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


  • Potential conflict of interest: Nothing to report.

  • This work was supported by a grant (Z01 BC 010313) from the Intramural Research Program of the Center for Cancer Research, National Cancer Institute (Bethesda, MD).


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. (Hepatology 2014;60:872–883)




chromatin immunoprecipitation




CpG island methylator phenotype


conditioned media


DNA methyltransferase 3A


enzyme-linked immunosorbent assay


false discovery rate


hepatitis B virus


hepatocellular carcinoma


Ingenuity Pathway Analysis






messenger RNA


National Institutes of Health


Ohio State University Comprehensive Cancer Center


quantitative reverse-transcriptase polymerase chain reaction




short hairpin


small interfering RNA


tumor node metastasis


University of California, Santa Cruz

The worldwide incidence of hepatocellular carcinoma (HCC) is currently estimated at nearly 750,000 new cases each year, resulting in over 600,000 deaths annually, and remains on the rise.[1] Patients are typically diagnosed with late-stage disease leading to poor survival rates. Two major risk factors are chronic hepatitis B virus (HBV) and hepatitis C virus infections, which are responsible for 93% of cases in developing countries and 53% of cases in developed countries.[1] Additional risk factors include chronic alcohol consumption, aflatoxin-B1-contaminated foods, and other conditions that cause cirrhosis.[2]

Patients at risk for HCC are screened and monitored for serum alpha-fetoprotein (AFP) levels. AFP is a molecular marker elevated (>1,000 ng/mL) in 60%-75% of HCC patients, making it the key biomarker used for HCC surveillance.[3] Though it is used for surveillance and to assess patient risk, its low sensitivity makes it inadequate to detect all patients that will develop cancer.[3] In fact, many patents with cirrhosis develop HCC without any increase in AFP. In the cohort of HCC patients that we study, 40% have normal levels (<20 ng/mL) of the protein. Currently, the function of AFP is not well understood and it is mainly thought of clinically as a diagnostic marker.

AFP is an oncofetal protein highly elevated during embryogenesis and detected mainly in the fetal liver and yolk sac.[3] It is secreted through the cell membrane and part of the albuminoid gene family, which also includes serum albumin, vitamin D binding protein, and alpha-albumin.[3-5] The synthesis of AFP decreases rapidly after birth and levels remain below 20 ng/mL in adults.[3, 6] It has been shown that AFP binds and transports unsaturated fatty acids, estrogen, retinoids, steroids, flavanoids, heavy metals, dioxins, and bilirubin.[4, 5] AFP also interacts with macrophages and inhibits natural killer cells.[4] In addition, oncofetal protein plays a role in regulation of cell proliferation and tumor growth. However, evidence for both stimulatory and inhibitory effects on cell growth remains contradictory and may be estrogen dependent.[4, 7]

Though the physical, chemical, and immunological properties of AFP have been well studied, the mechanisms underlying its biological function and its role in carcinogenesis remain unclear.[3, 4] AFP is elevated in many HCC patients; however, levels are heterogeneous, suggesting that the biology of AFP+ and AFP tumors may be different.[8] For example, AFP levels may be low in patients with early HCC, but very high in those who have cirrhosis without HCC.[9]

Aberrant microRNA (miRNA) expression is a ubiquitous feature in an increasing number of cancers, including HCC.[10] Studies indicate that these miRNAs are directly connected with epigenetic factors that regulate gene expression.[11] MiRNAs are short, noncoding RNAs approximately 22 nucleotides in length that regulate the function of messenger RNA (mRNA).[12] Partial sequence homology allows miRNAs to bind to the 3'-untranslated region of target mRNAs inhibiting translation or causing mRNA degradation.[11, 12] Recently, a specific group of miRNAs have been designated “epi-miRNAs” because they target effectors of epigenetic machinery.[13] In addition to their functional role, miRNAs show promise as biomarkers for early detection, prognosis, diagnosis, and treatment subgroups.[13-16]

In this study, we found that AFP+ and AFP HCC cases were biologically different according to miRNA, mRNA, and methylation expression patterns. In addition, we reveal an important functional role of AFP in HCC. Not only is AFP inversely correlated with miR-29 in HCC tumor tissue, but we demonstrate that AFP transcriptionally down-regulates miR-29a through c-MYC. Identifying the molecular mechanisms underlying AFP+ tumors will help build an understanding of heterogeneity in HCC as well as our understanding of AFP's functional role in HCC progression.

