Long non-coding RNA HULC modulates abnormal lipid ... · 15/01/2015 · 1 Long non-coding RNA HULC...

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Long non-coding RNA HULC modulates abnormal lipid metabolism in hepatoma cells

through an miRNA-9 mediated RXRA signaling pathway

Ming Cui1, Zelin Xiao1, Yue Wang2, Minying Zheng1, Tianqiang Song3,4, Xiaoli Cai2, Baodi

Sun1, Lihong Ye2 and Xiaodong Zhang1

Authors' Affiliations: 1State Key Laboratory of Medicinal Chemical Biology Department of

Cancer Research, College of life sciences, Nankai University, Tianjin, China; 2State Key

Laboratory of Medicinal Chemical Biology, Department of Biochemistry, College of Life

Sciences, Nankai University, Tianjin, China; 3Tianjin Medical University Cancer Institute and

Hospital, National Clinical Research Center for Cancer, Tianjin, China; 4Key Laboratory of

Cancer Prevention and Therapy, Tianjin Department of Hepatobiliary Tumor, Tianjin, China.

Corresponding Authors: Xiaodong Zhang, M.D., Ph.D., State Key Laboratory of Medicinal

Chemical Biology, Department of Cancer Research, College of Life Sciences, Nankai

University, 94 Weijin Road, Tianjin 300071, China. Phone: +86-22-23506830; fax:

+86-22-23501385; E-mail: [email protected].

Lihong Ye, M.D., Ph.D., State Key Laboratory of Medicinal Chemical Biology, Department of

Biochemistry, College of Life Sciences, Nankai University, Tianjin, 94 Weijin Road, Tianjin

300071, China. Phone: +86-22-23501385; fax: +86-22-23501385; E-mail:

[email protected].

on April 22, 2021. © 2015 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on January 15, 2015; DOI: 10.1158/0008-5472.CAN-14-1192

http://cancerres.aacrjournals.org/

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Abstract

HULC is a long non-coding RNA overexpressed in hepatocellular carcinoma (HCC),

but its functional contributions in this setting have not been determined. In this study,

we explored the hypothesis that HULC contributes to malignant development by

supporting abnormal lipid metabolism in hepatoma cells. HULC modulated the

deregulation of lipid metabolism in HCC by activating the acyl-CoA synthetase subunit

ACSL1. Immunohistochemical analysis of tissue microarrays revealed that ~77%

(180/233) of HCC tissues were positive for ACSL1. Moreover, HULC mRNA levels

correlated positively with ACSL1 levels in 60 HCC cases according to real-time PCR

analysis. Mechanistic investigations showed that HULC up-regulated the transcriptional

factor PPARA which activated the ACSL1 promoter in hepatoma cells. HULC also

suppressed miR-9 targeting of PPARA mRNA by eliciting methylation of CpG islands in

the miR-9 promoter. We documented the ability of HULC to promote lipogenesis,

thereby stimulating accumulation of intracellular triglycerides and cholesterol in vitro

and in vivo. Strikingly, ACSL1 overexpression which generates cholesterol was sufficient

to enhance the proliferation of hepatoma cells. Further, cholesterol addition was

sufficient to up-regulate HULC expression through a positive feedback loop involving

the retinoid receptor RXRA which activated the HULC promoter. Overall, we concluded

that HULC functions as an oncogene in hepatoma cells, acting mechanistically by

deregulating lipid metabolism through a signaling pathway involving miR-9, PPARA

and ACSL1 that is reinforced by a feed-forward pathway involving cholesterol and

RXRA to drive HULC signaling.




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Introduction

Growing evidence indicates that many noncoding regulatory elements are transcribed into

non-coding RNAs (ncRNAs) (1,2). Several types of ncRNAs are regarded as regulatory

RNAs which possess orchestrated functions involved in the control of genome dynamics, cell

biology, and developmental programming (3). NcRNA is habitually divided into two groups

on the basis of transcript size: long ncRNA (lncRNA, >200nt-long) and small ncRNA (4). A

spot of characterized human lncRNAs have been associated with a spectrum of biological

functions and the disruption of these functions play a critical role in the development of

cancer (5). Highly up-regulated in liver cancer (HULC) is the first identified lncRNA

specifically overexpressed in hepatocellular carcinoma (HCC) (6). HULC is transactivated by

CREB, and sequesters miR-372 by acting as a sponge (7), and insulin-like growth factors 2

mRNA-binding proteins (IGF2BPs) are able to govern the expression of HULC (8).

Previously, our group reported that hepatitis B virus X protein (HBx)-elevated HULC could

accelerate the growth of hepatoma cells by down-regulating p18 (9). However, the role of

HULC in abnormal lipid metabolism remains poorly understood.

HCC is the fifth-most common cancer worldwide and the third largest cause of cancer

death globally (10). Recently, mounting clinical and epidemiological studies have reported

that high-risk cancer is linked to metabolic syndromes, such as obesity, type diabetes and

atherosclerosis (11). Lipids, which represent a diverse group of water-insoluble molecules,

play essential roles in these processes. Meanwhile, high rates of lipid uptake and de novo lipid

synthesis are frequently exhibited by cancer cells. For example, various tumors and their




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precursor lesions undergo exacerbated endogenous fatty acid biosynthesis irrespective of the

levels of extracellular lipids (12). The changes in lipid metabolism can alter numerous cellular

processes, including proliferation, motility and tumourigenesis. As a central organ of energy

metabolism, the liver synthesizes most plasma apolipoproteins, endogenous lipids and

lipoproteins. Thus, the advent of HCC is accompanied by metabolic reprogramming, which is

reflected in changes in gene expression and microRNA profiles as well as altered levels of

circulating proteins and small metabolites (13). The AKT/mTOR pathway and insulin

signaling have been reported to contribute to the deregulation of lipid metabolism in

hepatoma cells thus far (14,15).

