EGFR Regulating PLOD2 Expression Promotes Proliferation And Invasion Through STAT3 In Glioma

Objective:PLOD2 plays a pro-cancer role in the progression of multiple solid tumors,but the biological behavior and molecular mechanisms in gliomas have not been clarified.This project aims to clarify the expression of PLOD2 in gliomas and its effect on prognosis,to study the biological function of PLOD2 in vitro cells,and to explore the molecular mechanism of EGFR regulation and PLOD2 expression.Methods:(1)IHC analysis of PLOD2 protein expression levels in Grade Ⅱ-Ⅳ gliomas from clinical gilomas specimens.Analysis of expression levels of PLOD2 m RNA in Grade Ⅱ-Ⅳ gliomas from CGGA dataset.Kaplan-Meier was used to analyze of effect of PLOD2 expression on clinical prognosis of gliomas patients from CGGA dataset.Analysis of correlation between EGFR and PLOD2 m RNA levels in gliomas from CGGA dataset.WB and q RT-PCR analysis of effect of EGFR on PLOD2 protein and m RNA expression in glioma cells.WB analysis of effect of Erlotinib treament inhibited growth factor receptor EGF promotes phosphorylation EGFR and PLOD2 protein expression in U87 cell stably expressing EGFR.(2)Lentivirus sh-PLOD2 was transfected into overexpressed EGFR vⅢ glioma cell to analyze the biological function of PLOD2 in glioma through in vitro cell proliferation,clonal formation,cell migration and cell invasion.(3)Analysis of correlation between STAT3 and PLOD2 m RNA levels in gilomas from CGGA dataset.WB and q RT-PCR analysis of effect of lentiviral sh-STAT3 on PLOD2 protein and m RNA expression in glioma cells.Dual-luciferase reporter genes analysis effect of transcription factor STAT3 on transcription levels of PLOD2 Promoter.WB analysis of effect of Cryptotanshinone or Erlotinib treated inhibited growth factor receptor EGF promotes phosphorylation STAT3 and PLOD2 protein expression in U87 cell stably expressing EGFR.Effect of co-expression of p-EGFR or p-STAT3 and PLOD2 on prognosis of patients.Results:(1)The protein expression of PLOD2 in the pathological tissue specimens of gliomas and the m RNA expression of PLOD2 in the CGGA dataset increased with the increase of glioma grade.High expression of PLOD2 in gliomas indicates poor prognosis(P<0.05).The m RNA expression levels of EGFR and PLOD2 were positively correlated in the CGGA dataset(P<0.05).The protein expression of PLOD2 was significantly increased in U87 vⅢ and LN229 vⅢ glioma cells.The inhibitor Erlotinib can effectively inhibit the growth factor EGF activation of EGFR Phosphorylation and PLOD2 protein expression.(2)The results of CCK8,clone formation,wound healing and cell invasion suggest that the proliferation and invasion ability of glioma cells knocked down the expression of PLOD2 is significantly weakened compared with the control group(P<0.05).(3)The m RNA expression levels of STAT3 and PLOD2 were positively correlated in the CGGA dataset(P<0.05).The expression of PLOD2 protein and m RNA in the glioma cell line of knock-down STAT3 was significantly decreased compared with control group.The double luciferase reporter gene suggested that transcription factor STAT3 could significantly up-regulate PLOD2 transcription level(P<0.05).The inhibitor Cryptotanshinone or Erlotinib can effectively inhibit the growth factor EGF activation of STAT3 Phosphorylation and PLOD2 protein expression.The co-expression of p-EGFR or p-STAT3 and PLOD2 correlates with worse prognosis of glioma patients(P<0.05).Conclusions:The abnormally high expression of PLOD2 in gliomas is associated with poor clinical outcome.EGFR up-regulated PLOD2 expression in gliomas.The mechanism may be EGFR activating transcription factors STAT3,impel the activation type p-STAT3 into the nucleus bind to the PLOD2 promoter region,enhance the transcriptional level of the PLOD2 to promote its expression,and then drive the occurrence and development of gliomas.