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Using RNA sequencing to identify a putative lncRNA-associated ceRNA network in laryngeal squamous cell carcinoma.

ABSTRACT: Accumulating evidence indicates that lncRNAs can interact with miRNAs to regulate target mRNAs through competitive interactions. However, this mechanism remains largely unexplored in laryngeal squamous cell carcinoma (LSCC). In this study, transcriptome-wide RNA sequencing was performed on 3 pairs of LSCC tissues and adjacent normal tissues to investigate the expression profiles of lncRNAs, miRNAs and mRNAs, with differential expression of 171 lncRNAs, 36 miRNAs and 1709 mRNAs detected. Seven lncRNAs, eight mRNAs and three miRNAs were identified to be dysregulated in patients' tissues by using qRT-PCR. GO and KEGG pathway enrichment analyses were performed to elucidate the potential functions of these differentially expressed genes in LSCC. Subsequently, a ceRNA (lncRNA-miRNA-mRNA) network including 4631 ceRNA pairs was constructed based on predicted miRNAs shared by lncRNAs and mRNAs. Cis- and transregulatory lncRNAs were analysed by bioinformatics-based methods. Importantly, mRNA-related ceRNA networks (mRCNs) were further obtained based on potential cancer-related coding genes. Coexpression between lncRNAs and downstream mRNAs was used as a criterion for the validation of mRCNs, with the ZNF561-AS1-miR217-WNT5A and SATB1-AS1-miR1299-SAV1/CCNG2/SH3 KBP1/JADE1/HIPK2 ceRNA regulatory interactions determined, followed by experimental validation after siRNA transfection. Moreover, ceRNA activity analysis revealed that different activities of ceRNA modules existing in specific pathological environments may contribute to the tumorigenesis of LSCC. Consistently, both downregulated SATB1-AS1 and ZNF561-AS1 significantly promoted laryngeal cancer cell migration and invasion, indicating their important roles in LSCC via a ceRNA regulatory mechanism. Taken together, the results of this investigation uncovered and systemically characterized a lncRNA-related ceRNA regulatory network that may be valuable for the diagnosis and treatment of LSCC.

SUBMITTER: Lyu K

PROVIDER: S-EPMC7549682 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:ObjectiveThis study aims to construct a systematic mRNA-miRNA-lncRNA network to identify novel lncRNAs and miRNAs biomarkers for laryngeal squamous cell carcinoma (LSCC).MethodsThe mRNA, miRNA and lncRNA expression profiles of LSCC were obtained from Gene Expression Omnibus (GEO) database. The differentially expressed mRNAs, miRNAs and lncRNAs (DEmRNAs, DEmiRNAs and DElncRNAs) were screened between LSCC tissues and controls. Functional analysis of DEmRNAs, DEmRNAs targeted by DEmiRNAs and DEmRNAs targeted by DElncRNAs were respectively performed. The miRWalk, starbase and DIANA-LncBase were respectively used to predict DEmiRNAs-DEmRNAs, DElncRNAs-DEmRNAs and DElncRNAs-DEmiRNAs pairs. ceRNA network was built by DEmiRNAs-DEmRNAs and DElncRNAs-DEmiRNAs pairs. LncRNA subcellular localization was predicted using lncLocator. Using published The Cancer Genome Atlas (TCGA) and external datasets (GSE127165 and GSE133632), we also validated the expression of key DElncRNAs and DEmiRNAs in ceRNA network. The diagnostic and prognostic value of candidate genes was evaluated by ROC curve analysis and survival analysis, respectively.ResultsThere were 5 mRNA datasets, 3 miRNA datasets and 2 lncRNA datasets in this study. Totally, 2957 DEmRNAs, 61 DElncRNAs and 23 DEmiRNAs were identified. Functional analysis of DEmRNAs shows that they were significantly enriched in cancer-related pathways, such as DNA replication and extracellular matrix organization. There were 11 DEmiRNAs, 17 DElncRNAs and 967 DEmRNAs in the ceRNA network. Notably, up-regulated lncRNA DGCR5-down-regulated has-miR-338-3p/has-miR-139-5p pairs in this network were experimentally validated. Moreover, down-regulated AL121839.2, down-regulated LINC02147, up-regulated AC079328.2, up-regulated AC004943.2 and up-regulated HMGA2-AS1 were located in the cytoplasm. AL121839.2 and LINC02147 interacted with has-miR-1246. AC004943.2, AC079328.2 and HMGA2-AS1 targeted has-miR-3185, has-miR-3137 and has-miR-582-5p, respectively. Based on the TCGA and external datasets (GSE127165 and GSE133632), DGCR5 and AC004943.2 were significantly up-regulated while AL121839.2 and LINC02147, has-miR-338-3p, has-miR-139-5p and has-miR-582-5p were significantly down-regulated, which were consistent with our integration analysis. DGCR5, AL121839.2, LINC02147, AC004943.2, has-miR-338-3p, has-miR-139-5p and has-miR-582-5p could predict the occurrence of LSCC. Survival analysis suggested that only, AL121839.2 has potential prognostic value for LSCC.ConclusionThis study provided novel insights into the ceRNA network and uncovered novel lncRNAs and miRNAs with diagnostic value in LSCC.

