The Mechanism Of PPFIA4 Promotes Colon Cancer Cell Proliferation And Migration By Enhancing Tumor Glycolysis

BACKGROUND:Colon cancer（CC）is one of the most common malignant diseases and is associated with an enormous and increasing public concern in the world.Thus,novel biomarkers and target treatment is urgently required to improve efficacy and efficiency of diagnosis and treatment for CC.For decades,increasing evidence confirmed glycolysis is the crucial mechanism that tumor growth and metastases,and plays a pivotal role in treatment resistance.To identify biomarkers for CC diagnosis and treatment,we performed a systematic bioinformatics analysis,and we found that the protein tyrosine phosphatase receptor type F polypeptide interacting protein a-4（PPFIA4）gene demonstrated significant differential expression in CC and correlated with prognosis of CC patients closely.We also confirmed the overexpression of PPFIA4 in CC tissues by molecular biology experiments.Though being rarely studied,PPFIA4 has been reported in several studies to be potentially associated with aberrant metabolic processes,making it a gene of interest in cancer glycolysis.This study intends to take CC as the research background,combining with bioinformatics and molecular biology experiments to explore the biological function of PPFIA4 in CC,and study the molecular mechanism of CC cell-mediated glycolysis affecting tumor progression,to provide experimental basis and scientific experimental basis for the development of novel CC biomarkers and effective targets.PART Ⅰ:Identification of the Glycolysis-related Gene Signature for Predicting Colon Cancer SurvivalOBJECTIVE:To establish a glycolysis-related gene risk score model and nomogram which may be a robust prognostic indicator for clinical use in CC.METHODS:1)The expressed gene RNA-sequencing dataset and corresponding TCGA clinical information of CC were extracted1.The glycolysis-related genes were identified by gene set enrichment analysis（GSEA）of glycolysis-related Pathways;2)Considering the association between glycosis-related gene expression level by univariate Cox regression analysis the least absolute shrinkage and selection operator（LASSO）logistic regression.And the glycolysis-related risk model was finally established by a multivariate Cox regression analysis;3)CC patients were divided into highand low-risk groups through the median score as a cutoff.The Kaplan-Meier method was applied to evaluate the significant difference of overall survival using“survival”R package between high-and low-risk groups.The receiver operating characteristic（ROC）analysis was applied to estimate the sensitivity and specificity of the Prediction model;4)Based on the glycosis-related gene and the results of TCGA clinical data analysis,a nomogram for predicting CC patients’survival were constructed,and then A calibration plot for predicting the accuracy of the nomogram.RESULTS:1)The primary screening and analysis of TCGA samples by GSEA identified 226 glycolysis-related genes in COAD samples compared with normal tissues.Of these,138 were overexpressed（FDR<0.01,logFC<0）and 56 were under-expressed（FDR<0.01,logFC>0）;2)A univariate Cox regression and LASSO regression were used to explore the interaction of the glycolysis-related genes with the overall survival of CC patients and determined 7 survival-related genes in CC patients when the P<0.01;3)Three genes（PPFIA4,PPP2CB and PMM2）comprised the risk score model,which was an independent prognostic indicator in CC patients determined by multivariate Cox regression analyses（P<0.05）;5)According to the specific gene signature,patients were categorized into high-and low-risk subgroups.The AUC of ROC analysis for the risk score was 0.675（1-year）,0.700（2-year）and 0.694（3-year）,implying excellent performance for survival prediction;5)Based on risk score,glycolysis-related genes（PPFIA4,PPP2CB and PMM2）,render,age and TNM stage,we construct a nomogram model and calibration plots indicated that the nomogram versus an ideal model showed high consistency.CONCLUSIONS:We provide novel insights into the relationship between glycolysis and CRC and established a glycolysis-related gene signature that could be applied to analyze patients’prognosis with CC.Furthermore,the nomogram models provided an insightful and applicative tool to evaluate CRC prognosis.In addition,this further demonstrates PPFIA4could be a promising therapeutic target in CRC patients with poor prognosis.PART Ⅱ:Expression and Clinical Significance of PPFIA4 in Colon Cancer Cells and TissuesOBJECTIVE:The expression and clinical significance of PPFIA4 in CC were verified combined with molecular biology experiments.METHODS:1)The expression level of PPFIA4 in CC tissues and normal tissues was analyzed by combining multiple bioinformatics databases;3)EXPRESSION levels of PPFIA4 in CC tissues and normal tissues are assessed by immunohistochemistry（IHC）;4)Quantitative real-time PCR（QRT-PCR）was used to detect the expression level of PPFIA4 in CC cells and normal colon cells.5)The correlation between PPFIA4 expression level and clinicopathological stage（TNM stage）and prognosis（OS and progression-free survival（DFS）of CC patients was analyzed based on clinical data.6）GO analysis of functional enrichment of PPFIA4 in colon cancer cells;7)Combined with the results of GO analysis,the correlation between PPFIA4expression level and glycoly-related gene expression level,angiogenic factor VEGFA and methylation level was analyzed.RESULTS:1)Combined with multiple bioinformatics databases,the mRNA expression of PPFIA4 in CC tissues was significantly higher than that in normal