Acyl-CoA synthetase long-chain family members (ACSLs) catalyze the initial step in

cellular long-chain fatty acid metabolism in mammals (16). There are five members in this

family. Among them, ACSL1 is one of the major isoforms, with high levels in the liver, and

can be regulated by the transcriptional factor peroxisome proliferator-activated receptor alpha

(PPARA) (17,18). Some studies have reported that the overexpression of ACSL1 increases the

uptake of fatty acids in hepatoma cells (19). However, whether ACSL1 contributes to the

development of HCC has been ill-documented. The retinoid X receptors (RXRs) were

identified in 1990 as orphan receptors that exhibited diverse transcriptional responses (20).

RXRs play a vital role in the nuclear receptor superfamily, forming heterodimers with many

other family members; as a result, RXRs are implicated in the control of various physiologic

processes (21). RXRA is a member of the RXR family that can be activated by sterol (22). In

humans, cholesterol homeostasis is maintained by the precise interactions between intestinal

uptake, de novo synthesis, hepatic output, and fecal disposal (23), and excessive or deficient




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cholesterol can result in pathophysiological sequelae (24). Secreted apoA-I binding protein

(AIBP) positively regulates cholesterol efflux from endothelial cells, and effective cholesterol

efflux is critical for proper angiogenesis (25). In addition, the primary metabolite of

cholesterol, 27-hydroxycholesterol (27HC), contributes to estrogen receptor-dependent

growth and liver X receptor-dependent metastasis in mouse models of breast cancer (26), and

cholesteryl ester accumulation induced by PTEN loss and PI3K/AKT activation underlies

human prostate cancer aggressiveness (27). However, the general mechanism responsible for

aberrant lipid metabolism during the development of HCC is not well understood.

In the present study, we investigated the role of lncRNAs in the abnormal lipid

metabolism of HCC. Intriguingly, our data demonstrate that the lncRNA HULC facilitates the

deregulation of lipid metabolism through miRNA-9/PPARA/ACSL1/cholesterol/RXRA/

HULC signaling. Thus, our finding provides new insights into the mechanism of aberrant

lipid metabolism in HCC.

Materials and Methods

Patient samples

Sixty HCC tissue samples and their corresponding adjacent non-tumorous liver tissues

were obtained from Tianjin First Center Hospital and Tianjin Tumor Hospital (Tianjin, China)

after surgical resection. Fifty-five out of 60 patients had a history of hepatitis B virus infection.

Written consent approving the use of tissue samples for research purposes was obtained from

patients. The information of HCC patients is presented in Supplementary Table S1. The study

protocol was approved by the Institute Research Ethics Committee at Nankai University.




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Cell Lines and Cell Culture

The human hepatoma H7402 cell line, human immortalized liver L-O2 cell line and

Chang liver cell line were cultured in RPMI-1640 medium (Gibco, CA, USA). The human

hepatoma cell lines, Huh7 (obtained from Shanghai Institutes for Biological Sciences),

HepG2 and HepG2.2.15 (a hepatoma HepG2 cell line with integrated full-length HBV DNA)

and human kidney epithelial (HEK) 293T cells were maintained in Dulbecco’s Modified

Eagle’s medium (Gibco, CA, USA). All cell lines were supplemented with heat-inactivated 10%

fetal bovine serum (FBS, Gibco, CA, USA), 100 U/ml penicillin and 100 mg/ml streptomycin

and grown at 5% CO2 and 37 .

In vivo Tumorigenicity Assay

Nude mice were housed and treated according to the guidelines established by the

National Institutes of Health Guide for the Care and Use of Laboratory Animals. We

conducted animal transplantations according to the Declaration of Helsinki. Briefly, HepG2

(or Huh7) cells were harvested and re-suspended at 2× 107 cells per ml in sterile

phosphate-buffered saline. Groups of 4-week-old male BALB/c athymic nude mice

(Experiment Animal Center of Peking, China) (each group, n=6) were subcutaneously

injected at the shoulder with 0.2 ml of the cell suspensions. According to the protocol (28),

cholesterol (Solarbio, China) was subcutaneously injected into the appropriate mice once in

proximity to the tumor after injection of 5 days. Group one, the control group was injected

with 100 μl acetone. Group two and three were experimental groups and were injected with




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300 μM/kg cholesterol in 100 μl acetone. All cells transplanted in mice in group three were

pre-treated with 100 nM HULC siRNA. Tumor growth was measured beginning 5 days after

injection of hepatoma cells. Tumor volume (V) was monitored by measuring the length (L)

and width (W) of the tumors with calipers and was calculated using the formula (L × W2) ×

0.5. After 25 days, tumor-bearing mice and controls were sacrificed, and the tumors were

excised and measured.

