Long Noncoding RNA HULC Modulates Abnormal Lipid ......Molecular and Cellular Pathobiology Long Noncoding RNA HULC Modulates Abnormal Lipid Metabolism in Hepatoma Cells through an

Molecular and Cellular Pathobiology

Long Noncoding RNA HULC Modulates AbnormalLipid Metabolism in Hepatoma Cells through anmiR-9–Mediated RXRA Signaling PathwayMing Cui1, Zelin Xiao1, Yue Wang2, Minying Zheng1, Tianqiang Song3,4, Xiaoli Cai2,Baodi Sun1, Lihong Ye2, and Xiaodong Zhang1

Abstract

HULC is a long noncoding RNA overexpressed in hepatocel-lular carcinoma (HCC), but its functional contributions in thissetting have not been determined. In this study, we explored thehypothesis that HULC contributes to malignant development bysupporting abnormal lipid metabolism in hepatoma cells. HULCmodulated the deregulation of lipid metabolism in HCC byactivating the acyl-CoA synthetase subunit ACSL1. Immunohis-tochemical analysis of tissue microarrays revealed that approxi-mately 77% (180/233) of HCC tissues were positive for ACSL1.Moreover, HULC mRNA levels correlated positively with ACSL1levels in 60 HCC cases according to real-time PCR analysis.Mechanistic investigations showed that HULC upregulated thetranscriptional factor PPARA, which activated the ACSL1 promot-er in hepatoma cells. HULC also suppressed miR-9 targeting of

PPARAmRNAby elicitingmethylation of CpG islands in themiR-9 promoter. We documented the ability of HULC to promotelipogenesis, thereby stimulating accumulation of intracellulartriglycerides and cholesterol in vitro and in vivo. Strikingly, ACSL1overexpression that generates cholesterol was sufficient toenhance the proliferation of hepatoma cells. Further, cholesteroladdition was sufficient to upregulate HULC expression through apositive feedback loop involving the retinoid receptor RXRA,which activated the HULC promoter. Overall, we concluded thatHULC functions as an oncogene in hepatoma cells, acting mech-anistically by deregulating lipid metabolism through a signalingpathway involvingmiR-9, PPARA, andACSL1 that is reinforced bya feed-forward pathway involving cholesterol and RXRA to driveHULC signaling. Cancer Res; 75(5); 846–57. �2015 AACR.

IntroductionGrowing evidence indicates that many noncoding regulatory

elements are transcribed into noncoding RNAs (ncRNA;refs. 1, 2). Several types of ncRNAs are regarded as regulatoryRNAs that possess orchestrated functions involved in thecontrol of genome dynamics, cell biology, and developmentalprogramming (3). NcRNA is habitually divided into twogroups on the basis of transcript size: long ncRNA (lncRNA,>200 nt long) and small ncRNA (4). A spot of characterizedhuman lncRNAs has been associated with a spectrum ofbiologic functions, and the disruption of these functions playsa critical role in the development of cancer (5). Highly upre-gulated in liver cancer (HULC) is the first identified lncRNA

specifically overexpressed in hepatocellular carcinoma (HCC;ref. 6). HULC is transactivated by CREB and sequestersmiR-372 by acting as a sponge (7), and insulin-like growthfactors 2 mRNA-binding proteins (IGF2BP) are able to governthe expression of HULC (8). Previously, our group reportedthat hepatitis B virus X protein (HBx)–elevated HULC couldaccelerate the growth of hepatoma cells by downregulatingp18 (9). However, the role of HULC in abnormal lipid metab-olism remains poorly understood.

HCC is the fifth-most common cancer worldwide and the thirdlargest cause of cancer death globally (10). Recently, mountingclinical and epidemiologic studies have reported that high-riskcancer is linked to metabolic syndromes, such as obesity, type IIdiabetes, and atherosclerosis (11). Lipids, which represent adiverse group of water-insoluble molecules, play essential rolesin these processes. Meanwhile, high rates of lipid uptake andde novo lipid synthesis are frequently exhibited by cancer cells. Forexample, various tumors and their precursor lesions undergoexacerbated endogenous fatty acid biosynthesis irrespective ofthe levels of extracellular lipids (12). The changes in lipid metab-olism can alter numerous cellular processes, including prolifer-ation, motility, and tumorigenesis. As a central organ of energymetabolism, the liver synthesizes most plasma apolipoproteins,endogenous lipids, and lipoproteins. Thus, the advent of HCC isaccompanied by metabolic reprogramming, which is reflectedin changes in gene expression and miRNA profiles as well asaltered levels of circulating proteins and small metabolites (13).The AKT/mTOR pathway and insulin signaling have beenreported to contribute to the deregulation of lipid metabolismin hepatoma cells thus far (14, 15).

1State Key Laboratory of Medicinal Chemical Biology, Department ofCancer Research, College of Life Sciences, Nankai University, Tianjin,China. 2State Key Laboratory of Medicinal Chemical Biology, Depart-ment of Biochemistry, College of Life Sciences, Nankai University,Tianjin, China. 3Tianjin Medical University Cancer Institute and Hospi-tal, National Clinical Research Center for Cancer, Tianjin, China. 4KeyLaboratory of Cancer Prevention and Therapy, Tianjin Department ofHepatobiliary Tumor, Tianjin, China.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Xiaodong Zhang, State Key Laboratory of MedicinalChemical Biology, College of Life Sciences, Nankai University, 94 Weijin Road,Tianjin 300071, China. Phone: 86-22-23506830; Fax: 86-22-23501385; E-mail:[email protected]; and Lihong Ye, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-14-1192

�2015 American Association for Cancer Research.

