Molecular Mechanism Of Liver X Receptor-mediated Downregulation Of FOXM1Expression And The Significance Of The Nuclear Receptor In The Growth Suppression Of Hepatocellular Carcinoma Cells

Background: Forkhead Box M1(FOXM1) is a member of the family of evolutionaryconserved transcriptional regulators, which possess a DNA binding-domain called theForkhead domain. The Forkhead domain, also known as winged helix DNA binding domain,contains110amio acids. FOXM1plays an important role in the proliferation and invasion ofvarious cancer cells. It has been reported that FOXM1is a key cell cycle regulator, whichupregulates the transcription of Cyclin B1and Cyclin D1, resulting in the enhancement of thecell cycle progression and cell proliferation. However, the regulatory mechanism of FOXM1expression is less understood. Liver X receptor (LXR) is a typical nuclear receptor. As amultiple functional transcriptional factor, it is closely involved in the metabolic regulation ofglucose, cholesterol and lipid. Moreover, it can play anti-inflammatory function. Recently, theanti-tumor function of LXR attracted much attention, but the relevant mechanism still need tobe clarified. Both LXR and FOXM1are highly expressed in liver cells, and both of them areassociated with tumors. However, it is unclear whether LXR could regulate FOXM1expression.Objective: To investigate the effects of LXR on FOXM1expression and the relevantmechanism, and on the cell proliferation and cell cycle of hepatocellular carcinoma (HCC)cells.Methods:.(1) Two human HCC cell lines HepG2and Hep3B were separately treatedwith different concentration of LXR ligand TO901317. Then the expression of FOXM1andits target genes Cyclin B1and Cyclin D1were examined by RT-PCR, Real-time PCR andWestern blot.(2) The potential LXR binding sites in FOXM1gene promoter region waspredicted by bioinformatics in website http://www.nubiscan.unibas.ch/. Subsequently,the influence of LXR on the transcriptional activity of FOXM1gene promoter was evaluated by luciferase reporter assay in the presence or absence of LXR ligand TO901317.(3) It wasanalyzed whether the nuclear proteins of HepG2cells could bind to the specific probesequence derived from5′regulatory region of FOXM1gene by electrophoresis mobilityshift assay(EMSA). Moreover, it was examined whether LXR could bind to LXR responseelements in FOXM1gene promoter region by chromatin immunoprecipitation (ChIP).(4) Theeffect of LXR ligand TO901317on HCC cells proliferation was assayed by CCK-8and3H-TdR incorporation tests, and the influence of the ligand on the cell cycle was analyzed byflow cytometry.Results:(1)LXR ligand dose-dependently downregulated the expression of FOXM1andits target genes Cyclin B1and Cyclin D1in HepG2and Hep3B cells at both mRNA andprotein levels;(2) LXR reduced the transcriptional activity of FOXM1gene promoter inHepG2cells;(3)LXR could bind to LXR response element IR2(CCGTCAcgTGACCT,-52～-38) in FOXM1gene promoter region in HepG2cells;(4)Activation of LXR in HepG2cellsinhibited proliferation and induced the cell cycle arrest at G1phase.Conclusion:(1)FOXM1was a novel target gene of LXR.(2)Activation of LXR in HCCcells suppressed FXOM1expression and cell proliferation, and induced the cell cycle arrest atG1phase, which suggest that downregulation of FXOM1may be a novel mechanism ofLXR-mediated anti-HCC effects, and LXR-FXOM1pathway may be a novel target for thetreatment of HCC.