LXRa-mediated downregulation of FOXM1 suppresses the proliferation of hepatocellular carcinoma cells

C Hu, D Liu1,2,3, Y Zhang1,3, G Lou1, G Huang1, B Chen1, X Shen1, M Gao1, W Gong1, P Zhou1, S Dai1, Y Zeng1 and F He1

Abstract

Liver X receptors (LXRs), including LXRa and LXRb isoforms, have important roles in the metabolic regulation of glucose, cholesterol and lipid. Moreover, activation of LXRs also represses the expression of cyclin D1 and cyclin B1, and thus suppresses the proliferation of multiple cancer cells, but the relevant mechanism is not well known. Forkhead box M1 (FOXM1) is a proliferationspecific member of forkhead box family, which is highly expressed in proliferating normal cells and numerous cancer cells. FOXM1 directly activates transcription of cyclin D1 and cyclin B1, resulting in the enhancement of cell cycle progression and cell proliferation. However, it is unclear whether LXRs are involved in the regulation of FOXM1. In this study, we demonstrated that specific LXRs agonists downregulated expression of FOXM1, cyclin D1 and cyclin B1 in hepatocellular carcinoma (HCC) cells, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly alleviated LXRs activation-mediated cell cycle arrest and cell growth suppression. Reporter assays showed that the activation of LXRs significantly reduced the transcriptional activity of FOXM1 promoter. Electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that LXRa but not LXRb could bind to an inverted repeat IR2 (-52CCGTCAcgTGACCT-39) in the promoter region of FOXM1 gene. Moreover, the xenograft tumor growth and the corresponding FOXM1 expression in nude mice were dramatically repressed by LXRs agonists. Taken together, we conclude that LXRa but not LXRb functions as a transcriptional repressor for FOXM1 expression. The pathway ‘LXRa–FOXM1–cyclin D1/cyclin B1’ is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for HCC treatment.

Keywords: LXRs; FOXM1; cell proliferation; cell cycle; HCC

INTRODUCTION

Liver X receptors (LXRs) are important nuclear receptors, which have two isoforms, LXRa (NR1H3) and LXRb (NR1H2). LXRa is highly expressed in liver, intestine, lung, kidney, whereas LXRb is ubiquitously expressed.1,2 LXRs can be activated by natural ligands, such as 22(R)-hydroxycholesterol, 20(S)hydroxycholesterol, 24(S)-hydroxycholesterol, and synthetic ligands including GW3965 and TO901317.3 Ligand-activated LXRs bind to response elements of target genes as a LXR–RXR (retinoid X receptor) heterodimer.4 Many genes related with cholesterol efflux (ABCA1, ABCG1, apoE), conversion of cholesterol to bile acids (CYP7A1) and cholesterol secretion (ABCG5/G8) are directly regulated by LXRs.5–9 Therefore, LXRs are considered as physiological sensors of sterol.10
Interestingly, accumulating studies demonstrated that LXRs activation inhibits multiple human cancer cells proliferation by suppressing the expression of cell cycle-related proteins, such as cyclin D1 and cyclin B1.11–13 However, bioinformatics analysis revealed that there are no classical LXREs (LXRs response elements) in the promoter regions of these genes, suggesting that the inhibition of cyclin D1 and cyclin B1 expression by LXRs might be mediated by other molecules.
Forkhead box M1 (FOXM1), a transcriptional regulator, is one member of the family with an evolutionary conserved DNA-binding domain called the forkhead domain.14,15 It is highly expressed in proliferating normal cells and various cancer cells.16 Overexpression of FOXM1 is associated with aggressive tumor feature and poor prognosis of hepatocellular carcinoma (HCC) and pancreatic ductal adenocarcinoma.17,18 Studies have demonstrated that FOXM1 was essential for development of HCC in mouse.19 FOXM1 upregulation was associated with the acquisition of a susceptible phenotype of HCC in rats, and also influenced human HCC development and prognosis.20 The level of FOXM1 protein can combine with the Milan criteria to predict the prognosis of HCC patients accepting orthotopic liver transplantation.21 Moreover, FOXM1 is also a key cell cycle regulator, which upregulates the transcription of cyclin D1 and cyclin B1 genes, resulting in the enhancement of cell cycle progression and proliferation.22 Knockdown of FOXM1 by small interfering RNA (siRNA) inhibits cell proliferation, induces cell cycle arrest and suppresses cell invasion in MHCC-97H cells in vitro.23 Therefore, FOXM1 is considered as a potential therapeutic target for the management of many tumors.24,25 Previous reports have showed that FOXM1 expression could be upregulated by several members of the nuclear receptor superfamily, including farnesoid X receptor and estrogen receptor a.26,27 However, it has never been reported whether LXRs could regulate the expression of FOXM1. In this study, we for the first time demonstrated via various means that LXRa, but not LXRb, is responsible for the downregulation of FOXM1 by directly binding to an inverted repeat IR2 in FOXM1 gene promoter region. LXRa-mediated suppression of FOXM1 repressed the expression of cyclin D1 and cyclin B1 in HCC cells, which led to cell cycle and cell proliferation arrest. The pathway ‘LXRa–FOXM1–cyclin D1/cyclin B1’ is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for the treatment of HCC.

