Non-small Cell Lung Cancer Associated MRNA Expression Signature, A Bioinformatics Analysis And Biological Effect And Mechanism Of CENPF

Part ?ABSTRACT ? NON-SMALL CELL LUNG CANCER ASSOCIATED MRNA EXPRESSION SIGNATURE:A BIOINFORMATICS ANALYSIS AND CLINICAL SIGNIFICANCEObjective:Lung cancer is one of the most lethal malignancies,and it is still one of the most common malignancies in China and even in the world,and non-small cell lung cancer(NSCLC)accounted 85%of lung caner.Most lung cancers can be removed in early stage.But even patients with stage I lung cancer have a 20%to 39%chance of recurrence within five years,leading to a poor prognosis.In recent years,the rapid development of genome sequencing technology has deepened people's understanding of tumor heterogeneity.At present,many studies have found the value of tumor molecular markers in cancer diagnosis,treatment and prognosis evaluation.However,the clinical application value of lung cancer-related molecular markers has not been confirmed.The main objectives of this study are as follows:1.screening the abnormal expression genes in lung cancer tissues to find the differential genes with high sensitivity and specificity for lung cancer diagnosis;2.looking for differential genes that can indicate the prognosis of lung cancer.The study of lung cancer tissue samples was designed in two stages.In the discovery phase,mRNAs related differential expressions of NSCLC were obtained by analyzing the mRNAs microarray data of tissues in the database.The differential mRNAs were analyzed by bioinformatics,GO analysis and Pathway analysis.RRA method was used to sequence differential mRNAs,and 32 differential genes were screened out,among which 15 were upregulated and 17 were down-regulated.In our study,lung cancer and adjacent tissues samples and clinical information of patients with adenocarcinoma and squamous cell carcinoma in TCGA database were collected to further verify the differential expression of candidate mRNAs and analyze the relationship between the differential mRNAs and clinical indicators.Methods:1.Three databases,Gene Expression Omnibus(GEO),ArrayExpress and Pubmed database,were used for literature retrieval,and the gene expression profiles meeting the inclusion criteria were obtained.The up-regulated and down-regulated genes were obtained by R language and RRA method(FDR<0.05).2.GO pathway analysis and KEGG pathway analysis were conducted online with GeneCodis network tools for the screened differential genes.3.The TCGA database(Cancer Genome Atlas)was used to search for 32 differential genes and biological verification was carried out.The mRNA expression of 32 differential genes in lung adenocarcinoma and paracancerous tissues,lung squamous cell carcinoma and paracancerous tissues and their clinical significance were analyzed.4.ROC analysis was performed on the differential mRNAs.5.The relationships between differential genes and clinical stage,lymph node metastasis of lung cancer were analyzed.6.K-M survival analysis was performed on the differential mRNAs.Results:1.Through searching,22 published gene expression profiles met the inclusion criteria of the study(table 1).Through RRA analysis,we found 643 differential genes,including 315 up-regulated genes and 328 down-regulated genes.According to the size of FDR value and the number of gene expression profiles involved,the first 15 genes were selected from 315 up-regulated genes and the first 17 genes were selected from 328 down-regulated genes.All of these genes have been reported in at least 8 gene expression profiles(table 2).2.315 up-regulated genes and 328 down-regulated genes were screened for GO pathway analysis and KEGG pathway analysis.GO pathway analysis is divided into biological processes,cell components and molecular functions.The results showed that biological processes were related to mitotic cell cycle,cell division,cell cycle,mitosis and multicellular organismal development.The cell components were enriched in cytoplasm,extracellular region,plasma membrane,extracellular space