| Objective: Lung cancer has been the major cause of cancer-related death in human for the reasons of high incidence of morbidity,metastasis,and drug resistance,and meanwhile also is one of the most common tumor types for brain metastasis.Despite a great advance in the treatment of lung cancer has been made in recent years the survival status of patients still not encouraging particularly as the occurrence of brain metastasis the life quality and survival time of patients suffered devastating impact.Additionally,due to the heterogeneity of lung cancer and the unique feature of intracranial microenvironment,almost all the treatment regimens and drugs have no good effect on the therapy of brain metastasis,and the life quality of vast majority of patients suffered severe damaging resulted from a series of cerebral dysfunction caused by the intracranial metastatic tumor.Therefore,research regarding the mechanism of brain metastasis of lung cancer will be of great help for clinical treatment of lung cancer with brain metastasis,offering novel strategy and direction.By now in the field of cancer research regarding the tumor microenvironment(TME)dating from the initial “seed-soil” theory to the latest applied tumor immunotherapy,scientists have reached the consensus that the TME has significant influence on the survival,immunity,and metastasis of tumor cells,even playing an decisive role,for which reason,the further research towards TME would be beneficial for systematically exploring the progression of tumorigenesis and advancing.The brain plays a fundamental role in the activity of organism and the physiological context,anatomical structure,metabolic and immune conditions dramatically differ from tissues in other extracranial organs.The unique physiological conditions in brain promoted the alteration of gene expression profile of tumor cells.Studies showed that this alteration of gene expression profile of brain metastatic tumor not only differed from that of the primary tumor,but also quite dissimilar with the other extracranial metastatic organs.Accordingly,the intracranial microenvironment is a complete fresh extracellular environment for tumor cells,applying an external force influencing the genetic inheritance and expression signature of tumor cells,which modulate the phenotype of tumor cells further enable them adapt to the completely new environment.Consequently,study on the interaction between tumor cells and the brain metastatic microenvironment would favor the further exploration of tumor brain metastasis mechanism,offering theoretical basis and drug targets.Brain microvascular endothelial cell(BMEC)is one of the primary cells consisting the brain metastasis microenvironment,meanwhile also is one of the essential cells forming the blood-brain barrier(BBB),neurovascular unit,and intracranial metastatic niche.During the whole process of brain metastasis,the BMEC plays a crucial role in the vascular and intracranial microenvironment.However,the mechanism of interaction between lung tumor cells and BMEC still an open question,and the study towards the interaction mechanism and mutual influence on the gene expression profile of each other still not enough.Thus,study towards the interaction between lung tumor cells and BMEC,particularly,regarding the alteration of gene expression profile would be of benefit for further study and exploration of the brain metastasis of lung cancer.As the development of RNA-Seq sequencing technology,the research regarding the transcriptome of genes has entered the era of big data,which also greatly fostered the application of bioinformatics to the field of medicine research.Through the bioinformatic analysis to large amounts of data regarding the gene transcription,viewing from the overall regulation network of genes,to mine the genes significantly correlated with the genesis and development of diseases,which provide novel strategy and theory basis of clinical treatment of diseases.Thus,by bioinformatic analysis to the transcriptome of brain metastatic lung tumor cells would be favoring for further study and exploration of the mechanism underlying the brain metastasis of lung cancer.Methods: In this study,comprehensive bioinformatic analysis of the multiple databases and disease types was performed to mine the significant factors associated with the interaction between lung tumor cells and brain metastatic microenvironment,which comprised the combination of the transcriptomic RNA-Seq data of brain metastatic lung tumor cells deriving from Gene Expression Omnibus(GEO)database and sequencing data of clinical samples of lung adenocarcinoma(LUAD)from The Cancer Genome Atlas(TCGA)database.Sequencing with RNA-Seq was perform to the transcriptome of the co-cultured human small cell lung cancer(SCLC)cell line NCIH209 with the human brain microvascular endothelial cell(HBMEC)aiming to determine the differentially expressed gene(DEG)associated with the alteration of gene expression profile under the influence of HBMEC.At the same time,the RNASeq data of transcriptome of the brain metastatic lung tumor cells deriving from human and mouse were downloaded from GEO database,which followed by the subsequential bioinformatic analysis after the quality control.By the conjoint analysis with the three transcriptomic sequencing data,to explore the rule of the altered gene expression profile,mining the factors associated with the brain metastasis of lung cancer.By the conjoint analysis with the transcriptomic sequencing data of clinical samples of LUAD patients from TCGA database and clinicopathological data,exploring the rules of the gene expression alteration of the associated factors in the primary and metastatic tumors.Utilizing the lentivirus-mediated RNA interference technology to downregulate the expression of specific gene in the tumor cells for studying the roles of the gene in related cellular functions.Establishing the mouse model of brain metastasis of lung cancer with internal carotid artery(ICA)injection.Intracranial metastatic tumor formation experiment with the mouse model was used to observe the roles of brain metastasisrelated factors in the intracranial metastatic tumorigenesis.Real-time PCR and Western Blot were performed to validate the expression of genes in the levels of m RNA and protein.Flowcytometry and MTS proliferation experiments were used to analyze the cell cycle progression and proliferative capability.Results: In this study,through the conjoint analysis to the transcriptome sequencing data of the co-cultured NCI-H209 cell,brain metastatic PC9 cells,and the brain metastatic LLC cells,it revealed that during the interaction between lung tumor cells and brain metastatic microenvironment,the lung tumor cells presented with the enrichment of gene sets associated with the functions of neural stem cell and embryonic stem cell,suggesting that it may underwent the transition of neural-stem-cell-like phenotype.Further analysis to the DEGs showed that CTGF/HOXB9 may be correlated with the alteration of metabolic phenotype of the brain metastatic lung tumor cells and the brain metastasis.The experimental results of intracranial tumorigenesis of the mouse model and cell cycle analysis showed that CTGF was the upstream regulator of HOXB9 and CTGF/HOXB9 promoted cell cycle progression of the brain metastatic lung tumor cells.Conclusion: 1.During the interaction with the brain metastatic microenvironment,the lung tumor cells primarily manifested with metabolic status characterized by hypoxia response,meanwhile showed the expression features of the neural-stem-cell-like functional genes,suggesting the presented plasticity of tumor cells during the interaction with the brain metastatic microenvironment.2.The brain metastatic microenvironment upregulated the expression of HOXB9 and CTGF,which promoted the proliferation and intracranial tumorigenic capability of the brain metastatic lung tumor cells.3.CTGF served as the upstream regulator of HOXB9,and through the regulation of HOXB9 expression to mediate the proliferation and cell cycle progression of the brain metastatic lung tumor cells. |
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