RESULTS

LXRs agonists inhibit HCC cells growth and induce cell cycle arrested at G1 phase

ATP-binding cassette transporter (ABCA1) is one of the LXRs target genes that can be induced after LXRs activation. As shown in Supplementary Figure 1, treatment of HCC cell lines HepG2 and Hep3B with the specific LXRs agonist GW3965 upregulated the mRNA level of ABCA1 in a concentration-dependent manner, which demonstrated that LXRs in these cells are functional. Subsequently, the cell counting kit-8 (CCK-8) test (Figure 1a) and H-TdR incorporation assay (Figure 1b) showed that the LXRs agonists GW3965 and T0901317 inhibited the proliferation of both HepG2 and Hep3B cells in a dose-dependent manner compared with the corresponding controls. Meanwhile, the cell cycle of these treated HCC cells was arrested at G1 phase by the LXRs agonists (Figure 1c).

LXRa expression is inversely correlated with FOXM1 expression in hepatocytes

The protein levels of LXRa, LXRb and FOXM1 in seven human HCC cell lines and hepatic cell L02 were examined by western blot in triplicate (Figure 2a, marked as samples 1–3). Then the relative expression ratio of target protein/b-actin was calculated respectively (Figure 2b), and Pearson’s test was used to detect the correlationship between the mean value of the genes expression. As shown in Figure 2c, there was a significant inverse correlation between the expression of LXRa and FOXM1 (Po0.05). However, there was no correlation between the expression of LXRb and FOXM1 (P40.05). These results suggested that LXRa might be involved in the regulation of FOXM1.

Activation of LXRs downregulates the expression of FOXM1, cyclin D1 and cyclin B1 in HCC cells

To evaluate whether LXRs are involved in the regulation of FOXM1, the real-time reverse transcriptase–PCR (RT–PCR) and western blot assays were performed. As shown in Figure 3, the LXRs agonist GW3965 dose-dependently downregulated FOXM1 at both mRNA (Figures 3a and b) and protein levels (Figures 3c and d). Furthermore, the expression of cyclin D1 and cyclin B1, two target genes of FOXM1,22 were also downregulated at both transcriptional (Figures 3a and b) and translational levels (Figures 3c and d) with the treatment of GW3965, suggesting that the suppression of cyclin D1 and cyclin B1 might be mediated by LXRs-induced downregulation of FOXM1.

