Non-small Cell Lung Cancer NCI-H460 Cell Driven Receptor Tyrosine Kinase High-Expression Molecular Regulation Mechansim Research

Epidermal growth factor receptor EGFR is a member of the ErbB family,with tyrosine kinase activity,is an important transmembrane receptor.EGFR is frequently overexpressed and/or abnormal expression in many tumors,which is also closely correlated with tumor occurrence and development.EGFR is regarded as a typical tumor marker and as well as the definitive target for the development of anticancer drugs.Therefore,finding the molecular mechanism of EGFR is essential for the interpretation of tumor pathogenesis and the development of anticancer drugs.The mutation of EGFR could continuously activate the signal pathway of the EGFR mediation,and promote the activating of cell survival signal pathway and anti-apoptotic signal pathway.The amplification of the signal pathways cascading results in the survival of EGFR mutant cells,which only rely on the function of EGFR,and then on EGFR "addiction".Non-small cell lung cancer manifest asthe EGFR mutation and highly express to the EGFR"addition",Whether EGFR is mutated needs the whole genome sequencing to characterize and if mutated,and it is irreversible.On the contrary,the form of high expression can be more easily detected and studied,the high expression of this "addiction" will gradually disappear as well,once the "active mutation" disappears.Therefore,our project puts forward using non-small cell lung cancer to drive EGFR to highly express this form of "addiction" as the model.The transcription factor as the bridge of"active mutation" and "passive mutation" could accurately find out the exact pathway of mutation which leads to "mutant addiction of EGFR high expression".Based on the above analysis,we use the high expression of EGFR as the researching model.Combined with the related data of the projects of tumor cell sequence,which is announced,study the molecular regulation of the non-small cell lung cancer leading EGFR to highly express,and find the concurrently co-activating "active mutation" with EGFR "passive mutation".The study by Real Time-PCR,Western Blotting and other methods of verification,the specific experimental schemes follow below:(1)Screened model cell lines with high expression of EGFR from gastric cancer MGC803,liver cancer Bel-7404,non small cell lung cancer NCI-H460,NCI-H446,lung cancer A549,human liver cells HL-7702,human embryonic lung fibroblasts WI-38;(2)Regulate the transcription factor of EGFR expression sphere by methods like bioinformatics prediction and screening literature and then use experimental verification by real-time fluorescence quantitative PCR(3)Localize the mutant gene in the specific signal pathway,which is announced by the lung cancer cells sequencing plan and buy some mutation pathway inhibitors(buy 11 signal pathway inhibitors from Selleck),after closing these mutation pathways,research transcription factors and EGFR expression situation,according to transcription factors and expressing situation,find which mutation pathway is the one drives transcription factors and activates EGFR.We determine that non-small cell lung cancer NCI-H460 is the cell line of high-expression of EGFR and screened the upstream transcription factor Egrl of EGFR high-expression,discover the specific signaling pathway position through the mutant gene announced by lung cancer single cell sequencing plan,then study the expressing situation of transcription factor Egrl and EGFR after signal pathway inhibitors suppressing these mutation,found only No.ninth drug AZD5438 can inhibit EGFR and Egrl,because AZD5438 is a valid inhibitor of CDK1,CDK2,CDK9,infer the path of variation that drives Egrl to activates EGFR mutation is related to CDK1/2/9 pathway.In order to explore non-small cell lung cancer NCI-H460 cells actuate tyrosine kinase high-expression molecular regulation mechanism in the future.