Statistical analysis

Each experiment was repeated at least three times. Statistical significance was assessed by

comparing mean values (6 standard deviation; SD) using Student’s t test for independent

groups as follow: *P < 0.05, **P < 0.01, ***P <0.001 and not significant (NS). Pearson’s

correlation coefficient was used to determine the correlations among gene expression in

tumorous tissues. ACSL1 expression in tumor tissues and matched adjacent non-tumor tissues

was compared using Wilcoxon’s signed-rank test.

Results

HULC is positively correlated with ACSL1 in clinical HCC tissues and up-regulates

ACSL1 in hepatoma cells

To demonstrate the role of HULC in the deregulation of lipid metabolism in HCC, we

examined the effects of HULC on several lipid metabolic enzymes in hepatoma cells. ACSL1

stood out as being noticeably up-regulated by HULC (Supplementary Fig. S1A). Thus, we

evaluated the expression of ACSL1 by immunohistochemical (IHC) staining in clinical HCC




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tissues using tissue microarrays and found that 77.3% (180/233) of HCC tissues were positive

for ACSL1 compared with 12.5% (2/16) of peritumoral liver tissues, in which the expression

of ACSL1 was stronger in HCC tissues than that in their peritumoral liver tissues (Fig. 1A).

Moreover, quantitative real-time PCR (qRT-PCR) revealed that the mRNA levels of ACSL1

were higher in HCC tissues compared with their adjacent non-tumorous liver tissues in 60

paired clinical HCC samples (Fig. 1B). ACSL1 has been reported to participate in the

formation of triglycerides and cholesterol in the liver (29). Our data demonstrated that the

increased-triglycerides/cholesterol was accumulated in HCC tissues relative to their

corresponding peritumoral tissues (Supplementary Fig. S1B). Furthermore, we observed that

the levels of HULC were positively associated with those of ACSL1, triglycerides and

cholesterol in the aforementioned clinical samples (Fig. 1C). Moreover, we found that

overexpression of HULC up-regulated ACSL1 in HepG2 and Huh7 (or L-O2) cells at the

mRNA and protein levels in a dose-dependent manner (Supplementary Fig. S1C, Fig. 1D, 1E

and S1D). Similarly, depletion of HULC led to a decrease in ACSL1 levels in HepG2.2.15

cells expressing high levels of endogenous HULC (Supplementary Fig. S1E and Fig. 1F).

Meanwhile, the transfection (or interference) efficiency of HULC (or HULC siRNA) was

validated by qRT-PCR or RT-PCR analysis (Fig. 1D-F and Supplementary Fig. S1C-E).

Strikingly, HULC was capable of enriching triglycerides and cholesterol in HepG2 cells in a

dose-dependent manner (Supplementary Fig. S1F) but failed to increase their levels in the

conditioned medium (Supplementary Fig. S1G). Taken together, these results show that

HULC is positively associated with ACSL1 in clinical HCC tissues and up-regulates ACSL1

in hepatoma cells.




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HULC up-regulates the transcriptional factor PPARA to activate ACSL1

Given that ACSL1 is activated by the transcriptional factor PPARA in the liver (17), we

speculated that HULC might modulate ACSL1 through PPARA. Interestingly, qRT-PCR

analysis demonstrated that the expression levels of HULC were positively associated with

those of PPARA in the 60 clinical HCC samples (Fig. 2A). However, inhibition of PPARA

expression abrogated the HULC-induced up-regulation of ACSL1 in HepG2 cells (Fig. 2B),

suggesting that PPARA is responsible for the up-regulation of ACSL1 mediated by HULC.

The efficiency of PPARA siRNA (or PPARA siRNA*) was validated in these cells

(Supplementary Fig. S2A). Moreover, the overexpression of HULC was able to up-regulate

PPARA at mRNA and protein levels in HepG2 and Huh7 cells (or L-O2 cells) in a

dose-dependent manner (Fig. 2C-E and Supplementary information S2B, S2C), whereas

HULC siRNA reversed these effects in HepG2.2.15 cells (Fig. 2F and 2G). The transfection

(or interference) efficiency of HULC (or HULC siRNA) was confirmed in these cells (Fig.

2C-G and Supplementary Fig. S2B, S2C). However, luciferase reporter assays demonstrated

that HULC failed to active the promoter of PPARA in HepG2 cells (Supplementary Fig. S2D

and S2E), implying that HULC might up-regulate PPARA at the post-transcriptional step.

Thus, we conclude that HULC activates ACSL1 by up-regulating the transcription factor

PPARA in hepatoma cells.

MiR-9 inhibits the expression of PPARA by targeting the 3��UTR of PPARA

Next, we identified several miRNAs that could potentially bind to the 3�untranslated




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region (UTR) of PPARA using TargetScan and microrna.org (http://www.targetscan.org/,

http://www. microrna.org/microrna/home.do). Because miR-9 has been reported to be

down-regulated in cancer (30-32), we focused our investigation on this miRNA. Three miR-9

binding sites in the 3�UTR of PPARA mRNA were constructed (Fig. 3A and Supplementary

Fig. S3A, S3B), and luciferase reporter assays revealed that miR-9 could directly bind to the

conserved seed region of the PPARA 3�UTR (position 7624-7631, pGL3-PPARA-7624) (Fig.

3B), rather than the poorly conserved seed regions (position 5684-5690, position 4375-4381)

(Supplementary Fig. S3C and S3D). However, the PPARA 3�UTR conserved seed region

mutant (position 7624-7631, pGL3-PPARA-mut) failed to work in these cells (Fig. 3B).

