modulation of mir-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma...

Revision for Hepatology; HEP-13-2227R1

Modulation of miR-29 Expression by Alpha-fetoprotein is linked to the Hepatocellular

Carcinoma Epigenome

Sonya Parpart1,2, Stephanie Roessler

1,†, Fei Dong

1,†, Vinay Rao

1,†, Atsushi Takai

1, Junfang Ji

1,†,

Lun–Xiu Qin3, Qing–Hai Ye

3, Hu–Liang Jia

3, Zhao–You Tang

3, Xin Wei Wang

1,¥

1Laboratory of Human Carcinogenesis, NCI, Bethesda, MD

2Tumor Biology Department, Georgetown University, Washington, DC

3Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, China

Keywords: DNMT3A, c-MYC, Epigenetics, Liver cancer, DNA methylation

This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/hep.27200

2

Footnote Page

†Current addresses: S.R.: Institute of Pathology, University Hospital Heidelberg, Heidelberg,

Germany

F.D.: Department of Pathology, Massachusetts General Hospital, 55 Fruit St, Boston, MA 02114

V.R.: Department of Internal Medicine, UConn Health Center, 263 Farmington Avenue,

Farmington, CT 06030-1235

J.J.: University of Hawaii Cancer Center, 701 Ilalo street, Honolulu, HI 96813.

¥Correspondence: Xin Wei Wang, National Cancer Institute, 37 Convent Drive, Bethesda,

Maryland 20892; Email: [email protected]; Phone: 301-496-2099; Fax: 301-496-0497

Financial Support:

This work was supported by the grant (Z01 BC 010313) from the Intramural Research

Program of the Center for Cancer Research, National Cancer Institute.

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Abstract

Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers

and ranks second in the 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 profiling, we found that 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.

Over expression 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. Further, AFP expression promotes tumor growth of AFP- HCC cells in nude mice.

Conclusion: our findings indicate that tumor biology differs considerably between AFP+ HCC

and AFP- HCC and that AFP is a functional antagonist of miR-29, which may contribute to

global epigenetic alterations and poor prognosis in HCC.

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Introduction

The worldwide incidence of 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 (HBV) and hepatitis C virus (HCV) 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 (>1000ng/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 cirrhotic patents develop HCC without any increase in AFP. In the cohort of

HCC patients that we study, 40% have normal levels (<20ng/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, the oncofetal

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protein plays a role in the 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 patients who have cirrhosis

without HCC (9).

Aberrant microRNA expression is a ubiquitous feature in an increasing number of

cancers including HCC (10). Studies indicate that these microRNAs (miRNAs) are directly

connected with epigenetic factors that regulate gene expression (11). MiRNAs are short, non-

coding RNAs about 22 nucleotides in length that regulate the function of messenger RNA

(mRNA) (12). Partial sequence homology allows miRNAs to bind to the 3’UTR of target

mRNAs inhibiting translation or causing mRNA degradation (11, 12). Recently, a specific group

of miRNAs have been designated “epi-miRNAs” as 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 the miRNA, mRNA and methylation expression patterns. In addition, we reveal an

important functional role of AFP in HCC. Not only is AFP inversely correlated to miR-29 in

HCC tumor tissue, we demonstrate that AFP transcriptionally down regulates miR-29a through

c-MYC. Identifying the molecular mechanisms underlying AFP+ tumors will help build an

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understanding of heterogeneity in HCC as well as our understanding of AFP’s functional role in

HCC progression.

Experimental Procedures

Patient studies and tumor specimens

Paired tumor and non-tumor 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 Experimental Procedures in the Supplemental

Material for details.

Xenograft study

The animal study protocol was approved by the NIH Animal Care and Use Committee

and all animals received human care according to the NIH “Guide for Care and Use of

Laboratory Animals.” Refer to Experimental Procedures in the Supplemental Material for

details.

Plasmids, lentiviral vectors, siRNA, qRT-PCR, conditioned media study design, protein

expression analysis, cell proliferation assays, ChiP assay, and all statistical analysis

Please refer to Experimental Procedures in the Supplementary Materials.

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Results

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 (<20ng/ml) and increasing ten-fold (i.e., 20-200, 200-2000, and

>2000ng/ml) where patients with >2000ng/ml AFP were considered to be extremely high cases.

Overall survival data confirms that poor clinical outcome is associated with increasing serum

AFP suggesting that the biological make up of these HCCs differ from those that are AFP-

(Figure 1A, Mantel-Cox p<0.05; log-rank p<0.005). Additional clinical characteristics of HCC

cases in each subgroup can be found in Suppl. 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 TNM staging, the only two variables from the univariable analysis that passed a

stepwise selection process using both forward addition and backwards subtraction with a p-value

cut-off 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).

Affymetrix mRNA expression data are available for 237 paired tumor and non-tumor

tissue samples as described in earlier studies (17-19), while miRNA expression data are available

on a subset of patients (n=223) in our cohort (Figure 1B). Global DNA methylation profiles were

also obtained for a subset of HCC patients who have mRNA expression data.