CancerResearch

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Acyl-CoA synthetase long-chain family members (ACSL) cat-alyze the initial step in cellular long-chain fatty acid metabolismin mammals (16). There are five members in this family. Amongthem, ACSL1 is one of the major isoforms, with high levels in theliver, and can be regulated by the transcriptional factor peroxi-some proliferator–activated receptor alpha (PPARA; refs. 17, 18).Some studies have reported that the overexpression of ACSL1increases the uptake of fatty acids in hepatoma cells (19). How-ever, whether ACSL1 contributes to the development of HCC hasbeen ill-documented. The retinoid X receptors (RXR) were iden-tified in 1990 as orphan receptors that exhibited diverse tran-scriptional responses (20). RXRs play a vital role in the nuclearreceptor superfamily, forming heterodimers with many otherfamily members; as a result, RXRs are implicated in the controlof various physiologic processes (21). RXRA is a member of theRXR family that can be activated by sterol (22). In humans,cholesterol homeostasis is maintained by the precise interactionsbetween intestinal uptake, de novo synthesis, hepatic output, andfecal disposal (23), and excessive or deficient cholesterol canresult in pathophysiological sequelae (24). Secreted apoA-I bind-ing protein (AIBP) positively regulates cholesterol efflux fromendothelial cells, and effective cholesterol efflux is critical forproper angiogenesis (25). In addition, the primary metabolite ofcholesterol, 27-hydroxycholesterol (27HC), contributes to estro-gen receptor–dependent growth and liver X receptor–dependentmetastasis inmousemodels of breast cancer (26), and cholesterylester accumulation induced by PTEN loss and PI3K/AKT activa-tion underlies human prostate cancer aggressiveness (27). How-ever, the generalmechanism responsible for aberrant lipidmetab-olism during the development of HCC is not well understood.

In the present study, we investigated the role of lncRNAs in theabnormal lipid metabolism of HCC. Intriguingly, our data dem-onstrate that the lncRNA HULC facilitates the deregulation oflipid metabolism through miR-9/PPARA/ACSL1/cholesterol/RXRA/HULC signaling. Thus, our finding provides new insightsinto the mechanism of aberrant lipid metabolism in HCC.

Materials and MethodsPatient samples

Sixty HCC tissue samples and their corresponding adjacentnontumorous liver tissueswere obtained fromTianjin First CenterHospital and Tianjin Tumor Hospital (Tianjin, China) aftersurgical resection. Fifty-five of 60 patients had a history of hep-atitis B virus infection.Written consent approving the use of tissuesamples for research purposes was obtained from patients. Theinformation of patients with HCC is presented in SupplementaryTable S1. The study protocol was approved by the InstituteResearch Ethics Committee at Nankai University.

Cell lines and cell cultureThe human hepatoma H7402 cell line, human immortalized

liver L-O2 cell line, and Chang liver cell line were cultured inRPMI-1640 medium (Gibco). The human hepatoma cell lines,Huh7 (obtained fromShanghai Institutes for Biological Sciences),HepG2, and HepG2.2.15 (a hepatoma HepG2 cell line withintegrated full-length hepatitis B virus DNA), and human kidneyepithelial (HEK) 293T cells were maintained in DMEM (Gibco).All cell lines were supplemented with heat-inactivated 10% FBS(Gibco), 100 U/mL penicillin, and 100mg/mL streptomycin andgrown at 5% CO2 and 37�C.

In vivo tumorigenicity assayNude mice were housed and treated according to the guide-

lines established by the National Institutes of Health Guide forthe Care and Use of Laboratory Animals. We conducted animaltransplantations according to the Declaration of Helsinki.Briefly, HepG2 (or Huh7) cells were harvested and resuspendedat 2 � 107 cells per mL in sterile PBS. Groups of 4-week-oldmale BALB/c athymic nude mice (Experiment Animal Center ofPeking, China; each group, n¼ 6) were subcutaneously injectedat the shoulder with 0.2 mL of the cell suspensions. Accordingto the protocol (28), cholesterol (Solarbio) was subcutaneouslyinjected into the appropriate mice once in proximity to thetumor after injection of 5 days. Group one, the control group,was injected with 100 mL acetone. Group two and three wereexperimental groups and were injected with 300 mmol/L/kgcholesterol in 100 mL acetone. All cells transplanted in mice ingroup three were pretreated with 100 nmol/L HULC siRNA.Tumor growth was measured beginning 5 days after injection ofhepatoma cells. Tumor volume (V) was monitored by measur-ing the length (L) and width (W) of the tumors with calipersand was calculated using the formula (L � W2) � 0.5. After25 days, tumor-bearing mice and controls were sacrificed, andthe tumors were excised and measured.