and cytosol.The molecular functions were enriched in protein binding,ATP binding,calcium ion binding,nucleotide binding and identical protein binding.KEGG pathway analysis showed that these genes were involved in cell cycle,ECM-receptor interaction,protein digestion and absorption,focal adhesion,and p53 signaling pathways(table 3,figure 1).3.32 differential genes in adenocarcinoma and adjacent tissues were analyzed.RNA-seq data of adenocarcinoma were downloaded from TCGA database.There were 58 adjacent tissues and 511 tumor tissues,among which 56 pairs of adenocarcinoma and adjacent tissues were included.Through 56 pairs of adenocarcinoma and paracancer tissues,the differential expressions of these 32 genes in lung cancer and paracancer tissues were statistically significant.Box plots were used to plot the differences between the above genes in lung cancer and adjacent tissues(table 5,figure 2).32 differential genes in squamous cell carcinoma and adjacent tissues were analyzed.RNA-seq data of squamous cell carcinoma were downloaded from TCGA database.There were 51 adjacent tissues and 502 tumor tissues,among which 51 pairs of squamous cell carcinoma and adjacent tissues were included.Through the verification of 51 pairs of squamous cell carcinoma and adjacent tissues,the differential expressions of these 32 genes in lung squamous cell carcinoma and corresponding adjacent tissues were statistically significant.Box plots were used to plot the differences between the above genes in lung cancer and adjacent tissues(table 6,figure 3).4.ROC analysis was conducted for mRNAs expression of 32 differential genes.In adenocarcinoma,the AUC area of the single mRNA was PYCR1,and the AUC was 0.996(figure 4A).In squamous cell carcinoma,the AUC areas of the single mRNA were TEK,CLIC5,CDH5,and 0.999(figure 4B).5.498 adenocarcinoma specimens and 485 squamous cell carcinoma specimens from TCGA database were selected to analyze the correlation between the above genes in tumor staging and lymph node metastasis.Among the 32 genes,TOP2A,ANLN,KIAA0101,UBE2T,TPX2,CCNB1,CENPF were highly expressed,while FAM107A,CLIC5,AGER,ADAMTS8,TCF21 and LIMS2 were poorly expressed in advanced lung adenocarcinoma(?,? VS ?,?)(table 7).UBE2T,CENPF were highly expressed,while AGER and LIMS2 were poorly expressed in advanced lung squamous cell carcinoma(?,? VS ?,?)(table 8).We used independent samples T test to study the correlation between differential genes and lymph node metastasis of lung cancer.The results showed that SPP1,TOP2A,ANLN,MMP11,KIAA0101,UBE2T,PYCR1,PAFAH1B3,TPX2,CCNB1,CENPF,FHL1,CLIC5,AGER and ADAMTS8mRNA expression levels were correlated with lymph node metastasis of lung adenocarcinoma(table 9).TOP2A,ANLN,CENPF,GPM6A,FHL1,FAM107A?CA4,S1PR1,FABP4,CLIC5,ADAMTS8,TCF21,HIGD1B and FCN3mRNA expression levels were closely related to lymph node metastasis of lung squamous cell carcinoma(table10).6.Survival data were downloaded from TCGA,and the survival data and differential gene expression were integrated together for the following survival analysis.In addition,484 adenocarcinoma specimens and 474 squamous cell carcinoma specimens from TCGA were selected to conduct K-M survival analysis and survival curve of the above 32 genes.By analysis,Kaplan-Meier analysis were used to construct a new independent cohort of 484 lung adenocarcinom and 474 lung squamous cell carcinoma,350 lung adenocarcinoma and 324 lung squamous cell carcinoma from the TCGA database.The results showed that high expression of SPP1,TOP2A,ANLN,KIAA0101 UBE2T,TPX2,CCNB1,CENPF and low expression of CA4,AGER had shorter median survival of lung adenocarcinoma(table 11,figure 5).High expression of SPP1,low expression of CLIC5,CA4,AGER,TCF21,HIGD1B,FCN3 were found to have shorter median survival of lung squamous cell carcinoma(table 13,figure 7).In addition,high expression levels of ANLN,KIAAA0101,OCIAD2,UBE2T,PAFAH1B3,TPX2,CCNB1 are closely related to shorter RFS of lung adenocarcinoma(table 12,figure 6),while high expression level of KIA0101 and low expression level of