Modulation of miR-29 expression by α-fetoprotein is linked to the hepatocellular carcinoma epigenome

Abstract

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.

© 2014 by the American Association for the Study of Liver Diseases. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Figures

Figure 1

AFP is inversely correlated with miR-29 and associated with increased DNA methyl transferase expression. A) HCC patients with high levels of serum AFP are associated with poor survival. Mantel-Cox p<0.05; log-rank p<0.05; n=237. B) The LCS cohort includes 274 HCC patients with matched tumor and non-tumor tissue samples. 186 patients (shown in green) have both mRNA expression and miRNA expression achieved by the Affymatrix gene expression array and OSU-CCC miRNA array, respectively. An additional 51 patients have gene expression data only (blue) and 37 patients have miRNA expression data only (yellow). C) miR-29a significantly decreases (top panel, n=223) and DNMT3A significantly increases (bottom panel, n=237) as serum AFP expression increases in HCC patients. D) Low expression of miR-29a (top panel, n=223) and increased DNMT3A (bottom panel, n=237) are associated with poor survival, respectively. E) Unsupervised hierarchical clustering of 48 HCC tumor samples reveals a unique methylation profile in patients with high AFP and DNMT3A gene expression. Manhattan clustering was used with a standard deviation cut-off of 2, which showed 211 probes to be differentially methylated. A median cut-off was used to determine high or low gene expression of AFP and DNMT3A for patient labeling. Fisher’s exact test confirms AFP high/DNMT3A high patients are enriched in cluster #1 (p<0.05). F) The three clusters observed in the unsupervised hierarchical clustering exhibit distinct differences in overall survival. Cluster #1, with predominantly AFP high and DNMT3A high patients, shows significantly worse overall survival than patients in cluster #2 or #3. Mantel-Cox p<0.05; n=48.

Figure 2

AFP transcriptionally regulates miR-29. A) Over expression of AFP significantly increases cell proliferation compared to control HLE cells after a 48hr transient transfection (p<0.0005 days 3–6 and p<0.005 days 7 and 8). Cell growth was monitored using a CalceinAM assay with 4 replicates per time point and error bars represent standard deviation. B) HLE cells in the presence of conditioned media taken from AFP over expressing HLE cells (AFP OE CM) proliferate faster than those in AFP− CM (taken from HLE cells transfected with an empty vector, Control CM). Cells were seeded on day 0, CM was applied on day 1 and cell growth was monitored by xCELLigence technology over a period of five days. There are four replicates per time point and error bars represent standard deviation. The growth rate of HLE cells growing in the presence of AFP is significantly faster on days 1–5 (p<0.05). C) Over expression of AFP in HLE cells leads to decreased mature miR-29a expression compared to control HLE cells (48 hour transient transfection). Top panel shows AFP protein expression by western blot. miR-29a expression was quantified using qRT-PCR in the bottom panel. D) DNMT3A and DNMT3B levels significantly increased after transient AFP over expression (48 hours) in HLE cells. E) Conditioned media taken from transfected cells was applied to HLE cells. AFP OE CM led to a significant decrease in mature miR-29a measured by qRT-PCR as compared to CM from control cells. ELISA data in the top panel shows that media taken from cells over expressing AFP has >500ng/ml of AFP present. F) AFP+ CM taken from HUH-7 cells led to a decrease in both the mature (top panel) and primary transcript (bottom panel) of miR-29a in HLE cells as measured by qRT-PCR. G) AFP+ CM from HUH-7 cells also lead to a decrease in miR-29a mature (top panel) and primary transcript (bottom panel) expression in SNU-475 cells.

Figure 3

AFP mediates the down regulation of miR-29a through c-MYC. A) AFP is over expressed and c-MYC silenced 72 hours post-transfection in HLE cells. B) miR-29a expression is only silenced when AFP is present and c-MYC is functionally active. Upon c-MYC silencing, miR-29a expression is induced even in the presence of AFP. C) The miR-29a/b-1 transcript is shown with two sets of primers designed around c-MYC binding sites. P-1 is in the promoter region and P-2 located in the intron of the transcript. Transcript schematic is modified from that created by Chang et al (22). D) Chromatin Immunoprecipitation was performed to pull down c-MYC bound to genomic regions in HLE cells after AFP+ or AFP− CM was applied (rabbit IgG was used as a control). Both P-1 and P-2 primer sets were used to quantify the amount of c-MYC bound to two specified regions on the miR-29a transcript using qRT-PCR. Results show that c-MYC bound to both regions of miR-29a/b-1 only in the presence of AFP.

Figure 4

AFP expression promotes cell growth, cell migration and invasion in vitro and tumorigenesis in vivo. A) AFP expression detected by western (top) and ELISA (bottom) in stable AFP+ versus control HLE cells. AFP is only expressed and secreted in cells infected with a lentivirus incorporating the AFP gene. B) Stable AFP+ cells show more rapid proliferation compared to control cells. The growth rate of AFP+ cells is significantly faster on days 1–3 (p<0.01). There are three replicates per time point and error bars represent standard deviation. C) AFP expression induces increased migratory capacity, significantly different from control cells starting at 8 hours (p<0.01). There are three replicates per times point and error bars represent standard deviation. D) AFP expression also leads to increased invasion significantly different from control cells starting at 12 hours (p<0.01). There are three replicates per time point and error bars represent standard deviation. E) Tumor incidence is significantly faster in nude mice injected with 0.5×106 AFP+ cells AFP+ cells compared to 0.5×106 AFP− control HLE cells (p<0.05). After 1 week, all mice in the AFP+ group carried a tumor compared to less than half of the control group. F) Quantification of tumor volume (mm3) shows a significant difference between the size of control versus AFP+ tumors (p<0.05). Error bars represent mean plus standard error. Image of tumors extracted from the control and AFP+ groups after 4 weeks.

Figure 5

Schematic illustrating the mechanism by which AFP transcriptionally down regulates miR-29a and drives poor prognosis in HCC.