We next determined if AFP+ HCC may have different miRNA expression. We performed

a class comparison analysis between 74 patients with normal serum AFP (<20ng/ml) and 39

patients with extremely high AFP levels (>2000ng/ml) and found that 18 miRNAs were

differentially expressed using a p-value cut-off of 0.001 and a false discovery rate of 5% (Table

2). Of the twelve down regulated miRNAs (ranked by fold change), seven were in the miR-29

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family. All members of the miR-29 family were also associated with poor survival in HCC

(Suppl. Table 2). Interestingly, when examining available array CGH 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).

We found that there is an inverse correlation between miR-29 and AFP where miR-29a

decreased significantly as serum AFP levels increased (Figure 1C, top panel; miR-29b/c data in

Suppl. Figure 1A). This coincides with an association between low levels of miR-29a and poor

overall survival (Figure 1D, top panel; miR-29b/c data in Suppl. Figure 1B). Since miR-29

targets 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, -0.35 respectively; p<0.0001; Suppl. Figure 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 (bottom panels of Figure 1C and 1D). 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, Suppl. Figure 2D). In addition, 10% of patients in the

miRNA expression dataset were randomly selected to validate their miR-29a expression by

quantitative RT-PCR. Suppl. Figure 2E shows a positive correlation between miR-29a

expression in the microarray data and the expression detected by RT-PCR (r= 0.75, p<0.001).

Since 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

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AFP low, DNMT3A low (LL) cases (n=28) reveals that tumors with high AFP/DNMT3A

expression (HH) are enriched in cluster #1 and have similar methylation profiles (Figure 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 (Figure 1C

and 1D, 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; Figure 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 (Suppl. Table 3). Using a quartile cut-off 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 (Suppl. Figure 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 <20ng/mL in adult

serum. The same trend was observed when fetal tissue was compared to adult normal liver tissue

(Suppl. Figure 3A). Conversely, the miR-29 family was hardly detectable in the fetal liver but

highly expressed in adult normal liver tissue (Suppl. Figure 3B) whereas DNMT3A expression

showed an increase in the fetal liver (Suppl. Figure 3C). These trends were also apparent in

mouse fetal and adult normal liver tissue over time (Suppl. Figure 3D). In the 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

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week. 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 over

expressed in AFP- HLE cells (Figure 2A). In addition, HLE cells proliferate faster in the

presence of conditioned media (CM) taken from HLE cells over expressing AFP compared to

cells growing in CM taken from HLE cells transfected with an empty vector (Figure 2B,

additional AFP negative cell line included in Suppl. Figure 4A).

Next, to determine if AFP functionally modifies miR-29a expression we transiently over

expressed AFP in HLE cells (Figure 2C, top panel). A fifty percent reduction in mature miR-29a

expression was observed in HCC cells over expressing AFP (Figure 2C, bottom panel; additional

AFP negative cell line included in Suppl. Figure 4B). We also analyzed DNMT3A and

DNMT3B expression in these cells and the levels of both DNMTs significantly increased after

transient AFP over expression compared to control cells (Figure 2D and Suppl. Figure 4C). Next,

we silenced AFP expression in HUH-7 cells using a lentiviral-shRNA construct. After a 96 hour

transient infection with lentivirus, AFP protein expression decreased and we observed an

induction of miR-29a expression (Suppl. Figure 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 (Suppl. Figure 4E). Though the AFP protein level was

reduced in the shAFP infected cells, abundant AFP remained in the media and was detected by

ELISA as long as one week after antibiotic selection (Suppl. Figure 4F, media changed every

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three 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 over expression or AFP

+ conditioned media. First, conditioned media was

collected from AFP- cells over expressing AFP (referred to as AFP OE CM) and placed on

freshly seeded HLE cells. Compared to HLE cells in the presence of AFP- conditioned media

(referred to as Control CM), taken from HLE cells transfected with an empty vector, the AFP OE

CM led to a significant decrease in mature miR-29a expression (Figure 2E, bottom panel). In

addition to AFP OE CM, we applied conditioned media from HUH-7 cells, which highly express

AFP (Suppl. Figure 4G), to two AFP negative HCC cell lines, HLE and SNU-475. The mature

miR-29a level decreased at least fifty percent in each cell line (Figure 2F and 2G, top panel).

Then we questioned whether the regulation of miR-29 was transcriptional or through the

processing of the miRNA. Though the 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 (Figure

2F and 2G, bottom panel; additional AFP negative cell line included in Suppl. Figure 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. Since 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

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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 cut-off. 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 cut-off 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 expression of transcriptional regulators in

the input dataset and IPA then assigns each transcriptional regulator a z-score (more detailed

information in Supplemental Experimental Procedures). In the gene list derived from our class

comparison analysis, a total of eight transcription factors were assigned activated (Table 3) while

twelve 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 36kb upstream of

the miR-29a/b-1 cluster (22, 23). In addition to MYC and GLI1, the UCSC Genome Browser

displays chromatin immunoprecipitation (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 as it ranked second most activated by z-score and

had been previously shown to inhibit miR-29a promoter expression experimentally.

