Long Noncoding RNA HULC Modulates Abnormal Lipid

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Published OnlineFirst January 15, 2015; DOI: 10.1158/0008-5472.CAN-14-1192 Cancer Molecular and Cellular Pathobiology Research

Long Noncoding RNA HULC Modulates Abnormal Lipid Metabolism in Hepatoma Cells through an miR-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

Abstract

HULC is a long noncoding RNA overexpressed in hepatocel- PPARA mRNA by eliciting methylation of CpG islands in the miR- lular carcinoma (HCC), but its functional contributions in this 9 promoter. We documented the ability of HULC to promote setting have not been determined. In this study, we explored the lipogenesis, thereby stimulating accumulation of intracellular hypothesis that HULC contributes to malignant development by triglycerides and cholesterol in vitro and in vivo. Strikingly, ACSL1 supporting abnormal lipid metabolism in hepatoma cells. HULC overexpression that generates cholesterol was sufﬁcient to modulated the deregulation of lipid metabolism in HCC by enhance the proliferation of hepatoma cells. Further, cholesterol activating the acyl-CoA synthetase subunit ACSL1. Immunohis- addition was sufﬁcient to upregulate HULC expression through a tochemical analysis of tissue microarrays revealed that approxi- positive feedback loop involving the retinoid receptor RXRA, mately 77% (180/233) of HCC tissues were positive for ACSL1. which activated the HULC promoter. Overall, we concluded that Moreover, HULC mRNA levels correlated positively with ACSL1 HULC functions as an oncogene in hepatoma cells, acting mech- levels in 60 HCC cases according to real-time PCR analysis. anistically by deregulating lipid metabolism through a signaling Mechanistic investigations showed that HULC upregulated the pathway involving miR-9, PPARA, and ACSL1 that is reinforced by transcriptional factor PPARA, which activated the ACSL1 promot- a feed-forward pathway involving cholesterol and RXRA to drive er in hepatoma cells. HULC also suppressed miR-9 targeting of HULC signaling. Cancer Res; 75(5); 1–12. 2015 AACR.

Introduction specifically overexpressed in hepatocellular carcinoma (HCC; ref. 6). HULC is transactivated by CREB and sequesters Growing evidence indicates that many noncoding regulatory miR-372 by acting as a sponge (7), and insulin-like growth elements are transcribed into noncoding RNAs (ncRNA; factors 2 mRNA-binding proteins (IGF2BP) are able to govern refs. 1, 2). Several types of ncRNAs are regarded as regulatory the expression of HULC (8). Previously, our group reported RNAs that possess orchestrated functions involved in the that hepatitis B virus X protein (HBx)–elevated HULC could control of genome dynamics, cell biology, and developmental accelerate the growth of hepatoma cells by downregulating programming (3). NcRNA is habitually divided into two p18 (9). However, the role of HULC in abnormal lipid metab- groups on the basis of transcript size: long ncRNA (lncRNA, olism remains poorly understood. >200 nt long) and small ncRNA (4). A spot of characterized HCC is the fifth-most common cancer worldwide and the third human lncRNAs has been associated with a spectrum of largest cause of cancer death globally (10). Recently, mounting biologic functions, and the disruption of these functions plays clinical and epidemiologic studies have reported that high-risk a critical role in the development of cancer (5). Highly upre- cancer is linked to metabolic syndromes, such as obesity, type II gulated in liver cancer (HULC) is the first identified lncRNA diabetes, and atherosclerosis (11). Lipids, which represent a diverse group of water-insoluble molecules, play essential roles 1State Key Laboratory of Medicinal Chemical Biology, Department of in these processes. Meanwhile, high rates of lipid uptake and Cancer Research, College of Life Sciences, Nankai University, Tianjin, de novo lipid synthesis are frequently exhibited by cancer cells. For China. 2State Key Laboratory of Medicinal Chemical Biology, Depart- example, various tumors and their precursor lesions undergo ment of Biochemistry, College of Life Sciences, Nankai University, Tianjin, China. 3Tianjin Medical University Cancer Institute and Hospi- exacerbated endogenous fatty acid biosynthesis irrespective of tal, National Clinical Research Center for Cancer, Tianjin, China. 4Key the levels of extracellular lipids (12). The changes in lipid metab- Laboratory of Cancer Prevention and Therapy, Tianjin Department of olism can alter numerous cellular processes, including prolifer- Hepatobiliary Tumor, Tianjin, China. ation, motility, and tumorigenesis. As a central organ of energy Note: Supplementary data for this article are available at Cancer Research metabolism, the liver synthesizes most plasma apolipoproteins, Online (http://cancerres.aacrjournals.org/). endogenous lipids, and lipoproteins. Thus, the advent of HCC is Corresponding Authors: Xiaodong Zhang, State Key Laboratory of Medicinal accompanied by metabolic reprogramming, which is reflected Chemical Biology, College of Life Sciences, Nankai University, 94 Weijin Road, in changes in gene expression and miRNA profiles as well as Tianjin 300071, China. Phone: 86-22-23506830; Fax: 86-22-23501385; E-mail: altered levels of circulating proteins and small metabolites (13). [email protected]; and Lihong Ye, E-mail: [email protected] The AKT/mTOR pathway and insulin signaling have been doi: 10.1158/0008-5472.CAN-14-1192 reported to contribute to the deregulation of lipid metabolism 2015 American Association for Cancer Research. in hepatoma cells thus far (14, 15).

