| Introduction: The latest report released by the International Agency for Research on Cancer has stated that lung cancer remains the most common and deadly form of malignancy.In general,surgery is the best option for treating patients with early stage disease because the five-year survival rate of pathological stage I non-small cell lung cancer(NSCLC)after lobectomy is 45%–65%.However,approximately 70% of patients are diagnosed in the late stage of the disease;therefore,the five-year survival rate of these patients is only 16.38%.Lung adenocarcinoma(LUAD)is the most common type of NSCLC,accounting for approximately 40% of cases.Therefore,the focus of the present study was limited to the complex molecular mechanisms leading to the onset and poor prognosis of LUAD.Dysregulation of the cell cycle result in increased cell proliferation,and the abnormal expression of cell cycle regulators can lead to tumor formation.Various chemotherapeutic agents have been developed to target the cell cycle.For example,cisplatin is one of the most successful anticancer drugs used to nonspecifically block the cell cycle.By focusing on the complex gene networks that cause dysregulation of cell cycle regulators,a potential strategy for the treatment of lung cancer could be developed.Previous studies have reported that noncoding RNAs,such as long noncoding RNAs(lnc RNAs)and micro RNAs(mi RNAs)are involved in cell cycle processes.Furthermore,it has been widely reported that lnc RNAs functioning as the competing endogenous RNAs(ce RNAs)could regulate cancer by sponging mi RNAs.Despite the rapid evolution of genomic technologies and analytical tools,the identification of novel lnc RNA-related ce RNA networks affecting the cell cycle and ultimately influencing LUAD remains challenging.Therefore,the present study aimed to investigate lnc RNA expression profiles of The Cancer Genome Atlas(TCGA)database via complex bioinformatics analysis to identify novel lnc RNAs and related biological functions,which initially identified that lnc RNA RAET1 K was significantly upregulated.Furthermore,we revealed that the upregulated expression of lnc RNA RAET1 K was correlated with poor prognosis in LUAD patients and facilitated cell cycle arrest at the G1 phase by functioning as a ce RNA to upregulate cyclin E1(CCNE1).Materials and Methods: The RNA sequence(RNA-seq)data of LUAD and corresponding clinical information were downloaded from the TCGA database.The gene symbol and type were converted from transcript IDs of RNA-seq data with the Ensembl datasets.The edge R package was used to normalize raw data sets and identify differentially expressed genes(DIFF-genes).The R package for weighted gene correlation network analysis(WGCNA)was used to build co-expression networks and define the gene modules.Database for Annotation,Visualization,and Integration Discovery(DAVID)and Clue GO plug-in of Cytoscape were performed to identify the biological processes(BPs)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway ontologies of the genes in blue module.A nomogram was generated using a multivariate Cox regression model to evaluate the potential prognostic signature of lnc RNA RAET1 K for OS of LUAD patients by nomogram and survival packages of R language.According to the gene expression level,GSEA was performed to identify the BPs and biological functions of lnc RNA RAET1 K expression level clustered into the modules.The ce RNA expression module of lnc RNA RAET1K/mi R-135a-5p/CCNE1 was constructed based on the correlation coefficient and the binding sites between the mi RNAs and target genes identified by mi Rcode database.To investigate the validity and potential biological mechanisms of the effects of the RAET1K/mi R-135a-5p axis on CCNE1 expression,in vitro experiments with A549 and H1299 cells were performed.The lnc RNA RAET1 K overexpression lentivirus and a negative control(NC)lentivirus were transfected in A549 and H1299 cells,while also transiently transfected with a group of mi R-135a-5p mimics and inhibitors.Real-time PCR and Western blot analysis were performed to evaluate the relative gene expression of CCNE1 based on lnc RNA