The Role And Mechanism Of Long Non-coding RNA Ftx In The Aerobic Glycolysis And Progression In Hepatocellular Carcinoma

Background and AimsHepatocellular carcinoma(HCC)is the principal type of primary hepatic carcinoma and one of the most popular malignancies around the world.Though there are various treatment strategies,HCC is still the second leading cause of cancer-associated mortality worldwide;the morbidity and mortality rates of HCC are particularly high in China.The lack of reliable biomarkers for tumorigenesis and the unclear clarification of the heterogeneous genetic and epigenetic alterations contribute to the poor progno sis of HCC.Aerobic glycolysis,also termed the Warburg effect,is a phenomenon by which highly proliferative malignant cells preferentially utilize glycolysis rather than oxidative phosphorylation,even in the presence of sufficient oxygen,to satisfy their high nutrient requirements.This effect is characterized by the consumption of glucose at a higher rate and the production of more lactate compared with normal differentiated cells.Accumulating evidence suggests that metabolic alteration,which is one of the most consistent hallmarks of cancer,exerts critical effects on tumor progression,and the aberrant expression of glycolysis-associated molecules contributes to tumorigenesis.Therefore,aerobic glycolysis is pivotal to producing energy in cancer cells,indicating that the molecules involved may be potential biomarkers and therapeutic targets for HCC.Long non-coding RNAs(lncRNAs),which do not have protein-coding ability,are a class of functional RNAs of>200 nucleotides(nt)in length and are involved in various biological processes.LncRNAs modulate target gene expression at the epigenetic,transcriptional and posttranscriptional levels.Recently,lncRNAs have emerged as important regulators of HCC apoptosis,invasion,metastasis,immune escape and development,as well as carbohydrate metabolism and lipid metabolism.Ftx is a well-conserved noncoding gene encoded within the X-inactivation center on the X chromosome.Except for the two clusters of microRNAs(miRs),Ftx also encodes a highly conserved transcript of 2,356 nt that is termed IncRNA Ftx.It has been demonstrated that lncRNA Ftx could inhibit HCC proliferation and metastasis by binding MCM2 and miR-374a.However,another research showed that lncRNA Ftx/miR-545 could contribute significantly to the tumorigenesis of HCC through activation of PI3K/AKT by targeting RIG-1.Thus,the specific effect of lncRNA Ftx in the progression of HCC,the association between lncRNA Ftx and aerobic glycolysis,and the underlying mechanism,remain unclear.Therefore,further investigations are needed to analyze the specific effect of lncRNA Ftx in the HCC progression and aerobic glycolysis and its intrinsic mechanism.Once activated by ligands,peroxisome proliferator-activated receptor-γ(PPARy)heterodimerizes with the retinoid X receptor and combines with PPAR response elements to regulate the transcription of target genes.It has been demonstrated that PPAR y serves a vital role in steatosis-associated hepatic tumorigenesis,in addition to increasing cell sensitivity to insulin and reversing insulin resistance.PPARy activation is additionally involved in the regulation of a number of crucial enzymes in carbohydrate metabolism;for example,PPARy activation promotes insulin-responsive glucose transporter 4(GLUT4)expression and inhibits pyruvate dehydrogenase kinase 1(PDK1)expression.Furthermore,PPARy activation may reduce tumor necrosis factor(TNF)a and leptin production,thus facilitating glucose utilization and improving insulin sensitivity in liver cells.However,the role of IncRNA Ftx in PPARy-mediated tumor metabolism remains poorly understood.The present study might provide a novel insight into therapeutic interventions for HCC.The present study investigated the aberrant status of lncRNA Ftx and its potential target gene PPARy to examine the possible signaling pathway that regulates aerobic glycolysis,and to identify a novel therapeutic target for HCC treatment.It was identified that lncRNA Ftx was upregulated in human HCC tissues and cell lines and,notably,was associated with aggressive clinicopathological features.In addition,the upregulation of IncRNA Ftx was an independent risk factor of poor prognosis.LncRNA Ftx knockdown inhibited the proliferation,invasion and migration of HCC cells,whereas IncRNA Ftx overexpression resulted in the opposite effects.Furthermore,IncRNA Ftx affected the glucose consumption and lactate production,suggesting that IncRNA Ftx might be involved in aerobic glycolysis in HCC.The measurement of glucose transporter expression,the activity and expression of key enzymes in carbohydrate metabolism further supported this assumption.Mechanistically,IncRNA Ftx expression in human HCC tissues was positively correlated with PPARγ,and promoted the expression of PPARγ and regulated its pathway effectors in HCC cells.PPARy activation in Bel-7402 cells partially rescued the alterations in glucose uptake,lactate production and relative glycolytic enzyme