Patients and Methods

Patient Studies and Tumor Specimens

Paired tumor and nontumor hepatic samples were obtained with informed consent, and the collection of these samples for research were approved by the institutional review board of the Liver Cancer Institute and Zhongshan Hospital (Fudan University, Shanghai, China), as described in previous studies.[17, 18] Refer to the Experimental Procedures section in the Supporting Information for details.

Xenograft Study

The animal study protocol was approved by the National Institutes of Health (NIH) Animal Care and Use Committee, and all animals received humane care according to the NIH Guide for Care and Use of Laboratory Animals. Refer to the Experimental Procedures section in the Supporting Information for details.

For details on plasmids, lentiviral vectors, small interfering RNA (siRNA), quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), conditioned media (CM) study design, protein expression analysis, cell proliferation assays, chromatin immunoprecipitation (ChIP) assay, and all statistical analysis, please refer to the Experimental Procedures section in the Supporting Information.


AFP+ HCC Patients Have a Distinct Genomic Profile That Is Linked to Poor Survival

In our cohort, we subdivided HCC patients into four groups based on serum AFP levels, starting at a normal level (<20 ng/mL) and increasing 10-fold (i.e., 20-200, 200-2,000, and >2,000 ng/mL), where patients with >2,000 ng/mL AFP were considered to be extremely high cases. Overall survival data confirm that poor clinical outcome is associated with increasing serum AFP, suggesting that the biological makeup of these HCCs differ from those that are AFP (Mantel-Cox, P < 0.05; log-rank, P < 0.005; Fig. 1A). Additional clinical characteristics of HCC cases in each subgroup can be found in Supporting Table 1). To test whether AFP identifies a unique molecular subclass, rather than late-stage tumors, we performed a multivariable analysis between AFP level and tumor node metastasis (TNM) staging, the only two variables from the univariable analysis that passed a stepwise selection process using both forward addition and backward subtraction with a P value cutoff of <0.05. Both AFP level and TNM staging were significant in the multivariable analysis, indicating that AFP predicts poor overall survival independent of tumor stage (Table 1).

Figure 1.

AFP is inversely correlated with miR-29 and associated with increased DNA methyltransferase 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 nontumor tissue samples. Overall, 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 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 cutoff of 2, which showed 211 probes to be differentially methylated. A median cutoff 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).

Table 1. Uni- and Multivariable Analyses of Factors Associated With Disease Stage, Aggressive HCC, and Survivala
 Univariable AnalysisbMultivariable Analysisc
Clinical VariableHazard Ratio (95% CI)P ValueHazard Ratio (95% CI)P Value
  1. a

    n = 237.

  2. b

    Univariable analysis, Cox's proportional hazards regression.

  3. c

    Multivariable analysis, Cox's proportional hazards regression.

  4. d

    Stages II and III were combined because of the presence of vascular invasion at these stages.

  5. Abbreviations: CI, confidence interval; n.a., not applicable.

AFP (>300 vs. ≤300 ng/mL)1.7 (1.1-2.5)0.0111.6 (1.0-2.4)0.043
Age (≥50 vs. <50 years)0.8 (0.5-1.2)0.26n.a. 
Sex (male vs. female)1.8 (0.9-3.7)0.116n.a. 
Cirrhosis (yes vs. no)5.2 (1.3-20.9)0.022n.a. 
Tumor size (>3 vs. ≤3 cm)2.4 (1.5-3.8)<0.001n.a. 
Multinodularity1.6 (1.0-2.5)0.046n.a. 
Vascular invasion1.8 (1.2-2.9)0.007n.a. 
TNM staging (II-III vs. I)d2.9 (1.8-4.8)<0.0012.9 (1.8-4.8)<0.001

Affymetrix mRNA expression data are available for 237 paired tumor and nontumor tissue samples, as described in earlier studies,[17-19] whereas miRNA expression data are available on a subset of patients (n = 223) in our cohort (Fig. 1B). Global DNA methylation profiles were also obtained for a subset of HCC patients who have mRNA expression data.