RAET1K/mi R-135a-5p compared with NC group.Dual-Luciferase Reporter assay was performed the interaction of co-transfected lnc RNA RAET1 K and mi R-135a-5p luciferase plasmids in A549 and H1299 cells.The distribution of cell cycle phase was analyzed by Flow Cytometry Analysis,while the counting kit-8(CCK-8)and cell colony formation assay were for the cell proliferation in the co-transfected lnc RNA RAET1 K and mi R-135a-5p LUAD cell lines.Results: The LUAD database included 564 tissue samples detected 991 DIFF-genes in both early and advanced clinical stages.WGCNA was performed for 991 DIFF-genes detected that four modules highly correlated with LUAD.The genes in the blue and brown modules were significantly and positively correlated with advanced stage disease,whereas the genes in the blue module showed strong associations with each other and were chosen for subsequent analyses.The GO functional enrichment analysis of 203 DIFF-genes in the blue module were performed using DAVID and KEGG pathways using Clue GO enriched in the cell cycle pathway.Cox proportional hazard and Kaplan-Meier analyses of the 12 lnc RNA and 191 m RNA in the blue module were performed,141 highly expressed hub genes were significantly associated with poor prognosis.RAET1K(HR = 1.428;95% CI = 1.052–1.939;P = 0.022)was the only lnc RNA among the 141 hub genes that was significantly upregulated in tumor tissue compared with normal tissue.A nomogram was constructed to predict 1-and 3-year survival rates in patients with LUAD by showing the risk score of clinical stage,age,sex,and RAET1 K expression level,supported the predictive prognostic signature of lnc RNA RAET1 K in LUAD OS.The GSEA results also indicated that among the genes in the blue module,lnc RNA RAET1 K expression was enriched in the cell cycle.The interaction of the ce RNA network and lnc RNA RAET1 K was combined with the expressional correlation and target sites,lnc RNA RAET1 K may function as a sponge to absorb mi R-135a-5p to modulate CCNE1 expression.To investigate the synergistic effect of the lnc RNA RAET1K/mi R-135a-5p axis on CCNE1 expression,A549 and H1299 cells were transfected with lentiviral vectors stably overexpressing lnc RNA RAET1 K and an empty control and also mi R-135a-5p mimics,an inhibitor,an NC,or an NC inhibitor.Real-time PCR and Western blot analysis showed that lnc RNA RAET1 K inhibited CCNE1 m RNA expression probably via the downregulation of mi R-135a-5p expression.The luciferase reporter assay was performed to validate the interactions between mi R-135a-5p and lnc RNA RAET1 K 3’UTR in A549 and H1299 cells.Cell cycle distributions were investigated following the co-transfection of lnc RNA RAET1 K and mi R-135a-5p mimics or an inhibitor in A549 and H1299 cells,the results showed that lnc RNA RAET1 K overexpression with decreased mi R-135a-5p could synergistically arrest the A549 and H1299 cells in the G1 phase and hinder cell cycle transformation from the G1 to S phase.The CCK-8 and cell colony formation assays indicated that lnc RNA RAET1 K overexpression with decreased mi R-135a-5p promoted cell proliferation ability compared to the NC group in A549 and H1299 cells.Conclusions: 1.WGCNA is a systematic biological method to identify synergistically altered gene clusters,which can explore the crucial molecular mechanism and candidate biomarkers involved in LUAD tumorigenesis.2.The lnc RNA RAET1 K,which is significantly upregulated in LUAD tissue compared with normal tissue of the TCGA database,can be used as an independent predictive prognostic signature of lnc RNA RAET1 K in LUAD OS,and may play a malignant biological role by affecting the cell cycle.3.In the LUAD lnc RNA RAET1 K acted as a ce RNA and increased the expression of CCNE1 by directly competing with mi RNA-135a-5p,which influenced the function of the cyclin E1 protein.4.Furthermore,the lnc RNA RAET1K/mi R-135a-5p/CCNE1 axis as ce RNA network,which accumulated of cells arrested at the G1/S phase boundary drives,was influenced cell cycle progression and promoted cell proliferation ability in LUAD onset and progression. |
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