Statistical analysisEach experiment was repeated at least three times. Statisti-

cal significance was assessed by comparing mean values(6 SD) using the Student t test for independent groups asfollow: �, P < 0.05; ��, P < 0.01; ��, P < 0.001 and not significant(NS). The Pearson correlation coefficient was used to determinethe correlations among gene expression in tumorous tissues.ACSL1 expression in tumor tissues and matched adjacent non-tumor tissues was compared using the Wilcoxon signed-ranktest.

ResultsHULC is positively correlated with ACSL1 in clinical HCCtissues and upregulates ACSL1 in hepatoma cells

To demonstrate the role of HULC in the deregulation oflipid metabolism in HCC, we examined the effects of HULCon several lipid metabolic enzymes in hepatoma cells. ACSL1stood out as being noticeably upregulated by HULC (Supple-mentary Fig. S1A). Thus, we evaluated the expression of ACSL1by immunohistochemical (IHC) staining in clinical HCC tissuesusing tissue microarrays and found that 77.3% (180/233) ofHCC tissues were positive for ACSL1 compared with 12.5%(2/16) of peritumoral liver tissues, in which the expression ofACSL1 was stronger in HCC tissues than that in their peritu-moral liver tissues (Fig. 1A). Moreover, quantitative real-timePCR (qRT-PCR) revealed that the mRNA levels of ACSL1 werehigher in HCC tissues compared with their adjacent nontumor-ous liver tissues in 60 paired clinical HCC samples (Fig. 1B).ACSL1 has been reported to participate in the formation oftriglycerides and cholesterol in the liver (29). Our data dem-onstrated that the increased triglycerides/cholesterol was accu-mulated in HCC tissues relative to their corresponding peritu-moral tissues (Supplementary Fig. S1B). Furthermore, weobserved that the levels of HULC were positively associatedwith those of ACSL1, triglycerides, and cholesterol in the afore-mentioned clinical samples (Fig. 1C). Moreover, we found that

LncRNA HULC Facilitates Abnormal Lipid Metabolism in HCC

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overexpression of HULC upregulated ACSL1 in HepG2 andHuh7 (or L-O2) cells at the mRNA and protein levels in adose-dependent manner (Supplementary Fig. S1C; Fig. 1D and1E; and Supplementary Fig. S1D). Similarly, depletion of HULCled to a decrease in ACSL1 levels in HepG2.2.15 cells expressinghigh levels of endogenous HULC (Supplementary Fig. S1Eand Fig. 1F). Meanwhile, the transfection (or interference)efficiency of HULC (or HULC siRNA) was validated by qRT-

PCR or RT-PCR analysis (Supplementary Fig. S1C–S1E and Fig.1D–F). Strikingly, HULC was capable of enriching triglyceridesand cholesterol in HepG2 cells in a dose-dependent manner(Supplementary Fig. S1F), but failed to increase their levels inthe conditioned medium (Supplementary Fig. S1G). Takentogether, these results show that HULC is positively associatedwith ACSL1 in clinical HCC tissues and upregulates ACSL1 inhepatoma cells.

Figure 1.HULC is positively correlated with ACSL1 in clinical HCC tissues and upregulates ACSL1 in hepatoma cells. A, the expression of ACSL1 was examined byIHC staining in clinical HCC tissues and peritumoral tissues. The expression of ACSL1 was stronger in HCC tissues than that in their peritumoral livertissues (a) and its amplification (b). B, relative mRNA levels of ACSL1 were assessed by qRT-PCR in 60 pairs of clinical HCC tissues and correspondingnontumorous tissues (P < 0.01; Wilcoxon signed-rank test). C, the correlation between HULC mRNA levels and ACSL1 mRNA levels (or levels of triglycerides orcholesterol) 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 correlation coefficient, r ¼ 0.7444, 0.7099, 0.6501). D and E, the expression of ACSL1 was examined by Western blotting after transfection ofHepG2 or Huh7 cells with the pcDNA3.1-HULC plasmid. The transfection efficiency of HULC was detected by qRT-PCR. F, the expression of ACSL1 wasexamined by Western blotting in HepG2.2.15 cells transfected with HULC siRNA. The interference efficiency of HULC was detected by qRT-PCR. Statisticallysignificant differences are indicated: �� , P < 0.01; Student t test.

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HULC upregulates the transcriptional factor PPARA to activateACSL1

Given that ACSL1 is activated by the transcriptional factorPPARA in the liver (17), we speculated that HULC mightmodulate ACSL1 through PPARA. Interestingly, qRT-PCR ana-lysis demonstrated that the expression levels of HULC werepositively associated with those of PPARA in the 60 clinicalHCC samples (Fig. 2A). However, inhibition of PPARA expres-sion abrogated the HULC-induced upregulation of ACSL1 inHepG2 cells (Fig. 2B), suggesting that PPARA is responsiblefor the upregulation of ACSL1 mediated by HULC. The effi-ciency of PPARA siRNA (or PPARA siRNA�) was validated inthese cells (Supplementary Fig. S2A). Moreover, the over-expression of HULC was able to upregulate PPARA at mRNAand protein levels in HepG2 and Huh7 cells (or L-O2 cells)in a dose-dependent manner (Fig. 2C–E and Supplementary

information; Supplementary Fig. S2B and S2C), whereas HULCsiRNA reversed these effects inHepG2.2.15 cells (Fig. 2F andG) .The transfection (or interference) efficiency of HULC (or HULCsiRNA) was confirmed in these cells (Fig. 2C–G and Supple-mentary Fig. S2B and S2C). However, luciferase reporter geneassays showed that HULC failed to activate the promoter ofPPARA in HepG2 cells (Supplementary Fig. S2D and S2E),implying that HULC might upregulate PPARA at the posttran-scriptional step. Thus, we conclude that HULC activates ACSL1by upregulating the transcription factor PPARA in hepatomacells.