We next determined whether AFP+ HCC may have different miRNA expression. We performed a class comparison analysis between 74 patients with normal serum AFP (<20 ng/mL) and 39 with extremely high AFP levels (>2,000 ng/mL) and found that 18 miRNAs were differentially expressed using a P value cutoff of 0.001 and a false discovery rate (FDR) of 5% (Table 2). Of the 13 down-regulated miRNAs (ranked by fold change), seven were in the miR-29 family. All members of the miR-29 family were also associated with poor survival in HCC (Supporting Table 2). Interestingly, when examining available array comparative genomic hybridization data,[20] none of the miR-29 family members are located in frequent genomic areas of loss, suggesting that miR-29 repression in HCC may be at the epigenetic or transcriptional level (Table 2).

Table 2. The miR-29 Family Is Differentially Expressed Between Patients With Normal Levels Versus Those With Extremely High Levels of Serum AFP
 Raw Intensity Valueb      
miRNA ProbeaAFP >2,000 ng/uLcAFP <20 ng/uLdFold ChangeParametric P ValueFDRChromosome LocationGain (%)eLoss (%)e
  1. a

    The miRNA array includes multiple spots for some miRNAs; therefore, there are multiple probe readouts. Additional probes for miRNAs are numbered in parenthesis after the probe name.

  2. b

    Mean values for miRNA expression are shown for AFP high and normal cases and were used to calculate fold change.

  3. c

    High AFP (n = 40).

  4. d

    Low AFP (n = 74).

  5. e

    Percent gain and loss were estimated by copy number variation (CNV) data from 36 HCC tumor tissue samples. In cases where the miRNA is not in a gene, the genes up- and downstream of the miRNA were used to estimate CNV.

miR-29a (1)1230.142193.450.56<0.000010.00017q326.61.3-2.6
miR-29a (2)1542.242730.720.56<0.000010.00047q326.61.3-2.6
miR-29b-1 (1)1477.042545.820.58<0.000010.00047q326.61.3-2.6
miR-122a (1)16063.1227088.340.59<0.000010.005918q2106.6
miR-29b-1 (2)1377.042242.490.61<0.000010.01007q326.61.3-2.6
miR-29b-1 (2)960.321559.230.62<0.000010.00407q326.61.3-2.6
miR-125b-1 (1)1613.312482.490.650.001100.032811q241.36.6
miR-125b-1 (2)1448.152194.680.660.001200.031511q241.36.6
miR-122a (2)29564.1540839.460.720.000700.025018q2106.6
miR-181b-2 (1)2216.11622.011.370.000100.00599q335.310.5
miR-181b-2 (2)2289.771470.031.56<0.000010.00019q335.310.5

We found that there is an inverse correlation between miR-29 and AFP, where miR-29a decreased significantly as serum AFP levels increased (Fig. 1C, top panel; miR-29b/c data in Supporting Fig. 1A). This coincides with an association between low levels of miR-29a and poor overall survival (Fig. 1D, top panel; miR-29b/c data in Supporting Fig. 1B). Because miR-29 targets DNA methyltransferase 3A (DNMT3A),[21] we also examined DNMT3A expression in all 237 HCC cases included in the gene expression microarray. We found that DNMT3A is negatively correlated with the miR-29 family (r = −0.41, −0.36, and −0.35, respectively; P < 0.0001; Supporting Fig. 2A-C). DNMT3A showed the opposite trend of miR-29 as it increased significantly with serum AFP and high levels are associated with poor overall survival (Fig. 1C,D, bottom panels). As a control, we compared AFP gene expression levels to AFP serum levels in patients and found a positive correlation (r = 0.66; P < 0.0001; Supporting Fig. 2D). In addition, 10% of patients in the miRNA expression data set were randomly selected to validate their miR-29a expression by qRT-PCR. Supporting Fig. 2E shows a positive correlation between miR-29a expression in the microarray data and the expression detected by qRT-PCR (r = 0.75; P < 0.001).

Because DNMT3A expression is positively correlated with AFP, and associated with poor overall survival, we hypothesized that the global methylation profile of HCC patients with high AFP and DNMT3A levels would differ from patients with low AFP and DNMT3A. To test our hypothesis, we analyzed a subset of HCC cases (n = 48) with Illumina 27k DNA methylation arrays. Unsupervised hierarchical clustering of AFP high, DNMT3A high (HH) cases (n = 20) and AFP low, DNMT3A low (LL) cases (n = 28) revealed that tumors with high AFP/DNMT3A expression (HH) were enriched in cluster 1 and had similar methylation profiles (Fig. 1E). We performed a Fisher's exact test and found that HH cases were significantly enriched in cluster 1 (P = 0.012), compared to clusters 2 and 3. Consistent with AFP and DNMT3A data (Fig. 1C,D, bottom panels), the patients in cluster 1 with high AFP/DNMT3A expression show significant survival differences, when compared to patients in clusters 2 and 3 with predominantly low AFP/DNMT3A expression (Mantel-Cox, P < 0.05; Fig. 1F). Here, we questioned whether AFP mRNA levels were associated with aberrant methylation. To test this, we analyzed CpG island methylation on the AFP promoter in all 48 HCC patients in the methylation array (Supporting Table 3). Using a quartile cutoff of tumor-specific AFP methylation, we compared low and high AFP methylation status to AFP mRNA expression in the same HCC cases and found no significant difference (Supporting Fig. 2F).