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Acyl-CoA synthetase long-chain family members (ACSL) cat- In vivo tumorigenicity assay alyze the initial step in cellular long-chain fatty acid metabolism Nude mice were housed and treated according to the guide- in mammals (16). There are five members in this family. Among lines established by the National Institutes of Health Guide for them, ACSL1 is one of the major isoforms, with high levels in the theCareandUseofLaboratoryAnimals.Weconductedanimal liver, and can be regulated by the transcriptional factor peroxi- transplantations according to the Declaration of Helsinki. some proliferator–activated receptor alpha (PPARA; refs. 17, 18). Briefly, HepG2 (or Huh7) cells were harvested and resuspended Some studies have reported that the overexpression of ACSL1 at 2 107 cells per mL in sterile PBS. Groups of 4-week-old increases the uptake of fatty acids in hepatoma cells (19). How- male BALB/c athymic nude mice (Experiment Animal Center of ever, whether ACSL1 contributes to the development of HCC has Peking, China; each group, n ¼ 6) were subcutaneously injected been ill-documented. The retinoid X receptors (RXR) were iden- at the shoulder with 0.2 mL of the cell suspensions. According tified in 1990 as orphan receptors that exhibited diverse tran- to the protocol (28), cholesterol (Solarbio) was subcutaneously scriptional responses (20). RXRs play a vital role in the nuclear injected into the appropriate mice once in proximity to the receptor superfamily, forming heterodimers with many other tumor after injection of 5 days. Group one, the control group, family members; as a result, RXRs are implicated in the control was injected with 100 mL acetone. Group two and three were of various physiologic processes (21). RXRA is a member of the experimental groups and were injected with 300 mmol/L/kg RXR family that can be activated by sterol (22). In humans, cholesterol in 100 mL acetone. All cells transplanted in mice in cholesterol homeostasis is maintained by the precise interactions group three were pretreated with 100 nmol/L HULC siRNA. between intestinal uptake, de novo synthesis, hepatic output, and Tumor growth was measured beginning 5 days after injection of fecal disposal (23), and excessive or deficient cholesterol can hepatoma cells. Tumor volume (V) was monitored by measur- result in pathophysiological sequelae (24). Secreted apoA-I binding the length (L) and width (W) of the tumors with calipers ing protein (AIBP) positively regulates cholesterol efflux from and was calculated using the formula (L W2) 0.5. After endothelial cells, and effective cholesterol efflux is critical for 25 days, tumor-bearing mice and controls were sacrificed, and proper angiogenesis (25). In addition, the primary metabolite of the tumors were excised and measured. cholesterol, 27-hydroxycholesterol (27HC), contributes to estro- gen receptor–dependent growth and liver X receptor–dependent Statistical analysis metastasis in mouse models of breast cancer (26), and cholesteryl Each experiment was repeated at least three times. Statisti- ester accumulation induced by PTEN loss and PI3K/AKT activa- cal significance was assessed by comparing mean values tion underlies human prostate cancer aggressiveness (27). How- (6 SD) using the Student t test for independent groups as ever, the general mechanism responsible for aberrant lipid metab- follow: , P < 0.05; , P < 0.01; , P < 0.001 and not significant olism during the development of HCC is not well understood. (NS). The Pearson correlation coefficient was used to determine In the present study, we investigated the role of lncRNAs in the the correlations among gene expression in tumorous tissues. abnormal lipid metabolism of HCC. Intriguingly, our data dem- ACSL1 expression in tumor tissues and matched adjacent non- onstrate that the lncRNA HULC facilitates the deregulation of tumor tissues was compared using the Wilcoxon signed-rank lipid metabolism through miR-9/PPARA/ACSL1/cholesterol/ test. RXRA/HULC signaling. Thus, our finding provides new insights into the mechanism of aberrant lipid metabolism in HCC. Results Materials and Methods HULC is positively correlated with ACSL1 in clinical HCC tissues and upregulates ACSL1 in hepatoma cells Patient samples To demonstrate the role of HULC in the deregulation of Sixty HCC tissue samples and their corresponding adjacent lipid metabolism in HCC, we examined the effects of HULC nontumorous liver tissues were obtained from Tianjin First Center on several lipid metabolic enzymes in hepatoma cells. ACSL1 Hospital and Tianjin Tumor Hospital (Tianjin, China) after stood out as being noticeably upregulated by HULC (Supple- surgical resection. Fifty-five of 60 patients had a history of hep- mentary Fig. S1A). Thus, we evaluated the expression of ACSL1 atitis B virus infection. Written consent approving the use of tissue by immunohistochemical (IHC) staining in clinical HCC tissues samples for research purposes was obtained from patients. The using tissue microarrays and found that 77.3% (180/233) of information of patients with HCC is presented in Supplementary HCC tissues were positive for ACSL1 compared with 12.5% Table S1. The study protocol was approved by the Institute (2/16) of peritumoral liver tissues, in which the expression of Research Ethics Committee at Nankai University. ACSL1 was stronger in HCC tissues than that in their peritumoral liver tissues (Fig. 1A). Moreover, quantitative real-time Cell lines and cell culture PCR (qRT-PCR) revealed that the mRNA levels of ACSL1 were The human hepatoma H7402 cell line, human immortalized higher in HCC tissues compared with their adjacent nontumor- liver L-O2 cell line, and Chang liver cell line were cultured in ous liver tissues in 60 paired clinical HCC samples (Fig. 1B). RPMI-1640 medium (Gibco). The human hepatoma cell lines, ACSL1 has been reported to participate in the formation of Huh7 (obtained from Shanghai Institutes for Biological Sciences), triglycerides and cholesterol in the liver (29). Our data dem- HepG2, and HepG2.2.15 (a hepatoma HepG2 cell line with onstrated that the increased triglycerides/cholesterol was accu- integrated full-length hepatitis B virus DNA), and human kidney mulated in HCC tissues relative to their corresponding peritu- epithelial (HEK) 293T cells were maintained in DMEM (Gibco). moral tissues (Supplementary Fig. S1B). Furthermore, we All cell lines were supplemented with heat-inactivated 10% FBS observed that the levels of HULC were positively associated (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin and with those of ACSL1, triglycerides, and cholesterol in the afore- grown at 5% CO2 and 37 C. mentioned clinical samples (Fig. 1C). Moreover, we found that

