Bioinformatics Analysis Of Lung Adenocarcinoma And TMCO1 And The Mechanism Of TMCO1 Regulating Proliferation And Migration Of A549

Lung cancer is the most malignant tumor with the highest morbidity and mortality in the world and in China.Lung adenocarcinoma is a common patholosical type of lung cancer,accounting for 40%of all lung cancers.Its metastatic ability is strong and its survival rate is very low.At present,the pathogenesis of LUAD metastasis is still unclear.Therefore,the search for differentially expressed genes(DEGs)and potential biomarkers of disease can help elucidate the molecular mechanisms involved in LUAD metastasis and promote the development of gene-targeted therapies.Over the years.bioinformatics analysis of high-throughput sequencing(eg.microarray)data has been widely used to find differential genes in lung cancer.Many differentially expressed genes play an important role in the progression of lung adenocarcinoma(LUAD)and can be considered as a potential molecular target or an effective diagnostic marker.This study will search the original mRNA and miRNA expression profiles of LUAD metastasis patients and their adjacent normal tissues from the GEO database,screen DEGs related to lung adenocarcinoma metastasis,construct ceRNA networks and perform GO and KEGG enrichment analysis involved in DEGs,which would provide some reference for the molecular mechanism of LUAD metastasis and for future research directions.Intracellular Ca2+homeostasis is essential for the cells to play a normal physiological function.The destruction of Ca2+homeostasis is closely related to the occurrence and development of tumors.It has been widely confirmed that many functional proteins involved in calcium homeostasis(Ca2+channels,calcium pumps.etc.)undergo changes in the expression level of malignant tumors.TMCOl is a newly discovered endoplasmic reticulum calcium channel.The loss of TMCOl gene or down-regulation of protein expression leads to the destruction of intracellular Ca2+homeostasis and abnormal Ca2+signaling.Therefore.TMCOl is a potential novel tumor regulatory factor,but its role in tumors is currently rarely involved.Therefore.this study will analyze the TMCOl gene based on TCGA database mining,explore its expression in each tumor,and cany out GO and KEGG enrichment analysis of its interacting proteins,which will lay a Theoretical foundation and direction for further research on the relationship between TMCO1 and tumor in the future.Based on the results of the bioinformatics analysis of the relationship between TMCO1 and lung adenocarcinoma,the effects of TMCO1 on the proliferation and migration of A549 cell line were preliminarily verified by cell functional experiments.At the same time,the A549 cells were intervened by the Guben Jiedu Decoction(clinically effective treatment of lung cancer of Professor Bingkui Piao of Guang’anmen Hospital of China Academy of Chinese Medical Sciences for the)to verify the therapeutic effect.Purpose:1.Through gene expression profiles analysis of patients with lung adenocarcinoma metastasis based on bioinformatics,screen differentially expressed genes related to metastasis of lung adenocarcinoma,constructing relevant ceRNA networks,exploring biological processes and signaling pathways involved in differential genes,which aims to provide some reference for molecular biology of LADC metastasis and for future research directions.2.Using the database analysis technology to systematically analysis TMCO1 to explore its expression in various tumors,and to find out the related pathways involved in TMCO1 interaction proteins,which aims to provide ideas and directions for further research on the relationship of TMCO1 and tumor and lay the theoretical foundation for in vitro experimental verification in the next step.3.TO preliminarily reveal the effects of TMCOl Rene on the proliferation and migration of A549 lung adenocarcinoma cells by in vitro experiments.4.To investigate the intervention effect of traditional Chinese medicine Guben Jiedu Decoction on proliferation and migration of A549 cells by in vitro experiments.Method:1.The original mRNA and miRNA expression profiles of LUAD metastasis patients and their adjacent normal tissues were retrieved from the GEO database,and differentially expressed mRNAs and miRNAs were screened.Subsequently,some databases were used to predict the relevant miRNAs of differential mRNAs,as well as the IncRNAs associated with these miRNAs.then construct a ceRNA interaction network map.In addition,the protein interaction network map(PPI)of DEGs was constructed,and GO and KEGG enrichment analysis were performed on DEGs,and the main biological processes,molecular functions,cytological components and involved signaling pathways were selected.2.The Gepia database(data from TCGA)was used to find out the difference in the expression of TMCO1 gene in each tumor and the relationship with the survival rate of tumor patients,which can find a tumor with significant correlation with TMCO1.Then.the interaction protein network of TMCO1 was analyzed by String database,and then GO functional enrichment analysis and KEGG pathway enrichment analysis were performed on these interacting proteins.3.The effects of TMCO1 on the proliferation and migration of lung adenocarcinoma cells(A549 cell line)were preliminarily verified,including preparation of cell lines and validation of TMCO1 knockout,MTT cell proliferation assay,and scratch assay to verify cell migration.At the same time.Guben Jiedu Decoction was added to confirm its effect on the proliferation and migration of A549 cells.Result:1.Data analysis of lung adenocarcinoma patients showed that 932 differentially expressed mRNAs(DEGs)and 101 differentially expressed miRNAs were screened respectively.A ceRNA network