To determine if c-MYC regulated the transcript of miR-29a, we silenced c-MYC protein

expression using siRNA in HLE cells (Figure 3A). HLE cells endogenously express c-MYC but

are negative for AFP. Indeed, the absence of c-MYC induced expression of the miR-29a

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transcript compared to cells transfected only with an empty vector (Figure 3B). As a control we

over expressed AFP and found that pri-miR-29a decreased as expected (Figure 3B).

Interestingly, in the absence of c-MYC protein, AFP was unable to decrease miR-29a transcript

expression (Figure 3B). To further test if AFP inhibits miR-29a through c-MYC, we designed

primers around predicted c-MYC binding sites on the miR-29a/b-1 transcript (Figure 3C). A

ChIP assay was used with anti-c-MYC to pull down genomic regions bound by c-MYC protein.

Following 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 (Figure 3A). To better understanding 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+ conditioned media

(eight hours) then with CHX for 15 minutes, we found an accumulation of c-MYC protein

(Suppl. Figure 5A), suggesting that AFP regulates the half-life of c-MYC.

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

(Figure 4A). Consistently, stable AFP+ HLE cells proliferated much faster than control cells

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(Figure 4B). These cells also had faster migratory and invasive capacities than control cells

(Figure 4C-D). Upon subcutaneous injection in nude mice, the group with subcutaneous injection

of 0.5x106 AFP

+ cells showed faster tumor incidence (Figure 4E) and enhanced tumor growth

(Figure 4F). Similar results were obtained with 1x106 cells (data not shown).

Discussion

AFP was first detected as a fetal-associated protein in 1957 and found to be tumor-

associated six years later (24, 25). Almost a decade past before the protein was isolated and

purified and it took yet another decade before biological studies were initiated (4). Currently,

more than fifty 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 to bind

and transport estrogen, its molecular mechanism in HCC is not well understood (4).

Recently, an interest in AFP and methylation has sparked. Zhang et. al examined the

promoter methylation status of nine genes in 50 paired tumor and non-tumor HCC tissue samples

(26). They defined a CpG island methylator phenotype (CIMP+) if five of the 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

(>30ng/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 3 or more genes

were methylated) and found that CIMP+ was more prevalent in patients with >400ng/ml serum

AFP. Furthermore, they observed that patients with CIMP+ often had multiple tumors and worse

recurrence-free survival compared to CIMP- patients (27).

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In our study, we find 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 the gene is not epigenetically regulated in

our HCC cohort and a feedback loop between the DNMT 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 to increase the migratory and invasive

properties of HCC cells indicating the oncofetal protein has a functional role in addition to its

role as a biomarker. We have found 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 (DNMTs), 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 upregulated (28, 29). Though not much is known about the

relationship between DNMTs and miRNA, a study by Croce and colleagues showed that miR-29

specifically targets both DNMT3A and DNMT3B in lung cancer (21). The regulation of DNMTs

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by miR-29 may contribute to the transcriptional silencing of tumor suppressors leading to poor

prognosis of cancer patients. Additionally, the 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 DNMTs in HCC.

It is interesting that c-MYC acts as the mediator between AFP and the miR-29a/b-1

transcript. In the early 1990’s 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 (>320ng/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, HBx, 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 don’t 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 due to 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 non-secreted form of AFP, which may have the ability to

interact with transcription factors, co-activators, 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+ conditioned media followed by a short

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treatment with cycloheximide 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, for 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 regards 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 (Figure 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 regards 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 the heterogeneous disease.

Acknowledgements

We appreciate the mouse-specific AFP primers given to us by Agnus Holczbauer in

Snorri Thorgeirrson’s Laboratory of Experimental Carcinogenesis, NCI. We also acknowledge

the SAIC- Protein Expression Lab for virus production.

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Figure Legend

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

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(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,

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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 0.5x106 AFP

+ cells AFP

+ cells compared to 0.5x10

6 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

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21

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.

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Table 1. Univariable and multivariable analyses of factors associated with disease stage, aggressive HCC and

survival (n= 237)

Univariable analysisa Multivariable analysis

b

Clinical Variable

Hazard ratio

(95% CI) p-value Hazard ratio

(95% CI) p-value

AFP (> 300 ng/mL vs ≤ 300 ng/mL) 1.7 (1.1 - 2.5) 0.011 1.6 (1.0 - 2.4) 0.043

Age (≥ 50 yr vs <50 yr) 0.8 (0.5 - 1.2) 0.26 n.a.

Sex ( male vs female) 1.8 ( 0.9 - 3.7) 0.116 n.a.

Cirrhosis (yes vs no) 5.2 (1.3 - 20.9) 0.022 n.a.

Tumor size (> 3 cm vs ≤ 3 cm) 2.4 (1.5 - 3.8) < 0.001 n.a.

Multinodularity 1.6 (1.0 - 2.5) 0.046 n.a.

Vascular invasion 1.8 (1.2 - 2.9) 0.007 n.a.

TNM staging (II-III vs I)c 2.9 (1.8 - 4.8) < 0.001 2.9 (1.8 - 4.8) < 0.001

aUnivariable analysis, Cox proportional hazards regression.

bMultivariable analysis, Cox proportional hazards regression.

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

CI, confidence interval.

n.a., not applicable.

