Role And Mechanism Of SLC7A5 In The Progression Of Lung Adenocarcinoma

Purpose:The incidence rate of lung cancer ranks second in the world and the mortality ranks first in the world.The average incidence rate of lung cancer in Yunnan Province is40/100000,about twice the national average incidence rate.The incidence rate of lung cancer in Xuanwei City is as high as 91/100000,about 4.11 times the national average.Gejiu City,like Xuanwei,is a high incidence area of lung cancer.The data show that the incidence of lung cancer among Yunxi miners is 531.91/100000 and the mortality is517.49/100000.The study found that environmental pollution is one of the important causes of lung cancer in these two areas.The high incidence of lung cancer in Xuanwei is related to the polycyclic aromatic hydrocarbons released from the local combustion of bituminous coal,while the main cause of Gejiu lung cancer is the influence of radon and mineral dust in the air of the workplace.In addition,a large number of studies have confirmed that some metabolites produced by polycyclic aromatic hydrocarbons and radon after being inhaled into the human body can form covalent adducts,damage the body’s DNA,cause some gene mutations,such as KRAS mutation or abnormal expression,and then cause a high incidence of cancer.Therefore,it is more urgent to study the pathogenesis of lung cancer,especially the lung cancer induced by environmental pollution factors,and find new targets for the treatment of this kind of lung cancer in Yunnan.SLC7A5 is highly expressed in a variety of tumors,such as breast cancer,gastric cancer,liver cancer,colorectal cancer and pancreatic cancer.It is negatively correlated with prognosis and is an independent prognostic factor.There are few studies on the expression and prognosis of SLC7A5 in lung cancer,but there is no report on whether the abnormal expression of SLC7A5 is caused by environmental pollution such as radon or polycyclic aromatic hydrocarbons.In addition,the regulatory mechanism of SLC7A5abnormal expression is not clear.It has been reported that in colorectal cancer,the abnormal expression of SLC7A5 is related to the activation of Ras signal pathway.KRAS mutation up regulates the expression of SLC7A5,and knockdown of SLC7A5 can significantly inhibit the growth of KRAS mutant tumors.However,the author has not clarified the specific regulation mechanism of SLC7A5 in Ras signal pathway.Which genes in Ras signaling pathway can regulate SLC7A5 expression still need to be further explored.This subject intends to analyze the expression and prognosis of SLC7A5 in Xuanwei lung adenocarcinoma,Gejiu lung adenocarcinoma and large sample tissues in the database by using basic experimental techniques and bioinformatics methods,find out the key factors regulating SLC7A5 expression in Ras pathway,and explore the regulatory mechanism of KRAS gene on SLC7A5 in lung adenocarcinoma.Methods:（1）Expression and prognosis of SLC7A5 in lung adenocarcinoma.Xuanwei lung adenocarcinoma and Gejiu lung adenocarcinoma samples were selected to detect the expression of SLC7A5 in cancer tissues and adjacent tissues and its correlation with clinicopathological features by immunohistochemistry.TCGA data set,GTEX data set and geo data set further verified the expression of SLC7A5 in lung adenocarcinoma;The expression of SLC7A5 in lung adenocarcinoma cell line was detected by q PCR and Western blot;Prognoscan website was used to analyze the prognosis of SLC7A5 expression in lung adenocarcinoma.（2）Mechanism of KRAS mutation regulating SLC7A5 expression.The differentially expressed up-regulated genes in A549(KRAS^G12S)cells and Calu-3（KRASwild）cells were screened by transcriptome sequencing.The differentially expressed up-regulated genes were screened by differential expression analysis of 288cases of KRAS wild-type lung adenocarcinoma and 154 cases of KRAS mutant lung adenocarcinoma in geo data set;WGCNA method was selected to analyze the weighted co expression of geo data set,and the genes related to SLC7A5 expression were screened.For the above three groups of data,ZNF24,which is closely related to SLC7A5expression,was screened by drawing Venn map;By interfering with RAS expression or inhibiting Ras activity and blocking MEK/ERK and PI3K/Akt signal pathways downstream of RAS,to explore which signal pathway regulates SLC7A5 and ZNF24;QPCR and Western blot were used to verify the regulatory relationship between SLC7A5and ZNF24;The effect of ZNF24 on the stability of SLC7A5 protein was investigated by adding cycloheximide;The interaction between SLC7A5 and ZNF24 was explored through online interaction prediction website,immunofluorescence,Immunocoprecipitation,pull down and yeast two hybrid experiment.（3）ZNF24 affects the biological behavior of lung adenocarcinoma through SLC7A5.The effects of knockdown of SLC7A5 and ZNF24 or knockdown of ZNF24 and transfection of overexpressing SLC7A5 plasmid on the proliferation,invasion and migration of lung adenocarcinoma cells were investigated in vitro by establishing stable cell lines.The nude mouse transplanted tumor model was established through in vivo animal experiments to explore the effect of knockdown of SLC7A5,ZNF24 or ZNF24and transfection of SLC7A5 plasmid on the growth of lung adenocarcinoma transplanted tumor.Results:（1）SLC7A5 was highly expressed in Xuanwei and Gejiu lung adenocarcinoma tissue samples and KRAS mutant lung adenocarcinoma cell lines,which confirmed that the prognosis of patients with high expression of SLC7A5 in lung adenocarcinoma data samples was poor;（2）ZNF24,a new regulatory factor of SLC7A5,was found,which confirmed that both ZNF24 and SLC7A5 were regulated by Ras/MEK/ERK and RAS/PI3K/Akt signal axes,and ZNF24 positively regulated the expression of SLC7A5 protein at the protein level and enhanced the stability of SLC7A5 protein;（3）Knockdown of SLC7A5 and ZNF24 significantly inhibited the growth of KRAS mutant lung adenocarcinoma,suggesting that ZNF24 promotes the growth of lung adenocarcinoma through SLC7A5.Conclusion:In this study,aiming at the up regulation of SLC7A5 gene caused by KRAS mutation in lung adenocarcinoma caused by environmental pollution,the expression level and molecular mechanism of SLC7A5 gene in lung adenocarcinoma were studied,and the above innovative research results were obtained.Conclusion SLC7A5 and ZNF24 are potential therapeutic targets for KRAS mutant lung adenocarcinoma.This study also elucidates the regulatory mechanism between SLC7A5 and ZNF24 in KRAS mutant lung adenocarcinoma.This study will not only provide new ideas for the pathogenesis of lung adenocarcinoma,but also provide new targets and strategies for the treatment of lung adenocarcinoma.