Study On Molecular Mechanism Of GALNT6-mediated Metastasis Of Lung Adenocarcinoma Promoted By Cancer-associated Fibroblasts

BackgroundLung cancer is the most common malignant tumor in the world and the leading cause of cancer-related death.Because of the strong invasive ability of lung cancer cells,lung cancer patients are prone to recurrence and metastasis,and once metastasis occurs,the mortality rate of patients is very high.Therefore,the control of cancer metastasis is still a major problem in the field of lung cancer research.Numerous studies have shown that tumor metastasis includes the processes of cell dissociation,migration,invasion,adaptation and re-adhesion.These processes are closely related to the microenvironment of tumor cells.Tumor microenvironment mainly consists of three parts: tumor cells,stromal cells and extracellular matrix.Interstitial cells play an important role in the metastasis of tumors.On the one hand,tumor cells are transformed directly or indirectly into cancer-related stromal cells.On the other hand,the transformed stromal cells promote the growth,development and metastasis of cancer cells.Fibroblasts are the most abundant cells in mesenchymal cells,and their great role in tumor metastasis has attracted much attention.However,the research on the role of cancer-related fibroblasts in promoting lung adenocarcinoma metastasis is still in its infancy.Some of the interaction processes have not been fully confirmed,and some of the in-depth molecularmechanisms need to be further explored.In recent years,with the advent of the post-genomic era,transcriptome has been widely used in cancer mechanism research.Transcriptionomics refers to the study of gene transcription and regulation in cells under specific conditions at the overall level.The purpose of transcriptome research is to quantitatively and qualitatively analyze the genes in specific cells,to study the differential gene expression and gene structure among different cells,so as to discover the genes,molecular mechanisms and regulatory networks related to the occurrence and development of diseases.At present,the molecular mechanism of cancer-related fibroblasts promoting lung adenocarcinoma metastasis based on transcriptome technology is relatively rare.Therefore,this study is based on transcriptome technology to study the molecular mechanism of cancer-related fibroblasts promoting lung adenocarcinoma metastasis.The results of this study are expected to provide a possibility for the discovery of potential EMT biomarkers and therapeutic targets for lung cancer metastasis,and further explore the application value of transcriptome technology in lung cancer and microenvironment-related research.Methods1.Establishment of Cell Model and Transcriptionomic Database of EMT Induced by CAF in Lung AdenocarcinomaIn this part,we firstly construct a cell model of lung adenocarcinoma EMT(Epithelial-Mesenchymal Transition)induced by CAF(Carcinoma-Associated Fibroblasts)to simulate the real process of interaction between lung cancer cells and fibroblast cells in vivo.Human fetal lung fibroblast WI38 was transformed into CAF by the secretion of human lung adenocarcinoma cell A549.Then,the secretion of CAF was collected to induce A549 to produce EMT.The cell model was identified by immunoblotting,immunofluorescence and invasion test.Then,RNA-seq technology was used to sequence lung adenocarcinoma cells at different time points in EMT process,and a complete transcriptome database was established,which provided a possibility to identify potential EMT biomarkers and therapeutic targets for lung cancermetastasis.2.Bioinformatics analysis of transcriptome data and screening and preliminary validation of key moleculesIn this part,we firstly use WGCNA(Weighted Gene Co-expression Network Analysis)bioinformatics analysis to analyze the transcriptome data of EMT in lung adenocarcinoma,and to identify the key molecules related to the occurrence of EMT.Then,the key molecules locked in bioinformatics analysis were preliminarily validated at cell model level and TCGA(The Cancer Genome Atlas)clinical data level to provide more reliable candidate molecules for the follow-up study of complex molecular mechanisms.3.Molecular mechanisms of GALNT6 promoting EMT in lung adenocarcinoma