miR-9 inhibits the expression of PPARA by targeting the 30UTRof PPARA

Next, we identified several miRNAs that could potentiallybind to the 30untranslated region (UTR) of PPARA using

Figure 2.HULC upregulates the transcriptional factor PPARA to activate ACSL1. A, the correlation between HULC mRNA levels and PPARA mRNA levels was examinedby qRT-PCR in 60 cases of clinical HCC tissues (P < 0.01; Pearson correlation coefficient, r ¼ 0.8517). B, the effect of PPARA siRNA on HULC-enhancedACSL1 was examined by Western blotting in HepG2 cells. The transfection efficiency of HULC and the interference efficiency of PPARA were detected byqRT-PCR and Western blotting, respectively. C–E, HepG2 and Huh7 cells were transfected with pcDNA3.1-HULC, and the mRNA (or protein) levels of PPARAwere examined by RT-PCR (or Western blotting). The transfection efficiency of HULC was detected by RT-PCR (or qRT-PCR). F and G, HULC siRNA wastransfected into HepG2.2.15 cells, and the mRNA (or protein) levels of PPARA were assessed by RT-PCR (or Western blotting). The interference efficiency ofHULC was detected by RT-PCR (or qRT-PCR). Statistically significant differences are indicated: �� , P < 0.01; Student t test.






TargetScan and microrna.org (http://www.targetscan.org/,http://www. microrna.org/microrna/home.do). Because miR-9has been reported to be downregulated in cancer (30–32), wefocused our investigation on this miRNA. Three miR-9 bindingsites in the 30UTR of PPARA mRNA were constructed (Fig. 3Aand Supplementary Fig. S3A and S3B), and luciferase reporterassays revealed that miR-9 could directly bind to the conservedseed region of the PPARA 30UTR (position 7624-7631, pGL3-PPARA-7624; Fig. 3B), rather than the poorly conserved seedregions (position 5684-5690, position 4375-4381; Supplemen-tary Fig. S3C and S3D). However, the PPARA 30UTR conservedseed region mutant (position 7624-7631, pGL3-PPARA-mut)failed to work in these cells (Fig. 3B). Conversely, anti–miR-9increased the luciferase activities of pGL3-PPARA-7624 butfailed to influence the mutant (Fig. 3C), suggesting that miR-9 is able to directly bind to the 30UTR of PPARA. These effectswere also observed in 293T cells (Supplementary Fig. S3C–S3F). Furthermore, the overexpression of miR-9 suppressed theexpression of PPARA in HepG2 cells in a dose-dependentmanner (Fig. 3D), and the reverse outcome was obtained whenthe cells were treated with anti–miR-9 (Fig. 3E). Together, ourdata indicate that miR-9 suppresses the expression of PPARAby targeting its 30UTR.

HULC downregulates miR-9 through inducing methylation ofCpG islands in its promoter

Next, we validated the anticorrelation between HULC andmiR-9 in the 60 clinical HCC samples (Fig. 4A). Then, our datashowed that theoverexpressionofHULCdownregulatedmiR-9 inHepG2 cells in a dose-dependent manner (Fig. 4B). Interestingly,we found that treatmentwith 5-Aza-20-deoxycytidine (Aza, aDNAmethylation inhibitor) heightened the levels of miR-9 in HepG2cells in a dose-dependentmanner (Fig. 4C), suggesting thatHULCmight influence the epigenetic regulation of the miR-9 promoter(33, 34). Then, we examined the methylation status of miR-9using both methylation-specific PCR (MSP) and bisulfite-sequencing analysis (BSP). As shown in Fig. 4D, miR-9 comprisesthree members, termed miR-9-1, miR-9-2 and miR-9-3. MSPassays revealed that the CpG sites of miR-9-1 (or miR-9-2,miR-9-3) were highly methylated following the overexpressionof HULC in L-O2 [or Chang liver (Chang), HepG2, and H7402]cells. BSP assays further validated the above observations in L-O2(orHepG2) cells (Fig. 4E and Supplementary Fig. S4A).Moreover,we confirmed these data in two pairs of clinical samples (Fig. 4Fand Supplementary Fig. S4B). It has also been reported that DNA(cytosine-5-)-methyltransferase 1 (DNMT1), amethylase, is capa-ble of regulating the expression of miRNAs through inducing

Figure 3.MiR-9 inhibits the expression ofPPARA by targeting the 30UTR ofPPARA. A, a model demonstrating thepredicted conserved miR-9 bindingsite at nucleotides 7624-7631 of thePPARA 30UTR. The generated mutantsites at the PPARA 30UTR seed regionare indicated. The wild-type PPARA30UTR (or mutant) was inserted intothe downstream of luciferase reportergene in the pGL3-control vector. B andC, the effect of miR-9 (or anti–miR-9)on the pGL3-PPARA-7624 and pGL3-PPARA-mut reporters in HepG2 cellswas measured by luciferase reporterassays. D and E, the effect of miR-9 (oranti–miR-9) on the expression ofPPARA in HepG2 cells was measuredby Western blotting. The transfectionefficiency of miR-9 (or anti–miR-9)was detected by qRT-PCR. Eachexperiment was repeated at leastthree times. Statistically significantdifferences are indicated: �� , P < 0.01;NS, nonsignificant; Student t test.

