstrated significantly elevated SMAD3 in male compared to female mice in response to EtOH, DEN-initiation and DEN+EtOH-DW. Conclusions These data demonstrate male mice are more susceptible to HCC incidence and progression in the setting of chronic ethanol feeding than females. Differences in markers of hepatic immune response in male mice suggest increased TGFβ-SMAD3 signaling may enhance promotion in this model of HCC progression, effects modulated by chronic ethanol feeding.

+ T cells and macrophages. DILI cases had significantly fewer B lymphocytes than AIH and VH and significantly fewer NK cells than VH. Prominent plasma cells were unusual in IAD (three of 10 cases), but were associated strongly with AIH (eight of nine) and also observed in most with AID (six of nine). They were also found in five of 10 cases with VH. Liver biopsies from subjects with DILI were characterized by low counts of mature B cells and NK cells in portal triads in contrast to VH. NK cells were found only in cases of VH, whereas AIH and VH both showed higher counts of B cells than DILI. Plasma cells were associated most strongly with AIH and less so with AID, but were uncommon in IAD.

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