Conversely, anti-miR-9 increased the luciferase activities of pGL3-PPARA-7624 but failed to

influence the mutant (Fig. 3C), suggesting that miR-9 is able to directly bind to the 3�UTR of

PPARA. These effects were also observed in 293T cells (Supplementary Fig. S3C-F).

Furthermore, the overexpression of miR-9 suppressed the expression of PPARA in HepG2

cells in a dose-dependent manner (Fig. 3D), and the reverse outcome was obtained when the

cells were treated with anti-miR-9 (Fig. 3E). Together, our data indicate that miR-9 suppresses

the expression of PPARA by targeting its 3�UTR.

HULC down-regulates miR-9 through inducing methylation of CpG islands in its

promoter

Next, we validated the anticorrelation between HULC and miR-9 in the 60 clinical HCC

samples (Fig. 4A). Then, our data showed that the overexpression of HULC down-regulated

miR-9 in HepG2 cells in a dose-dependent manner (Fig. 4B). Interestingly, we found that




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treatment with 5-Aza-2’-deoxycytidine (Aza, a DNA methylation inhibitor) heightened the

levels of miR-9 in HepG2 cells in a dose-dependent manner (Fig. 4C), suggesting that HULC

might influence the epigenetic regulation of the miR-9 promoter (33,34). Then, we examined

the methylation status of miR-9 using both methylation-specific PCR (MSP) and bisulfite

-sequencing analysis (BSP). As shown in Figure 4D, miR-9 comprises three members, termed

miR-9-1, miR-9-2 and miR-9-3. MSP assays revealed that the CpG sites of miR-9-1 (or

miR-9-2, miR-9-3) were highly methylated following the overexpression of HULC in L-O2

(or Chang liver (Chang), HepG2 and H7402) cells. BSP assays further validated the above

observations in L-O2 (or HepG2) cells (Fig. 4E and Supplementary Fig. S4A). Moreover, we

confirmed these data in two pairs of clinical samples (Fig. 4F and Supplementary Fig. S4B).

It has also been reported that DNA (cytosine-5-)-methyltransferase 1 (DNMT1), a methylase,

is capable of regulating the expression of miRNAs through inducing methylation of their CpG

islands (35). Interestingly, we observed that HULC was able to up-regulate DNMT1 in

HepG2 cells (Supplementary Fig. S4C), hinting that HULC might induce methylation of CpG

islands in the miR-9 promoter through up-regulation of DNMT1. Therefore, we conclude that

HULC inhibits the expression of miR-9 through eliciting methylation of CpG islands in the

miR-9 promoter.

The product cholesterol of ACSL1 is able to up-regulate HULC by activating RXRA in

hepatoma cells

Given that the positive feedback loops in signaling pathways are involved in the

progression of cancer, we evaluated whether HULC facilitates aberrant lipid metabolism in




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liver cancer via a feedback mechanism. Surprisingly, we found that the promoter activities of

HULC were dose-dependently decreased in HepG2 cells after treatment with Triacsin C (an

inhibitor of ACSL1) or ACSL1 siRNA, although this was not observed in L-O2 cells (Fig. 5A

and Supplementary Fig. S5A). Meanwhile, we observed that the expression levels of HULC

were also down-regulated in HepG2 cells (Fig. 5B and Supplementary Fig. S5B), suggesting

that a positive feedback loop involving HULC/miR-9/PPARA/ACSL1/HULC is established

in hepatoma cells but not in normal liver cells. Next, we utilized acetone as the solvent to

deliver triglyceride (or cholesterol) to the cells. Although some triglyceride (or cholesterol)

dissolved out, the cells responded to the stimulation. Strikingly, we found that cholesterol was

able to stimulate the activity of the HULC promoter in HepG2 (or Huh7) cells in a

dose-dependent manner, although triglyceride failed to have an effect in these cells (Fig. 5C,

5D, Supplementary information S5C and S5D). According to the report (22), we cloned the

HULC promoter including the mutant in the RXRA binding site (Fig. 5E). Intriguingly, the

treatment with cholesterol failed to activate the above mutant in HepG2 cells (Fig. 5E),

suggesting that RXRA may be implicated in the regulation of HULC mediated by cholesterol.

Moreover, we verified that RXRA siRNA could abolish the cholesterol-increased HULC

promoter activity (Fig. 5F). However, the treatment with cholesterol failed to influence the

expression of RXRA (Supplementary Fig. S5E), suggesting that cholesterol, as a type of

sterol, might be able to activate the HULC promoter by stimulating RXRA, rather than

up-regulating RXRA. Meanwhile, the efficiencies of ACSL1 siRNA (or ACSL1 siRNA*) and

RXRA siRNA were validated by western blotting analysis in these cells (Supplementary Fig.

S5F and S5G). As a result, we conclude that the product cholesterol of ACSL1 up-regulates




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HULC by activating RXRA in hepatoma cells.

