ZHX2 Inhibits SREBP1c-mediated De Novo Lipogenesis In Hepatocellular Carcinoma Via MiR-24-3p

Hepatocellular carcinoma(HCC)is a primary liver tumor and one of the most common malignant tumors worldwide.Its global morbidity and mortality are ranked sixth and fourth in cancer,respectively.More and more clinical and epidemiology research demonstrate metabolic associated fatty liver disease(MAFLD)as an independent risk factor for HCC.Due to its rapidly growing incidence,MAFLD is becoming a major cause of HCC.Related studies have found that increased liver de novo lipogenesis(DNL)is a key contributing factor in the development of MAFLD and many cancers,including HCC.These findings indicate that altered lipid metabolism play critical roles in HCC.However,the molecular mechanism has not yet been fully elucidated.Key enzymes responsible for de novo lipogenesis,such as fatty acid synthase(FASN),Acetyl-CoA carboxylase 1(ACC1),ATP citrate lyase(ACL)and stearoyl-Coenzyme A desaturase 1(SCD1),are highly expressed in HCC.Moreover,SREBPs(sterol regulatory element-binding proteins),the master regulators of de novo lipogenesis,regulate a variety of key DNL enzymes.SREBP1c is mainly expressed in the liver and regulates hepatic lipid metabolism.Recent studies have found that high expression of SREBP1c promoted the development of HCC.Therefore,identifying new regulators of SREBP1c will provide potential targets for HCC therapy.ZHX2(Zinc fingers and homobox 2),a transcriptional factor and a member of the ZHX family,exerts anti-cancer effect by transcriptionally inhibiting cell cycle genes,multidrug resistance gene and HBx in HCC.Genetic analysis of familial hyperlipidemia identified Zhx2 as a hyperlipidemia susceptibility gene.Consistently,ZHX2 reduces liver lipid levels and protects mice from high-fat diet-induced liver injury,which strongly suggests that ZHX2 is involved in lipids metabolic regulation.Recently we reported the role of ZHX2 in hepatic lipid hemostasis.ZHX2 inhibits LPL(Lipoprotein lipase)-mediated exogenous lipid uptake in hepatocytes leading to retardation in MAFLD and consequent HCC.However,whether ZHX2 is involved in de novo lipogenesis remains largely unknownIn this study,we investigated whether ZHX2 inhibit the development of HCC by regulating de novo lipogenesis.Our data may provide new potential strategies for HCC therapy.1.ZHX2 inhibits de novo lipogenesis in HCC cellsTo evaluate whether ZHX2 regulates de novo lipogenesis in HCC cells,content of neutral lipid was measured in HepG2 and Huh7 cells cultured in medium,with charcoal stripped fetal bovine serum(FBS).BODIPY staining showed that ZHX2 overexpression significantly decreased the intensity of neutral lipid staining,whereas knockdown of ZHX2 increased lipid level.Consistently,ZHX2 inhibited level of intracellular free fatty acids(FFAs)and triglyceride(TG)in HCC cells.D-Glucose-13C6 tracer technology is the most direct method to study de novo lipogenesis.To further clarify the role of ZHX2 in de novo lipogenesis,D-Glucose-13C6 was used to trace the newly synthesized lipids in HepG2 cells with inducible ZHX2 overexpression.Since saturated fatty acids and monounsaturated fatty acids are mainly derived from de novo lipogenesis,13C-labeled saturated FFAs and monounsaturated FFAs were analyzed by UHPLC-QTOF-MS.Compared to that of control cells,HepG2 cells with ZHX2 overexpression showed significantly reduced 13C-labeled newly synthetic FFAs.In addition,RT-qPCR demonstrated that ZHX2 inhibited expression of lipogenesis key enzyme genes,such as ACL,ACC1,FASN and SCD1 in HepG2 and Huh7 cells.Collectively,the above data suggest that ZHX2 inhibits de novo lipogenesis in HCC cells2.ZHX2 suppresses HCC cell proliferation depends on de novo lipogenesisMounting evidences have demonstrated de novo lipogenesis as a hallmark of cancers which is essential for cell proliferation.ZHX2,a novel HCC suppressor,inhibits cell proliferation and colony formation in HCC cell lines.To test whether ZHX2 exerts its tumor suppressor function