tissues（all P<0.05）;2)IHC showed that the expression level of PPFIA4 was increased in CC tissues,and QRT-PCR showed that PPFIA4 was highly expressed in CC cells（all P<0.05）.3)Increased expression of PPFIA4 was associated with higher TNM staging and poorer OS and DFS in CC patients;4)GO analysis suggested that PPFIA4 was involved in glycolysis and angiogenesis of CC cells,and affected the activity of DNA-binding transcription activator,etc.5)The increased expression level of PPFIA4 was associated with hypermethylation,increased expression level of key glycolysis genes,and increased expression level of angiogenic factor（VEGFA）（all P<0.05）.CONCLUSIONS:PPFIA4 is overexpression in CC tissues and cells,and it’s related to progression of CC.It is speculated that PPFIA4 can be used as a potential molecular marker and therapeutic target.PART Ⅲ:PPFIA4 Promotes Colon Cancer Cell Proliferation and MigrationOBJECTIVE:Detection of the effect of PPFIA4 gene on the proliferation,invasion and migration of CC,and to explore its mechanism,to provide a new idea for the study of the pathogenesis of CC and the search for an effective diagnostic and therapeutic target.METHODS:1)The PPFIA4 overexpression Plasmid was constructed by inserting the PPFIA4 c DNA into p DNA-3.1 plasmid;2)Short-hairpin RNAs（shRNAs）for PPFIA4 knockdown were designed;3)The proliferative capacity of colon cancer with PPFIA 4 expression and inhibition of PPFIA4were detected by cell accounting experiments,CCK-8 and cell clone experiments;4)The migration,invasion,proliferation of colon cancer cell PPFIA4 expression and inhibition of PPFIA4 were detected by Transwell experiments and qRT-PCR expreiments;5)Changes in mRNA expression levels of markers related to epithelial mesenchymal transformation after overexpression of PPFIA4 and inhibition of PPFIA4 expression were detected by qRT-PCR.RESULTS:1)qRT-PCR showed that the expression of PPFIA4 increased in SW403 cells and DLD1 cells transfected with overexpressed PPFIA4 plasmid,and the expression of PPFIA4 was effectively silenced in HCT116 cells and DLD1 cells transfected with PPFIA4 shRNA plasmid（all P<0.001）.2)The proliferation of SW403 cells and DLD1 cells was enhanced after transfection of PPFIA4 overexpressed plasmid by cell counting experiment,CCK-8 and clone formation experiment.The proliferation of HCT116 cells and DLD1cells was decreased after transfection with PPFIA4 shRNA(^*P<0.05,^**P<0.01,^***P<0.001).3)Transwell experiment found that the invasion and migration of SW403 cells and DLD1 cells were enhanced after transfection with overexpressed PPFIA4 plasmid.Inhibition of PPFIA4 expression reduced the invasion and migration ability of HCT116 cells and DLD1 cells(^*P<0.05,^**P<0.01,^***P<0.001).4)qRT-PCR analysis showed that the expression levels of mesenchymal markers in SW403 cells overexpressing PPFIA4 were up-regulated,including vimentin,SNAI1,ZEB1 and TWIST1,and the epithelial marker CDH1 was down-regulated.In contrast,in HCT116 cells transfected with PPFIA4 shRNA,PPFIA4 knockout up-regulated CDH1 and down-regulated Vimentin,SNAI1,ZEB1 and TWIST1(^*P<0.05,^**P<0.01,^***P<0.001).CONCLUSIONS:PPFIA4 promotes the proliferation and migration and invasion of CC cells,and plays an important role in the development of CC.PART Ⅳ:PPFIA4 Promotes Colon Cancer Cell Proliferation and Migration by Enhancing Tumor GlycolysisOBJECTIVE:We aimed to clarify the role of PPFIA4 in regulation of glycolysis,whether and how PPFIA4 affects glycolysis and biological behavior of CC.METHODS:1)Sea-horse extracellular flux analyzer expressed detections of PPFIA4 or changes of ATP,ECAE and lactic acid level;2)Bioinformatics explored the mechanism of Promote the migration,invasion,proliferation of colon cancer cell;3)qRT-PCR and western blot measure the expressions level of the Protein levels of PFKFB3/ENO2 in HCT116 cells transfected with PPFIA4 shRNA;4)Cell viability of SW403 cells was determined by cell count assay,transwell analysis of the migration and invasion of SW403 cells,and total ATP levels were measured in SW403 cells.After transfect with PPFIA4 expression plasmid and with or without PFKFB3/ENO2 siRNA.RESULTS:1)Here we showed that overexpression of PPFIA4significantly upregulated the levels of ATP（P<0.01）and ECAR,which detects the extracellular acid-producing capacity of cells and indirectly shows the glycolysis ability（P<0.01）.The Product of glycolysis,lactate（P<0.001）,in SW403 cells was also increased.Consistently,inhibiting PPFIA4 in HCT116 cells decreased the levels of ATP,ECAR and lactate production;2)PPFIA4 showed significant positive correlations with expression levels of PFKFB3（6-phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3）and ENO2（enolase 2）in the TCGA colon cancer database;3)The Positive correlation between PPFIA4 and PFKFB3/ENO2 was then validated by q PCR and Western blot experiments,which showed that PPFIA4 overexpression in SW403 cells led to upregulated PFKFB3 and ENO2.On the other hand,knocking down PPFIA4 in HCT116 cells led to downregulation of PEKFB3and ENO2;4)We found that knocking down PFKFB3 or ENO2 indeed attenuated the effects of PPFIA4 overexpression on promoting cell proliferation（cell counting assay）,migration,invasion（transwell assay）and glycolysis（measured by ATP levels）(^*P<0.05,^**P<0.01,^***P<0.001).CONCLUSIONS:Our study reports PPFIA4 as a potential novel therapeutic target in CC.Knocking down the expression of PPFIA4 in CC cells inhibited cell proliferation,migration,invasion,and glycolysis activities.Thus,PPFIA4silencing could be a strategy for reprogramming glycolysis of CC,and its efficacy is dependent on ENO2/PFKFB3.