HULC disrupts the lipid metabolism of hepatoma cells through miR-9/PPARA/ACSL1

signaling in vitro

Next, we investigated the effect of HULC on lipogenesis in hepatoma cells using oil red O

staining. These results showed that the overexpression of HULC was able to accelerate

lipogenesis in HepG2 and Huh7 cells, whereas ACSL1 siRNA (or miR-9, PPARA siRNA,

Triacsin C) could block this event. Inversely, anti-miR-9 was capable of enhancing

lipogenesis in the cells (Fig. 6A). In addition, we validated that the treatment with HULC

siRNA (or ACSL1 siRNA, miR-9, PPARA siRNA, and Triacsin C) could attenuate the

lipogenesis in HepG2.2.15 cells. However, anti-miR-9 was able to rescue the HULC

siRNA-repressed lipogenesis (Fig. 6B), and we obtained a similar effect of HULC on

intracellular triglyceride and cholesterol in this system (Fig. 6C, 6D, Supplementary Fig. S6A

and S6B). The role of ACSL1 in the growth of hepatoma cells remains enigmatic. In this

study we showed that the overexpression of ACSL1 could facilitate the proliferation of

HepG2 and Huh7 cells, as demonstrated by MTT assays and cloning formation assays (Fig.

6E and 6F). Thus, we sought to evaluate whether the product cholesterol of ACSL1 may be

responsible for the promotion of cell proliferation. As expected, MTT assays further

corroborated that the treatment with cholesterol was able to promote the proliferation of

hepatoma cells, which could be eliminated by HULC siRNA (Supplementary Fig. S6C),

suggesting that cholesterol might enhance the proliferation of hepatoma cells by up-regulating

HULC. In addition, we assessed whether HULC affected other signaling pathways and factors




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involved in lipid metabolism in hepatoma cells, such as the AKT/mTOR pathway, SREBP1,

SREBP2, chREBP, HMGCR, FASN, ACLY, SQS and miR-122 (14,36). However, we did not

observe alterations in these factors at the levels of mRNA and protein in HepG2 and Huh7

cells (Supplementary Fig. S6D-G). Thus, we conclude that HULC contributes to aberrant

lipid metabolism in hepatoma cells through miR-9/PPARA/ACSL1 signaling.

HULC-modulated abnormal lipid metabolism facilitates tumor growth in vivo

To better understand the role of HULC in abnormal lipid metabolism, we subcutaneously

injected pretreated cells into 4-week-old BALB/c athymic nude mice. We confirmed that the

levels of HULC and ACSL1 were preserved in the tumor tissues (Supplementary Fig. S7A).

We observed that treatment with ACSL1 siRNA abolished the HUCL-accelerated

proliferation of HepG2 (or Huh7) cells in mice (Fig. 7A, 7B and Supplementary Fig. S7B),

further supporting the conclusion that ACSL1 is responsible for the promotion of tumor

growth mediated by HULC. IHC staining further confirmed that the expression of Ki-67, a

marker of proliferation, as well as BrdU incorporation in the tumor tissues was consistent

with tumor growth among the different groups (Supplementary Fig. S7C). Interestingly, oil

red O staining revealed that lipid droplets were increased in the tumor tissues overexpressing

HULC, whereas silencing of ACSL1 reversed this effect (Fig. 7C and Supplementary Fig.

S7C). The levels of triglycerides and cholesterol were consistent with the oil red O staining in

the tumor tissues (Fig. 7D and 7E), suggesting that abnormal lipid metabolism contributes to

the growth of hepatoma cells. To better evaluate the effect of cholesterol on the expression of

HULC in hepatoma cells in vivo, we injected supraphysiological cholesterol in proximity to




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the tumor tissues. As expected, the injection significantly increased the levels of tissue HULC

(Supplementary Fig. S7D). Notably, we observed that the pre-treatment with HULC siRNA

remarkably abolished the cholesterol-increased growth of hepatoma cells (Fig. 7F, 7G and

Supplementary Fig. S7E), suggesting that cholesterol is able to up-regulate HULC in

hepatoma cells. Together, we conclude that HULC-modulated abnormal lipid metabolism

contributes to tumor growth, and this process requires the metabolic enzyme ACSL1 and its

product cholesterol.

Discussion

LncRNAs play crucial roles in cancer (37), and we previously reported that

HBx-enhanced HULC is able to promote the growth of hepatoma cells (9). Metabolism

deregulation and, exacerbated lipid biosynthesis and accumulation early during cancer

development accelerate cell growth and transformation (38). In this study, we assessed

whether HULC participates in abnormal lipid metabolism in HCC.

To better understand the roles of HULC in modulating lipid metabolism, we first

measured the effect of HULC on lipid metabolic enzymes in hepatoma cells. Interestingly,

ACSL1 drew our attention because ACSL1 and its intracellular products, such as triglycerides

and cholesterol, were remarkably up-regulated and increased by HULC expression. However,

the levels of triglycerides and cholesterol in the conditioned medium were unaltered in

HULC-treated cells relative to controls, which may be the result of the ability of ACSL1 to

inhibit cholesterol efflux (39). Moreover, we validated that the expression levels of HULC

were positively correlated with those of ACSL1 and its products in clinical HCC samples (Fig.