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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

Probea

AFP

>2000ng/ulc

AFP

<20ng/uld

Fold

Change

Parametric

p-value FDR

Chromosome

Location

Gain

(%)e

Loss (%)e

miR-29a (1) 1230.14 2193.45 0.56 < 0.00001 0.0001 7q32 6.6 1.3 - 2.6

miR-29a (2) 1542.24 2730.72 0.56 < 0.00001 0.0004 7q32 6.6 1.3 - 2.6

miR-29b-2 1756 3106.81 0.57 < 0.00001 0.0001 1q32 34.2 1.3

miR-29b-1 (1) 1477.04 2545.82 0.58 < 0.00001 0.0004 7q32 6.6 1.3 - 2.6

miR-122a (1) 16063.12 27088.34 0.59 < 0.00001 0.0059 18q21 0 6.6

miR-29b-1 (2) 1377.04 2242.49 0.61 < 0.00001 0.0100 7q32 6.6 1.3 - 2.6

miR-29b-1 (2) 960.32 1559.23 0.62 < 0.00001 0.0040 7q32 6.6 1.3 - 2.6

miR-29c 1418.1 2247.65 0.63 < 0.00001 0.0059 1q32 34.2 1.3

miR-125b-1 (1) 1613.31 2482.49 0.65 0.00110 0.0328 11q24 1.3 6.6

miR-125b-1 (2) 1448.15 2194.68 0.66 0.00120 0.0315 11q24 1.3 6.6

miR-122a (2) 29564.15 40839.46 0.72 0.00070 0.0250 18q21 0 6.6

miR-let-7d 1116.67 1429.47 0.78 0.00030 0.0233 9q22 2.6 13.2

miR-let-7f 679.58 862.59 0.79 0.00060 0.0250 9q22 2.6 13.2

miR-181b-2 (1) 2216.1 1622.01 1.37 0.00010 0.0059 9q33 5.3 10.5

miR-181c 1279.19 930.72 1.37 0.00010 0.0093 19q13 9.2 - 10.5 3.9

miR-181b-1 1807.52 1193.32 1.51 0.00010 0.0044 1q32 34.2 1.3

miR-181b-2 (2) 2289.77 1470.03 1.56 < 0.00001 0.0001 9q33 5.3 10.5

miR-32 1891.71 1143.94 1.65 0.00010 0.0059 9q31 2.6 13.2

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

cHigh AFP, n=40

dLow AFP, n=74

ePercent 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 upstream and downstream of the miRNA were used to estimate CNV.

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Table 3. Eight transcription factors are activated in patients with high AFP, high DNMT3A and low miR-29a

expression.

Transcription Factora

Ingenuity Regulation

z-score

Predicted

Activation State

p-value of

Overlap

UCSC ChIP-

Seq binding

sites? (#)

FOXM1 3.437 Activated 1.42E-07 Yes (3)

MYC 3.022 Activated 3.67E-11 Yes (5)

TBX2 2.624 Activated 2.22E-05 n.s

SREBF1 2.466 Activated 1.40E-06 n.s

E2F 2.398 Activated 1.53E-07 Yes (1)

FOXO1 2.301 Activated 1.08E-13 n.s.

SP1 2.165 Activated 2.24E-04 Yes (6)

GLI1 2.138 Activated 1.18E-04 n.s. aTranscription factors were predicted in Ingenuity Pathway Analysis from 379 differentially expressed genes

between HCC cases with high AFP/DNMT3A and low miR-29a expression versus cases with the opposite

expression pattern.

n.s. = none shown

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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.

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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

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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.

143x268mm (300 x 300 DPI)

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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. 124x197mm (300 x 300 DPI)

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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 0.5x106 AFP+ cells compared to 0.5x106 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) at 4 weeks shows a difference between the size of control versus AFP+ tumors (p<0.04). Error bars represent mean plus standard error. Image of tumors extracted from the control and AFP+ groups after 4 weeks.

196x228mm (300 x 300 DPI)

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Figure 5. Schematic illustrating the mechanism by which AFP transcriptionally down regulates miR-29a and

drives poor prognosis in HCC.

179x120mm (300 x 300 DPI)

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Supplemental Material

Modulation of miR-29 Expression by Alpha-fetoprotein is linked to the Hepatocellular Carcinoma Epigenome

Sonya Parpart1,2, Stephanie Roessler1,†, Fei Dong1,†, Vinay Rao1,†, Atsushi Takai1, Junfang Ji1,†, Lun–Xiu Qin3, Qing–Hai Ye3, Hu–Liang Jia3, Zhao–You Tang3, Xin Wei Wang1,¥

1Laboratory of Human Carcinogenesis, NCI, Bethesda, MD

2Tumor Biology Department, Georgetown University, Washington, DC

3Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, China

Supplemental Experimental Procedures

Patient studies and tumor specimens

Affymetrix U133A 2.0 microarrays were completed on paired tumor and non-tumor HCC tissues for 237

patients and deposited into GEO Omnibus with accession number GSE14520 (16). OSU-CCC miRNA

microarrays version 2.0 were completed on 223 paired tumor and non-tumor patient HCC tissue samples

with accession number GSE6857 (17). Illumina Human BeadChip Methylation microarrays were

completed on 48 paired tumor and non-tumor HCC tissues and deposited into GEO Omnibus (accession

number available soon). Class comparison analysis for differentially expressed genes and miRNAs was

done using BRB Array Tools (version 4.3). Hierarchical clustering and analysis of Illumina methylation

microarrays was performed in R (version 2.15).