LXRa but not LXRb is responsible for the downregulation of FOXM1

Both LXRa and LXRb can be activated by LXRs agonists and there is no specific agonist for each one isoform. To distinguish which isoform is responsible for the downregulation of FOXM1, LXRa and LXRb were interfered separately using siRNAs. As shown in Figure 4a, both siRNAs for LXRa and LXRb were efficient. Moreover, knockdown of LXRa by siRNA decreased the GW3965mediated downregulation of FOXM1, cyclin D1 and cyclin B1 (Figure 4b), and thus partly relieved the GW3965-induced cell proliferation inhibition and cell cycle arrest (Figures 4c, d and e). However, knockdown of LXRb by siRNA did not affect the GW3965-mediated repression of FOXM1. Interestingly, the siRNA-mediated repression of LXRb also partly relieved the LXRs anti-proliferative effect (Supplementary Figure 2). These results showed that both LXRa and LXRb possess antitumor function, but only LXRa is responsible for the downregulation of FOXM1.

Knockdown of FOXM1 significantly attenuates GW3965-mediated antitumor effect

To investigate whether the antitumor effect of LXRs is truly mediated by FOXM1, three siRNAs against FOXM1 mRNA were synthesized, and the one (numbered as si565) with the best silence effect was selected to be used in the subsequent experiments (Figure 5a). Figure 5b showed that knockdown of FOXM1 by si565 dramatically attenuated the GW3965-mediated repression of cyclin D1 and cyclin B1 in HepG2 cells, and thus alleviated GW3965-induced cell growth inhibition and cell cycle arrest (Figures 5c, d and e). These results showed that the antitumor effect of LXRs is, at least partially, mediated by downregulation of FOXM1.

Activation of LXRs represses the transcriptional activity of FOXM1 gene promoter

The expression of FOXM1 mRNA was suppressed by GW3965 suggested that activation of LXRs may modulate FOXM1 expression at transcriptional level. Moreover, bioinformatics analysis predicted that there are several potential LXRs binding sites in 50 regulatory region of FOXM1 gene (Figure 6a). So we hypothesized that LXRs suppress FOXM1 via exerting their inhibitory activities on FOXM1 promoter. We constructed a series of luciferase reporter expression vectors (pGL3-FOXM1s, pFs) including pF1 ( 1975 to þ 21), pF2 ( 916 to þ 21), pF3 ( 673 to þ 21), pF4 ( 581 to þ 21), pF5 ( 442 to þ 21) and pF6 ( 250 to þ 21). These vectors were driven by different length of FOXM1 promoter regions. The transfection assays showed that GW3965 and TO901317 significantly inhibited FOXM1 promoter activity (Figure 6b). Moreover, the mutated pF3 without IR2 element (-52CCGTCAcgTGACCT-39) showed no response to LXRs agonists (Figure 6c), suggesting that the putative LXRE/IR2 ( 52 to 39) was required for the LXRa-mediated suppression of FOXM1 promoter activity.

LXRa but not LXRb binds to LXRE/IR2 in human FOXM1 promoter region

To determine whether LXRs could directly bind to the IR2 element in the promoter region of FOXM1, the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assays were performed. The sequences of the probes used in EMSA were summarized in Figure 7a. The EMSA showed a DNA/ protein band of expected mobility, which reflected the interaction between the probe containing IR2 ( 52 to 39) and the nuclear extract of HepG2 cells (Figures 7b and c). The interaction was increased after treatment with GW3965 (Figure 7b), but competitively inhibited by high concentration of cold (unlabeled) IR2 probe (not by cold mutated IR2 probe; Figures 7b and c). Once the labeled IR2 probe was mutated, the interaction was almost extinguished (Figure 7b). Moreover, the ChIP assays showed that LXRa, but not LXRb, directly bound to the FOXM1 promoter region ( 121 to þ 21) containing the LXRE/IR2 ( 52 to 39) in HepG2 cells, and this binding was enhanced by GW3965 and TO901317 in a dose-dependent manner (Figure 7d).

