Effect Of KDM5D On The Occurrence Of Lung Adenocarcinoma And Its Mechanism

Lung adenocarcinoma is one of the most common malignant tumors leading to a great threat to human health.In recent years,the incidence of lung adenocarcinoma has gradually increased because it is difficult for patients to cure in the early stage.To date,the death caused by lung adenocarcinoma has accounted for more than 50%of lung cancer.Therefore,searching for effective strategies for diagnosis and treatment of lung adenocarcinoma has being attracted more attentions.According to reports,histone methylation modification plays an important role in the occurrence and development of malignant tumors.Histone Demethylase KDM5D(JARID1D or SMCY)is a member of histone demethylase,which is located on the Y chromosome and usually plays a transcriptional repressive role.There are four members in the KDM5s family,namely KDM5A/B/C/D,and the current research mainly focuses on KDM5 A/B/C.Although there are reports that KDM5D is involved in the invasion and metastasis of prostate cancer,the role of KDM5D-encoded protein in the development and progression of lung cancer,especially lung adenocarcinoma have not been reported Aim of this study was to investigate whether the molecular mechanism of KDM5D in the metastasis of lung adenocarcinoma.Firstly,detecting the protein expression level of KDM5D in mouse tissues and mRNA and protein expression in lung adenocarcinoma cell lines by Realtime-PCR and Western blot.The results show that KDM5D is abundantly expressed in mouse lung tissue,however,KDM5D is lower expressed in lung adenocarcinoma cell lines.Then constructing KDM5D CRISPR/Cas9 knockout plasmid and futher infect A549 cells to obtain stable transfected cell lines;Meanwhile,constructing pCDH-KDM5D overexpression plasmid to obtain stable cell lines by infecting NCI-H1299 cells.The results show that successfully constructed the KDM5D CRISPR/Cas9 knockout plasmid to obtain A549-Ctrl,A549-sgRNA1 and A549-sgRNA2 stable transfected cell lines.At the same time,the pCDH-KDM5D overexpression plasmid was successfully constructed,and stable transfected cell lines NCI-H1299-Ctrl and NCI-H1299-KDM5D overexpressing KDM5D were obtained.Through in vitro experiment,we detected the abilities of KDM5D in proliferation,migration,and invasion in the knockout/overexpression of KDM5D cell lines.The influences of KDM5D were evaluated through in vitro scratch healing,Transwell,plate cloning formation experiment,etc.The results demonstrated that,comparing to A549-Ctrl,the abilities of A549-sgRNA1 and A549-sgRNA2 cells in proliferation,migration and invasion were significantly accelerated.However,The proliferation and migration abilities of KDM5D cells was significantly reduced in the overexpressed NCI-H1299 cell line.Detecting the effect of knockout and overexpression of KDM5D on the activity of related proteins in the MAPK signaling pathway by Western Blot.Western Blot results show that after knocking out KDM5D,the phosphorylation levels of p38 and ERK were significantly increased.The phosphorylation levels of p38 and ERK were significantly decreased in the KDM5D overexpressed cell line.Finally,the specific molecular mechanism of KDM5D regulating p38 MAPK signaling pathway was detected by Co-IP experiment and GST pull-down experiment,and the key site of KDM5D removal of p38 methylation was found.The results of Co-IP experiments show that KDM5D interacts with MEK3,MEK6,moreover,we find that KDM5D interacts with p38 via the PHD2 domain.GST Pull-down experiments show that KDM5D dose not directly interact with p38,but could significantly inhibit the binding of MEK3 to p38 through the PHD domain.The results of Co-IP experiments indicate that overexpressing KDM5D can down-regulate the p38 methylation level and reduce the activation of p38 by MEK3 by removing the methylation at the K165 site of p38,as well as down-regulate the p38 phosphorylation level;Our experimental results show that knockout and overexpression of KDM5D could regulate the proliferation,migration and invasion of lung adenocarcinoma cells,and the migration changes are more obvious.KDM5D could binds to p38 through the PHD2 domain and inhibits the binding of MEK3 to p38 KDM5D affects p38 phosphorylation by removing the methylation of p38 K165,and thereby inhibie the migration of lung adenocarcinoma cells.This paper systematically studies the role of KDM5D in the metastasis of lung adenocarcinoma,and preliminary explores the molecular mechanism of KDM5D in the metastasis of lung adenocarcinoma.We hope this research will provide a new target and ideas for the diagnosis and treatment of clinical lung adenocarcinoma.