| Background and Objective(s):The detection rate of multiple primary lung cancers(MPLCs)is increasing year by year,and with the wide application of high-throughput sequencing,molecular genetic characteristics are expected to help identify the relationship between multiple cancer foci.This study aims to bioinformatically analyze the gene expression differences between simultaneous multiple primary lung adenocarcinoma and single primary lung adenocarcinoma by whole transcriptome sequencing,explore the effects of differential genes on biological functions such as proliferation,migration and invasion of lung adenocarcinoma cells,analyze the possible mechanisms,and provide new ideas for the diagnosis of MPLCs.Methods:1.Cancer tissue specimens from patients with synchronous multiple primary lung adenocarcinoma(s MP-LUAD)who underwent surgery between January 2021 and November 2021 and single primary lung adenocarcinoma(SP-LUAD),transcriptome sequencing using NGS technology,and bioinformatics software for differentially expressed gene identification,GO function enrichment,and KEGG pathway enrichment.2.Screening of differentially expressed genes: genes were classified into low,medium and high expression groups according to their FPKM values.SMP-LUAD and SP-LUAD samples with FPKM values falling in the two expression groups were used as the differentially expressed genes to be selected,and the top 5 differentially expressed genes were selected as the target genes;validated at the m RNA level by q RT-PCR,target genes were screened by the P<0.05 criterion(m RNADUOX1).3.GO functional enrichment and KEGG pathway enrichment analysis were performed on m RNADUOX1 using bioinformatics software to screen for its possible involvement in biological processes or pathways.4.Effect of DUOX1 on the biofunctionality of lung adenocarcinoma cells4.1 The expression levels of DUOX1 in A549,H292,H1975 and H2347 were compared using Western blot and q RT-PCR,and the lung adenocarcinoma cell lines with the highest(H292)and lowest(A549)expression levels were screened as the tool cells for subsequent experiments,respectively.4.2 The DUOX1 overexpression lentiviral vector was constructed by CRISPR-SAM technology,and the infected cells were co-cultured with A549,and the stable strain was recorded as A549-p DUOX1 by puromycin screening,and the empty vector A549-NC was used as the control group;the DUOX1 knockdown lentiviral vector was constructed by RNAi technology,and the infected cells were co-cultured with H292,and the puromycin screening The stable strain was recorded as H292-Si,and the empty vector H292-NC was used as the control group;Western blot technique was used to detect the effect of A549 overexpression of DUOX1 and H292 knockdown of DUOX1 to determine whether the construction was successful.4.3 The effects of DUOX1 overexpression and knockdown on the proliferation ability of lung adenocarcinoma cells were detected by CCk8;the effects of DUOX1 overexpression and knockdown on the clonogenic ability of lung adenocarcinoma cells were detected by plate clone formation assay;the effects of DUOX1 overexpression and knockdown on the invasive ability of lung adenocarcinoma cells were detected by Transwell assay;the effects of DUOX1 overexpression and knockdown on the invasive ability of lung adenocarcinoma cells were detected by scratch assay.The effect of DUOX1 overexpression and knockdown on the migration ability of lung adenocarcinoma cells was examined by Transwell assay.5.Western blot to detect the effects of DUOX1 overexpression and knockdown on the expression of E-cadherin and vimentin,key proteins of epithelial mesenchymal transition.Results:1.Bioinformatics analysis showed that: 194 differentially expressed m RNAs were identified,22 were up-regulated and 172 were down-regulated;GO functional enrichment of up-regulated m RNA differentially expressed genes showed that the most significant enrichment in biological processes was in the c AMP response,the most significant enrichment in cellular composition was in membranes,and the most significant enrichment in molecular function was in protein binding;the GO functional enrichment of down-regulated m RNA differentially expressed genes showed that the most significant enrichment in biological processes was in extracellular matrix organization,the most significant enrichment in cellular composition was in membranes,and the most significant enrichment in molecular function was in protein binding;KEGG pathway enrichment analysis showed that the main enriched pathway for upregulated m RNA differentially expressed genes was the arrhythmogenic right ventricular cardiomyopathy pathway and for downregulated m RNA differentially expressed genes was the PI3K-Akt signaling pathway.2.The 5 differentially expressed m RNAs were 2 up-regulated m RNADUOX1,CACNA2D2 and 3 down-regulated m RNAGPX8,COL1A2,COL1A1;q RT-PCR results showed that the expression of m RNADUOX1 was higher in the s MP-LUAD group than in the SP-LUAD group(P<0.05),m RNACACNA2D2,GPX8,COL1A2,and COL1A1 in the s MP-LUAD group were not statistically different from those in the SP-LUAD group(P>0.05).3.The GO functional enrichment analysis of m RNADUOX1 showed that biological processes were enriched in 15 GO terms including regulation of wound healing,redox process and regulation of cell motility,which may be related to epithelial mesenchymal transition.4.Effect of DUOX1 on the biofunctional science of lung adenocarcinoma cells4.1 H292 with the highest expression of DUOX1 and A549 with the lowest expression were selected as the tool cells for the follow-up experiments.4.2 Western blot results showed that the expression of DUOX1 in the A549-p DUOX1 group was higher than that in the A549-NC group,suggesting successful overexpression of DUOX1 in A549;the expression of DUOX1 in the H292-Si group was lower than that in the H292-NC group,suggesting successful knockdown of DUOX1 in H292.4.3 Experiments on cellular biofunctions show: in the DUOX1 overexpression group,A549-p DUOX1 cells showed reduced proliferation,clonogenesis,invasion and migration ability compared to the A549-NC group(P<0.05);in the DUOX1 knockdown group,the proliferation,clonogenesis,invasion and migration abilities of H292-Si cells were stronger than those of H292-NC group(P<0.05).5.Western blot results show: overexpression of DUOX1 upregulates E-cadherin expression and downregulates vimentin expression in lung adenocarcinoma cells,suggesting that overexpression of DUOX1 may inhibit epithelial mesenchymal transition;knockdown of DUOX1 downregulated the expression of E-cadherin and upregulated the expression of vimentin in lung adenocarcinoma cells,suggesting that knockdown of DUOX1 may promote epithelial mesenchymal transition.Conclusion(s):1.The differential expression of genes in the transcriptome profiles of concurrent multiple primary lung adenocarcinomas and single primary lung adenocarcinomas suggests that transcriptome sequencing may provide a new approach to the diagnosis of multiple primary lung adenocarcinomas.2.Functional enrichment analysis of m RNADUOX1 suggests a possible association with epithelial mesenchymal transition and involvement in the malignant biological phenotype of multiple primary lung cancers.3.Overexpression of DUOX1 can inhibit the proliferation,clonal formation,invasion and migration of lung adenocarcinoma cells,and can inhibit the epithelial-mesenchymal transformation of lung adenocarcinoma cells,suggesting that DUOX1 plays a cancer-inhibiting role in lung adenocarcinoma and may be a potential molecular marker to assist in the diagnosis and treatment of lung adenocarcinoma. |