expression mediated by lncRNA Ftx knockdown;similarly,inhibiting PPARy in Huh7 cells partially abrogated the IncRNA Ftx-induced alterations.In conclusio n,lncRNA Ftx is a promoter of the Warburg effect and tumor progression,partly via the PPARy pathway,and may serve as a promising therapeutic target for HCC treatment.Part 1 The expression and clinical significance of lncRNA Ftx in hepatocellular carcinoma tissuesAimTo examine the expression status of IncRNA Ftx in HCC tissues and adj acent tissues,analyze its correlations with several progression or prognosis-associated clinicopathological features,and analyze its effect on the prognosis in HCC patients.Methods1.A total of 73 patients with HCC were recruited between February 2012 and January 2013 at Shandong Provincial Hospital Affiliated to Shandong University.For each patient,paired HCC tissues and adjacent non-tumor tissues(as a control)were fresh-frozen in liquid nitrogen immediately following surgical resection and stored at-80℃.2.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was utilized to examine the expression level of IncRNA Ftx in HCC tissues and adjacent non-tumor tissues.3.The clinicopathological characteristics,including tumor size,histological grade,metastasis and tumor capsule,were collected.The patients were followed-up by telephone for overall survival and disease-free survival.4.The associations between the expression level of IncRNA Ftx and clinicopathological characteristics,such as metastasis and histological grade were evaluated by x2 test.Kaplan-Meier survival analyses were utilized to evaluate the prognostic value of IncRNA Ftx in HCC.Multivariate Cox analyses were utilized to evaluate the prognostic value of IncRNA Ftx and other factors.Results1.Compared with the 73 non-tumorous control tissues,lncRNA Ftx expression was markedly higher in the 73 HCC tissues.2.Although there was no significant association between IncRNA Ftx expression and age,sex or tumor size,high lncRNA Ftx expression levels were positively associated with histological grade,tumor capsule and metastasis.3.For patients with poor histological grade,metastasis or incomplete capsule,the expression level of IncRNA Ftx was relatively high.4.Kaplan-Meier survival analyses suggested that IncRNA Ftx was associated with poor prognosis.5.Multivariate analyses showed that the high expression level of IncRNA Ftx was an independent risk factor for the prognosis of HCC.ConclusionLncRNA Ftx is upregulated in human HCC tissues and significantly associated with histological grade,metastasis and tumor capsule.The high expression level of IncRNAFtx is an independent risk factor for the poor prognosis of HCC,indicating that lncRNA Ftx might be involved in HCC tumorigenesis and thus resulting in the poor prognosis.Part 2 The effect of lncRNA Ftx on the biological behaviors and aerobic glycolysis in hepatocellular carcinoma cellsAimTo investigate the effect and regulation of lncRNA Ftx on the biological behaviors including proliferation,clone formation,migration and invasion,and aerobic glycolysis in HCC cell lines.Methods1.To choose suitable cell lines for functional research,lncRNA Ftx expression was screened in a panel of HCC cell lines(Huh7,SMMC-7721 and Bel-7402)and a non-neoplastic hepatic cell line(LO2).2.Lentivirus(LV)design and construction:Construct short hairpin RNA(ShRNA)lentivirus mediated LV-Ftx-RNAi and its negative control,and overexpressing lentivirus mediated LV-Ftx and its negative control.3.Lentiviral transfections:According to multiplicity of infection(MOI)value,the LV-Ftx-RNAi and its negative control,polybrene and enhanced infection solution were transfected into the Bel-7402 cells,while the LV-Ftx and its negative control,polybrene and enhanced infection solution were transfected into the Huh7 cells.4.Construction of stable cell lines:To produce cell lines with stably interfered expression of lncRNA Ftx or stably expressing IncRNA Ftx,HCC cells were selected with puromycin after transfection.5.Verify efficiency of lentivirus transfection:In order to make sure the ratio of green fluorescent cells and fluorescence intensity,the cells were observed by fluorescence microscope after transfection.The efficiency of knockdown and overexpression of IncRNA Ftx mediated by lentivirus was verified through RT-qPCR on mRNA level.6.Cell functional experiments:CCK-8 assays were performed to determine whether lncRNA Ftx has an impact on tumor cell proliferation.Clone formation assays were performed to evaluate the effect of IncRNA Ftx on the ability to form colonies.In addition,to assess the effect of IncRNA Ftx on HCC cell invasion and migration ability,cell invasion and migration assays(Transwell assays with or without Matrigel,respectively)were performed using stably transfected cells.7.The measurement of the Warburg effect comprises three parts:The analysis of glucose consumption(glucose consumption,GLUT1 and GLUT4 expression),the measurement of the glycolysis(lactate production and lactate dehydrogenase(LDH)activity,phosphofructokinase(PFKL)activity and expression),and