Receptor for advanced glycation end products (RAGE) was studied in different stages of carbon tetrachloride induced hepatic fibrosis (HF), and effect of its gene silencing in the HF development was evaluated in rats. Silencing RAGE expression by specific siRNA effectively suppressed NF-κB activity, hepatic stellate cell activation, and accumulation of extracellular matrix proteins in the fibrotic liver, and also greatly improved the histopathology and the ultrastructure of liver cells. These effects may be partially mediated by the inhibition on IκBα degradation. RAGE gene silencing effectively prevented liver from fibrosis, therefore it offers a potential pharmacological tool for anti-HF gene therapy.

Since the receptor for advanced glycation end products (RAGE)-ligand axis has been demonstrated to be important in fibrogenesis, rat models may be used to assess whether specific small interfering RNAs (siRNAs) that target RAGE are able to reduce the progression of hepatic fibrosis. However, the effect of RAGE-targeted siRNA on established hepatic fibrosis remains to be elucidated. In the present study, RAGE-specific siRNA expression vectors were constructed prior to the animal experiment. Sprague-Dawley rats were treated initially with olive oil (2 ml/kg) or 50% CCl4 (2 ml/kg; CCl4/olive oil=1:1) twice per week for six weeks to generate the fibrosis model. The rats were then treated with phosphate‑buffered saline, a RAGE-specific siRNA expression vector, at different doses or a non-specific siRNA expression vector twice weekly via tail vein injection for up to six weeks, and were sacrificed at week two, four or six. Compared with the control groups, RAGE‑specific siRNA therapy significantly decreased RAGE mRNA and protein expression in rat livers (P<0.01). Following six weeks of RAGE gene-silencing treatment, the liver function, which was assessed by analyzing serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin (TBIL), improved to varying degrees (P<0.01). The expression of nuclear factor-κB (NF-κB) significantly decreased following RAGE gene‑silencing therapy (P<0.01). In addition, the serum levels of inflammatory cytokines, including tumor necrosis factor‑α (TNF-α) and interleukin-6 (IL-6), and extracellular matrix (ECM) components, including hyaluronic acid (HA), laminin (LN) and procollagen type III (PCIII) also decreased (P<0.01). Furthermore, the expression of α-smooth muscle actin (α-SMA) and collagen I, which indicate the activation of hepatic stellate cells (HSCs), were downregulated following RAGE gene-silencing therapy (P<0.01). Furthermore, the inflammatory activity grade and fibrosis stage of rat livers also significantly improved compared with the control groups following RAGE gene-silencing therapy. Specific targeting of RAGE using siRNA may inhibit RAGE gene expression effectively in the rat hepatic fibrosis model and attenuate the progression of established hepatic fibrosis. This therapeutic effect may be mediated via inhibition of the expression of NF-κB. These findings suggest that RAGE may be a new target to prevent hepatic fibrosis.

Cholangiocarcinoma (CCA) is a rare but devastating malignancy. Up to 90% of patients presenting with CCA have no identifiable risk factors. The base excision repair (BER) pathway has a principal role in the repair of mutations caused by oxidized or reduced bases. The MutY homolog (MUTYH, MYH) is one of the key proteins in the BER pathway, but the role of MYH in the tumorigenesis of CCA is largely unknown. In this study, we investigated the influence of MYH rs3219476 and rs3219472 polymorphisms on CCA incidence. MYH genotypes were detected using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. We found that for rs3219472, compared with subjects carrying the MYH G/G genotype, those with the A/A genotype had a 2.816-fold higher risk of CCA [odds ratio (OR)=2.816, 95% confidence interval (CI)=0.992-7.999, P=0.047). For rs3219476, compared with subjects carrying the MYH T/T genotype, those with the T/G genotype had a reduced risk of CCA (OR=0.359, 95% CI=0.17-0.758, P=0.006). Our findings suggest that since significantly increased CCA risk was found in individuals with a homozygous variant genotype for rs3219472, it may be a biomarker for screening individuals at high risk of developing the disease.

Specific small interfering RNAs (siRNAs) targeting receptor for advanced glycation end products (RAGE) inhibit the expression of RAGE, α-smooth muscle actin and type I collagen in the T6 hepatic stellate cells (HSCs), indicating that RAGE is important for the activation of HSCs and the expression of collagen. The present study aimed to investigate the effect of specific siRNAs targeting RAGE on the development of hepatic fibrosis (HF), using primary rat HSCs, which were isolated and cultured in vitro. The expression vectors for specific siRNAs targeting RAGE were constructed and transfected into primary rat HSCs. Untreated and nonspecific siRNA-transfected primary rat HSCs served as controls. The expression levels of RAGE, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), laminin (LN), hyaluronic acid (HA) and N-terminal procollagen III propeptide (PIIINP) in primary HSCs were detected by reverse transcription quantitative polymerase chain reaction and western blotting. The mRNA and 42 kD protein expression of RAGE in the pAKD-GR126-transfected primary HSCs were significantly downregulated compared with those in the untreated and the pAKD-negative control (NC)-transfected controls. The mRNA and protein expression levels of IL-6, TNF-α, TGF-β1, CTGF, LN, HA and PIIINP in the pAKD-GR126-transfected primary HSCs were also markedly downregulated compared with those in the untreated and pAKD-NC-transfected controls. Therefore, RAGE-specific siRNAs inhibited the expression of RAGE in primary rat HSCs and inhibited the development of HF.

Increasing evidence has suggested that hepatic lipid accumulation is associated with hepatic insulin resistance; however, the underlying mechanism is yet to be determined. It was demonstrated that the levels of microRNA-215 (miR-215) expression in the liver of rats fed a high-fat diet were significantly increased compared with rats on a control diet. Additionally, it was revealed via luciferase assays and western blotting that miR-215 targets rapamycin-insensitive companion of mammalian target of rapamycin (Rictor), an important protein in the hepatic insulin signalling pathway. Following overexpression of miR-215 in the H4IIE rat hepatocarcinoma cell line, it was reported that the intracellular insulin signalling pathway was inhibited; conversely, inhibition of miR-215 expression induced this pathway. Furthermore, it was demonstrated via reverse transcription-quantitative polymerase chain reaction analysis that free fatty acids promoted the expression of miR-215. The present study provided a novel mechanistic insight into the association between nonalcoholic fatty liver and hepatic insulin resistance.