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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 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 nontumorous tissues (P < 0.01; Wilcoxon 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 correlation coefficient, r ¼ 0.7444, 0.7099, 0.6501). D and 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 t test. overexpression of HULC upregulated ACSL1 in HepG2 and PCR or RT-PCR analysis (Supplementary Fig. S1C–S1E and Fig. Huh7 (or L-O2) cells at the mRNA and protein levels in a 1D–F). Strikingly, HULC was capable of enriching triglycerides dose-dependent manner (Supplementary Fig. S1C; Fig. 1D and and cholesterol in HepG2 cells in a dose-dependent manner 1E; and Supplementary Fig. S1D). Similarly, depletion of HULC (Supplementary Fig. S1F), but failed to increase their levels in led to a decrease in ACSL1 levels in HepG2.2.15 cells expressing the conditioned medium (Supplementary Fig. S1G). Taken high levels of endogenous HULC (Supplementary Fig. S1E together, these results show that HULC is positively associated and Fig. 1F). Meanwhile, the transfection (or interference) with ACSL1 in clinical HCC tissues and upregulates ACSL1 in efficiency of HULC (or HULC siRNA) was validated by qRT- hepatoma cells.

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HULC upregulates the transcriptional factor PPARA to activate information; Supplementary Fig. S2B and S2C), whereas HULC ACSL1 siRNA reversed these effects in HepG2.2.15 cells (Fig. 2F and G) . Given that ACSL1 is activated by the transcriptional factor The transfection (or interference) efficiency of HULC (or HULC PPARA in the liver (17), we speculated that HULC might siRNA) was confirmed in these cells (Fig. 2C–GandSupple- modulate ACSL1 through PPARA. Interestingly, qRT-PCR ana- mentary Fig. S2B and S2C). However, luciferase reporter gene lysis demonstrated that the expression levels of HULC were assays showed that HULC failed to activate the promoter of positively associated with those of PPARA in the 60 clinical PPARA in HepG2 cells (Supplementary Fig. S2D and S2E), HCC samples (Fig. 2A). However, inhibition of PPARA expres- implying that HULC might upregulate PPARA at the posttran- sion abrogated the HULC-induced upregulation of ACSL1 in scriptional step. Thus, we conclude that HULC activates ACSL1 HepG2 cells (Fig. 2B), suggesting that PPARA is responsible by upregulating the transcription factor PPARA in hepatoma for the upregulation of ACSL1 mediated by HULC. The effi- cells. ciency of PPARA siRNA (or PPARA siRNA) was validated in these cells (Supplementary Fig. S2A). Moreover, the over- miR-9 inhibits the expression of PPARA by targeting the 30UTR expression of HULC was able to upregulate PPARA at mRNA of PPARA and protein levels in HepG2 and Huh7 cells (or L-O2 cells) Next, we identified several miRNAs that could potentially in a dose-dependent manner (Fig. 2C–EandSupplementary bind 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 examined by 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-enhanced ACSL1 was examined by 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 and 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 t test.

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Figure 3. MiR-9 inhibits the expression of PPARA by targeting the 30UTR of PPARA. A, a model demonstrating the predicted conserved miR-9 binding site at nucleotides 7624-7631 of the PPARA 30UTR. The generated mutant sites at the PPARA 30UTR seed region are indicated. The wild-type PPARA 30UTR (or mutant) was inserted into the downstream of luciferase reporter gene in the pGL3-control vector. B and 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 and 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, nonsignificant; Student t test.

TargetScan and microrna.org (http://www.targetscan.org/, HULC downregulates miR-9 through inducing methylation of http://www. microrna.org/microrna/home.do). Because miR-9 CpG islands in its promoter has been reported to be downregulated in cancer (30–32), we Next, we validated the anticorrelation between HULC and focused our investigation on this miRNA. Three miR-9 binding miR-9 in the 60 clinical HCC samples (Fig. 4A). Then, our data sites in the 30UTR of PPARA mRNA were constructed (Fig. 3A showed that the overexpression of HULC downregulated miR-9 in and Supplementary Fig. S3A and S3B), and luciferase reporter HepG2 cells in a dose-dependent manner (Fig. 4B). Interestingly, assays revealed that miR-9 could directly bind to the conserved we found that treatment with 5-Aza-20-deoxycytidine (Aza, a DNA seed region of the PPARA 30UTR (position 7624-7631, pGL3- methylation inhibitor) heightened the levels of miR-9 in HepG2 PPARA-7624; Fig. 3B), rather than the poorly conserved seed cells in a dose-dependent manner (Fig. 4C), suggesting that HULC regions (position 5684-5690, position 4375-4381; Supplemen- might influence the epigenetic regulation of the miR-9 promoter tary Fig. S3C and S3D). However, the PPARA 30UTR conserved (33, 34). Then, we examined the methylation status of miR-9 seed region mutant (position 7624-7631, pGL3-PPARA-mut) using both methylation-specific PCR (MSP) and bisulfite- failed to work in these cells (Fig. 3B). Conversely, anti–miR-9 sequencing analysis (BSP). As shown in Fig. 4D, miR-9 comprises increased the luciferase activities of pGL3-PPARA-7624 but three members, termed miR-9-1, miR-9-2 and miR-9-3. MSP failedtoinfluence the mutant (Fig. 3C), suggesting that miR- assays revealed that the CpG sites of miR-9-1 (or miR-9-2, 9isabletodirectlybindtothe30UTR of PPARA. These effects miR-9-3) were highly methylated following the overexpression were also observed in 293T cells (Supplementary Fig. S3C– of HULC in L-O2 [or Chang liver (Chang), HepG2, and H7402] S3F). Furthermore, the overexpression of miR-9 suppressed the cells. BSP assays further validated the above observations in L-O2 expression of PPARA in HepG2 cells in a dose-dependent (or HepG2) cells (Fig. 4E and Supplementary Fig. S4A). Moreover, manner (Fig. 3D), and the reverse outcome was obtained when we confirmed these data in two pairs of clinical samples (Fig. 4F the cells were treated with anti–miR-9 (Fig. 3E). Together, our and Supplementary Fig. S4B). It has also been reported that DNA data indicate that miR-9 suppresses the expression of PPARA (cytosine-5-)-methyltransferase 1 (DNMT1), a methylase, is capa- by targeting its 30UTR. ble of regulating the expression of miRNAs through inducing