LXRs agonists repress HCC xenograft tumor growth and FOXM1 expression in vivo

The above studies clearly showed that the LXRs agonists repressed FOXM1 expression in vitro, we then investigated whether the agonists can modulate FOXM1 in vivo. First, we compared the sequences of LXRE/IR2 in FOXM1 promoter regions among several mammals including homo sapiens, mus musculus and rattus norvegicus by ClustalW serve (www.ch.mbnet.org/software/ ClustalW. html). The results showed that the sequences of LXRE/IR2 are highly conserved in these three mammals (Figure 8a), suggesting that they may have similar biological functions. Subsequently, we determined the effect of GW3965 and TO901317 at blocking tumor growth in vivo. Figure 8b showed that both GW3965 and TO901317 decreased the tumor size compared with the vehicle group. Remarkably, this was accompanied by the downregulation of FOXM1 mRNA (Figure 8c) and protein (Figure 8d) expression in the xenografts. These data demonstrate that LXRs agonists potently inhibit HCC growth and FOXM1 expression in vivo. However, whether the regulatory mechanism by which LXRs suppress FOXM1 expression in mice is similar as that in human needs more studies.

DISCUSSION

Activation of LXRs inhibit the proliferation of multiple human cancer cell lines, such as prostate, breast, colon, lung, bone, skin, cervical, liver, ovarian and leukemia cancer cells.11,12,28–31 Moreover, LXRs activation downregulate cyclin D1 expression in vascular smooth muscle cells,32 breast and colon cancer cells,30,31 as well as cyclin B1 expression in macrophage.33 However, the mechanism by which LXRs regulates cyclin D1 and cyclin B1 remains not well known. Uno et al.31 have demonstrated that LXRs could suppress cyclin D1 via inhibiting b-catenin. In this study, we found that the LXRa-mediated downregulation of FOXM1 is a novel mechanism by which LXRs suppress cyclin D1 and cyclin B1 in HCC cells.
FOXM1 regulates transcription of cell cycle genes essential for G1-S and G2-M progression, including Cdc25A, Cdc25B, cyclin B1, cyclin D1, p21 and p27.22,34,35 Therefore, it is possible to inhibit tumorigenesis, proliferation and metastasis by inhibiting FOXM1.36 So far, it has been reported that both chemical drugs and peptides could be used to antagonize the role of FOXM1 for the treatment of cancers. The chemical drugs include docetaxel,37 siomycin A,37 gefitinib,38 as well as the proteasome inhibitors such as MG115, MG132 and bortezomib.39 Moreover, a cell-penetrating alternative reading frame (ARF26–44) peptide was reported as an inhibitor of FOXM1 in the inhibition of proliferations of human HCC and mice HCC cells.40 In this study, we confirmed that LXRa directly inhibited FOXM1 expression in HCC cells, and thus suppressed the expression of cyclin D1 and cyclin B1, which led to cell cycle and cell proliferation arrest. Knockdown of FOXM1 significantly attenuated the antitumor effect of GW3965, suggesting that the antitumor effect of LXRs is, at least partially, mediated by LXRa-induced repression of FOXM1. As for the reason why the siRNA-mediated knockdown of FOXM1 did not completely abolish the antitumor affect of LXRs, it may be because that the silence efficiency of siRNA for FOXM1 is not 100%. In addition, there may exist other mechanism(s) by which LXRs suppress the proliferation of HCC cells. For example, previous study has demonstrated that both LXRa and LXRb could interact with b-catenin protein, and both of them bound to the promoter region of b-catenin to inhibit colon cancer cells and mouse embryo fibroblasts proliferation.31 Another group reported that activated LXRb repressed lymphocyte proliferation via abcg1-dependent alteration of cholesterol homeostasis.41 These results indicate that the antitumor mechanisms of LXRs might be diverse in different cells, so more studies are warranted.