with 17 key mRNAs.28 key miRNAs and 60 related IncRNAs was constructed.The intersection of DEGs and differentially expressed miRNA target genes found 104 common genes,which are both DEGs associated with lung adenocarcinoma metastasis and may be regulated by differentially expressed miRNAs associated with lung adenocarcinoma metastasis.Genes with a high degree of association can be considered as key genes in LUAD.GO analysis results showed that these DEGs were mainly involved in cell-substrate adhesion,ameboidal-type cell migration,extracellular structure organization,cell junction organization and other biological processes(BP),enriched in focal adhesion,cell-substrate adherens junction,cell-cell junction,actin cytoskeleton and other cellular components(CC).and involved in growth factor binding,transmembrane receptor protein kinase activity,actin binding.glycosaminoglycan binding and other molecular functions(MF).KEGG analysis results show that these DEGs were mainly involved in ECM-receptor interaction,focal adhesion,axon guidance,PI3K-Akt,cGMP-PKG and other signaling pathways,which indicate that DEGs are closely related to pathways common in cancer,so these DEGs may play an important role in the development of lung cancer.2.TMCO1 gene analysis showed that TMCO1 gene has significantly high expression in BRCA(invasive breast cancer),COAD(colon adenocarcinoma).DLBC(diffuse large B-cell lymphoma),ESCA(esophageal cancer),GBM(polymorphic glioblastoma)Tumor),LGG(low brain glioma),LIHC(hepatocellular carcinoma),LUAD(lung adenocarcinoma).LUSC(lung squamous cell carcinoma),OV(ovarian serous cystadenoma),PAAD(pancreatic cancer),in PRAD(prostate cancer),READ(rectal adenocarcinoma).SKCM(skin melanoma),STAD(stomach adenocarcinoma),and THYM(thymoma).The expression level of TMCO1 was found to be significantly correlated to the survival rate of CESC(cervical squamous cell carcinoma and adenocarcinoma),HNSC(head and neck squamous cell carcinoma),KIRC(kidney clear cell carcinoma).LGG(low brain glioma),and LUAD(lung adenocarcinoma).There were 10 proteins Directly interact with TMCO1,including UQCRQ,SSR3,SLC25A3.PPP2R1A,HSD17B12,EMC4,CYC1,CDKN2B,ATP1A1 and AFG3L.GO analysis results show that these interactive proteins are mainly involved in oxidative phosphorylation.mitochondrial ATP synthesis coupled electron transport,respiratory electron transport chain,purine nucleotide metabolism and other biological processes(BP).enriched in protein phosphatase type 2A complex,respiratory chain.inner mitochondrial membrane protein complex and other cellular components(CC),and involved in protein phosphatase regular activity,electron transfer activity,ubiquinol-cytochrome C reductase activity and other molecular functions(MF).KEGG analysis results show that TMCOl is mainly enriched Signaling pathways such as Parkinson’s disease,oxidative phosphorylation,Alzheimer’s disease,non-alcoholic fatty liver disease.PI3K-Akt and other pathways.3.The results of MTT assay showed that the inhiibition rate of KD#29 and KD#33 cell lines on proliferation of A549 cells was significantly different from that of Control#7(P<0.05),indicating that TMCO1 gene knockdown can inhibit the proliferation of A549 cells,in the Control#7 cell line group,the inhibition rate of cell proliferation in each drug-containing serum group was significantly different from that in Control#7 control group(P<0.05),and the difference between 1%and 2%Chinese medicine groups and the Western medicine group was not statistically significant,indicating that low-concentration Chinese medicine-containng serum has more obvious inhibitory effect.In the KD#29 and KD#33 cell line groups,the inhibition rate of cell proliferation in each drus-containing serum group was compared with the control group.There was no statistical significance,indicating that Western medicine and Chinese medicine did not inhibit proliferation of A549 cells after TCMCO1 knockout.4.The results of the scratch test showed that the healing rates of KD#29 and KD#33 were significantly lower than that of Control#7,indicating that knockdown of TMCO1 gene can inhibit the proliferation of A549 cells to some extent,compared with Control#7 group,the healing rate of Control#7 Western medicine group and Control#7 Chinese medicine group was also reduced,and there was no significant difference between Chinese medicine and Western medicine.It can be seen that Chinese medicine has a certain inhibitory effect on the migration of A549 cells.Conclusion:1.In this study,we systematically analyzed the gene expression profiles of patients with LUAD metastasis,and identified the differentially expressed mRNA,miRNA and related ceRNA interaction networks of LUAD metastasis.Functional annotation and pathway enrichment of DEGs were done.The results of this study provide a useful set of reference indicators for the future study of the molecular mechanisms of luns adenocarcinoma and the presentation of new biomarkers.2.In this study,a systematic analysis of TMCO1 gene vas done.It is found to be highly expressed in most tumors and was siniificantly associated with the survival rate of some tumor patients.Functional annotation and pathway enrichment of TMCOl interacting proteins were also performed.The results of this study preliminarily prove that TMCO1 plays an important role in the occurrence and development of tumors and provides new possible molecular targets for clinical anticancer treatment.3.In this study.MTT and cell scratch assays were performed using wildtype and TMCO1 knockout A549 cell lines and Gubenjiedu Chinese Decoction was added.The results showed that TMCO1 can regulate the proliferation and migration of A549 cells to some extent.GubenJiedu Decoction has a certain intervention effect on the proliferation and migration of A549 cells.