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Figure 4. HULC downregulates miR-9 through inducing methylation of CpG islands in its promoter. A, the correlation between HULC mRNA levels and miR-9 levels was examined by qRT- PCR in 60 cases of clinical HCC tissues (P < 0.01; Pearson correlation coefficient, r ¼ –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 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, adjacent peritumoral tissues; T, HCC tissues. For the bisulfite-sequencing analysis, 20 CpG sites were analyzed. Three clones were sequenced for each sample. Statistically significant differences are indicated: , P < 0.01; , P < 0.001; Student t test.

methylation of their CpG islands (35). Interestingly, we observed cells after treatment with Triacsin C (an inhibitor of ACSL1) or that HULC was able to upregulate DNMT1 in HepG2 cells ACSL1 siRNA, although this was not observed in L-O2 cells (Supplementary Fig. S4C), hinting that HULC might induce (Fig. 5A and Supplementary Fig. S5A). Meanwhile, we observed methylation of CpG islands in the miR-9 promoter through that the expression levels of HULC were also downregulated in upregulation of DNMT1. Therefore, we conclude that HULC HepG2 cells (Fig. 5B and Supplementary Fig. S5B), suggesting that inhibits the expression of miR-9 through eliciting methylation a positive feedback loop involving HULC/miR-9/PPARA/ACSL1/ of CpG islands in the miR-9 promoter. HULC is established in hepatoma cells but not in normal liver cells. Next, we utilized acetone as the solvent to deliver triglyce- The product cholesterol of ACSL1 is able to upregulate HULC by ride (or cholesterol) to the cells. Although some triglyceride (or activating RXRA in hepatoma cells cholesterol) dissolved out, the cells responded to the stimulation. Given that the positive feedback loops in signaling pathways Strikingly, we found that cholesterol was able to stimulate the are involved in the progression of cancer, we evaluated whether activity of the HULC promoter in HepG2 (or Huh7) cells in a HULC facilitates aberrant lipid metabolism in liver cancer via a dose-dependent manner, although triglyceride failed to have an feedback mechanism. Surprisingly, we found that the promoter effect in these cells (Fig. 5C and D, Supplementary information, activities of HULC were dose-dependently decreased in HepG2 S5C and S5D). According to the report (22), we cloned the HULC

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Figure 5. The product cholesterol of ACSL1 is able to upregulate 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 in HepG2 cells. The expression levels of ACSL1 were detected by Western blotting in the cells pretreated with Triacsin C. C and 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, nonsigniﬁcant; Student t test.

promoter including the mutant in the RXRA-binding site (Fig. 5E). the product cholesterol of ACSL1 upregulates HULC by activating Intriguingly, the treatment with cholesterol failed to activate the RXRA in hepatoma cells. above mutant in HepG2 cells (Fig. 5E), suggesting that RXRA may be implicated in the regulation of HULC mediated by cholesterol. HULC disrupts the lipid metabolism of hepatoma cells through Moreover, we verified that RXRA siRNA could abolish the cho- miR-9/PPARA/ACSL1 signaling in vitro lesterol-increased HULC promoter activity (Fig. 5F). However, the Next, we investigated the effect of HULC on lipogenesis in treatment with cholesterol failed to influence the expression of hepatoma cells using Oil Red O staining. These results showed RXRA (Supplementary Fig. S5E), suggesting that cholesterol, as a that the overexpression of HULC was able to accelerate lipogen- type of sterol, might be able to activate the HULC promoter by esis in HepG2 and Huh7 cells, whereas ACSL1 siRNA (or miR-9, stimulating RXRA, rather than upregulating RXRA. Meanwhile, PPARA siRNA, Triacsin C) could block this event. Inversely, the efficiencies of ACSL1 siRNA (or ACSL1 siRNA ) and RXRA anti–miR-9 was capable of enhancing lipogenesis in the cells siRNA were validated by Western blot analysis in these cells (Fig. 6A). In addition, we validated that the treatment with HULC (Supplementary Fig. S5F and S5G). As a result, we conclude that siRNA (or ACSL1 siRNA, miR-9, PPARA siRNA, and Triacsin C)