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methylation of their CpG islands (35). Interestingly, we observedthat HULC was able to upregulate DNMT1 in HepG2 cells(Supplementary Fig. S4C), hinting that HULC might inducemethylation of CpG islands in the miR-9 promoter throughupregulation of DNMT1. Therefore, we conclude that HULCinhibits the expression of miR-9 through eliciting methylationof CpG islands in the miR-9 promoter.

Theproduct cholesterol of ACSL1 is able toupregulateHULCbyactivating RXRA in hepatoma cells

Given that the positive feedback loops in signaling pathwaysare involved in the progression of cancer, we evaluated whetherHULC facilitates aberrant lipid metabolism in liver cancer via afeedback mechanism. Surprisingly, we found that the promoteractivities of HULC were dose-dependently decreased in HepG2

cells after treatment with Triacsin C (an inhibitor of ACSL1) orACSL1 siRNA, although this was not observed in L-O2 cells(Fig. 5A and Supplementary Fig. S5A). Meanwhile, we observedthat the expression levels of HULC were also downregulated inHepG2 cells (Fig. 5B and Supplementary Fig. S5B), suggesting thata positive feedback loop involving HULC/miR-9/PPARA/ACSL1/HULC is established in hepatoma cells but not in normal livercells. Next, we utilized acetone as the solvent to deliver triglyce-ride (or cholesterol) to the cells. Although some triglyceride (orcholesterol) dissolved out, the cells responded to the stimulation.Strikingly, we found that cholesterol was able to stimulate theactivity of the HULC promoter in HepG2 (or Huh7) cells in adose-dependent manner, although triglyceride failed to have aneffect in these cells (Fig. 5C and D, Supplementary information,S5C and S5D). According to the report (22), we cloned the HULC

Figure 4.HULC downregulates miR-9 throughinducingmethylation of CpG islands inits promoter. A, the correlationbetween HULC mRNA levels andmiR-9 levels was examined by qRT-PCR in 60 cases of clinical HCC tissues(P < 0.01; Pearson correlationcoefficient, r¼ –0.6575) . B, the effectof HULC on miR-9 was assessed byqRT-PCR in HepG2 cells. Thetransfection efficiency of HULC wasdetected by RT-PCR. C, the effect ofAZA on miR-9 was examined byqRT-PCR in HepG2 cells. D, schematicshowing the three types of miR-9genomic loci. CpG islands assayed formethylation are depicted in black. E,the methylation of miR-9-1 CpG siteswas examined by MSP analysis in L-O2, Chang liver (Chang), HepG2, andH7402 cells. Bands in the "U" or "M"lanes represent PCR productsobtained with unmethylation-specificor methylation-specific primers,respectively. L-O2 and HepG2 cellswere transfected with HULC. Bisulfite-sequencing analysis was used toexamine the status of miR-9-1 CpGsites in the treated cells. Ten CpG siteswere analyzed, and three clones weresequenced for each sample. Closedandopen circles representmethylatedand unmethylated CpG sites,respectively. F, MSP analysis andbisulfite-sequencing analysis of miR-9-1 CpG sites in HCC tissues and theirperitumoral tissues. P, adjacentperitumoral tissues; T, HCC tissues.For the bisulfite-sequencing analysis,20 CpG sites were analyzed. Threeclones were sequenced for eachsample. Statistically significantdifferences are indicated: �� , P < 0.01;�� , P < 0.001; Student t test.






promoter including themutant in theRXRA-binding site (Fig. 5E).Intriguingly, the treatment with cholesterol failed to activate theabovemutant in HepG2 cells (Fig. 5E), suggesting that RXRAmaybe implicated in the regulation of HULCmediated by cholesterol.Moreover, we verified that RXRA siRNA could abolish the cho-lesterol-increasedHULCpromoter activity (Fig. 5F).However, thetreatment with cholesterol failed to influence the expression ofRXRA (Supplementary Fig. S5E), suggesting that cholesterol, as atype of sterol, might be able to activate the HULC promoter bystimulating RXRA, rather than upregulating RXRA. Meanwhile,the efficiencies of ACSL1 siRNA (or ACSL1 siRNA�) and RXRAsiRNA were validated by Western blot analysis in these cells(Supplementary Fig. S5F and S5G). As a result, we conclude that

the product cholesterol of ACSL1 upregulates HULC by activatingRXRA in hepatoma cells.

