Modulation of miR-29 Expression by Alpha-Fetoprotein Is Linked to the Hepatocellular Carcinoma Epigenome

Globally, hepatocellular carcinoma (HCC) accounts for 70%-85% of primary liver cancers and ranks as the second leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally highly expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP(+) and AFP(-) tumors differ biologically. Multivariable analysis in 237 HCC cases demonstrates that AFP level predicts poor survival independent of tumor stage (P < 0.043). Using microarray-based global microRNA (miRNA) profiling, we found that miRNA-29 (miR-29) family members were the most significantly (P < 0.001) down-regulated miRNAs in AFP(+) tumors. Consistent with miR-29's role in targeting DNA methyltransferase 3A (DNMT3A), a key enzyme regulating DNA methylation, we found a significant inverse correlation (P < 0.001) between miR-29 and DNMT3A gene expression, suggesting that they might be functionally antagonistic. Moreover, global DNA methylation profiling reveals that AFP(+) and AFP(-) HCC tumors have distinct global DNA methylation patterns and that increased DNA methylation is associated with AFP(+) HCC. Experimentally, we found that AFP expression in AFP(-) HCC cells induces cell proliferation, migration, and invasion. Overexpression of AFP, or conditioned media from AFP(+) cells, inhibits miR-29a expression and induces DNMT3A expression in AFP(-) HCC cells. AFP also inhibited transcription of the miR-29a/b-1 locus, and this effect is mediated through c-MYC binding to the transcript of miR-29a/b-1. Furthermore, AFP expression promotes tumor growth of AFP(-) HCC cells in nude mice. Conclusion: Tumor biology differs considerably between AFP(+) HCC and AFP(-) HCC; AFP is a functional antagonist of miR-29, which may contribute to global epigenetic alterations and poor prognosis in HCC.