Aberrant Expression Profile of Long Noncoding RNA in Human Sinonasal Squamous Cell Carcinoma by Microarray Analysis

Abstract

Objectives. This study aimed to identify aberrantly expressed long noncoding RNAs (lncRNAs) profile of sinonasal squamous cell carcinoma (SSCC) and explore their potential functions. Methods. We investigated lncRNA and mRNA expression in SSCC and paired adjacent noncancerous tissues obtained from 6 patients with microarrays. Gene ontology (GO) analysis and pathway analysis were utilized to investigate the gene function. Gene signal-network and lncRNA-mRNA network were depicted. Quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to validate 5 lncRNAs in a second set of paired SSCC and adjacent noncancerous tissues obtained from 22 additional patients. Results. We identified significantly differentially expressed lncRNAs (n = 3146) and mRNAs (n = 2208) in SSCC relative to noncancerous tissues. The GO annotation indicated that there are some core gene products that may be attributed to the progress of SSCC. The pathway analysis identified many pathways associated with cancer. The results of lncRNA-mRNA network and gene signal-network implied some core lncRNAs/mRNAs might play important roles in SSCC pathogenesis. The results of qRT-PCR showed that all of the 5 lncRNAs were differentially expressed and consistent with the microarray results. Conclusion. Our study is the first screening and analysis of lncRNAs expression profile in SSCC and may offer new insights into pathogenesis of this disease.

Figure 1

(a) Brief microarray results of lncRNAs. Expression levels of 78,243 lncRNAs were assessed in 6 pairs of SSCC tissues and paired adjacent noncancerous sinonasal tissues using Agilent Human lncRNA 4∗180 K microarrays. Compared with paired adjacent noncancerous tissues, 3146 lncRNAs (4.02%) had significant changes in expression levels (fold change >2, p < 0.05). A total of 874 lncRNAs were excluded due to low expression levels. A total of 3146 lncRNAs were then identified from the screen, with 1174 upregulated and 1098 downregulated. (b) Brief microarray results of mRNAs. Expression levels of 32,776 mRNAs were assessed in 6 pairs of SSCC tissues and paired adjacent noncancerous sinonasal tissues using Agilent Human lncRNA 4∗180 K microarrays. Compared with paired adjacent noncancerous tissues, 2208 mRNAs (6.73%) had significant changes in expression levels (fold change >2, p < 0.05). A total of 284 mRNAs were precluded due to low expression levels. A total of 2208 lncRNAs were then identified from the screen, with 821 upregulated and 1103 downregulated.

Figure 2

Heat map of lncRNAs and mRNAs that were often aberrantly expressed in SSCC compared with paired adjacent noncancerous sinonasal tissues. (a) Hierarchical clustering analysis of the top 100 dysregulated lncRNAs. (b) Hierarchical clustering analysis of the top 100 dysregulated mRNAs. Each row represents one lncRNA or mRNA, and each column represents one tissue sample. The relative lncRNA or mRNA expression is depicted according to the color scale. Red indicates elevated expression and green indicates reduced expression. 2, 0 and −2 are fold changes in the corresponding spectrum. Carcinoma group (A3, F5, D6, F2, F9, and D2). Paired adjacent noncancerous group (D9, F1, G10, B2, G2, and H2).

Figure 3

lncRNA-mRNA coexpression network. The SSCC consisted of coexpression relationships between lncRNAs and mRNAs. The red circles denote mRNAs and the blue circles denote lncRNAs. The node degree is indicated by the circle size. An edge represents a coexpression relationship between mRNA and a lncRNA in the context of SSCC progression.

Figure 4

GO and KEGG pathway analysis. (a)–(c) Top 10 enrichment GO terms for differentially expressed mRNAs. The bar plot shows the enrichment scores (−lg(p value)) of the significant enrichment GO terms. (a) GO terms of biological process (BP); (b) GO terms of cellular component (CC); (c) GO terms of molecular function (MF). (d) Top 10 pathways of differentially expressed mRNAs in SSCC. The vertical axis represents the pathway category and the horizontal axis represents the enrichment score (−lg(p value)) of the pathway.

Figure 5

Signal-net. The interaction network of differentially expressed genes (signal-net). The circles represent important functional genes in SSCC (blue: downregulated genes; red: upregulated genes); the circle size represents the degree of interaction (betweenness centrality); the lines indicate the interactions.

Figure 6

qRT-PCR validation. qRT-PCR verification of 5 candidate lncRNAs in 22 pairs of SSCC tissues. The y-axis represents the relative expression levels of lncRNAs. Paired t-tests (2-tailed) were performed to compare the expression levels between carcinoma (C) and noncancerous tissues (N), and a p value <0.05 indicated statistical significance.