The most common LXRs response element is DR4 (direct repeat separated by 4 bp).42 Besides, ER7 (everted repeat separated by 7 bp) and IR1 (inverted repeat separated by 1 bp) have been reported to be capable of conferring responsiveness on the LXR target gene.43,44 In this study, the potential LXREs (including two IR2 elements) in human FOXM1 promoter region were predicted (Figure 6a), but only one IR2 element ( 52 to 39) was identified to interact with LXRa, which mediated the downregulation of FOXM1. Furthermore, studies have revealed that mouse hepatocytes with conditionally deleted FOXM1 failed to proliferate and were highly resistant to develop HCC in response to a diethylnitrosamine/phenobarbital liver tumor induction protocol.19 Our study showed that the activation of LXRs inhibited FOXM1 expression in xenograft tumors in nude mice, suggesting that the LXRs agonists-mediated downregulation of FOXM1 might also repress the tumorigenesis in vivo, which needs further studies.
LXRs agonists received accumulating attention for their benefits in correcting metabolic disorders characterized by cholesterol accumulation.10 However, they also increased hepatic lipogenesis via upregulation of fatty acid synthase,45 sterol regulatory element-binding protein 1c and carbohydrate response element-binding protein.46–48 These side-effects dampened the enthusiasm in developing LXRs ligands for the treatment of sterol disorders, so the development of selective LXRs modulators targeting certain genes for anticancer therapy with minimal sideeffects is quite necessary. Interestingly, knockdown of fatty acid synthase or sterol regulatory element-binding protein 1 reduced lipogenesis in RWPE1 cells, which partly relieved activated LXRinduced cell growth arrest. So Kim et al.49 concluded that the increased lipogenic activity would be one of the key mechanisms to mediate the anti-proliferative effect by LXRs activation. As for the detailed molecular mechanism by which LXR-mediated lipogenesis suppresses cell proliferation, it remains to be elucidated.
In this study, we observed that the knockdown of LXRb could also decrease GW3965-mediated anti-proliferative effect, although it did not affect the expression of FOXM1 (Supplementary Figure 2), suggesting that both LXRa and LXRb possess antitumor function, but only LXRa is responsible for the downregulation of FOXM1. As for the mechanism(s) by which LXRb represses proliferation of HCC cells, it needs more studies.
Taken together, we have clearly demonstrated for the first time in this study that LXRa (but not LXRb) repressed FOXM1 expression in HCC cells via directly binding to the LXRE/IR2 ( 52 to 39) in human FOXM1 gene promoter region. The pathway ‘LXRa–FOXM1–cyclin D1/cyclin B1’ is a novel mechanism by which LXRs suppress the proliferation of HCC cells, suggesting that the pathway may be a novel target for the treatment of HCC.

MATERIALS AND METHODS

Materials

GW3965 and TO901317 were purchased from Sigma (St Louis, MO, USA). TRIzol reagent, Lipofectamin2000, Moloney Murine Leukemia Virus reverse transcriptase and cell culture medium (Dulbecco’s modified Eagle’s medium, DMEM) were bought from Invitrogen (Carlsbad, CA, USA). SiRNA pools for LXRa (sc-38828), LXRb (sc-45316) and control siRNA (sc-37007) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). SiRNAs for FOXM1 were designed and synthesized by Genephama (Shanghai, China). Dual luciferase assay systems were from Promega (Madison, WI, USA). LightShift chemiluminescent EMSA kit was from Pierce (Rockford, IL, USA). ChIP kit was from Upstate (Lake Placid, NY, USA).

Cell culture

All the cell lines were from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM supplemented with 10% fetal bovine serum, streptomycin (100 mg/ml) and penicillin (100 U/ml). Cells were serum deprived for 6 h before treatment with GW3965 or TO901317 and then cultured in DMEM containing 0.5% fetal bovine serum (conditioned medium).