HULCdisrupts the lipidmetabolism of hepatoma cells throughmiR-9/PPARA/ACSL1 signaling in vitro

Next, we investigated the effect of HULC on lipogenesis inhepatoma cells using Oil Red O staining. These results showedthat the overexpression of HULC was able to accelerate lipogen-esis 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 HULCsiRNA (or ACSL1 siRNA, miR-9, PPARA siRNA, and Triacsin C)

Figure 5.The product cholesterol of ACSL1 isable to upregulate HULC by activatingRXRA in hepatoma cells. A, the effectof ACSL1 on the HULC promoter wasmeasured by luciferase reporterassays in HepG2 and L-O2 cells. Thecells were treated with increasingdose of Triacsin C. B, the effect ofACSL1 on HULC expression wasmeasured by qRT-PCR in HepG2 cells.The expression levels of ACSL1 weredetected by Western blotting in thecells pretreated with Triacsin C. C andD, the effect of triglycerides (orcholesterol) on the HULC promoterwas measured in HepG2 cells byluciferase reporter assays. E, the HULCpromoter, including the mutant in theRXRA binding site (pGL3-HULC-mut),was generated as indicated. The effectof cholesterol on this mutant wasmeasured in HepG2 cells by luciferasereporter assays. F, the effect of RXRAsiRNA on the cholesterol-treatedHULC promoter was measured inHepG2 cells by luciferase reporterassays. Each experiment wasrepeated at least three times.Statistically significant differencesare indicated: ��, P < 0.01; NS,nonsignificant; Student t test.

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could attenuate the lipogenesis in HepG2.2.15 cells. However,anti–miR-9 was able to rescue the HULC siRNA-repressed lipo-genesis (Fig. 6B), and we obtained a similar effect of HULC onintracellular triglyceride and cholesterol in this system (Fig. 6CandD; Supplementary Fig. S6A and S6B). The role of ACSL1 in thegrowth of hepatoma cells remains enigmatic. In this study, weshowed that the overexpression of ACSL1 could facilitate theproliferation of HepG2 and Huh7 cells, as demonstrated by MTTassays and cloning formation assays (Fig. 6E and F). Thus, we

sought to evaluate whether the product cholesterol of ACSL1maybe responsible for the promotion of cell proliferation. Asexpected,MTT assays further corroborated that the treatment withcholesterol was able to promote the proliferation of hepatomacells, which could be eliminated byHULC siRNA (SupplementaryFig. S6C), suggesting that cholesterol might enhance the pro-liferation of hepatoma cells by upregulating HULC. In addition,we assessed whether HULC affected other signaling pathwaysand factors involved in lipid metabolism in hepatoma cells, such

Figure 6.HULC disrupts the lipid metabolism ofhepatoma cells through miR-9/PPARA/ACSL1 signaling in vitro. A, theeffect of HULC (or anti–miR-9, miR-9,PPARA siRNA, ACSL1 siRNA, andTriacsin C) on lipogenesis wasdetermined by Oil Red O staining inHepG2 (or Huh7) cells. B, the effect ofHULC siRNA (or anti–miR-9, miR-9,PPARA siRNA, ACSL1 siRNA, andTriacsin C) on lipogenesis wasdetermined by Oil Red O staining inHepG2.2.15 cells. C and D, the effect ofHULC (or anti–miR-9, miR-9, PPARAsiRNA, ACSL1 siRNA, and Triacsin C)on cellular triglycerides (orcholesterol) was measured in HepG2cells using the tissue triglycerideassay kit (or tissue total cholesterolkit). E and F, the effect of ACSL1 onthe proliferation of hepatoma cellswas assessed by MTT assays andcloning formation assays in HepG2and Huh7 cells, respectively.Statistically significant differencesare indicated: � , P < 0.05; �� , P < 0.01;NS, nonsignificant; Student t test.






as the AKT/mTOR pathway, SREBP1, SREBP2, chREBP, HMGCR,FASN, ACLY, SQS, and miR-122 (14, 36). However, we did notobserve alterations in these factors at the levels of mRNA andprotein in HepG2 and Huh7 cells (Supplementary Fig. S6D–

S6G). Thus, we conclude that HULC contributes to aberrant lipidmetabolism in hepatoma cells through miR-9/PPARA/ACSL1signaling.

HULC-modulated abnormal lipid metabolism facilitatestumor growth in vivo

To better understand the role of HULC in abnormal lipidmetabolism, we subcutaneously injected pretreated cells into4-week-old BALB/c athymic nude mice. We confirmed that thelevels of HULC and ACSL1 were preserved in the tumor tissues(Supplementary Fig. S7A). We observed that treatment with

ACSL1 siRNA abolished the HUCL-accelerated proliferationof HepG2 (or Huh7) cells in mice (Fig. 7A and B and Supple-mentary Fig. S7B), further supporting the conclusion that ACSL1is responsible for the promotion of tumor growth mediatedby HULC. IHC staining further confirmed that the expression ofKi-67, a marker of proliferation, as well as BrdUrd incorpora-tion in the tumor tissues was consistent with tumor growthamong the different groups (Supplementary Fig. S7C). Interest-ingly, oil redO staining revealed that lipid droplets were increasedin the tumor tissues overexpressing HULC, whereas silencingof ACSL1 reversed this effect (Fig. 7C and SupplementaryFig. S7C). The levels of triglycerides and cholesterol were consis-tent with the Oil Red O staining in the tumor tissues (Fig. 7Dand E), suggesting that abnormal lipid metabolism contributesto the growth of hepatoma cells. To better evaluate the effect of