Univariable and multivariable analysis

A univariable analysis employing Cox proportional hazards regression was performed to

determine the effect of clinical variables on HCC patient survival and includes clinical variables that

reflect features of aggressive or late stage tumors. A multivariable analysis was performed considering

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only the variables from the univariable analysis that passed a stepwise selection process using both

forward addition and backwards subtraction selection with a p-value cut-off of <0.05. The final, most

prudent survival model includes the clinical group (high vs. low AFP) and TNM staging as AFP level

outperformed the other clinical variables with the same significance. No multi-colinearity between the

covariates was found.

Human and mouse liver studies

Human fetal liver total RNA was purchased from Clontech and contained RNA from normal

pooled fetal livers from 63 spontaneously aborted male and female Caucasian fetus ages 22-40 weeks.

Adult liver RNA was taken from a pool of 8 healthy donor livers. Mouse fetal liver RNA was collected as

described previously (18).

PCR amplification

AFP, DNMT3A and miR-29 levels were detected by quantitative RT-PCR in both human and

mouse RNA. For the human tissue, the following probes were purchased from Life Technologies: AFP

#Hs00173490, DNMT3A #Hs01027166, DNMT3B #Hs00171876, miR-29a #002112, miR-29b #000413,

and miR-29c #000587. 18S and U6B were used as endogenous control for gene expression and miRNA

expression, respectively (Life Technologies, 18S: #4319413E, U6B: #001093). For the mouse tissue,

miRNA expression was detected by the same method as human RNA, however, AFP gene expression was

detected using mouse specific probes and SYBR green master mix (Life Technologies, catalog

#4367659). Mouse-AFP primer set: 5’- CCA GGA AGT CTG TTT CAC AGA AG -3’ (forward), 5’-

CAA AAG GCT CAC ACC AAA GAG -3’ (reverse) and b-actin as a control 5’- TTG CTG ACA GGA

TGC AGA AG -3’(forward), 5’- ACA TCT GCT GGA AGG TGG AC- 3’ (reverse).

For quantification of miRNA-29a, Taqman quantitative real-time PCR kits were followed. First,

reverse transcription of 10ng RNA was performed using the Taqman miRNA reverse transcription kit

with primers for miR-29a and U6B, as an endogenous control (Life Technologies, catalog #4366597).

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Following reverse transcription, PCR reactions were prepared using the probes for both miR-29a and

U6B then qRT-PCR was run in duplicate on the ABI 7500 thermocycler. For quantification of the

primary transcript of miR-29a (pri-miR-29a), reverse transcription of 1µg RNA was carried out using a

Taqman high capacity cDNA reverse transcription kit (Life Technologies, catalog #4368813) and gene

expression assays for pri-miR-29a and Actin, as an endogenous control (Life Technologies, pri-miR-29a:

#Hs03302672_pri, Actin: #4352935E). PCR reactions were prepared following reverse transcription and

qRT-PCR was run in duplicate on the ABI 7500 thermocycler.

Cell proliferation assays

Calcein AM was used to measure cell proliferation differences in HLE cells transfected with a

plasmid to over express AFP versus control cells. 2000 cells per well were seeded into 96-well plates 24

hours after transient transfection and cell proliferation was measured over an eight-day period. To

measure cell proliferation, migration and invasion of stable AFP+ or control cells, or HLE cells in the

presence of conditioned media, the xCELLigence system was employed (Roche Applied Science,

Indianapolis, IN). xCELLigence monitored electrical impedance to distinguish the growth, migration or

invasion differences of HLE cells or growth of SNU-182 cells over a period of five days in the presence

of AFP+ and AFP- CM. AFP+ CM was taken from HLE cells over expressing AFP for 48 hours while

AFP- CM was taken from HLE cells transfected with a control plasmid. CM was diluted 1:1 with fresh,

complete DMEM prior to treatment. HLE cells were only treated with CM one time, 24 hours after cells

were seeded onto the xCELLigence plate.

Lentiviral vectors, plasmids and siRNA

Lentiviruses were packaged using a Trans-Lenti shRNA Packaging Kit also purchased from Open

Biosystems (catalog #TLP5913; lot #LD 230901). A standard protocol was followed to transfect 1x106

HEK293T cells with a control or shRNA-AFP lentiviral construct. Supernatant of the HEK293T cells was

collected after 72 hours and filtered through Millex-HV 0.45um PVDF filters purchased from Millipore

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(catalog #SLHA033SS). 1x106 HUH-7 cells were seeded onto 10cm plates the day prior to infection.