H-TdR incorporation assay and CCK-8 test

HepG2 and Hep3B cells (2 105/ml, 100 ml per well) were seeded in triplicate in 96-well flat-bottom plates. After 12 h, the cultures were grown for 48 h in the presence of dimethylsulphoxide, GW3965 or TO901317. Then the cells were pulsed in the last 10 h with 1 mCi of 3H-TdR before collection and assessed for radioactivity using a scintillation counter TriCarb 2100 TR (Perkin Elmer Life Science, Boston, MA, USA). For the CCK-8 (Dojindo, Kumamoto, Japan) test, the cells were treated with dimethylsulphoxide or LXRs agonists for 48 h, and subsequently mixed with 10 ml of CCK-8 solutions per well and incubated for further 1 h at 37 1C. The amount of formazan dye generated by cellular dehydrogenase activity was measured for absorbance at 450 nm by a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The optical density values of each well represented the survival/proliferation of HCC cells.

Flow cytometry assay

After treatment with dimethylsulphoxide or LXRs agonists for 48 h, HepG2 and Hep3B cells were trypsinized and fixed in 70% ethanol at 4 1C for 24 h, then the cells were incubated with propidium iodide (40 mg/ml) and RNase A (100 mg/ml) (Sigma, St Louis, MO, USA) in phosphate-buffered saline at 37 1C for 1 h. Subsequently, the cells were re-suspended in phosphatebuffered saline for further analysis. Data were acquired using a Beckman Coulter EPICS Elite ESP apparatus (Hialeah, FL, USA) and then analyzed by Multicycle AV Phoenix Flow Systems (San Diego, CA, USA).

Real-time RT–PCR

The expression of mRNA for human ABCA1 (a target gene of LXRs), FOXM1, cyclin D1, cyclin B1 and mouse FOXM1 were examined by real-time RT–PCR using the SYBR Green Mix (Bio-Rad, Hercules, CA, USA) on iQ5 system (Bio-Rad). Each reaction was performed in triplicate. Results were normalized to human or mouse b-actin mRNA. The primer sequences were listed in Supplementary Table S1.

Western blot

After HCC, cells or mice xenograft tumors were lysed, the protein concentrations were measured by Bradford protein assay kit (Biyuntian, Beijing, China), and then western blot analysis was performed as described.50 Primary antibodies anti-LXRa (ab41902), anti-LXRb (ab56237), anti-FOXM1 (ab83097), anti-cyclin D1 (16663) were from Abcam (Cambridge, MA, USA), anti-cyclin B1 (BS1392) were from Bioworld Technology (Minneapolis, MN, USA) and anti-b-actin antibody (sc-47778) was from Santa Cruz Biotechnology. Horseradish peroxidaselabeled goat anti-rabbit or anti-mouse immunoglobulin G and the ECL chemiluminescence kit were from Pierce.

Knockdown of the target genes by siRNAs

After grown to 80–90% confluence, the HepG2 cells were transiently transfected using Lipofectamine2000 (Invitrogen) with siRNAs or siRNA pools. Six hours later, the cells were cultured in DMEM containing 0.5% fetal bovine serum in the presence or absence of LXRs agonists for 48 h. Then the cells were harvested and the assays were performed including real-time RT–PCR, western blot, 3H-TdR incorporation assay, CCK-8 test and flow cytometry. The sequences of siRNAs for FOXM1 were listed in Supplementary Table S2.

Construction of luciferase reporter expression vectors

Various length of human FOXM1 promoter regions containing different fragments were amplified by PCR using HepG2 cells genomic DNA as template, then the fragments were separately cloned into pGL3-Basic vector after digestion with KpnI and NheI, and the resulting plasmids were named respectively as pF1 ( 1975 to þ 21), pF2 ( 916 to þ 21), pF3 ( 673 to þ 21), pF4 ( 581 to þ 21), pF5 ( 442 to þ 21) and pF6 ( 250 to þ 21). The mutated pF3 without IR2 element ( 52 to 39) was generated by one-step opposite direction PCR with the Mutant Best Kit (Takara, Dalian, China). The primers for plasmid construction were listed in Supplementary Table S3.