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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 and 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 the tissue triglyceride assay kit (or tissue total cholesterol kit). E and 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, nonsigniﬁcant; Student t test.

could attenuate the lipogenesis in HepG2.2.15 cells. However, sought to evaluate whether the product cholesterol of ACSL1 may anti–miR-9 was able to rescue the HULC siRNA-repressed lipo- be responsible for the promotion of cell proliferation. As genesis (Fig. 6B), and we obtained a similar effect of HULC on expected, MTT assays further corroborated that the treatment with intracellular triglyceride and cholesterol in this system (Fig. 6C cholesterol was able to promote the proliferation of hepatoma and D; Supplementary Fig. S6A and S6B). The role of ACSL1 in the cells, which could be eliminated by HULC siRNA (Supplementary growth of hepatoma cells remains enigmatic. In this study, we Fig. S6C), suggesting that cholesterol might enhance the pro- showed that the overexpression of ACSL1 could facilitate the liferation of hepatoma cells by upregulating HULC. In addition, proliferation of HepG2 and Huh7 cells, as demonstrated by MTT we assessed whether HULC affected other signaling pathways assays and cloning formation assays (Fig. 6E and F). Thus, we and factors involved in lipid metabolism in hepatoma cells, such

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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 a 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 a 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 t test.

as the AKT/mTOR pathway, SREBP1, SREBP2, chREBP, HMGCR, ACSL1 siRNA abolished the HUCL-accelerated proliferation FASN, ACLY, SQS, and miR-122 (14, 36). However, we did not of HepG2 (or Huh7) cells in mice (Fig. 7A and B and Supple- observe alterations in these factors at the levels of mRNA and mentary Fig. S7B), further supporting the conclusion that ACSL1 protein in HepG2 and Huh7 cells (Supplementary Fig. S6D– is responsible for the promotion of tumor growth mediated S6G). Thus, we conclude that HULC contributes to aberrant lipid by HULC. IHC staining further conﬁrmed that the expression of metabolism in hepatoma cells through miR-9/PPARA/ACSL1 Ki-67, a marker of proliferation, as well as BrdUrd incorpora- signaling. tion in the tumor tissues was consistent with tumor growth among the different groups (Supplementary Fig. S7C). Interest- HULC-modulated abnormal lipid metabolism facilitates ingly, oil red O staining revealed that lipid droplets were increased tumor growth in vivo in the tumor tissues overexpressing HULC, whereas silencing To better understand the role of HULC in abnormal lipid of ACSL1 reversed this effect (Fig. 7C and Supplementary metabolism, we subcutaneously injected pretreated cells into Fig. S7C). The levels of triglycerides and cholesterol were consis- 4-week-old BALB/c athymic nude mice. We conﬁrmed that the tent with the Oil Red O staining in the tumor tissues (Fig. 7D levels of HULC and ACSL1 were preserved in the tumor tissues and E), suggesting that abnormal lipid metabolism contributes (Supplementary Fig. S7A). We observed that treatment with to the growth of hepatoma cells. To better evaluate the effect of