cellsIn this part,we firstly use immunohistochemical technique to detect the expression of GALNT6(polypeptide N-acetylgalactosaminyltransferase 6)in clinical samples of lung adenocarcinoma,and analyze the correlation between its expression and clinical indicators.Secondly,we use immunoblotting,immunofluorescence,scratch test,invasive test and animal experiments to detect the effects of GALNT6 expression on EMT phenotype,invasion and metastasis of lung adenocarcinoma cells in vitro and in vivo.Finally,we reveal its potential molecular mechanism through cell-level experiments,so as to provide strong evidence for GALNT6 as a biomarker of EMT and a new target for lung cancer treatment.Results1.Establishment of Cell Model and Transcriptionomic Database of EMT Induced by CAF in Lung AdenocarcinomaIn this part,firstly,we successfully constructed a cell model of CAF-induced EMT in lung adenocarcinoma.When the secretion of human lung adenocarcinoma cell A549 was used to induce human fetal lung fibroblasts WI38 for 24 hours,it was found that the expression of two cancer-related fibroblast-specific markers,alpha-SMA and FAP,wassignificantly increased,suggesting that normal fibroblasts transformed into CAF under the action of secretion of lung cancer cells.After collecting the secretion of CAF to induce human lung adenocarcinoma cell A549 for 72 hours,it was found that the EMT marker E-cadherin was significantly down-regulated and Vimentin was significantly up-regulated,indicating that EMT occurred in lung adenocarcinoma cell A549 under the action of CAF secretion,and the invasion and metastasis ability was enhanced.Next,RNA-seq technique was used to sequence transcriptomes of the experimental group and the control group at 6 time points(3 hours,6 hours,12 hours,24 hours,48 hours and 72hours)during EMT of lung adenocarcinoma,and a complete transcriptome database was successfully established,which provided the possibility for identifying potential early biomarkers of EMT and therapeutic targets for lung cancer metastasis.2.Bioinformatics analysis of transcriptome data and screening and preliminary validation of key moleculesIn this part of the study,we first analyzed the transcriptome data of EMT in lung adenocarcinoma by WGCNA method,and identified some key genes such as GBX,HES7,L3MBTL1,CITED1,NFE2,SPARC,TMEM37,GALNT6,CACNB2,which may serve as potential biomarkers for early EMT.Then,through the preliminary verification of cell model level and TCGA clinical data level,it was found that GALNT6,SPARC and HES7 may play a role in the early EMT initiation of lung adenocarcinoma,and provide new ideas for the further study of the molecular mechanism of metastasis.3.Molecular mechanisms of GALNT6 promoting EMT in lung adenocarcinoma cellsIn this study,we firstly found that GALNT6 was highly expressed in lung adenocarcinoma tissues,and the high expression of GALNT6 was significantly correlated with clinical T,N,TNM and adverse prognosis.Secondly,we discussed the effect of GALNT6 on EMT phenotype and function in vitro and in vivo.When GALNT6 was overexpressed,the ability of tumour cells to produce EMT increased,and the ability of tumour cells to invade and metastasize increased significantly.WhenGALNT6 was knocked down,the ability of tumour cells to generate EMT decreased,and the ability of tumour cells to invade and metastasize decreased significantly.Finally,we reveal the potential molecular mechanism.GALNT6 enhances the stability of GRP78(Glucose-Regulated Protein 78)through O-glycosylation,and stable GRP78 regulates EMT through MEK/ERK signaling pathway,which promotes the invasion and metastasis of tumors.Conclusions1.This study successfully constructed the cell model of EMT induced by CAF in lung adenocarcinoma,and successfully established the transcriptome database of EMT in lung adenocarcinoma.2.Through WGCNA bioinformatics analysis and preliminary validation,we found that GALNT6,SPARC and HES7 may play an important role in the early EMT initiation of lung adenocarcinoma.3.This study reveals the potential molecular mechanism of EMT in lung adenocarcinoma.GALNT6 promotes the invasion and metastasis of lung adenocarcinoma cells through GRP78-MEK-ERK pathway.Therefore,GALNT6-GRP78 may be a promising new target for anti-metastasis therapy.