Study On The Molecular Landscape Of Xuanwei Female Lung Adenocarcinoma And The Biological Effect Of CENPM In Lung Adenocarcinoma

Background and Objective:Lung cancer is one of the malignant tumors with the highest morbidity and mortality in humans,and its morbidity trend has gradually increased in recent years.In China,lung cancer is the main cause of cancer-related deaths,and its morbidity and mortality are increasing year by year.It is urgent to prevent and treat this cancer across the country.The common malignant tumor in Xuanwei,Yunnan Province,is lung cancer,and its morbidity and mortality rate ranks first in the country.In recent years,Xuanwei's lung cancer has been deeper researched.It has been revealed that the use of stoves,coal type,industrial pollution,smoking,eating habits,and genetic factors are essential reasons for the high incidence of lung cancer in Xuanwei.Although the research on Xuanwei lung cancer has made great progress,the prevention and diagnosis of Xuanwei lung cancer is still a difficult problem.With the continuous and rapid development of science and technology,especially the continuous innovation of high-throughput sequencing technology and gene chip technology,precision medicine has made significant progress in the field of tumor diagnosis and treatment.Therapies such as radiotherapy,chemotherapy,targeted therapy,and immunotherapy are widely used in clinical practice.Consequently,more and more relevant driver genes that promote and inhibit the occurrence and development of lung cancer have been discovered.Moreover,the signaling pathways regulating the biological phenotypes of lung cancer are constantly revealed.Under the combination of high-throughput sequencing,bioinformatics analysis,cell in vitro experiments,molecular biology techniques,and nude mouse tumor formation experiments,in this study,the Xuanwei female lung adenocarcinoma genome and immunological profile are comprehensively characterized,the differentially expressed genes and pathways with have research value are explored,and the biological function and mechanism of cyclin CENPM are investigated,so as to reveal clinically potential biomarkers for the treatment of lung adenocarcinoma.Section ?:Study on the Molecular Landscape of Xuanwei female LungAdenocarcinomaObjective:The Xuanwei area is a hot spot of lung adenocarcinoma in females in China,which is strongly associated with the consumption of local smoky coal.Comprehensive characterization of its genomic and immunological landscapes is crucial for cancer prevention and the development of precision therapy.Methods:This study reports extensive genomic,transcriptomic,and immunological profiles of 117 Xuanwei female lung adenocarcinoma(XWFA),comprising 112 pairs of tumour-normal whole-exon sequencing(WES)profiles and 33 normal and 115 tumour mRNA-seq profiles.Finally,the study establishes a rat lung cancer model induced with local smoky coal.Results:EGFR mutations are detected in 52.68%of the tumour samples,followed by TP53(41.07%),RBM10(10.71%),and KRAS(7.14%).It is suggested that the mutation spectrum and transcriptome pathway alterations show many similarities between XWFA and smoking lung tumour samples.Samples from the TRU-W subcluster highly express canonical Wnt signaling pathway genes and are lowly enriched with immune infiltration,which should be weighed in further immunotherapy.The rat lung cancer model reveals stepwise pathway alterations along with the immune infiltration status at the tumour initiation stage.Conclusion:These data elucidate a comprehensive genomic landscape of XWAF and establish a foundation for further investigation of its molecular pathogenesis.Section ?:Bioinformatics analysis of CENPM in lung adenocarcinoma Objective:Lung cancer is one of the malignant tumors with the highest morbidity and mortality in humans,and its morbidity trend has gradually increased in recent years.To further explore the molecular mechanism of the occurrence and development of lung adenocarcinoma,we used bioinformatics tools to screen and analyze the differentially expressed genes in lung adenocarcinoma,so as to explore genes and pathways that may have research value.Method:In this study,the R language package was used to screen the differentially expressed genes on the GSE lung adenocarcinoma chip dataset downloaded from the TCGA-LUAD and GEO(Gene Expression Omnibus)databases.Subsequently,the enrichment analysis for the selected differential genes was conducted regarding functions and pathways.Then,the expression difference verification,clinicopathological characteristics,and patient survival prognosis analysis were performed in the database and patient samples.Finally,the co-expression analysis,enrichment analysis,and protein interaction network analysis of differential genes are carried out.Results:In lung adenocarcinoma,there are 471 significantly up-regulated genes and 919 significantly down-regulated genes.Differentially expressed genes are significantly enriched in pathways related to cell cycle regulation,such as spindle checkpoints and centromere DNA replication.Compared with normal control tissues,CENPM expression was significantly up-regulated in lung adenocarcinoma.Its expression level is significantly related to the clinical TNM staging and classification of patients with lung adenocarcinoma.The high expression level of CENPM is a significant risk factor for the prognosis of patients with lung adenocarcinoma.As indicated in the differential co-expression analysis,715 genes were significantly positively correlated with CENPM,and 353 genes were significantly