Aberrant microRNA expression implicates on hepatocellular carcinoma (HCC) development. Conversely, coffee consumption reduces by ~40% the risk for fibrosis/cirrhosis and HCC, while decaffeinated coffee does not. It is currently unknown whether these protective effects are related to caffeine (CAF), or to its combination with other common and/or highly bioavailable coffee compounds, such as trigonelline (TRI) and chlorogenic acid (CGA). We evaluated whether CAF individually or combined with TRI and/or CGA alleviates fibrosis-associated hepatocarcinogenesis, examining the involvement of miRNA profile modulation. Then, male C3H/HeJ mice were submitted to a diethylnitrosamine/carbon tetrachloride-induced model. Animals received CAF (50 mg/kg), CAF+TRI (50 and 25 mg/kg), CAF+CGA (50 and 25 mg/kg) or CAF+TRI+CGA (50, 25 and 25 mg/kg), intragastrically, 5×/week, for 10 weeks. Only CAF+TRI+CGA combination reduced the incidence, number and proliferation (Ki-67) of hepatocellular preneoplastic foci while enhanced apoptosis (cleaved caspase-3) in adjacent parenchyma. CAF+TRI+CGA treatment also decreased hepatic oxidative stress and enhanced the antioxidant Nrf2 axis. CAF+TRI+CGA had the most pronounced effects on decreasing hepatic pro-inflammatory IL-17 and NFκB, contributing to reduce CD68-positive macrophage number, stellate cell activation, and collagen deposition. In agreement, CAF+TRI+CGA upregulated tumor suppressors miR-144-3p, miR-376a-3p and antifibrotic miR-15b-5p, frequently deregulated in human HCC. CAF+TRI+CGA reduced the hepatic protein levels of pro-proliferative EGFR (miR-144-3p target), antiapoptotic Bcl-2 family members (miR-15b-5p targets), and the number of PCNA (miR-376a-3p target) positive hepatocytes in preneoplastic foci. Our results suggest that the combination of most common and highly bioavailable coffee compounds, rather than CAF individually, attenuates fibrosis-associated hepatocarcinogenesis by modulating miRNA expression profile.

微小的microRNA表达与肝细胞癌（HCC）的发展有关。相反，咖啡消耗量减少了约40％的纤维化/肝硬化和HCC风险，而不含咖啡因的咖啡则没有。目前尚不清楚这些保护作用是否与咖啡因（CAF）有关，还是与咖啡因和其他常见的和/或生物利用度较高的咖啡化合物（例如藜芦啉（TRI）和绿原酸（CGA））结合使用。我们评估了CAF是单独使用还是与TRI和/或CGA联合使用减轻了纤维化相关的肝癌发生，研究了miRNA谱调节的参与。然后，将雄性C3H / HeJ小鼠置于二乙基亚硝胺/四氯化碳诱导的模型中。动物接受了CAF（50 mg / kg），CAF + TRI（50和25 mg / kg），CAF + CGA（50和25 mg / kg）或CAF + TRI + CGA（50、25和25 mg / kg），在胃内 5次/周，持续10周。只有CAF + TRI + CGA组合可以降低肝细胞癌前灶的发生率，数量和增殖（Ki-67），同时可以增强邻近实质中的细胞凋亡（裂解的caspase-3）。CAF + TRI + CGA处理还降低了肝脏的氧化应激并增强了抗氧化剂Nrf2轴。CAF + TRI + CGA对减少肝脏促炎性IL-17和NFκB具有最显着的作用，有助于减少CD68阳性巨噬细胞数量，星状细胞活化和胶原蛋白沉积。一致的是，CAF + TRI + CGA上调了人类肝癌中经常被失调的肿瘤抑制因子miR-144-3p，miR-376a-3p和抗纤维化miR-15b-5p。CAF + TRI + CGA降低了促增殖EGFR（miR-144-3p靶标），抗凋亡Bcl-2家族成员（miR-15b-5p靶标）的肝蛋白水平，肿瘤前灶中PCNA（miR-376a-3p靶标）阳性肝细胞的数量。我们的结果表明，最常见的和生物利用度最高的咖啡化合物的组合，而不是单独的CAF，可通过调节miRNA表达谱来减轻与纤维化相关的肝癌的发生。