Luciferase assay

HepG2 cells were grown to 80–90% confluences in 96-well plates. Then the cells were transiently transfected using Lipofectamine2000 (Invitrogen) with the above luciferase reporter expression vectors. Six hours later, the cells were cultured in DMEM containing 0.5% fetal bovine serum in the presence or absence of LXRs agonists for 18 h. Subsequently, the cell extracts were prepared and the luciferase assays were performed using the dual luciferase assay systems according to the manufacturer’s instruction (Promega). The transfection experiments were performed three times in triplicate, and the luciferase activity was normalized to pRL-TK activity.

Electrophoretic mobility shift assay

The biotin-labeled probes used are: IR2: ACGTTCCGTCAcgTGACCTTAACG or mutated IR2: ACGTTCCGCAGcgCTGCCTTAACG, where underlined nucleotides represent response element half-sites and bases in bold are mutated. The EMSA was performed using the LightShift chemiluminescent EMSA kit (Pierce) according to the manufacturer’s instructions. Briefly, the labeled probes were incubated with nuclear extracts from HepG2 cells for 10 min at 25 1C, and then the reaction samples were electrophoresed on a 6% non-denaturing acrylamide gel in a 0.5 Tris-borate-EDTA buffer. After transferring to the nylon membrane, the biotin-labeled probes were detected. For competition assays, the cold IR2 or cold mutated IR2 probes were included in the binding reaction at the 5 or 30 excess concentrations over the biotin-labeled probe before the addition of labeled oligonucleotides.

ChIP assays

The ChIP assays were performed using a ChIP assay kit (Upstate) according to the manufacturer’s instructions. Briefly, after treatment with different concentrations of GW3965 or TO901317 for 48 h, HepG2 cells were fixed for 10 min with 1% formaldehyde, and then disrupted in SDS lysis buffer. The chromatin was sonicated to shear DNA to an average length between 200 and 1000 bp as verified by agarose gel. Subsequently, the chromatin was immunoprecipitated with antibodies (2 mg) directed against LXRa or LXRb, taking equal amount of immunoglobulin G as negative control. Final DNA extractions were PCR amplified using primer pairs that cover the LXRE/IR2 sequence in the FOXM1 promoter region ( 121 to þ 21). For additional control of nonspecific binding, another PCR amplification reaction of the precipitated chromatin DNA was also performed to detect a 130-bp fragment located at 1348 to 1218 (1.1-kb upstream from the LXRE/IR2). Sequences of the primers for PCR were listed in the Supplementary Table S4.

Animal experiments

Eight-week-old male nude mice were maintained according to the local conventions for the care and use of laboratory animals (Chongqing, China). In all, 5 106 HepG2 cells in 150 ml phosphate-buffered saline were subcutaneously injected into the right axillae of the nude mice. When palpable tumors were formed, mice were injected intraperitoneally once a day with either vehicle alone (soyabean oil) or GW3965 (30 mg/kg) or TO901317 (30 mg/kg) for 2 weeks. Four weeks after the administration of the LXRs agonists, tumors were harvested and tumor volume was estimated using the following formula: volume ¼ width2 length 1/2, then real-time RT–PCR and western blot analyses were also performed to detect FOXM1 expression level.

Statistical analysis

All data are expressed as means±s.d. unless otherwise stated. Comparisons between two groups were determined using two-sided Student’s t-test, and comparisons between three or more groups were made with ANOVA followed by Tukey–Kramer post hoc analysis. The correlation between expression of LXRs and FOXM1 was carried out by Pearson’s test. In all cases, a difference was considered statistically significant if the P-value was o0.05 (‘*’ represents Po0.05, ‘#’ represents Po0.01).

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