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1 and Supplementary Fig. S1). Next, we explored the mechanism by which HULC activates

ACSL1 in hepatoma cells. ACSL1 has been reported to be a classic target gene of PPARA in

the liver (16). Accordingly, we observed that HULC modulated ACSL1 by up-regulating

PPARA (Fig. 2 and Supplementary Fig. S2). Furthermore, we determined that HULC could

increase PPARA expression by down-regulating the ability of miR-9 to target the PPARA

3�UTR at the post-transcriptional level, rather than by activating the transcription of PPARA

directly (Fig. 3 and Supplementary Fig. S3). Numerous studies have noted that the expression

of miR-9 is regulated by CpG island methylation, and lncRNAs are able to modulate the

expression of genes through epigenetic regulation (40,41). Hence, we validated that HULC

was capable of inducing the methylation of CpG islands in the promoter of miR-9 (Fig. 4 and

Supplementary Fig. S41). This finding suggests that HULC governs the expression of PPARA

through epigenetic regulation. The methylation of miR-9-3 is significantly associated with an

increased risk of recurrence, and high methylation levels of either miR-9-1 or miR-9-3 result

in a significant decrease in recurrence-free survival times in clear cell renal cell carcinoma

(30). Moreover, the inhibition of miR-9-mediated suppression of SOX2 is involved in

chemoresistance and cancer stemness in glioma cells (42), and miR-9 is able to target

MTHFD2 to inhibit the proliferation of breast cancer cells (43). Together with these findings,

our data imply that the methylation and inhibition of miR-9 might hijack other signaling

pathways to facilitate hepatocarcinogenesis. To better understand the underlying mechanism

by which HULC elicits the methylation of CpG islands in the miR-9 promoter, we assessed

the influence of HULC on methylase levels in hepatoma cells. Strikingly, our observations

indicated that HULC was able to up-regulate the expression of DNMT1 in hepatoma cells,




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suggesting that HULC induces the methylation of miR-9 promoter CpG islands possibly by

up-regulating DNMT1. The role of ACSL1 in the growth of hepatoma cells has not been

reported. Therefore, we examined the effect of ACSL1 overexpression on the proliferation of

HepG2 and Huh7 cells by MTT assays and cloning formation assays. Notably, we observed

that ACSL1 was able to promote the proliferation of hepatoma cells, suggesting that the role

of ACSL1 in the promotion of cell proliferation might be associated with the disturbance of

lipid metabolism mediated by ACSL1 in hepatoma cells. Additionally, it has been reported

that the AKT/mTOR cascade influences the growth, survival, metabolism, and migration of

liver cancer cells, and the extent of aberrant lipogenesis is correlated with the activation of the

AKT/mTOR signaling pathway (14,44). This relationship suggests that the AKT/mTOR

pathway plays a pivotal role in the deregulation of lipid metabolism in HCC. Therefore, we

wondered whether the AKT/mTOR pathway and other signaling pathway members implicated

in lipid metabolism, such as SREBP1/2, chREBP, HMGCK, MVK, FASN, ACLY, SQS and

miR-122 (14,36), were involved in this event. However, we failed to demonstrate that HULC

disturbed lipid metabolism in hepatoma cells through these signaling pathways. Therefore, we

conclude that HULC is able to induce aberrant lipid metabolism through ASCL1/miR-9/

PPARA/ASCL1 signaling in hepatoma cells.

Given that cancer disrupts cellular homeostasis and creates many new methods of

regulation, such as positive feedback loops (45), we are interested in whether the action of

HULC is involved in a feedback loop as well. Interestingly, we observed that ACSL1 could

influence the expression of HULC in hepatoma cells in a positive feedback fashion. Thus, we

hypothesized that the products of ACSL1 might be responsible for the activation of HULC.




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Indeed, luciferase reporter assays revealed that cholesterol was able to activate the promoter

of HULC, whereas triglycerides were not. Furthermore, we identified a binding site for

RXRA in the promoter of HULC using bioinformatics analysis. RXRA is an orphan receptor

that can be activated by sterol (22). Thus, we speculated that RXRA might participate in

cholesterol-activated HULC in hepatoma cells. As expected, we found that RXRA was

responsible for the activation of HULC mediated by cholesterol in hepatoma cells. It has been

reported that PPARA, liver X receptor (LXRA) and bile acid receptor (FXR) can form

heterodimers with RXRA (46-48). Here, we treated cells with siRNA targeting PPARA (or

LXRA and FXR) mRNA to assess the effect of PPARA (or LXRA and FXR) on HULC.

However, our data demonstrated that all treatments failed to influence the

cholesterol-enhanced luciferase reporter activity of the HULC promoter (data not shown),

implying that PPARA, LXRA and FXR are not implicated in cholesterol-induced RXRA

activation. Strikingly, we observed that cholesterol failed to up-regulate HULC in normal

liver cells (Fig. 5 and Supplementary Fig. S5) that possibly because RXRA is not

constitutively phosphorylated and thus fails to escape from Ub/proteasome-mediated

degradation in liver cells (49). Of particular note, this finding provides new insights into the

mechanism by which HULC is highly expressed in hepatoma cells.

Recent reports pinpoint that the growth-promoting effects of elevated levels of insulin,

glucose, or triglycerides are involved in insulin resistance-promoted colorectal cancer (50).

Heightened intracellular levels of triglyceride and their metabolites, such as diacylglycerol,

may activate the protein kinase-C and MAPK pathways with potentially mitogenic and

carcinogenic effects (51). Consistent with our study, these findings suggest that




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HULC-enhanced accumulation of triglycerides in hepatoma cells might emerge as a risk

factor for hepatocarcinogenesis. In this article, we found that cholesterol was involved in the

promotion of hepatoma cell growth mediated by HULC, which is consistent with other

reports (25-27).