Cells were infected with the control or shRNA-AFP virus at an MOI=10 for 24 hours then the media was

changed to complete DMEM. Cells continued incubating for 72 hours prior to collection and RNA

extraction by TRIzol.

pCMV6-XL5-AFP and pCMV6-entry plasmids were purchased from Origene. Transient

transfection of pCMV-AFP was carried out for AFP over expression in HLE and SNU-182 cells. A total

of 4µg DNA was used for transfections with 8µL lipofectamine in 10cm plates and a standard transfection

protocol was followed. pLKO.1 lentiviral constructs empty, for use as a control, or with shRNA targeting

AFP were purchased from Open Biosystems (catalog #RHS4533; clone ID: TRCN0000056862).

Precision LentiORF pLOC vectors, empty or including AFP, were purchased from Thermo

Scientific Open Biosystems to make stable AFP+ cell lines (catalog # OHS5897-101187109 and

OHS5899-101187109; clone ID: PLOHS_100003572). These vectors were sent to the SAIC- Frederick

Protein Expression lab for virus production. HLE cells were infected at an MOI of 10 then selected for

using 20ug/uL Blasticidine 72 hours after initial infection.

siRNA-MYC was purchased from Qiagen (Hs_MYC_5). In siRNA-MYC experiments, HLE cells

were seeded at 1x105 in 10cm plates and treated with 50pmol siRNA for 72 hours.

Conditioned media experiments

All conditioned media (CM) experiments were carried out in 6-well plates with HLE cells seeded

at 65,000 cells per well, SNU-475 cells seeded at 45,000 cells per well, and SNU-182 cells seeded at

100,000 cells per well to account for differences in cell size and to reach the same confluency at

collection points. Conditioned media was collected and prepared as follows: when cells reached 80%

confluence in culture (T-75 flask), media was changed to 10mL of fresh complete DMEM. After 24

hours, the media was collected from the confluent cells and mixed with the same volume of fresh,

complete DMEM (1:1) prior to placement on seeded, AFP- cells. Cells were seeded 24 hours prior to

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treatment with 2mL CM (diluted 1:1 with complete DMEM) and treatment remained on cells for 8 hours

prior to collection and RNA extraction by TRIzol. CM collected from cells over expressing AFP was

collected in the same manner.

In AFP over expression conditioned media experiments, AFP was transiently transfected in 4x105

HLE or SNU-182 cells for 48 hours in 10cm plates. After 24 hours, 6.5mL fresh, complete DMEM was

placed on HLE or SNU-182 cells then the cells were incubated at 37°C for an additional 24 hours prior to

collecting the CM. CM was again diluted 1:1 with fresh, complete DMEM and placed on HLE or SNU-

182 cells seeded in 6-well plates. Cells were collected and RNA extracted using TRIzol 8 hours after

treatment.

Chromatin Immunoprecipitation (ChIP) assay primers and PCR

ChIP assays were ordered from Millipore and performed per the manufacturer’s protocol (catalog

#17-295). A total of 4µg of a ChIP grade c-MYC antibody was used for the immunoprecipitation

(Epitomics, Rabbit monoclonal N-term, catalog #1472-1) and 4µg of rabbit IgG was used as a negative

control (Thermoscientific, catalog #26156). To quantify the amount of c-MYC bound to the miR-29a

transcript, two sets of primers were designed and ordered from Integrated DNA Technologies. The first,

P-1, in the promoter region (-100 bases from TSS) : 5’- GGT GGA GGG AGC GTT GAT TT-3’

(forward), 5’- TTG GTC ACT GCG TGT CAT CT-3’ (reverse) and the second, P-2, in the intron region

(+19kB from TSS): 5’- GTG CCG GTC TAC CTT GGA TG-3’ (forward), and 5’-AAG GCT TTC CTT

CTG GCA CT-3’ (reverse). PCR reactions of the purified chromatin for both sets of primers were

prepared using SYBR green master mix (Life Technologies, catalog #4367659) and run in triplicate using

the ABI 7500 thermocycler. Cycle threshold (Ct) values of each sample were normalized to those from

rabbit IgG.

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Protein expression analysis

Western blot was used to analyze protein abundance. All primary antibodies were incubated

overnight at 4°C in 5% milk. AFP antibody was diluted 1:1000 (Cell Signaling, Rabbit monoclonal

D12c1, catalog #4448S). c-MYC antibody was diluted 1:2000 (Epitomics, Rabbit monoclonal N-term,

catalog #1472-1). β-actin antibody was used as an endogenous control and diluted 1:10,000 (Sigma,

mouse monoclonal, catalog #A5441-2ML). Secondary antibodies were also diluted 1:10,000 and included

ECL anti-mouse (NA931) and ECL anti-rabbit (NA934) from Fisher Scientific (catalog #45000679 and

45000682, respectively).

Microarray analysis tools and statistics

IPA predicts the activation state of transcription factors by comparing gene expression patterns to

expression of transcriptional regulators in the input dataset and then assigns each transcriptional regulator

a z-score. For example, a transcription regulator is assigned “activated” if 1) the regulator is activating

and 2) genes associated with the transcriptional regulator are observed to be up regulated. Conversely, a

transcriptional regulator would be assigned “inhibited” if 1) the regulator is inhibiting and 2) genes

associated with the transcriptional regulator are observed to be down regulated. Z-scores are not assigned

unless the transcriptional regulator has significantly more “activated” predictions than “inhibited”

predictions or vice versa (Ingenuity ® Systems, www.ingenuity.com).