via regulating de novo lipogenesis,colony formation and cell proliferation assays were performed in HepG2 and Huh7 cells at low fatty FBS condition.Ectopic expression of ZHX2 significantly inhibited proliferation of HCC cells,displaying as reduced colony formation ability and lowered growth rate More importantly,this inhibition was almost completely abolished after supplement with oleic acid(OA),a monounsaturated fatty acid that is one of the major products of de novo lipogenesis.Reciprocally,silencing of ZHX2 promoted HCC cells proliferation and colony formation,and this enhancement was eliminated by inhibitors of ACL(BMS-303141)and SCD1(A939572).Above results suggested that ZHX2 decreases proliferation of HCC cells by inhibiting de novo lipogenesis3.ZHX2 represses expression of SREBPlc to inhibit de novo lipogenesisSREBP1 is a key regulator of DNL and transcriptionally regulates the expression of key enzymes of DNL.We then elucidated whether ZHX2 regulates SREBP1c,the hepatic isoform of SREBP1.To address that,SREBP1c levels were examined in HepG2 and Huh7 cells with overexpression or knockdown of ZHX2.Ectopic expression of ZHX2 dramatically decreased SREBP1c at both mRNA and protein levels in HepG2 and Huh7 cells,while silencing of ZHX2 clearly increased SREBP1c expression.In addition,results of RT-qPCR showed that ZHX2 mRNA levels were negatively correlated with SREBP1c mRNA levels in HCC specimensIn order to estimate the role of SREBP1c in ZHX2-mediated inhibition of de novo lipogenesis,the content of neutral lipid,FFAs and TG were assessed in HepG2 and Huh7 cells.As expected,ectopic expression of ZHX2 reduced levels of intracellular lipid together with cellular FFAs and TG in Huh7 and HepG2 cells,while overexpression of SREBP1c clearly abolished ZHX2-mediated repression of natural lipids.Reciprocally,knockdown of ZHX2 in HepG2 and Huh7 cells led to significantly increased level of cellular lipid and TG/FFAs which was clearly reversed by treatment of Fatostatin,the inhibitor of SREBP1c or siRNA of SREBP1c.Taken together,all these data supported the hypothesis that ZHX2 negatively regulates lipogenesis by repressing SREBPlc expression.4.ZHX2 inhibits HCC cell proliferation and tumor formation by down-regulating SREBPlcTo verify whether the inhibition of ZHX2 on cell proliferation was achieved by repressing SREBP1c,in vitro cell growth and in vivo spontaneous tumor formation assays were performed.ZHX2 overexpression suppressed colony formation and proliferation in HCC cells.More importantly,ectopic expression of SREBPlc not only increased colony formation and proliferation of HCC cells but also completely rescued ZHX2-mediated inhibition on HCC cell growth.Reciprocally,Fatostatin orsiSREBP1c inhibited HCC cell growth and abolished ZHX2 silencing-mediated enhancement in colony formation and cell proliferation of HepG2 and Huh7 cells.In order to validate the role of ZHX2-SREBP1c axis in liver cancer in vivo.6-8 weeks old male hepatic-specific Zhx2 knockout mice(Zhx2-KOhep)and control littermates(WT)were used to induce spontaneous liver cancer via hydrodynamic injection of SB(Sleeping Beauty)transposition system and AKT/Myc plasmids.Fatostatin was i.p.injected to retard lipogenesis.All the mice were sacrificed 8 weeks after hydrodynamic injection.As expected,the number of externally invisible tumors(>1 mm)in Zhx2-KOhep mice was more than WT mice.Consistently,liver weight,liver/body weight,FFAs and TG of liver homogenate in Zhx2-KOhep mice were significantly increased compared to WT mice.More importantly,fatostatin treatment significantly reduced the numbers of liver tumor nodules,levels of FFAs and TG,and abolished the difference of tumor nodules and levels of FFAs and TG in Zhx2-KOhep and WT miceAll the data suggest that ZHX2 inhibits de novo lipogenesis by down-regulating SREBP1c,thereby inhibits HCC cell growth and tumor progression.5.ZHX2 represses SREBPlc expression via miR-24-3pAs a previously reported transcriptional factor,ZHX2 might repress SREBP1c at transcriptional