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cholesterol on the expression of HULC in hepatoma cells in vivo, the methylation of CpG islands in the miR-9 promoter, we we injected supraphysiological cholesterol in proximity to the assessed the influence of HULC on methylase levels in hepa- tumor tissues. As expected, the injection significantly increased toma cells. Strikingly, our observations indicated that HULC the levels of tissue HULC (Supplementary Fig. S7D). Notably, was able to upregulate the expression of DNMT1 in hepatoma we observed that the pretreatment with HULC siRNA remark- cells, suggesting that HULC induces the methylation of miR-9 ably abolished the cholesterol-increased growth of hepatoma promoter CpG islands possibly by upregulating DNMT1. The cells (Fig. 7F and G and Supplementary Fig. S7E), suggesting that role of ACSL1 in the growth of hepatoma cells has not been cholesterol is able to upregulate HULC in hepatoma cells. Togeth- reported. Therefore, we examined the effect of ACSL1 over- er, we conclude that HULC-modulated abnormal lipid metabo- expression on the proliferation of HepG2 and Huh7 cells by lism contributes to tumor growth, and this process requires the MTT assays and cloning formation assays. Notably, we observ- metabolic enzyme ACSL1 and its product cholesterol. ed 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 Discussion of lipid metabolism mediated by ACSL1 in hepatoma cells. In LncRNAs play crucial roles in cancer (37), and we previously addition, it has been reported that the AKT/mTOR cascade reported that HBx-enhanced HULC is able to promote the growth influences the growth, survival, metabolism, and migration of of hepatoma cells (9). Metabolism deregulation, exacerbated liver cancer cells, and the extent of aberrant lipogenesis is lipid biosynthesis, and accumulation in the development of correlated with the activation of the AKT/mTOR signaling cancer accelerate cell growth and transformation (38). In this pathway (14, 44). This relationship suggests that the AKT/ study, we assessed whether HULC participates in abnormal lipid mTOR pathway plays a pivotal role in the deregulation of lipid metabolism in HCC. metabolism in HCC. Therefore, we wondered whether the To better understand the roles of HULC in modulating lipid AKT/mTOR pathway and other signaling pathway members metabolism, we first measured the effect of HULC on lipid implicated in lipid metabolism, such as SREBP1/2, chREBP, metabolic enzymes in hepatoma cells. Interestingly, ACSL1 HMGCK, MVK, FASN, ACLY, SQS, and miR-122 (14, 36), were drew our attention because ACSL1 and its intracellular products, involved in this event. However, we failed to demonstrate that such as triglycerides and cholesterol, were remarkably upregu- HULC disturbed lipid metabolism in hepatoma cells through lated and increased by HULC expression. However, the levels of these signaling pathways. Therefore, we conclude that HULC triglycerides and cholesterol in the conditioned medium were is able to induce aberrant lipid metabolism through ASCL1/ unaltered in HULC-treated cells relative to controls, which may miR-9/ PPARA/ASCL1 signaling in hepatoma cells. be the result of the ability of ACSL1 to inhibit cholesterol efflux Given that cancer disrupts cellular homeostasis and creates (39). Moreover, we validated that the expression levels of HULC many new methods of regulation, such as positive feedback loops were positively correlated with those of ACSL1 and its products (45), we are interested in whether the action of HULC is involved in clinical