In aggregate, our work demonstrates that HULC plays pivotal roles in aberrant lipid

metabolism in HCC through miR-9/PPARA/ACSL1/cholesterol/RXRA/HULC signaling, a

model for this role of HULC is represented in Figure S7F. In particular, our data show that

HULC elicits the methylation of CpG islands in the miR-9 promoter, resulting in the

suppression of miR-9 expression. MiR-9 is able to target the 3�UTR of transcription factor

PPARA, and the decrease in miR-9 leads to up-regulation of PPARA and the subsequent

transactivation of ACSL1, which enhances lipogenesis and enriches intracellular triglycerides

and cholesterol in hepatoma cells. Thus, HULC-enhanced abnormal lipid metabolism

accelerates the growth of liver cancer. Furthermore, the cholesterol product of ACSL1

up-regulates HULC by activating the transcription factor RXRA, forming a positive feedback

loop with HULC/miR-9/PPARA/ACSL1/cholesterol/RXRA/HULC in hepatoma cells. Thus,

our findings provide new insights into the mechanism of abnormal lipid metabolism mediated

by HULC in the development of HCC.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

Authors' Contributions




20

Conception and design: Ming Cui, Lihong Ye, Xiaodong Zhang

Development of methodology: Ming Cui, Lihong Ye, Xiaodong Zhang

Acquisition of data (provided animals, acquired and managed patients, provided

facilities, etc.): Ming Cui, Zelin Xiao, Yue Wang, Minying Zheng, Tianqiang Song, Xiaoli

Cai, Baodi Sun

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational

analysis): Ming Cui, Zelin Xiao, Yue Wang, Xiaodong Zhang

Writing, review, and/or revision of the manuscript: Ming Cui, Xiaodong Zhang

Study supervision: Lihong Ye, Xiaodong Zhang

Grant Support

This work was supported by grants from National Natural Science Foundation of China (No.

31470756, No. 81071624 and No. 81272218) and National Basic Research Program of China

(973 Program, No.2015CB553703, No.2015CB55905 and No.2011CB933100).

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Figure legends

Figure 1. HULC is positively correlated with ACSL1 in clinical HCC tissues and up-regulates

ACSL1 in hepatoma cells. (A) The expression of ACSL1 was examined by IHC staining in

clinical HCC tissues and peritumoral tissues. The expression of ACSL1 was stronger in HCC

tissues than that in their peritumoral liver tissues (a) and its amplification (b). (B) Relative

mRNA levels of ACSL1 were assessed by qRT-PCR in 60 pairs of clinical HCC tissues and

corresponding non-tumorous tissues (**P<0.01; Wilcoxon’s signed-rank test). (C) The

correlation between HULC mRNA levels and ACSL1 mRNA levels (or levels of triglycerides

or cholesterol) was examined by qRT-PCR (or Tissue triglyceride assay kit, Tissue total

cholesterol assay kit) in 60 cases of clinical HCC tissues (**P<0.01; Pearson’s correlation

coefficient, r=0.7444, 0.7099, 0.6501). (D, E) The expression of ACSL1 was examined by

western blotting after transfection of HepG2 or Huh7 cells with the pcDNA3.1-HULC

plasmid. The transfection efficiency of HULC was detected by qRT-PCR. (F) The expression

of ACSL1 was examined by western blotting in HepG2.2.15 cells transfected with HULC

siRNA. The interference efficiency of HULC was detected by qRT-PCR. Statistically

significant differences are indicated: **P<0.01; Student’s t test.

Figure 2. HULC up-regulates the transcriptional factor PPARA to activate ACSL1. (A) The

correlation between HULC mRNA levels and PPARA mRNA levels was examined by

qRT-PCR in 60 cases of clinical HCC tissues (**P<0.01; Pearson′s correlation coefficient,

r=0.8517). (B) The effect of PPARA siRNA on HULC-enhanced ACSL1 was examined by




29

western blotting in HepG2 cells. The transfection efficiency of HULC and the interference

efficiency of PPARA were detected by qRT-PCR and western blotting, respectively. (C-E)

HepG2 and Huh7 cells were transfected with pcDNA3.1-HULC, and the mRNA (or protein)

levels of PPARA were examined by RT-PCR (or western blotting). The transfection efficiency

of HULC was detected by RT-PCR (or qRT-PCR). (F, G) HULC siRNA was transfected into

HepG2.2.15 cells, and the mRNA (or protein) levels of PPARA were assessed by RT-PCR (or

western blotting). The interference efficiency of HULC was detected by RT-PCR (or

qRT-PCR). Statistically significant differences are indicated: **P<0.01; Student’s t test.

Figure 3. MiR-9 inhibits the expression of PPARA by targeting the 3�UTR of PPARA. (A) A

model demonstrating the predicted conserved miR-9 binding site at nucleotides 7624-7631 of

the PPARA 3�UTR. The generated mutant sites at the PPARA 3�UTR seed region is indicated.

The wild-type PPARA 3�UTR (or mutant) was inserted into the downstream of luciferase

reporter gene in the pGL3-control vector. (B, C) The effect of miR-9 (or anti-miR-9) on the

pGL3-PPARA-7624 and pGL3-PPARA-mut reporters in HepG2 cells was measured by

luciferase reporter assays. (D, E) The effect of miR-9 (or anti-miR-9) on the expression of

PPARA in HepG2 cells was measured by western blotting. The transfection efficiency of

miR-9 (or anti-miR-9) was detected by qRT-PCR. Each experiment was repeated at least three

times. Statistically significant differences are indicated: **P<0.01; NS; Student’s t test.