Figure 7.HULC-modulated abnormal lipidmetabolism facilitates the tumorgrowth in vivo. A, photographs ofdissected tumors from nude micetumor transplanted with HepG2 (orHuh7) cells pretreated with pcDNA3.1,pcDNA3.1-HULC, or pcDNA3.1-HULC,and ACSL1 siRNA together. B, theaverage weight of tumors fromexperimental groups of nude mice. C,lipogenesis in the tumor tissues frommice transplanted with HepG2 cellswas determined by Oil Red O stainingusing frozen sections. D, the levels oftriglycerides were individuallymeasured using a tissue triglycerideassay kit in the tumor tissues fromeach nude mouse transplanted withHepG2 cells. E, the levels of cholesterolwere individually measured using atissue total cholesterol kit in the tumortissues from each nude mousetransplanted with HepG2 cells. F,photographs of dissected tumorsfrom nude mice transplanted withHepG2 (or Huh7) cells treated withcholesterol or cholesterol/HULCsiRNA. G, the average weight of thetumors from the experimental groupsof nude mice. Statistically significantdifferences are indicated: � , P < 0.05;�� , P < 0.01; Student t test.

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cholesterol on the expression of HULC in hepatoma cells in vivo,we injected supraphysiological cholesterol in proximity to thetumor tissues. As expected, the injection significantly increasedthe levels of tissue HULC (Supplementary Fig. S7D). Notably,we observed that the pretreatment with HULC siRNA remark-ably abolished the cholesterol-increased growth of hepatomacells (Fig. 7F and G and Supplementary Fig. S7E), suggesting thatcholesterol is able to upregulate HULC in hepatoma cells. Togeth-er, we conclude that HULC-modulated abnormal lipid metabo-lism contributes to tumor growth, and this process requires themetabolic enzyme ACSL1 and its product cholesterol.

DiscussionLncRNAs play crucial roles in cancer (37), and we previously

reported that HBx-enhanced HULC is able to promote the growthof hepatoma cells (9). Metabolism deregulation, exacerbatedlipid biosynthesis, and accumulation in the development ofcancer accelerate cell growth and transformation (38). In thisstudy, we assessed whether HULC participates in abnormal lipidmetabolism in HCC.

To better understand the roles of HULC in modulating lipidmetabolism, we first measured the effect of HULC on lipidmetabolic enzymes in hepatoma cells. Interestingly, ACSL1drew our attention because ACSL1 and its intracellular products,such as triglycerides and cholesterol, were remarkably upregu-lated and increased by HULC expression. However, the levels oftriglycerides and cholesterol in the conditioned medium wereunaltered in HULC-treated cells relative to controls, which maybe the result of the ability of ACSL1 to inhibit cholesterol efflux(39). Moreover, we validated that the expression levels of HULCwere positively correlated with those of ACSL1 and its productsin clinical HCC samples (Fig. 1 and Supplementary Fig. S1).Next, we explored the mechanism by which HULC activatesACSL1 in hepatoma cells. ACSL1 has been reported to be aclassic target gene of PPARA in the liver (16). Accordingly, weobserved that HULC modulated ACSL1 by upregulating PPARA(Fig. 2 and Supplementary Fig. S2). Furthermore, we determin-ed that HULC could increase PPARA expression by downregu-lating the ability of miR-9 to target the PPARA 30UTR at theposttranscriptional level, rather than by activating the transcrip-tion of PPARA directly (Fig. 3 and Supplementary Fig. S3).Numerous studies have noted that the expression of miR-9 isregulated by CpG island methylation, and lncRNAs are able tomodulate the expression of genes through epigenetic regulation(40, 41). Hence, we validated that HULC was capable ofinducing the methylation of CpG islands in the promoter ofmiR-9 (Fig. 4 and Supplementary Fig. S41). This finding sug-gests that HULC governs the expression of PPARA throughepigenetic regulation. The methylation of miR-9-3 is signifi-cantly associated with an increased risk of recurrence, and highmethylation levels of either miR-9-1 or miR-9-3 result in asignificant decrease in recurrence-free survival times in clearcell renal cell carcinoma (30). Moreover, the inhibition of miR-9–mediated suppression of SOX2 is involved in chemoresis-tance and cancer stemness in glioma cells (42), and miR-9 isable to target MTHFD2 to inhibit the proliferation of breastcancer cells (43). Together with these findings, our data implythat the methylation and inhibition of miR-9 might hijack othersignaling pathways to facilitate hepatocarcinogenesis. To betterunderstand the underlying mechanism by which HULC elicits

the methylation of CpG islands in the miR-9 promoter, weassessed the influence of HULC on methylase levels in hepa-toma cells. Strikingly, our observations indicated that HULCwas able to upregulate the expression of DNMT1 in hepatomacells, suggesting that HULC induces the methylation of miR-9promoter CpG islands possibly by upregulating DNMT1. Therole of ACSL1 in the growth of hepatoma cells has not beenreported. Therefore, we examined the effect of ACSL1 over-expression on the proliferation of HepG2 and Huh7 cells byMTT assays and cloning formation assays. Notably, we observ-ed that ACSL1 was able to promote the proliferation of hepa-toma cells, suggesting that the role of ACSL1 in the promotionof cell proliferation might be associated with the disturbanceof lipid metabolism mediated by ACSL1 in hepatoma cells. Inaddition, it has been reported that the AKT/mTOR cascadeinfluences the growth, survival, metabolism, and migration ofliver cancer cells, and the extent of aberrant lipogenesis iscorrelated with the activation of the AKT/mTOR signalingpathway (14, 44). This relationship suggests that the AKT/mTOR pathway plays a pivotal role in the deregulation of lipidmetabolism in HCC. Therefore, we wondered whether theAKT/mTOR pathway and other signaling pathway membersimplicated in lipid metabolism, such as SREBP1/2, chREBP,HMGCK, MVK, FASN, ACLY, SQS, and miR-122 (14, 36), wereinvolved in this event. However, we failed to demonstrate thatHULC disturbed lipid metabolism in hepatoma cells throughthese signaling pathways. Therefore, we conclude that HULCis able to induce aberrant lipid metabolism through ASCL1/miR-9/ PPARA/ASCL1 signaling in hepatoma cells.