HCC samples (Fig. 1 and Supplementary Fig. S1). in a feedback loop as well. Interestingly, we observed that ACSL1 Next, we explored the mechanism by which HULC activates could influence the expression of HULC in hepatoma cells in a ACSL1 in hepatoma cells. ACSL1 has been reported to be a positive feedback fashion. Thus, we hypothesized that the pro- classic target gene of PPARA in the liver (16). Accordingly, we ducts of ACSL1 might be responsible for the activation of HULC. observed that HULC modulated ACSL1 by upregulating PPARA Indeed, luciferase reporter assays revealed that cholesterol was (Fig. 2 and Supplementary Fig. S2). Furthermore, we determin- able to activate the promoter of HULC, whereas triglycerides were ed that HULC could increase PPARA expression by downregu- not. Furthermore, we identified a binding site for RXRA in the lating the ability of miR-9 to target the PPARA 30UTR at the promoter of HULC using bioinformatics analysis. RXRA is an posttranscriptional level, rather than by activating the transcrip- orphan receptor that can be activated by sterol (22). Thus, we tion of PPARA directly (Fig. 3 and Supplementary Fig. S3). speculated that RXRA might participate in cholesterol-activated Numerous studies have noted that the expression of miR-9 is HULC in hepatoma cells. As expected, we found that RXRA was regulated by CpG island methylation, and lncRNAs are able to responsible for the activation of HULC mediated by cholesterol in modulate the expression of genes through epigenetic regulation hepatoma cells. It has been reported that PPARA, liver X receptor (40, 41). Hence, we validated that HULC was capable of (LXRA), and bile acid receptor (FXR) can form heterodimers with inducing the methylation of CpG islands in the promoter of RXRA (46–48). Here, we treated cells with siRNA targeting PPARA miR-9 (Fig. 4 and Supplementary Fig. S41). This finding sug- (or LXRA and FXR) mRNA to assess the effect of PPARA (or LXRA gests that HULC governs the expression of PPARA through and FXR) on HULC. However, our data demonstrated that all epigenetic regulation. The methylation of miR-9-3 is signifi- treatments failed to influence the cholesterol-enhanced luciferase cantly associated with an increased risk of recurrence, and high reporter activity of the HULC promoter (data not shown), imply- methylation levels of either miR-9-1 or miR-9-3 result in a ing that PPARA, LXRA, and FXR are not implicated in cholesterol- significant decrease in recurrence-free survival times in clear induced RXRA activation. Strikingly, we observed that cholesterol cell renal cell carcinoma (30). Moreover, the inhibition of miR- failed to upregulate HULC in normal liver cells (Fig. 5 and 9–mediated suppression of SOX2 is involved in chemoresis- Supplementary Fig. S5). The reason may be related to RXRA not tance and cancer stemness in glioma cells (42), and miR-9 is being constitutively phosphorylated, thus it fails to escape from able to target MTHFD2 to inhibit the proliferation of breast Ub/proteasome-mediated degradation in liver cells (49). Of cancer cells (43). Together with these findings, our data imply particular note, this finding provides new insights into the mech- that the methylation and inhibition of miR-9 might hijack other anism by which HULC is highly expressed in hepatoma cells. signaling pathways to facilitate hepatocarcinogenesis. To better Recent reports pinpoint that the growth-promoting effects of understand the underlying mechanism by which HULC elicits elevated levels of insulin, glucose, or triglycerides are involved in