Two-sided Student’s t-test was used to estimate significant difference between mean values and a

p<0.05 was considered a significant difference.

Xenograft study

Male athymic Nu/Nu nude mice were purchased from Charles River (Frederick, MD) at five

weeks old, maintained on antibiotics throughout the study and housed in pathogen-free conditions.

0.5x106 stable HLE cells (AFP+ or control) were suspended in 100uL complete DMEM then mixed 1:1

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with Matrigel HC (BD Matrigel Matrix High Concentration, Cat# 354248) and subcutaneously injected

(200uL total) into each flank of the mice (2 injections per mouse) at six weeks old. Each experimental

group consisted of 5 animals (ten injection sites). Xenograft tumors began to appear 7 days after injection

at which point the mice were measured twice per week for 28 days. The longest (L) and the shortest (W)

diameters as well as the height (H) of each tumor was measured with a caliper once excised from the

mouse and tumor volume was calculated using the following formula: Tumor volume (mm3) =

1/6(pLWH). The above experiment was repeated with the use of 1x106 cells per injection site.

Supplemental Figure Legends

Suppl. Figure 1. A) miR-29b (top) and -29c (bottom) decrease significantly as serum AFP increases

(n=223). B) Low miR-29b (top) and -29c (bottom) expression are related to poor outcome in HCC

(n=223).

Suppl. Figure 2. A-C) DNMT3A is inversely correlated with miR-29a (A), -29b (B), and -29c (C)

expression in 223 HCC patients. D) AFP gene expression positively correlated with serum AFP levels in

237 HCC patients. E) miR-29a expression in the array correlates with miR-29a expression measured by

qRT-PCR (n=19). F) AFP mRNA expression from the Affymatrix array (log2) is not significantly

different between HCC cases with low or high methylation status determined by a quartile cut-off.

Suppl. Figure 3. An inverse trend between the miR-29 family and AFP occurs during development in

normal physiology. A) AFP is highly expressed in the normal fetal liver tissue but not apparent in adult

normal liver tissue. B) miR-29 is highly expressed in the normal adult liver tissue compared to normal

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fetal liver tissue. C) DNMT3A expression is increased in normal fetal liver compared to normal adult

liver tissue. D) AFP and miR-29 expression have a switch point just after birth where AFP expression

significantly drops and miR-29 expression begins to increase. The liver bud typically forms at E10.5 and

the liver develops between E12-15. Both AFP and miR-29 levels during development were normalized to

that in 6 months old adult mice.

Suppl. Figure 4. A) SNU-182 cells proliferate significantly faster in the presence of CM taken from HLE

cells over expressing AFP as compared to SNU-182 cells in the presence of CM taken from HLE cells

transfected with an empty plasmid (days 1-5, p<0.01). There are four replicates per time point and error

bars represent standard deviation. B) Over expression of AFP in SNU-182 cells leads to decreased mature

miR-29a expression compared to control SNU-182 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. C) DNMT3A and DNMT3B levels significantly increased after a 48 hour transient AFP

over expression in SNU-182 cells. D) Silencing AFP in HUH-7 cells leads to an induction of miR-29a

compared to control cells (96 hour transient infection). Top panel shows AFP protein expression by

western blot while the bottom panel shows miR-29a expression quantified by RT-PCR. E) No significant

cell proliferation difference is observed between HUH-7 cells infected with shAFP and control (as

monitored by xCELLigence). Cells were infected for 96 hours prior to beginning the proliferation assay

and selected for, using an antibiotic, 24 hours prior to the proliferation assay. F) AFP protein level

detected by western blot (top panel) and ELISA (bottom panel) 1 week after lentiviral infection. Cell

selection using an antibiotic began 72 hours after infection and continued throughout the week. Media

was changed every three days. G) ELISA data shows AFP is only expressed in the media of HUH-7 cells.

SNU-182, SNU-475 and HLE cell media are all AFP-. H) 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 as measured by

qRT-PCR.

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Suppl. Figure 5. A) The accumulation of c-MYC in the presence and absence of AFP was estimated

using cycloheximide (CHX) treatment to block translation. HLE cells were treated with AFP+ or AFP-

conditioned media for 8 hours then CHX was applied for 15 minutes to stop protein translational

elongation. After treatment, c-MYC protein expression increased 1.8 fold in the presence of AFP.

