The Studies On Mechanism And Molecular Imaging Of PTPRH Regulating Glycolysis Via PI3K/AKT/mTOR Signal Pathway In Non-small Cell Lung Cancer

Objectives:The morbidity and mortality of lung cancer are increasing year by year.Rapid progress,easy metastasis and chemotherapy resistance are the treatment difficulty of NSCLC at this stage.The most common pathological type of lung cancer is non-small cell lung cancer（NSCLC）.Therefore,it is very important to explore new key targets.Targeted tumor energy metabolism therapy has attracted much attention and has potential clinical significance.Protein tyrosine phosphatase H receptor（PTPRH）as one member of protein tyrosine phosphatases family is highly expressed in NSCLC.Existing studies show that it is important of PTPRH by regulating cell migration,invasion and proliferation,but its correlation with the glycolysis of NSCLC is not clear.The purpose of this study are 1.To determine the differential expression of PTPRH through clinical patient tissue samples,index of ¹⁸F-FDG PET/CT imaging and the expression of glycolysis related protein were obtained,and analyze the correlation.2.The molecular mechanism of PTPRH regulating NSCLC glycolysis and its effect on tumor biological behavior were verified by cell experiments.3.Through constructing a nude mouse xenograft model of NSCLC,¹⁸F-FDG micro PET imaging was used for molecular imaging evaluation in vivo,providing methods and basis for the application of molecular imaging in clinical targeted therapy of NSCLC.Methods:1.From June 1,2018 to August 30,2020,¹⁸F-FDG PET/CT imaging was performed.Within one month after ¹⁸F-FDG PET/CT imaging,patients who underwent surgery and were pathologically confirmed as NSCLC after surgery were included.The expression of PTPRH was detected by IHC staining.PTPRH expression and the clinical characteristics of NSCLC patients were also analyzed.According to the GEPIA analysis and the correlation analysis with glycolysis related factors.IHC staining was used to detect the expression of glycolysis related protein in NSCLC tissues.The region of interest was delineated by ¹⁸F-FDG PET/CT imaging to obtain semi-quantitative indicators.The correlation between the IHC staining score of PTPRH and index of¹⁸F-FDG PET/CT imaging and the expression of glycolysis related protein in 80 NSCLC primary lesions was analyzed to study the relationship between the expression of PTPRH and glycolysis in NSCLC.2.q RT-PCR and Western blot detected expression of PTPRH;Selected NSCLC lines A549 and H460 to down-regulate and up-regulate the expression of PTPRH by lentivirus transfection;The effect of PTPRH on cell biological behavior was detected by colony assay,Ed U experiment,Transwell assay,wound healing assay and flow cytometry;The changes of ¹⁸F-FDG uptake and glucose metabolites after PTPRH regulation were detected by gamma counter and lactic acid test;The expression of glycolysis related protein in NSCLC cells was detected by Western blot after the regulation of PTPRH.Signal pathway enrichment analysis using Gene Set Enrichment Analysis（GSEA）,KEGG was used the for annotation to investigate the metabolic pathways involved in PTPRH in cells.After regulating PTPRH,detected the changes of the expression of pathway related proteins;740Y-P and LY294002 were used to activate and inhibit the PI3K/AKT/m TOR signal pathway,and phase recovery experiment was performed to verify that PTPRH can affect the proliferation ability,invasion ability,apoptosis,¹⁸F-FDG uptake,lactate production and glycolysis related protein expression of cells through the PI3K/AKT/m TOR signal pathway.3.Use subcutaneous xenograft model in nude mice,weight and volume of tumor body were monitored dynamically.Molecular image evaluation used ¹⁸F-FDG micro PET imaging.Detected the protein expression levels of PTPRH,glycolysis related protein,p-PI3K/PI3K and p-AKT/AKT using IHC staining analyses.Results:1.IHC results indicated that PTPRH is highly expressed in NSCLC（P<0.001）;The expression of PTPRH was closely related to the diameter and clinical stage of NSCLC（P<0.01）;The expression level of PTPRH in NSCLC was positively correlated with the glycometabolism indexes of ¹⁸F-FDG PET/CT imaging（r=0.312,P<0.01;r=0.205,P<0.05;r=0.207,P<0.05）;Bioinformatics analysis showed that the expression of PTPRH was positively correlated with glycolysis related factors;The expression level of PTPRH in NSCLC was positively correlated with glycolysis related protein（GLUT1r=0.331,P<0.05;HK2 r=0.260,P<0.05;PKM2 r=0.312,P<0.01;LDHA r=0.305,P<0.01）.2.The results of q RT-PCR and Western blot showed that PTPRH was highly expressed in seven NSCLC cell lines in varying degrees.Lentivirus transfection regulated the expression of PTPRH.q RT-PCR and Western blot detected transfection efficiency;Through colony assay,Ed U experiment,Transwell assay and wound healing assay,it was confirmed that down-regulation and up-regulation of PTPRH can reduce and improve the proliferation,invasion and migration of cells;Flow cytometry showed that down-regulation of PTPRH could induce apoptosis;Gamma counter and lactic acid test confirmed that down-regulation and up-regulation of PTPRH could reduce and increase ¹⁸F-FDG uptake and lactic acid production;Western blot confirmed that down-regulation and up-regulation of PTPRH could inhibit and promote the glycolysis related protein expression;Bioinformatics analysis suggested that PTPRH could be enriched into PI3K/AKT/m TOR signal pathway;Western blot results showed that down-regulating and up-regulating PTPRH could inhibit and promote the protein expression of pathway-associated protein in PI3K/AKT/m TOR pathway,and was verified by PI3K/AKT/m TOR pathway inhibitor LY294002 and agonist 740Y-P;The rescue experiment confirmed that PTPRH can promote cell proliferation,invasion,apoptosis,¹⁸F-FDG uptake,lactic acid production and glycolysis related protein expression through PI3K/AKT/m TOR.3.Construct subcutaneous xenograft model using lentivirus transfection in nude mice.It confirmed that down-regulation of PTPRH could inhibit the growth of transplanted tumor using the generation curve;Down-regulation of PTPRH could inhibit SUVmax was confirmed by ¹⁸F-FDG micro PET imaging（P<0.001）.IHC staining results confirmed that down-regulation of PTPRH could inhibit the expression of glycolysis related protein,p-PI3K/PI3K and p-AKT/AKT（P<0.001）.Conclusions:1.PTPRH is highly expressed,and is closely related to the tumor diameter and clinical stage;The expression of PTPRH was associated with SUVmax,MTV,TLG and the expression of glycolysis related protein（GLUT1,HK2,PKM2 and LDHA）.2.PTPRH can promote the proliferation,invasion,migration,and inhibit apoptosis of NSCLC cells,promote ¹⁸F-FDG uptake,lactate production and glycolysis related protein（GLUT1,HK2,PKM2 and LDHA）expression.PTPRH can regulate the glycolysis process of NSCLC through PI3K/AKT/m TOR signal pathway.3.Through xenograft tumor model in nude mice,it was verified that down-regulation of PTPRH can inhibit the growth of NSCLC xenograft tumor in nude mice,¹⁸F-FDG uptake reduced,and further validate in vivo through molecular imaging imaging.The relationship between PTPRH expression and NSCLC glycolysis process was explored.