MicroRNAs are conserved, small (20-25 nucleotide) noncoding RNAs that negatively regulate expression of mRNAs at the post-transcriptional level. MicroRNA signature is altered in different disease states including cancer and some microRNAs act as oncogenes or tumor suppressors. To identify microRNAs that may play a causal role in hepatocarcinogenesis we used an animal model in which C57BL/6 mice fed choline deficient and amino acid defined (CDAA) diet develop nonalcoholic steatohepatitis (NASH)-induced hepatocarcinogenesis after 70 weeks. Microarray analysis identified 30 hepatic microRNAs that are significantly (P<.01) altered in mice fed CDAA diet for 6, 18, 32 and 65 weeks compared to those fed choline sufficient and amino acid defined diet (CSAA). Real-time RT-PCR analysis demonstrated upregulation of oncogenic miR-155, miR-181b, miR-221/222 and miR-21 and downregulation of the most abundant liver specific miR-122 at early stages of hepatocarcinogenesis.
Western blot analysis showed reduced expression of hepatic PTEN, a target of miR-21, and C/EBPβ, a target of miR-155, in these mice at early stages. DNA binding activity of NF-κB that transactivates miR-155 gene was significantly (P=0.002) elevated in the liver nuclear extract of mice fed CDAA diet. Further, the expression of miR-155, as measured by in situ hybridization and real-time RT-
PCR, correlated with diet-induced histopathological changes in the liver. Ectopic expression of miR-155 promoted growth of hepatocellular carcinoma (HCC) cells whereas its depletion inhibited cell growth. Notably, miR-155 was significantly (P=0.0004) upregulated in primary human HCCs with concomitant decrease (P=0.02) in C/EBPβ level compared to matching liver tissues.
The expression of tissue inhibitor of metalloprotease 3 (TIMP3), a tumor suppressor and a validated miR-181 target, was markedly suppressed in the livers of mice fed CDAA diet. Upregulation of hepatic transforming growth factor β (TGFβ) and its downstream mediators Smad 2, 3 and 4 and increase in phospho-Smad2 in the liver nuclear extract correlated with elevated miR-181b/d in mice fed CDAA diet. The levels of the precursor and mature miR-181b were augmented on exposure of hepatic cells to TGFβ and were significantly reduced by small interference RNA-mediated depletion of Smad4, showing the involvement of TGFβ signaling pathway in miR-181b expression. Ectopic expression of miR-181b showed that miR-181b enhanced matrix metallopeptidases 2 (MMP2) and MMP9 activity and promoted growth, clonogenic survival, migration and invasion of HCC cells that could be reversed by modulating TIMP3 level. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice. miR-181b also enhanced resistance of HCC cells to the anticancer drug doxorubicin.
Conclusion: Temporal changes in microRNA profile occur at early stages of CDAA diet-induced hepatocarcinogenesis. Reciprocal regulation of specific oncomirs and their tumor suppressor targets implicate their role in NASH-induced hepatocarcinogenesis and suggest their use in the diagnosis and prognosis of liver cancer.