level.To test this hypothesis,SREBP1c promoter region was cloned to luciferase reporter vector for dual luciferase assays.Result showed that ZHX2 had no effect on the promoter activity of SREBP1c.In addition,ChIP and published ChIP-seq data show that ZHX does not bind to the SREBP1c promoterIncreasing data demonstrate the important role of miRNAs-mediated posttranscriptional gene silencing in regulation of lipid metabolism,we therefore evaluated whether ZHX2 represses SREBPlc through miRN As.To address that,miRNAs microarray analysis was performed in ZHX2 overexpressing HepG2 and control cells.Totally,61 miRNAs were up-regulated(>4 fold,ZHX2 vs control),and were predicated to target on SREBP1c(www.mircode.org and www.targetscan.org).We then verified the expression of these 6 miRNAs in HepG2 and Huh7 cells with ZHX2 overexpression.Results of RT-qPCR showed that miR-24-3p,but not other miRNAs,was significantly upregulated by ZHX2.Indeed,ectopic expression of ZHX2 significantly increased miR-24-3p,while knockdown of ZHX2 dramatically reduced miR-24-3p in HCC cells.Dual luciferase report assay showed that ZHX2 obviously enhanced miR-24-3p promoter activity,indicating that ZHX2 promoted expression of miR-24-3p by enhancing its promoter activityIn order to confirm SREBPlc as the target of miR-24-3p,miR-24-3p inhibitor,mimic or overexpression plasmid was transfected into HepG2 cells,respectively.miR-24-3p inhibitor enhanced while miR-24-3p mimic or overexpression plasmid inhibited expression of SREBP1c at both mRNA and protein level.The SREBP1c reporter plasmid containing the miR-24-3 binding site was constructed and dual luciferase assays results showed that inhibition of miR-24-3p increased luciferase levels,and overexpression of miR-24-3p decreased luciferase levels.Indicating that miR-24-3p can directly bind to SREBP1.To further validate that ZHX2 represses SREBPlc through upregulating miR-24-3p,a series of co-transfections were performed in HepG2 cells.As expected,SREBP1c mRNA and protein level were decreased in ZHX2 overexpressing HepG2 cells However,this inhibition was reversed when miR-24-3p inhibitor were co-transfected with ZHX2.Reciprocally,silencing of ZHX2 increased SREBP1c expression,while miR-24-3p mimic or overexpression plasmid co-transfected with ZHX2 siRNA reversed the increasing of SREBP1c at mRNA and protein level.All the data suggest that ZHX2 enhances the miR-24-3p promoter activity,leading to increased expression of miR-24-3p,which further inhibits the expression of SREBP1c.6.ZHX2 inhibits de novo lipogenesis and cell proliferation in HCC cells via miR-24-3pTo test whether ZHX2 inhibits de novo lipogenesis and cell proliferation via miR-24-3p,level of FFAs,colony formation and cell proliferation were assessed in HepG2 cells with co-transfection.ZHX2 overexpressed or knockdown HepG2 cells were transfected with miR-24-3p inhibitor,mimic or overexpression plasmid,respectively Consistent with previous data,overexpression of ZHX2 decreased FFAs level and inhibited cell growth and colony formation while knockdown of ZHX2 increased FFAs level and promoted cell proliferation in HepG2.And,these alterations were almost completely destroyed by miR-24-3p inhibitor,mimic or overexpression plasmid.Above all,ZHX2 promotes expression of miR-24-3p at transcriptional level,which leads to suppression of lipogenesis,and further suppresses HCC cell proliferationConclusion and significance:In summary,we identified,for the first time,that HCC suppressor ZHX2 is an important inhibitor of DNL.ZHX2 significantly inhibits SREBP1c,the key regulator of DNL,thereby inhibiting DNL in hepatocarcinoma cells,thereby slowing down the progression of HCC.Mechanically,ZHX2 transcriptionally increased expression of miR-24-3p,which targets on SREBPlc and leads to its decreased expression.Our data revealed a novel mechanism of HCC lipid metabolism reprogramming,and found a new target and new pathway for the HCC suppressor gene ZHX2,which may provide a new strategy for the treatment of HCC.