OF10 Cancer Res; 75(5) March 1, 2015 Cancer Research

LncRNA HULC Facilitates Abnormal Lipid Metabolism in HCC

insulin resistance–promoted colorectal cancer (50). Heightened of abnormal lipid metabolism mediated by HULC in the intracellular levels of triglyceride and their metabolites, such as development of HCC. diacylglycerol, may activate the protein kinase-C and MAPK pathways with potentially mitogenic and carcinogenic effects (51). Disclosure of Potential Conflicts of Interest Consistent with our study, these findings suggest that HULC- No potential conflicts of interest were disclosed. enhanced accumulation of triglycerides in hepatoma cells might emerge as a risk factor for hepatocarcinogenesis. In this article, we Authors' Contributions found that cholesterol was involved in the promotion of hepa- Conception and design: M. Cui, L. Ye, X. Zhang toma cell growth mediated by HULC, which is consistent with Development of methodology: M. Cui, X. Zhang other reports (25–27). Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Cui, Z. Xiao, Y. Wang, M. Zheng, T. Song, X. Cai, In aggregate, our work demonstrates that HULC plays pivotal B. Sun, L. Ye, X. Zhang roles in aberrant lipid metabolism in HCC through miR-9/ Analysis and interpretation of data (e.g., statistical analysis, biostatistics, PPARA/ACSL1/cholesterol/RXRA/HULC signaling, a model for computational analysis): M. Cui, Z. Xiao, Y. Wang, L. Ye, X. Zhang this role of HULC is represented in Supplementary Fig. S7F. Writing, review, and/or revision of the manuscript: M. Cui, L. Ye, X. Zhang In particular, our data show that HULC elicits the methylation Administrative, technical, or material support (i.e., reporting or organizing of CpG islands in the miR-9 promoter, resulting in the sup- data, constructing databases): M. Cui, L. Ye, X. Zhang Study supervision: L. Ye, X. Zhang pression of miR-9 expression. MiR-9 is able to target the 30UTR of transcription factor PPARA, and the decrease in miR-9 leads Grant Support to upregulation of PPARA and the subsequent transactivation This work was supported by grants from National Natural Science of ACSL1, which enhances lipogenesis and enriches intracellu- Foundation of China (nos. 31470756, 81071624, and 81272218) and lar triglycerides and cholesterol in hepatoma cells. Thus, HULC- National Basic Research Program of China (973 Program, nos. enhanced abnormal lipid metabolism accelerates the growth 2015CB553703, 2015CB55905, and 2011CB933100). The costs of publication of this article were defrayed in part by the payment of liver cancer. Furthermore, the cholesterol product of ACSL1 of page charges. This article must therefore be hereby marked advertisement in upregulates HULC by activating the transcription factor accordance with 18 U.S.C. Section 1734 solely to indicate this fact. RXRA, forming a positive feedback loop with HULC/miR-9/ PPARA/ACSL1/cholesterol/RXRA/HULC in hepatoma cells. Received April 21, 2014; revised November 15, 2014; accepted November 22, Thus, our findings provide new insights into the mechanism 2014; published OnlineFirst January 15, 2015.

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OF12 Cancer Res; 75(5) March 1, 2015 Cancer Research

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

Ming Cui, Zelin Xiao, Yue Wang, et al.

Cancer Res Published OnlineFirst January 15, 2015.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-14-1192

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