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Suppl. Table 1. Clinical Characteristics of Patients

Parameters

AFP <20ng/mL

(n=74)

AFP 20-200ng/mL

(n= 48)

AFP 200-2000ng/mL

(n= 76)

AFP >2000ng/mL

(n=39) P-value Age (year, mean ± SD) 53 ± 10 50 ± 11 49 ± 12 51 ± 10 0.25b Sex (Male / Female) 65/9 42/6 65/11 35/4 0.45a HBV positive (Yes / No / n.a.) 69/2/3 44/1/3 66/2/8 34/1/4 0.99b Liver cirrhosis (Yes / No) 69/5 43/5 67/9 39/0 0.14b ALT (>50U/L / ≤50U/L) 31/43 24/24 26/50 16/23 0.38a Tumor size (> 3cm / ≤ 3cm / n.a.) 48/26 27/21 51/24/1 36/3 < 0.01a

Multinodular (Yes / No) 12/62 9/39 17/59 13/26 0.19a Microvascular invasion (Yes / No / n.a.) 28/33/13 18/22/8 26/31/19 16/16/7 0.97a TNM classification (II-III / I / n.a.) 38/33/3 27/18/3 37/32/7 26/9/4 0.17a aFisher's exact test

bOne-way ANOVA

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Suppl. Table 2. Low miR-29 expression is associated with poor overall survival in HCC patients

miRNA Hazard Ratioa Parametric p-value

SD of log Intensities FDR

miR-338 3.14 < 0.00001 0.32 0.0003 miR-7b 1.88 0.00058 0.40 0.0257 miR-7-2 1.64 0.00108 0.52 0.0342

miR-181b-2 1.60 0.00005 0.62 0.0081 miR-346 1.58 0.00047 0.56 0.0242 miR-326 1.51 0.00146 0.55 0.0342 miR-7-3 1.51 0.00043 0.66 0.0242

miR-181b-2 1.39 0.00042 0.86 0.0242 miR-194 0.80 0.00029 1.28 0.0216 miR-148a 0.77 0.00138 0.95 0.0342 miR-29c 0.77 0.00165 1.04 0.0367 miR148a 0.77 0.00137 0.93 0.0342 miR-144 0.74 0.00140 0.80 0.0342 miR-29b 0.74 0.00031 0.96 0.0216 miR-148 0.73 0.00021 0.92 0.0216 miR-215 0.73 0.00071 0.81 0.0288 miR-193 0.73 0.00057 0.96 0.0257 miR-29a 0.72 0.00148 0.73 0.0342

miR-29b-1 0.71 0.00104 0.75 0.0342 miR-194-1 0.71 0.00142 0.75 0.0342 miR-124a-2 0.70 0.00125 0.79 0.0342

miR-340 0.62 0.00103 0.59 0.0342 miR-194-2 0.62 0.00002 0.76 0.0047 miR-152 0.59 0.00144 0.48 0.0342

miR-194-1 0.51 0.00018 0.49 0.0216 miR-296 0.50 0.00028 0.46 0.0216

miR-124a-1 0.44 0.00074 0.54 0.0288 miR-124a-3 0.39 0.00001 0.40 0.0020

aCox regression analysis completed on 223 patients reveals the most significant survival-related miRNAs sorted by hazard ratio.

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Suppl. Table 3. Methylation Status of HCC Tumors

Tumor ID Tumor

beta values Nontumor beta values

Fold difference (T-NT)

LCS-411 0.314 0.677 -0.363 LCS-196 0.382 0.661 -0.279 LCS-400 0.321 0.580 -0.259 LCS-342 0.396 0.645 -0.249 LCS-376 0.385 0.631 -0.245 LCS-036 0.438 0.658 -0.220 LCS-406 0.347 0.566 -0.219 LCS-420 0.405 0.620 -0.215 LCS-343 0.415 0.630 -0.215 LCS-387 0.419 0.631 -0.211 LCS-067 0.405 0.607 -0.203 LCS-424 0.382 0.561 -0.178 LCS-056 0.420 0.576 -0.156 LCS-385 0.580 0.706 -0.126 LCS-421 0.546 0.669 -0.123 LCS-357 0.468 0.575 -0.107 LCS-043 0.512 0.618 -0.106 LCS-395 0.568 0.674 -0.106 LCS-405 0.505 0.608 -0.103 LCS-379 0.507 0.606 -0.098 LCS-360 0.548 0.645 -0.097 LCS-413 0.495 0.592 -0.097 LCS-007 0.526 0.623 -0.096 LCS-396 0.531 0.621 -0.090 LCS-332 0.587 0.665 -0.078 LCS-330 0.589 0.664 -0.075 LCS-425 0.490 0.565 -0.075 LCS-346 0.571 0.643 -0.072 LCS-410 0.532 0.601 -0.068 LCS-382 0.506 0.572 -0.066 LCS-372 0.515 0.577 -0.061 LCS-063 0.527 0.587 -0.060 LCS-339 0.627 0.678 -0.051 LCS-045 0.573 0.623 -0.051 LCS-042 0.525 0.557 -0.032 LCS-295 0.526 0.557 -0.031 LCS-340 0.533 0.556 -0.024 LCS-328 0.601 0.619 -0.018 LCS-329 0.622 0.631 -0.009 LCS-345 0.575 0.584 -0.009 LCS-294 0.595 0.602 -0.007 LCS-069 0.443 0.435 0.009 LCS-033 0.658 0.638 0.020 LCS-401 0.609 0.578 0.031 LCS-009 0.969 0.817 0.153 LCS-347 0.716 0.555 0.160 LCS-038 0.496 0.240 0.256 LCS-049 0.455 0.007 0.448

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modulation of mir-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma...

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