negatively correlated with CENPM.CENPM differentially co-expressed genes are significantly enriched in mTORC1 regulation and PI3K-AKT-mTOR signal transduction pathway.The protein interaction network and enrichment analysis of CENPM confirmed that CENPM is closely related to other cell cycle genes.Conclusions:There are a large number of differentially expressed genes in lung adenocarcinoma,and they are significantly enriched in signal pathways related to cell cycle regulation,such as spindle checkpoints and centromere DNA replication.CENPM is abnormally highly expressed in lung adenocarcinoma owing to the clinicopathological characteristics and survival prognosis of patients.This indicates that CENPM may play a role in promoting cancer in lung adenocarcinoma and may be related to tumor cell proliferation and disease progression.Besides,CENPM differentially co-expressed genes are significantly enriched in mTORC1 regulation and PI3K-AKT-mTOR signal transduction pathway and significantly interact with other cyclins in the CCAN protein family.It demonstrates that CENPM and its protein family may play an essential role in the PI3K-AKT-mTOR signaling pathway.Section ?:Effect of CENPM on biological behavior of lung adenocarcinoma and its mechanismObjective:To explore the effects of up-regulation and down-regulation of CENPM on the proliferation,migration,invasion,and apoptosis of lung cancer cell lines A549,95D,NCI-H1975,and PC-9.Besides,the biological functions of CENPM in vivo are investigated through the results of tumor growth curve and immunohistochemistry.Moreover,biometric analysis is conducted to reveal signal pathways related to CENPM,detect the downstream signaling pathway molecules in animal models,and clarify the molecular mechanism that CENPM mediates the biological phenotypes of lung adenocarcinoma through signaling pathways.Method:(1)LV-sh CENPM and CENPM OE are transfected into lung cancer cells A549,95D,NCI-H1975,PC-9.Using qPCR and Western blot,the transfection efficiency is measured,and the proliferation,migration,invasion,and apoptosis of successfully transfected lung cancer cells are detected.(2)LV-sh CENPM and LV-sh NC are transfected into lung adenocarcinoma cells A549,and the transfection efficiency is determined by qPCR.A nude mouse model of lung adenocarcinoma is established by subcutaneous tumor implantation.The body weight and tumor size of nude mice are observed and measured every five days.After 32 days,they are put to death,weighed and photographed.Western blot and immunohistochemistry are used to detect the expression of CENPM,MCM2,PCNA,E-cad,and Vimentin in the tumors of each group of nude mice.(3)The R language package is employed to screen the differentially expressed genes on the GSE lung adenocarcinoma chip dataset downloaded from the TCGA and GEO(Gene Expression Omnibus)databases.Subsequently,enrichment analysis is conducted on the selected differential genes regarding functions and pathways.Then,the expression difference verification,clinicopathological characteristics,and patient survival prognosis analysis were performed on the database and patient samples.Next,the co-expression analysis,enrichment analysis,and protein interaction network analysis of differential genes are carried out.Finally,Western blot is used to verify the downstream signaling pathway molecules.Results:(1)After transfection of LV-sh CENPM and CENPM OE,CENPM was successfully down-regulated and up-regulated in lung cancer cells A549,95D,NCI-H1975,and PC-9.(2)Down-regulating CENPM can inhibit the proliferation of lung adenocarcinoma cells 95D and NCI-H1975;up-regulating CENPM can promote the proliferation of lung adenocarcinoma cells A549 and PC-9.(3)Down-regulating CENPM can inhibit the migration and invasion of lung adenocarcinoma cells 95D and NCI-H1975;up-regulating CENPM can promote the migration and invasion of lung adenocarcinoma cells H1299 and SPC-A1.(4)Down-regulating CENPM can promote the apoptosis of lung adenocarcinoma cells 95D and NCI-H1975;up-regulating CENPM can inhibit the apoptosis of lung adenocarcinoma cells A549 and PC-9.(5)Down-regulating CENPM can inhibit the expression of p-mTOR in lung adenocarcinoma cells,and up-regulating CENPM can promote the expression of p-mTOR in lung adenocarcinoma cells.However,the up-regulation and down-regulation of CENPM had no significant statistical significance on the expression of mTOR in lung cells.(6)The volume of subcutaneous tumors in nude mice in the LV-sh CENPM group is significantly smaller than that in the LV-sh NC group.(7)Western blot and immunohistochemical detection results demonstrate that the expression of MCM2,PCNA,and Vimentin in the LV-sh CENPM group is significantly lower than that in the LV-sh NC group,while the expression of E-cad is significantly higher than that in the LV-sh NC group.(8)GSEA analysis suggests that CENPM is closely related to the PI3K/AKT/mTOR signaling pathway.Western blot verifies that the expression of p-mTOR in the LV-sh CENPM group is significantly lower than that in the LV-sh NC group while there is no significant change in the expression of mTOR.Conclusion:CENPM can promote lung cell proliferation,migration,and invasion,inhibit their apoptosis,and promote the tumorigenic ability of lung adenocarcinoma cells in nude mice.This indicates that CENPM is likely to play a role in promoting cancer in lung cancer.CENPM is closely related to the PI3K/AKT/mTOR signaling pathway.Its mechanism of affecting the biological behavior of lung cancer cells may be achieved by regulating and promoting the expression of p-mTOR.