Project description:A cutaneous squamous cell carcinoma (cSCC) derived from keratinocytes is the second most common cause of non-melanoma skin cancer. The accumulation of the mutational burden of genes and cellular DNA damage caused by the risk factors (e.g., exposure to ultraviolet radiation) contribute to the aberrant proliferation of keratinocytes and the formation of a cSCC. A cSCC encompasses a spectrum of diseases that range from recursor actinic keratosis (AK) and squamous cell carcinoma (SCC) in situ (SCCIS) to invasive cSCCs and further metastatic SCCs. Emerging evidence has revealed that lncRNAs are involved in the biological process of a cSCC. According to the ceRNA regulatory theory, lncRNAs act as natural miRNA sponges and interact with miRNA response elements, thereby regulating the mRNA expression of their down-stream targets. This study was designed to search for the potential lncRNAs that may become potential therapeutic targets or biomarkers of a cSCC. Considering the spirit of the study to be adequately justified, we collected microarray-based datasets of 19 cSCC tissues and 12 normal skin samples from the GEO database (GSE42677 and GSE45164). After screening the differentially expressed genes via a limma package, we identified 24 differentially expressed lncRNAs (DElncRNAs) and 3221 differentially expressed mRNAs (DEmRNAs). The miRcode, miRTarBase, miRDB and TargetScan databases were used to predict miRNAs that could interact with DElncRNAs and DEmRNAs. A total of 137 miRNA-lncRNA and 221 miRNA-mRNA pairs were retained in the ceRNA network, consisting of 31 miRNAs, 11 DElncRNAs and 155 DEmRNAs. For the functional analysis, the top enriched biological process was enhancer sequence-specific DNA binding in Gene Ontology (GO) terms. The FoxO signaling pathway, autophagy and cellular senescence were the top enrichment terms based on a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The combination of a STRING tool and Cytoscape software (plug-in MCODE) identified five core mRNAs and built a core mRNA-associated ceRNA network. The expression for five identified core mRNAs and their related nine lncRNAs was validated using the external dataset GSE7553. Finally, one lncRNA HLA-F-AS1 and three mRNAs named AGO4, E2F1 and CCND1 were validated with the same expression patterns. We speculate that lncRNA HLA-F-AS1 may sponge miR-17-5p or miR-20b-5p to regulate the expression of CCND1 and E2F1 in the cSCC. The present study may provide potential diagnostic and therapeutic targets for cSCC patients.

Project description:BackgroundLaryngeal squamous cell carcinoma (LSCC) is the most common squamous cell carcinoma. Though significant effort has been focused on molecular pathogenesis, development, and recurrence of LSCC, little is known about its relationship with the immune-related long non-coding RNA (lncRNA) pairs.MethodsAfter obtaining the transcriptome profiling data sets and the corresponding clinical characteristics of LSCC patients and normal samples from The Cancer Genome Atlas (TCGA) database, a series of bioinformatic analysis was conducted to select the differently expressed immune-related lncRNAs and build a signature of immune-related lncRNA pairs. Then, the effectiveness of the signature was validated.ResultsA total of 111 LSCC patients and 12 normal samples' transcriptome profiling data sets were retrieved from TCGA. 301 differently expressed immune-related lncRNAs were identified and 35,225 lncRNA pairs were established. After univariate Cox analysis, LASSO regression and multivariate Cox analysis, 7 lncRNA pairs were eventually selected to construct a signature. The riskscore was computed using the following formula: Riskscore = 0.95 × (AL133330.1|AC132872.3) + (-1.23) × (LINC01094|LINC02154) + 0.65 × (LINC02575|AC122685.1) + (-1.15) × (MIR9-3HG|LINC01748) + 1.45 × (AC092687.3|SNHG12) + (-0.87) × (AC090204.1|AL158166.1) + 0.64 × (LINC01063|Z82243.1). Patients were classified into the high-risk group (> 1.366) and the low-risk group (< 1.366) according to the cutoff value (1.366), which is based on the 5-year riskscore ROC curve. The survival analysis showed that the low-risk group had a better prognosis (P < 0.001). The riskscore was better than other clinical characteristics in prognostic prediction and the area under the curves (AUCs) for the 1-, 3-, and 5-year survivals were 0.796, 0.946, and 0.895, respectively. Combining age, gender, grade, stage, and riskscore, a nomograph was developed to predict survival probability in LSCC patients. Then, the riskscore was confirmed to be related with the content of tumor-infiltration immune cells and the model could serve as a potential predictor for chemosensitivity.ConclusionWe successfully established a more stable signature of 7 immune-related lncRNA pairs, which has demonstrated a better prognostic ability for LSCC patients and may assist clinicians to precisely prescribe chemo drugs.

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Using RNA sequencing to identify a putative lncRNA-associated ceRNA network in laryngeal squamous cell carcinoma.

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