The inverse correlation between AFP and the miR-29 family provoked our curiosity as to whether the trend in cancer existed in normal physiological conditions. As an oncofetal protein, AFP is known to be highly expressed during development, then decrease to <20 ng/mL in adult serum. The same trend was observed when fetal tissue was compared to adult normal liver tissue (Supporting Fig. 3A). Conversely, the miR-29 family was hardly detectable in the fetal liver, but highly expressed in adult normal liver tissue (Supporting Fig. 3B), whereas DNMT3A expression showed an increase in fetal liver (Supporting Fig. 3C). These trends were also apparent in mouse fetal and adult normal liver tissue over time (Supporting Fig. 3D). In mouse liver, the miR-29 family was minimally expressed until after birth, when it began to rise significantly. Concurrent with the rise in miR-29, AFP begins to decrease and a switch point is observed at 1 week after birth. It is this switch point in expression that led us to explore AFP's role in functionally regulating miR-29 expression in HCC.

AFP Expression Transcriptionally Regulates miR-29a

AFP has previously been shown to have an effect on HCC cell growth, but the findings have been contradictory.[4] We show that AFP can promote cell proliferation when overexpressed in AFP HLE cells (Fig. 2A). In addition, HLE cells proliferate faster in the presence of CM taken from HLE cells overexpressing AFP, compared to cells growing in CM taken from HLE cells transfected with an empty vector (Fig. 2B; additional AFP negative cell line included in Supporting Fig. 4A).

Figure 2.

AFP transcriptionally regulates miR-29. (A) Overexpression of AFP significantly increases cell proliferation, compared to control HLE cells, after a 48-hour 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 four replicates per time point, and error bars represent standard deviation. (B) HLE cells in the presence of CM taken from AFP overexpressing 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 5 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) Overexpression 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 blotting. miR-29a expression was quantified using qRT-PCR in the bottom panel. (D) DNMT3A and 3B levels significantly increased after transient AFP overexpression (48 hours) in HLE cells. (E) CM 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 show that media taken from cells overexpressing AFP has >500 ng/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 led to a decrease in miR-29a mature (top panel) and primary transcript (bottom panel) expression in SNU-475 cells.

Next, to determine whether AFP functionally modifies miR-29a expression, we transiently overexpressed AFP in HLE cells (Fig. 2C, top panel). A 50% reduction in mature miR-29a expression was observed in HCC cells overexpressing AFP (Fig. 2C, bottom panel; additional AFP negative cell line included in Supporting Fig. 4B). We also analyzed DNMT3A and DNMT3B expression in these cells, and levels of both DNMTs significantly increased after transient AFP overexpression, compared to control cells (Fig. 2D and Supporting Fig. 4C). Next, we silenced AFP expression in HUH-7 cells using a lentiviral/short hairpin (sh)RNA construct. After a 96-hour transient infection with lentivirus, AFP protein expression decreased and we observed an induction of miR-29a expression (Supporting Fig. 4D). However, when we selected for cells that incorporated the control or shAFP lentiviral construct and compared their cell proliferation rate, we found no significant difference (Supporting Fig. 4E). Though AFP protein level was reduced in shAFP-infected cells, abundant AFP remained in media and was detected by enzyme-linked immunosorbent assay (ELISA) as long as 1 week after antibiotic selection (Supporting Fig. 4F; media changed every 3 days). We reasoned that the AFP in the media may still be functional. The lack of AFP depletion in cultured media upon knockdown prompted us to only focus on adding AFP protein to AFP cells either by overexpression or AFP+ CM. First, CM was collected from AFP cells overexpressing AFP (referred to as AFP OE CM) and placed on freshly seeded HLE cells. Compared to HLE cells in the presence of AFP CM (referred to as control CM), taken from HLE cells transfected with an empty vector, AFP OE CM led to a significant decrease in mature miR-29a expression (Fig. 2E, bottom panel). In addition to AFP OE CM, we applied CM from HUH-7 cells, which highly express AFP (Supporting Fig. 4G), to two AFP HCC cell lines (HLE and SNU-475). The mature miR-29a level decreased at least 50% in each cell line (Fig. 2F,G, top panel).

