The Role In MAPK Signaling Pathway Of A-Raf-S214C Mutant And The Functional Study Of Two A-Raf Kinase Binding Protein

Cancer devastates millions of people every year,and causes great distress to people's lives.And in all cancers,lung cancer is one of the most common and serious malignancies worldwide,with the highest morbidity and mortality.Additionally,the cost of the treatment for lung cancer is enormous,and the treatment cycle is long,lung cancer brings heavy burden to the family and society.Therefore,it is very important to study the pathogenesis of lung cancer and findnew targets for the treatment of lung cancer.Raf kinase(Rapidly Accelerated Fibrosarcoma)is a homologous protein of retroviral oncogene v-Raf,which is a key kinase of cancer occurrence,and directly related to the incidence,prognosis and treatment of lung cancer(Fibrosarcoma).A-Raf is one of the members of the Raf kinase family,and its biological functions are still unclear.It may be involved in the regulation of energy metabolism and cell transformation.It has been reported thatA-Raf mutations can cause lung,ovarian and colorectal cancer.Previous studies have reported that A-Raf-S214C mutant can transform bronchial epidermal cells to cause cancer,and A-Raf-S214C mutant was also identified in patients with Langerhans cell histiocytosis.Therefore,the regulation of A-Raf kinase is expected to become one of the most important means to treat cancer.Both GRP78 and PARP1 are the binding proteins of A-Raf kinase.The former is involved in the endoplasmic reticulum stress,maintaining endoplasmic reticulum homeostasis,and some studies found that GRP78 was highly expressed in some tumor cells,so it may be a potential target for the treatment of cancer;PARP1 is mainly involved in DNA damage repair and cell apoptosis.We are going to investigate its effects on the pathophysiological functions of tumor cell through the regulation of PARP1 expression or the inhibition of PARP1 activity.In this study,we investigated the A-Raf kinase(A-Raf,B-Raf,and C-Raf),one of the three members of the Raf kinase family.First,we constructed a cell line stablely expressing A-Raf and its mutant A-Raf S214C.Then,identified the A-Raf kinase binding proteins by Co-immunoprecipitation and mass spectrometry.For the study of newly discovered A-Rafkinase binding proteins with potential relation to tumorigenesis,we investigated their effects on pathophysiological functions of tumor cell through siRNA interference,overexpression,gene knockout.and tested their influence on the downstream signal of Raf kinase,especially on the phosphorylation level of ERK,and investigated the influence of their expression levels on tumor cells through testing their effects on lung cancer cells(A549).By investigating the effect of A-Raf S214C mutants on ERK signaling,we found that the A-Raf S214C mutant is an activated mutant of A-Raf,and A-Raf mutant showed a a stronger phosphorylation capacity for ERK than A-Raf widetype.In this study,we successfully constructed A-Raf kinase binding protein GRP78 and PARP1 gene knockdown stable cell lines and overexpression stable cell lines in A549 cells,respectively.Through the CCK8 experiment,cell scratches and transwell experiments,it was proved that the anti-apoptotic ability of A549 was increased after the knockdown of PARP1 and the migration ability of the cells was also increased.These results provide a new basis for the treatment of cancer by regulating PARP1.This study will be helpful for understanding carcinogenic mechanism of A-Raf kinase,and provide new targets and methods for the treatment of A-Raf related diseases,especially lung cancer.