The Role And Mechanism Of Polypyrimidine Tract Binding Protein In The Warburg Effect And Progression Of Keloid

BackgroundThe pathogenesis of keloid remains unclear,and the lack of effective and stable treatment options has always been regarded as a clinical challenge.Due to its invasion of the surrounding skin,keloid is also regarded as skin tumor.In our previous studies,we compared the energy metabolism of different types of scars with normal skin and found that only keloid exhibited metabolic patterns of aerobic glycolysis and oxidative phosphorylation.Aerobic glycolysis,also known as Warburg effect,is a classical energy metabolism pathway of tumor cells.Polypyrimidine tract binding protein(PTB)is expressed at high levels in various tumors and is an important RNA-binding protein.The main function of PTB is to regulate alternative splicing in cells and to regulate the Warburg effect in tumor cells.However,little is known about the expression characteristics of PTB in keloid and the molecular mechanisms of keloid energy metabolism.Objectives1.Investigate whether the increased expression of PTB in keloid is specific,and clarify the expression difference of PTB in keloids and surrounding normal skin.2.Clarify whether PTB regulates the Warburg effect and biological behavior of keloid fibroblasts.3.Investigate whether keloid has the characteristic of alternative splicing of pyruvate kinase M(PKM),and clarify whether pyruvate kinase M isoform 2(PKM2)regulates the Warburg effect of keloid fibroblasts.Clarify whether PTB regulates Warburg effect and biological behavior of keloid fibroblasts through alternative splicing of PKM.Methods and Results1 The expression of PTB in different types of scars Methods:Three keloid samples,three hypertrophic scar samples,and three atrophic scar samples were obtained.Tissue RNA and tissue protein were extracted,and the expression of PTB was detected by qPCR and Western blot.In addition,four clinical samples containing keloid with surrounding normal skin were used for immunohistochemical staining to detect the expression of PTB protein in different areas.Keloid fibroblasts(KFb)and normal skin fibroblasts(NFb)were cultured in vitro,and the expression of PTB in KFb and NFb was detected by qPCR and Western blot.Results:The relative expression of PTB mRNA and protein in keloid and hypertrophic scar was significantly higher than that in atrophic scar;There were more PTB-positive cells in keloid tissue than in the surrounding normal skin tissue;In the four samples tested,the relative expression of PTB mRNA and protein in three KFb samples increased.2 The effect of PTB on the Warburg effect and biological behavior of keloid fibroblastsMethods:①KFb and NFb were cultured in vitro,and RNA interference technology was used to knock down PTB expression in KFb.PTB overexpression was achieved in NFb through PTB lentivirus.Glucose and lactate levels in the cell culture medium were measured using glucose and lactate assay kits under two cell treatment conditions.The total RNA and total protein of KFb and NFb were extracted,and qPCR was used to detect the transcription of key enzymes of glycolysis,including hexokinaseⅡ(HKII)and phosphofructokinase-2/fructose-2,6-bisphosphatase 3(PFKFB3).Western Blot was used to detect the protein expression of HKII,PFKFB3,pyruvate kinase M1(PKM1),and pyruvate kinase M2(PKM2).The expression of key enzymes of the tricarboxylic acid cycle(TCA cycle),including citrate synthase(CS),isocitrate dehydrogenase 1(IDH1),and α-ketoglutarate dehydrogenase(OGDH),was detected by qPCR and Western Blot.②KFb and NFb were cultured in vitro,and RNA interference technology was used to knock down PTB expression in KFb.PTB overexpression was achieved in NFb through PTB lentivirus.The CCK-8 experiment was used to detect fibroblast proliferation under two cell treatment conditions.The scratch test was used to detect fibroblast migration under two cell treatment conditions.The acridine orange(AO)and Lyso-Tracker red staining were used to detect autophagy ability of fibroblasts under two treatment conditions.Results:① Knocking down PTB can inhibit glucose uptake and lactate generation in KFb,inhibit the expression of GLUT1 and LDHA,inhibit the expression of key enzymes such as PKM2,HKII,and PFKFB3 in the process of glycolysis,and inhibit the protein expression of three key limiting enzyme in the TCA cycle,including CS,IDH1,and OGDH.Overexpression of PTB in NFb can promote the glucose uptake and lactic acid production of NFb,promote the expression of GLUT 1 and LDHA,promote the expression of key enzymes such as PKM2,HKII and PFKFB3 in the process of glycolysis,and promote the expression of CS and OGDH in the TCA cycle while inhibit IDH1 protein expression.②After PTB was knocked down in KFb,cell proliferation and migration were inhibited,while autophagy was promoted.Overexpression of PTB in NFb promoted cell proliferation and migration,but inhibited autophagy.3 Study on the mechanism of PTB regulating the Warburg effect and progression in keloidMethods:① Immunohistochemistry staining of PKM1 and PKM2 was performed on keloid and surrounding normal skin to detect their protein expression.Total RNA was extracted from KFb and normal NFb,and PKM DNA fragments were amplified by RT-PCR.The amplified PKM DNA was then cut by the restriction enzyme Pstl and the expression of PKM1 and PKM2 DNA was detected by agarose gel electrophoresis.Total protein was extracted from KFb and normal NFb,and the expression of PKM1 and PKM2 in cells was detected by Western blot,and the PKM1/PKM2 ratio was compared.②KFb and NFb were cultured in vitro,and PKM2 expression in KFb was knocked down using RNA interference technology.PKM2 was overexpressed in NFb using PKM2 lentivirus.Glucose consumption and lactate production in the cell culture medium were measured using glucose and lactate assay kits.③PTB was overexpressed in the second-generation NFb using PTB lentivirus,and PKM2 expression was knocked down using RNA interference technology in the later passages of cells.Changes in the Warburg effect were measured.④PTB was overexpressed in the second-generation NFb using PTB lentivirus,and PKM2 expression was knocked down using RNA interference technology in the later passages of cells.Cell proliferation ability was measured using the CCK-8 assay,and cell migration ability was measured using the scratch assay.Results:①Immunohistochemical results showed that keloid expressed higher levels of PKM2 protein than surrounding normal skin tissues,while the surrounding normal skin tissues expressed more PKM1 protein.RT-PCR and restriction enzyme analysis showed that the relative expression of PKM1 DNA in NFb was higher than in KFb,while the relative expression of PKM2 DNA in KFb was higher than in NFb.After detecting the protein expression,it was found that KFb expressed higher levels of PKM2 than NFb.②Knockdown of PKM2 inhibited glucose uptake and lactate production in KFb.Overexpression of PKM2 in NFb can promote the glucose uptake and lactic acid production.③Overexpression of PTB in NFb and PKM2 knockdown resulted in a decrease in PTB’s ability to enhance the Warburg effect.④The ability of PTB to enhance cell proliferation and migration was also reduced after PKM2 knockdown.Conclusions1.PTB expression is upregulated in both hypertrophic scars and keloids,with higher expression in keloids compared to surrounding normal skin.2.PTB promotes the Warburg effect of keloid fibroblasts.PTB can regulate the expression of key enzymes in the TCA cycle.PTB promotes proliferation and migration of keloid fibroblasts,and inhibits cell autophagy.3.There is an alternative splicing of phenomenon in keloid with PKM2 upregulation and PKM1 downregulation in keloid.PKM2 promotes the Warburg effect of keloid fibroblasts.Knocking down the expression of PKM2 can partially inhibit the promoting effect of PTB on the Warburg effect and biological behavior of keloid fibroblasts,indicating that PTB at least partially regulates the Warburg effect and biological function of keloid through PKM selective splicing.