Given that cancer disrupts cellular homeostasis and createsmany newmethods of regulation, such as positive feedback loops(45), we are interested in whether the action of HULC is involvedin a feedback loop as well. Interestingly, we observed that ACSL1could influence the expression of HULC in hepatoma cells in apositive feedback fashion. Thus, we hypothesized that the pro-ducts of ACSL1 might be responsible for the activation of HULC.Indeed, luciferase reporter assays revealed that cholesterol wasable to activate the promoter of HULC, whereas triglycerides werenot. Furthermore, we identified a binding site for RXRA in thepromoter of HULC using bioinformatics analysis. RXRA is anorphan receptor that can be activated by sterol (22). Thus, wespeculated that RXRA might participate in cholesterol-activatedHULC in hepatoma cells. As expected, we found that RXRA wasresponsible for the activation ofHULCmediated by cholesterol inhepatoma cells. It has been reported that PPARA, liver X receptor(LXRA), and bile acid receptor (FXR) can form heterodimers withRXRA (46–48). Here, we treated cells with siRNA targeting PPARA(or LXRA and FXR) mRNA to assess the effect of PPARA (or LXRAand FXR) on HULC. However, our data demonstrated that alltreatments failed to influence the cholesterol-enhanced luciferasereporter activity of the HULC promoter (data not shown), imply-ing that PPARA, LXRA, and FXR are not implicated in cholesterol-induced RXRA activation. Strikingly, we observed that cholesterolfailed to upregulate HULC in normal liver cells (Fig. 5 andSupplementary Fig. S5). The reason may be related to RXRA notbeing constitutively phosphorylated, thus it fails to escape fromUb/proteasome-mediated degradation in liver cells (49). Ofparticular note, this finding provides new insights into the mech-anism by which HULC is highly expressed in hepatoma cells.

Recent reports pinpoint that the growth-promoting effects ofelevated levels of insulin, glucose, or triglycerides are involved in






insulin resistance–promoted colorectal cancer (50). Heightenedintracellular levels of triglyceride and their metabolites, such asdiacylglycerol, may activate the protein kinase-C andMAPK path-ways with potentially mitogenic and carcinogenic effects (51).Consistent with our study, these findings suggest that HULC-enhanced accumulation of triglycerides in hepatoma cells mightemerge as a risk factor for hepatocarcinogenesis. In this article, wefound that cholesterol was involved in the promotion of hepa-toma cell growth mediated by HULC, which is consistent withother reports (25–27).

In aggregate, our work demonstrates that HULC plays pivotalroles in aberrant lipid metabolism in HCC through miR-9/PPARA/ACSL1/cholesterol/RXRA/HULC signaling, a model forthis role of HULC is represented in Supplementary Fig. S7F.In particular, our data show that HULC elicits the methylationof CpG islands in the miR-9 promoter, resulting in the sup-pression of miR-9 expression. MiR-9 is able to target the 30UTRof transcription factor PPARA, and the decrease in miR-9 leadsto upregulation of PPARA and the subsequent transactivationof ACSL1, which enhances lipogenesis and enriches intracellu-lar triglycerides and cholesterol in hepatoma cells. Thus, HULC-enhanced abnormal lipid metabolism accelerates the growthof liver cancer. Furthermore, the cholesterol product of ACSL1upregulates HULC by activating the transcription factorRXRA, 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 thedevelopment of HCC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Cui, L. Ye, X. ZhangDevelopment of methodology: M. Cui, X. ZhangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): M. Cui, Z. Xiao, Y. Wang, M. Zheng, T. Song, X. Cai,B. Sun, L. Ye, X. ZhangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Cui, Z. Xiao, Y. Wang, L. Ye, X. ZhangWriting, review, and/or revision of the manuscript: M. Cui, L. Ye, X. ZhangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Cui, L. Ye, X. ZhangStudy supervision: L. Ye, X. Zhang

Grant SupportThis work was supported by grants from National Natural Science

Foundation of China (nos. 31470756, 81071624, and 81272218) andNational Basic Research Program of China (973 Program, nos.2015CB553703, 2015CB55905, and 2011CB933100).

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 21, 2014; revised November 15, 2014; acceptedNovember 22,2014; published OnlineFirst January 15, 2015.

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2015;75:846-857. Published OnlineFirst January 15, 2015.Cancer Res Ming Cui, Zelin Xiao, Yue Wang, et al. Pathway

Mediated RXRA Signaling−in Hepatoma Cells through an miR-9Long Noncoding RNA HULC Modulates Abnormal Lipid Metabolism

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