Figure 4. HULC down-regulates miR-9 through inducing methylation of CpG islands in its

promoter. (A) The correlation between HULC mRNA levels and miR-9 levels was examined




30

by qRT-PCR in 60 cases of clinical HCC tissues (**P<0.01; Pearson′s correlation coefficient,

r=-0.6575). (B) The effect of HULC on miR-9 was assessed by qRT-PCR in HepG2 cells. The

transfection efficiency of HULC was detected by RT-PCR. (C) The effect of

5-aza-2’deoxycytidine (AZA) on miR-9 was examined by qRT-PCR in HepG2 cells. (D)

Schematic showing the three types of miR-9 genomic loci. CpG islands assayed for

methylation are depicted in black. (E) The methylation of miR-9-1 CpG sites was examined

by MSP analysis in L-O2, Chang liver (Chang), HepG2, and H7402 cells. Bands in the ‘U’ or

‘M’ lanes represent PCR products obtained with unmethylation-specific or

methylation-specific primers, respectively. L-O2 and HepG2 cells were transfected with

HULC. Bisulfite-sequencing analysis was used to examine the status of miR-9-1 CpG sites in

the treated cells. Ten CpG sites were analyzed, and three clones were sequenced for each

sample. Closed and open circles represent methylated and unmethylated CpG sites,

respectively. (F) MSP analysis and bisulfite-sequencing analysis of miR-9-1 CpG sites in

HCC tissues and their peritumoral tissues. P represents adjacent peritumoral tissues; T

represents HCC tissues. For the bisulfite-sequencing analysis, twenty CpG sites were

analyzed. Three clones were sequenced for each sample. Statistically significant differences

are indicated: **P<0.01; ***P<0.001; Student’s t test.

Figure 5. The product cholesterol of ACSL1 is able to up-regulate HULC by activating

RXRA in hepatoma cells. (A) The effect of ACSL1 on the HULC promoter was measured by

luciferase reporter assays in HepG2 and L-O2 cells. The cells were treated with increasing

dose of Triacsin C. (B) The effect of ACSL1 on HULC expression was measured by qRT-PCR




31

in HepG2 cells. The expression levels of ACSL1 were detected by western blotting in the

cells pre-treated with Triacsin C. (C, D) The effect of triglycerides (or cholesterol) on the

HULC promoter was measured in HepG2 cells by luciferase reporter assays. (E) The HULC

promoter including the mutant in the RXRA binding site (pGL3-HULC-mut) was generated

as indicated. The effect of cholesterol on this mutant was measured in HepG2 cells by

luciferase reporter assays. (F) The effect of RXRA siRNA on the cholesterol-treated HULC

promoter was measured in HepG2 cells by luciferase reporter assays. Each experiment was

repeated at least three times. Statistically significant differences are indicated: **P<0.01; NS;

Student’s t test.

Figure 6. HULC disrupts the lipid metabolism of hepatoma cells through

miR-9/PPARA/ACSL1 signaling in vitro. (A) The effect of HULC (or anti-miR-9, miR-9,

PPARA siRNA, ACSL1 siRNA and Triacsin C) on lipogenesis was determined by oil red O

staining in HepG2 (or Huh7) cells. (B) The effect of HULC siRNA (or anti-miR-9, miR-9,

PPARA siRNA, ACSL1 siRNA and Triacsin C) on lipogenesis was determined by oil red O

staining in HepG2.2.15 cells. (C, D) The effect of HULC (or anti-miR-9, miR-9, PPARA

siRNA, ACSL1 siRNA and Triacsin C) on cellular triglycerides (or cholesterol) was measured

in HepG2 cells using Tissue triglyceride assay kit (or Tissue total cholesterol kit). (E, F) The

effect of ACSL1 on the proliferation of hepatoma cells was assessed by MTT assays and

cloning formation assays in HepG2 and Huh7 cells, respectively. Statistically significant

differences are indicated: *P<0.05; **P<0.01; NS; Student’s t test.




32

Figure 7. HULC-modulated abnormal lipid metabolism facilitates the tumor growth in vivo.

(A) Photographs of dissected tumors from nude mice tumor transplanted with HepG2 (or

Huh7) cells pretreated with pcDNA3.1, pcDNA3.1-HULC, or pcDNA3.1-HULC and ACSL1

siRNA together. (B) The average weight of tumors from experimental groups of nude mice.

(C) Lipogenesis in the tumor tissues from mice transplanted with HepG2 cells was

determined by oil red O staining using frozen sections. (D) The levels of triglycerides were

individually measured using Tissue triglyceride assay kit in the tumor tissues from each nude

mouse transplanted with HepG2 cells. (E) The levels of cholesterol were individually

measured using Tissue total cholesterol kit in the tumor tissues from each nude mouse

transplanted with HepG2 cells. (F) Photographs of dissected tumors from nude mice

transplanted with HepG2 (or Huh7) cells treated with cholesterol or cholesterol/HULC siRNA.

(G) The average weight of the tumors from the experimental groups of nude mice.

Statistically significant differences are indicated: *P<0.05; **P<0.01; Student’s t test.

























Published OnlineFirst January 15, 2015.Cancer Res Ming Cui, Zelin Xiao, Yue Wang, et al. miRNA-9/PPARA/ACSL1/cholesterol/RXRA/HULC signalingmetabolism in hepatoma cells through Long non-coding RNA HULC modulates abnormal lipid

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