the detection of the Krebs cycle(citrate synthase(CS),isocitrate dehydrogenase(IDH1)and a-ketoglutarate dehydrogenase(OGDH)activities and expressions).8.Statistical analysis.Statistical analyses were performed using SPSS 22.0 and GraphPad Prism 6.The significance of differences was evaluated by Student’s t-tests(two-tailed)for two-group comparisons,and the one-way analysis of variance and the Bonferroni post hoc test for multiple comparisons.P<0.05 was considered to indicate a statistically significant difference.Results1.It was identified that the expression levels of IncRNA Ftx were significantly increased in the HCC cell lines compared with the L02 cell line.Additionally,the expression level of lncRNA Ftx was the highest in Bel-7402 cells and the lowest in Huh7 cells.Thus,lncRNA Ftx was overexpressed in Huh7 cells as the base level of lncRNA Ftx was low,and it was knocked down in Bel-7402 cells as the base level was high.2.After the selection of puromycin,the efficiency of transfection was as high as 90%and cells grown well under the green fluorescence microscope.3.RT-qPCR was used to confirm the transfection efficiency.Huh7 clones with～12 times increased lncRNA Ftx expression levels compared with the normal control were chosen,and Bel-7402 clones with-74.68%decreased IncRNA Ftx expression levels compared with the normal control were selected.4.Biological behaviors:Knockdown of IncRNA Ftx could inhibit the proliferation,clone formation,migration and invasion in Bel-7402 cells,whereas overexpression of IncRNA Ftx could promote the proliferation,clone formation,migration and invasion in Huh7 cells.5.The analysis of glucose consumption:Knockdown of lncRNA Ftx decrease the glucose consumption through GLUTs in Bel-7402 cells,whereas overexpression of IncRNA Ftx could facilitate the glucose consumption through GLUTs in Huh7 cells.6.The measurement of the glycolysis:Knockdown of IncRNA Ftx could reduce the lactate production by regulating glycolytic enzyme including LDH and PFKL in Bel-7402 cells,whereas overexpression of lncRNA Ftx could favor the lactate production byregulating glycolytic enzymes including LDH and PFKL in Huh7 cells.7.The detection of the Krebs cycle:Knockdown of lncRNA Ftx could facilitate the Krebs-cycle-associated molecules including CS,IDH1 and OGDH in Bel-7402 cells,whereas overexpression of IncRNA Ftx could weaken the Krebs-cycle-associated molecules including CS,IDH1 and OGDH in Huh7 cells.ConclusionLncRNA could facilitate the glucose consumption through GLUTs,favor the lactate production by regulating glycolytic enzymes,and weaken oxidative phosphorylation by regulating the Krebs-cycle-associated molecules,thus promoting the proliferation,migration and invasion in HCC cells.Part 3 The molecular mechanism of IncRNA Ftx on the aerobic glycolysis and progression in vitroAimTo investigate the underlying mechanism of lncRNA Ftx on the aerobic glycolysis and progression in HCC.Methods1.LncRNA target prediction:Bioinformatics analysis of the predicted IncRNA targets was performed using the nucleotide BLASTn program.2.The mRNA expression levels of PPARy were compared in 73 HCC tissue samples using RT-qPCR.Pearson’s correlation was utilized to measure its correlation with IncRNA Ftx expression.3.The mRNA and protein expression levels of PPARy and PPARy pathway effectors(TNFa,leptin and PDK1)were assessed by RT-qPCR and western blot upon knocking down and overexpressing IncRNA Ftx in Bel-7402 cells and Huh7 cells,respectively.4.To confirm whether the promotion of aerobic glycolysis by IncRNA Ftx is mediated through the PPARy pathway,PPARγ was suppressed with its antagonist GW9662 in IncRNA Ftx-overexpressing Huh7 cells,and activated with its agonist pioglitazone in IncRNA Ftx-knockdown Bel-7402 cells.Subsequently,the glucose consumption,lactate production,LDH activity,mRNA expression levels of relative enzymes and molecules,and PPARy pathway effectors were analyzed.Results1.According to the nucleotide BLASTn program,the 940-1058 nt region of Ftx(a length of 118 nt)was highly homologous with PPARy.The present results suggested that lncRNA Ftx may directly target the PPARy gene and regulate the transcriptional and post-transcriptional expression of PPARy.2.A positive correlation between the expression levels of IncRNA Ftx and PPARy in HCC tissues was observed.3.It was observed that IncRNA Ftx overexpression could promote PPARy mRNA and protein expression levels,and decrease the mRNA expression levels of PPARy pathway effectors(TNFa,leptin and PDK1).4.PPARy activation in Bel-7402 cells partially rescued the IncRNA Ftx knockdown-mediated alterations in aerobic glycolysis and PPARy pathway effectors;similarly,inhibiting PPARγ in Huh7 cells partially abrogated the alterations in aerobic glycolysis and PPARy pathway effectors induced by IncRNA Ftx overexpression.ConclusionLncRNA Ftx acts as a novel promoter of HCC progression and glycolysis by partially targeting the PPARγ pathway.Targeting this aberrantly activated pathway may provide a novel approach for HCC therapy and merits further investigation.