Then, we questioned whether regulation of miR-29 was transcriptional or through processing of miRNA. Though the Ohio State University Comprehensive Cancer Center (OSU-CCC) microarray includes over 1,700 probes for miRNAs, only 18 were significantly differentially expressed with a fold change greater than 20% in our class comparison analysis. Using a Taqman assay specific to a region on the miR-29a transcript, we tested whether or not expression of miR-29a decreased at the transcriptional level when AFP was present. Indeed, when the primary transcript of miR-29a was tested, a decrease was apparent in the presence of AFP, suggesting a transcriptional method of regulation (Fig. 2F,G, bottom panel; additional AFP cell line included in Supporting Fig. 4H).

c-MYC Mediates the Transcriptional Down-Regulation of miR-29a

The above results reveal that AFP plays a functional role in down-regulating miR-29a expression. Because AFP is a membrane-secreted protein and not abundant in the cell nucleus, we hypothesized that it acts through a transcription factor of miR-29a. To determine which transcription factors were activated in the presence of AFP, we went back to the mRNA profiles of HCC patients and ran a class comparison analysis between 44 patients with high AFP, high DNMT3A, and low miR-29a (our phenotype of interest) versus 44 patients with low AFP, low DNMT3A, and high miR-29a expression using median cutoff. A total of 379 genes were differentially expressed with a fold change greater than 2 after 10,000 permutations were computed with an FDR of 1% and a P value cutoff of 0.001. The gene list was imported into Ingenuity Pathway Analysis (IPA) to find transcription factors significantly activated or deactivated based on altered gene expression. The activation state of transcription factors is determined by comparing gene expression patterns to the expression of transcriptional regulators in the input data set, and IPA then assigns each transcriptional regulator a z-score (more detailed information in the Experimental Procedures section of the Supporting Information). In the gene list derived from our class comparison analysis, a total of eight transcription factors were assigned activated (Table 3), whereas 12 were inhibited with statistical significance (not shown). Among those activated were MYC and GLI1, both shown previously to bind the promoter of miR-29a/b-1 and inhibit its expression.[22, 23] The miR-29a promoter region has been mapped to roughly 36 kilobases upstream of the miR-29a/b-1 cluster.[22, 23] In addition to MYC and GLI1, the University of California, Santa Cruz (UCSC) Genome Browser displays ChIP sequencing data showing that FOXM1, E2F, and SP1 also have binding sites on the miR-29a/b-1 locus (Table 3). In our analysis, c-MYC seemed a likely transcription factor involved because it ranked as the second-most activated by z-score and had been previously shown to inhibit miR-29a promoter expression experimentally.

Table 3. Eight Transcription Factors Are Activated in Patients With High AFP, High DNMT3A, and Low miR-29a Expression
Transcription FactoraIngenuity Regulation Z-ScorePredicted Activation StateP Value of OverlapUCSC ChIP Sequence Binding Sites? (No.)
  1. a

    Transcription factors were predicted in IPA from 379 differentially expressed genes between HCC cases with high AFP/DNMT3A and low miR-29a expression versus cases with the opposite expression pattern.

  2. Abbreviation: n.s., none shown.

FOXM13.437Activated1.42E-07Yes (3)
MYC3.022Activated3.67E-11Yes (5)
E2F2.398Activated1.53E-07Yes (1)
SP12.165Activated2.24E-04Yes (6)