TCF21 are closely related to shorter RFS of lung squamous cell carcinoma(table 14,figure 8).Conclusion:1.The 32 genes showed significant differences in lung cancer and paracancerous tissues.2.All the 32 genes showed significant differences in ROC analysis(P<0.05),showing certain diagnostic efficacy.3.The high expression of TOP2A,ANLN,KIAA0101,UBE2T,TPX2,CCNB1 and CENPF were significantly correlated with lymph node metastasis,clinical stage and prognosis of lung adenocarcinoma.Part?Abstract ? THE EFFECTS AND MECHANISMS OFCENPFIN LUNG ADENOCARCINOMA Objective:In the first part of the study,Kaplan-Meier survival analysis was performed on 483 adenocarcinoma samples from the TCGA database and we found that the survival time of the high-expression group ofSPPTOPAANNKIAA101UBE2T TPX2.CCNBl and CENPF was sienificantly lower than that of the low-expressior group. Among the above genes, excluded these genes whose gene function in lung cancer had been studied in the previous literature.whileTPX2UBE2T and CENPF have not been studied in lung adenocarcinoma yet.We further conducted MTT assay of these three genes, and found that the difference of CENPF was more significant and then CENPF was selected for the following functional experiment. CENPF has been found to be elevated in a variety of tumors and in some tumors it has been associated with clinical indicators such as staging and prognosis. We used cytofunctional and animal experiments to verify the role ofCENPF inlung adenocarcinoma and to explore the mechanism of action Methods:1.The impact of TPX2UBE2T and CENPF expression on cell proliferation were investigated by MTT assay, and then CENPF was selected.2.Expression levels of CENPF were investigated in public online databasethe prognosis of CENPF in LUAD was assessed by Kaplan-Meier analysis.3.Ouantitative reverse transcription-polymerase chain reaction(qRT-PCR)was performed using 13 matched pairs of clinical LUAD tissue samples.4. The expression abundance of CENPF in four commonlyused lung cancer cell lines was detected bygRT-PCR. and A549 cell line was selected in combination with MTT assay for subsequent tests.5. The impact of CENPF expression on cellproliferationcelleveleapoptosis colony formation was investigated by MTT assayflow cytometric analysis and colony formation assay.respectively.6.Experimental xenograft lung cancer model of nude mice armpit ofright forelimb to determine the effect of CENPF on LUAD tumorigenesis. Results:lThe results ofMTT assay ofTPX2UBE2T and CENPF genes showed a more significant difference in CENPF,and then CENPF was selected for the following functional experiment.2.CENPF mRNA expression was significantly elevated in LUAD tissues compared with adjacent non-tumor lung tissues in GEPIA(P<0.001).Up-regulated CENPF was significantly positively correlated with pathological stagerelapse-free survival(RFS) and overall survival(OS) of LUAD patients.3.qRT-PCR results showed that in 13 matched pairs ofLUAD tissues and adjacent non-tumor tissues,CENPF was highly upregulated in LUAD(P=0.010)which was consistent with the microarray analysis results.4.qRT-PCR results showed that CENPF expression was up-regulated in LUAD cel lines (A549 and H1299) compared with that in small cell lung cancer cell lines (H446 and H69).5.CENPF knockdown greatly suppressed A549 cell proliferation,inducedS phase arrest, promoted apoptosis, decreased colony numbers of LUAD cells.6.Furthermore, knockdown ofCENPF significantly inhibited the tumor growth of the LUAD cells in an experimental xenograft lung cancer model of nude mice armpit of right forelimb. Conelusion:1.CENPF knockdown inhibits the proliferation invasion and metastasis of lung cancer A549 cells, and promotes cell apoptosis.2.CENPF is highly expressed in LUAD tissues and is related to the prognosis of LUAD patients, thus playing an important regulatory role in the occurrence and development of LUAD3.CENPF plays a cancer-promoting effect and it may serve as a potential biomarker of prognostic relevance and a potential therapeutic target for LUAD.