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. 2022 Jul 7:12:809760.
doi: 10.3389/fonc.2022.809760. eCollection 2022.

Construction of a lncRNA-mRNA Co-Expression Network for Nasopharyngeal Carcinoma

Chunmei Fan  1   2   3 , Fang Xiong  1   2   3 , Yanyan Tang  1 , Panchun Li  4 , Kunjie Zhu  1 , Yongzhen Mo  5 , Yumin Wang  3 , Shanshan Zhang  3 , Zhaojiang Gong  4 , Qianjin Liao  1 , Guiyuan Li  1   5 , Zhaoyang Zeng  1   5 , Can Guo  1   5 , Wei Xiong  1   5 , He Huang  2
Affiliations

Construction of a lncRNA-mRNA Co-Expression Network for Nasopharyngeal Carcinoma

Chunmei Fan et al. Front Oncol. .

Abstract

Long non-coding RNAs (lncRNAs) widely regulate gene expression and play important roles in the pathogenesis of human diseases, including malignant tumors. However, the functions of most lncRNAs remain to be elucidated. In order to study and screen novel lncRNAs with important functions in the carcinogenesis of nasopharyngeal carcinoma (NPC), we constructed a lncRNA expression profile of 10 NPC tissues and 6 controls through a gene microarray. We identified 1,276 lncRNAs, of which most are unknown, with different expression levels in the healthy and NPC tissues. In order to shed light on the functions of these unknown lncRNAs, we first constructed a co-expression network of lncRNAs and mRNAs using bioinformatics and systematic biological approach. Moreover, mRNAs were clustered and enriched by their biological functions, and those lncRNAs have similar expression trends with mRNAs were defined as functional molecules with potential biological significance. The module may help identify key lncRNAs in the carcinogenesis of NPC and provide clues for in-depth study of their functions and associated signaling pathways. We suggest the newly identified lncRNAs may have clinic value as biomarkers and therapeutic targets for NPC diagnosis and treatment.

Keywords: MYC; genomic instability; long non-coding RNA; nasopharyngeal carcinoma; p53; weighted gene co-expression network analysis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Differentially expressed RNAs in the NPC and the control NPE samples. (A) Heatmap of all differentially expressed RNAs, including lncRNAs and mRNAs. (B) Differentially expressed mRNAs. (C) Differentially expressed lncRNAs. N: normal nasopharyngeal epithelium; T: nasopharyngeal carcinoma.
Figure 2
Figure 2
Differentially expressed lncRNAs were validated by qPCR. 10 normal and 26 cancerous tissues were used to detect expression levels of lncRNAs. Six upregulated and six downregulated lncRNAs were validated. * means p<05, ** means p<0.01, *** means p<0.001, **** means p<0.0001.
Figure 3
Figure 3
Biological function of differentially expressed lncRNAs. (A) The efficiency of knock down was detected by qPCR. (B) CCK8 was used to measure the proliferation ability of lncRNAs. (C) Transwell assays were performed to detect the invasion potential of lncRNAs after knock down. (D) Wound healing assays were employed to assess the migration rate. * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001.
Figure 4
Figure 4
GSEA revealed the concurrent up-regulation of a branch of lncRNAs and mRNAs located on the chromosome 12q24 region. (A) GSEA showed that genes in the chromosome 12q24 region were significantly enriched in NPC. (B) mRNAs in the chromosome 12q24 region were significantly up-regulated in NPC. (C) lncRNAs and mRNAs in the chromosome 12q24 region were concurrently up-regulated (red asterisks beside the right of the heatmap indicate lncRNAs; the rest of the rows represent mRNAs). N, the nasopharyngeal epithelium; T, nasopharyngeal carcinoma.
Figure 5
Figure 5
The lncRNA–mRNA co-expression network for NPC was constructed using WGCNA. (A) Heatmap of the topological overlap matrix of all the differentially expressed lncRNAs and mRNAs in NPC. The elements above and the left of the heatmap are hierarchical clustering trees. The different branches of the clustering tree represent different gene modules and have been displayed as different colored boxes. (B) Highly correlated, co-expressed lncRNAs and mRNAs with topological overlap greater than 0.09 were selected. They formed the basis of the lncRNA–mRNA co-expression network of NPC, which was illustrated using the Cytoscape software. There were 2,196 nodes (915 lncRNAs and 1,281 mRNAs) and 35,290 connections (or relationships) in the co-expression network.
Figure 6
Figure 6
A potential NPC lncRNA–mRNA ceRNA module based on the competition for miR-142-3p. (A) GSEA predicted that miR-142-3p target genes were significantly enriched among the RNAs that were differentially expressed in NPC. (B) Expression profiles of lncRNAs and mRNAs that may be targeted by miR142-3p in NPC (red asterisks indicate lncRNAs). N, the nasopharyngeal epithelium; T, nasopharyngeal carcinoma. (C) An NPC lncRNA–mRNA regulatory module based on the competition for miR142-3p, constructed through GSEA and WGCNA.
Figure 7
Figure 7
p53 pathway-related lncRNA-mRNA co-expression modules in NPC. (A) GSEA revealed that involvement in the p53 signal pathway was significantly enriched in the differentially expressed RNAs in NPC. (B) Many genes in the p53 pathway were significantly up-regulated in NPC (red asterisks indicate lncRNAs). N: the control nasopharyngeal epithelium, T, nasopharyngeal carcinoma. (C) We used GSEA and WGCNA to construct the lncRNA–mRNA co-expression module related to the p53 signaling pathway.
Figure 8
Figure 8
The MYC-driven lncRNA–mRNA co-expression network module in NPC. (A) The regulatory model for MYC and the other transcription factors involved in NPC (obtained using IPA). (B) Expression of the potential mRNAs and lncRNAs downstream of MYC in NPC (red asterisk indicates lncRNAs). N, the control nasopharyngeal epithelium; T, nasopharyngeal carcinoma. (C) We generated a MYC-driven lncRNA–mRNA co-expression network module for NPC by integrating the IPA and the WGCNA results.
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