To determine whether c-MYC regulated the transcript of miR-29a, we silenced c-MYC protein expression using siRNA in HLE cells (Fig. 3A). HLE cells endogenously express c-MYC, but are negative for AFP. Indeed, the absence of c-MYC induced expression of the miR-29a transcript, compared to cells transfected only with an empty vector (Fig. 3B). As a control, we overexpressed AFP and found that pri-miR-29a decreased as expected (Fig. 3B). Interestingly, in the absence of c-MYC protein, AFP was unable to decrease miR-29a transcript expression (Fig. 3B). To further test whether AFP inhibits miR-29a through c-MYC, we designed primers around predicted c-MYC-binding sites on the miR-29a/b-1 transcript (Fig. 3C). A ChIP assay was used with anti-c-MYC to pull down genomic regions bound by c-MYC protein. After ChIP, P-1 and P-2 primer sets were employed to quantify the amount of c-MYC bound to the specified regions on the miR-29a/b-1 transcript. Figure 3D shows that c-MYC was abundantly bound to the miR-29a/b-1 promoter and to the intron in the presence of AFP, but did not bind without AFP. Though the presence of AFP leads to increased binding of c-MYC to the miR-29a transcript, an increased steady-state level of c-MYC protein during AFP overexpression was minimal (Fig. 3A). To better understand the possible mechanism by which AFP facilitates MYC binding to the miR-29a transcript, we conducted a set of experiments with cycloheximide (CHX). CHX interferes with the translation step during protein synthesis and effectively stops translational elongation. By treating cells first with AFP+ CM (8 hours), then with CHX for 15 minutes, we found an accumulation of c-MYC protein (Supporting Fig. 5A), suggesting that AFP regulates the half-life of c-MYC.

Figure 3.

AFP mediates the down-regulation of miR-29a through c-MYC. (A) AFP is overexpressed 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 is located in the intron of the transcript. Transcript schematic is modified from that created by Chang et al.[22] (D) ChIP was performed to pull down c-MYC bound to genomic regions in HLE cells after AFP+ or AFP CM was applied (rabbit immunoglobulin G [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.

AFP Expression Promotes Tumor Growth In Vivo

A stable AFP+ HLE cell line was created for a xenograft study in nude (aythymic Nu/Nu) mice. Control and AFP+ HLE cells were infected with a lentiviral construct and selected using an antibiotic. Stable AFP+ HLE cells express AFP endogenously as well as in the media (Fig. 4A). Consistently, stable AFP+ HLE cells proliferated much faster than control cells (Fig. 4B). These cells also had faster migratory and invasive capacities than control cells (Fig. 4C,D). Upon subcutaneous (SC) injection in nude mice, the group with SC injection of 0.5 × 106 AFP+ cells showed faster tumor incidence (Fig. 4E) and enhanced tumor growth (Fig. 4F). Similar results were obtained with 1 × 106 cells (data not shown).

Figure 4.

AFP expression promotes cell growth, cell migration, and invasion in vitro and tumorigenesis in vivo. (A) AFP expression detected by western blotting (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.


AFP was first detected as a fetal-associated protein in 1957 and found to be tumor associated 6 years later.[24, 25] Almost a decade passed before the protein was isolated and purified, and it took yet another decade before biological studies were initiated.[4] Currently, more than 50 years after its discovery, AFP is thought to exist mainly as a transport protein and biomarker for HCC. Though it has been shown to have growth-regulatory properties and bind and transport estrogen, its molecular mechanism in HCC is not well understood.[4]

Recently, an interest in AFP and methylation has been sparked. Zhang et Al. examined the promoter methylation status of nine genes in 50 paired tumor and nontumor HCC tissue samples.[26] They defined a CpG island methylator phenotype (CIMP)+ if five out of nine genes were concordantly methylated. Not only did this group observe a higher frequency of CIMP+ in tumor than in nontumor tissue, they found that CIMP+ was most frequent in HCC with elevated AFP (>30 ng/mL).[26] In addition to this study, another group (Wu et al.) found similar results.[27] They analyzed promoter methylation of seven genes in 65 HCC cases (CIMP+ if three or more genes were methylated) and found that CIMP+ was more prevalent in patients with >400 ng/mL of serum AFP. Furthermore, they observed that patients with CIMP+ often had multiple tumors and worse recurrence-free survival, compared to CIMP patients.[27]

In our study, we found an association between AFP and methylation. Not only is AFP associated with increased expression of DNA methyltransferases, enzymes that catalyze the methylation process, AFP is also associated with increased methylation of many gene promoters. When we analyzed CpG island methylation of the AFP promoter itself, we found no association with mRNA expression in 48 HCC cases, suggesting that the gene is not epigenetically regulated in our HCC cohort and a feedback loop between the DNA methyltransferase enzymes and AFP is not evident. Global methylation profiling of the same 48 HCC cases showed that a significant number of patients with high AFP and DNMT3A expression cluster together, suggesting that their poor outcome is driven by a common mechanism. Moreover, AFP was observed to induce cell growth in vitro and in vivo, a trait ubiquitous in cancer, and increase the migratory and invasive properties of HCC cells, indicating that the oncofetal protein has a functional role in addition to its role as a biomarker. We have found that AFP works by transcriptionally inhibiting miR-29a expression, which leads to the induction of DNMT3A, and we propose that AFP drives these epigenetic changes to shape the microenvironment in a way that promotes tumorigenesis. Based on this evidence, AFP, as an extracellular protein circulating in blood, changes the cell fate and tumorigenic capacity of HCC cells, making it an ideal candidate to target therapeutically using pharmacological interventions.

DNA methylation is a key epigenetic component that regulates gene expression. DNA methyltransferases, enzymes that methylate DNA by binding to CpG dinucleotides on gene promoters, are associated with transcriptional silencing and may lead to aberrant methylation of genes when up-regulated.[28, 29] Though not much is known about the relationship between DNA methyltransferases and miRNA, a study by Croce et al. showed that miR-29 specifically targets both DNMT3A and 3B in lung cancer.[21] Regulation of DNA methyltransferases by miR-29 may contribute to the transcriptional silencing of tumor suppressors, leading to poor prognosis of cancer patients. Additionally, down-regulation of miR-29 has been shown in HCC and lung cancer, suggesting tumor-suppressor properties.[30, 31] In our study, we find that AFP-induced miR-29a suppression leads to increased expression of both DNA methyltransferases in HCC.

It is interesting that c-MYC acts as the mediator between AFP and the miR-29a/b-1 transcript. In the early 1990s, the association between c-MYC and HCC was first described.[32] Peng et al. found that the c-MYC gene was amplified (>1.5-fold) in nearly 40% of their HCC cases and showed that those patients not only had elevated serum AFP (>320 ng/mL), but were more likely to have hepatitis B infection.[32] They concluded that amplification of c-MYC was not uncommon in HCC and may be related to its biological behavior. Furthermore, hepatitis B X protein, a hepatitis B viral protein that transforms hepatocytes and has been implicated in HBV-driven HCC, has also been shown to activate c-MYC.[33-35] Though we do not see an amplification of the c-MYC gene when comparing AFP low to AFP high patients in our cohort (data not shown), that might be the result of the fact that all patients have hepatitis B infection.

In our molecular studies, we observe that c-MYC binds to the miR-29a/b-1 transcript in the presence of AFP. It is known that AFP does not localize to the nucleus; therefore, the mechanism by which AFP promotes c-MYC binding to the nuclear transcript is unclear, but there are several possibilities. For example, AFP may induce the nuclear localization of c-MYC by transporting a number of factors into tumor cells or may even bind to and transport c-MYC itself. There is also evidence of a nonsecreted form of AFP, which may have the ability to interact with transcription factors, coactivators, or regulators of cell cycle.[36-38] In addition, the half-life of c-MYC is short and fluctuates substantially in response to many cellular activities.[39] In our study, we found that treatment with AFP+ CM followed by a short treatment with CHX led to an accumulation of c-MYC protein in HLE cells. It is possible that AFP extends the half-life of the c-MYC protein by increasing its stability, because even a relatively short extension of c-MYC expression could greatly change the cellular microenvironment in favor of tumor growth. Undoubtedly, further functional analysis at the molecular level must be done to elucidate the function of AFP in regard to c-MYC signaling and epigenetic alterations that drive poor outcome in HCC.

We propose that AFP may regulate miR-29a expression, which, in turn, alters DNA methyltransferase expression and modulates the epigenome in HCC (Fig. 5). The increased methylation we observe may inhibit tumor-suppressor gene expression, provoking aggressive HCC and poor outcome. For the first time, striking differences at the molecular level are shown between AFP+ and AFP HCC patients. In light of these findings, it is our hope that further work is done to determine the molecular functions of AFP in regard to c-MYC signaling and the epigenome. The more insight we gain at the molecular level, the better able we are to subgroup HCC patients and appropriately treat this heterogeneous disease.

Figure 5.

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


The authors appreciate the mouse-specific AFP primers given to them by Agnus Holczbauer in Snorri Thorgeirrson's Laboratory of Experimental Carcinogenesis, NCI. The authors also acknowledge the SAIC-Protein Expression Lab for virus production.