An in silico analysis of dynamic changes in microRNA expression profiles in stepwise development of nasopharyngeal carcinoma

Abstract

Background: MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA (mRNA) and protein synthesis. Recent studies have shown that some miRNAs are involved in the progression of nasopharyngeal carcinoma (NPC). However, the aberrant miRNAs implicated in different clinical stages of NPC remain unknown and their functions have not been systematically studied.

Methods: In this study, miRNA microarray assay was performed on biopsies from different clinical stages of NPC. TargetScan was used to predict the target genes of the miRNAs. The target gene list was narrowed down by searching the data from the UniGene database to identify the nasopharyngeal-specific genes. The data reduction strategy was used to overlay with nasopharyngeal-specifically expressed miRNA target genes and complementary DNA (cDNA) expression data. The selected target genes were analyzed in the Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway. The microRNA-Gene-Network was build based on the interactions of miRNAs and target genes. miRNA promoters were analyzed for the transcription factor (TF) binding sites. UCSC Genome database was used to construct the TF-miRNAs interaction networks.

Results: Forty-eight miRNAs with significant change were obtained by Multi-Class Dif. The most enriched GO terms in the predicted target genes of miRNA were cell proliferation, cell migration and cell matrix adhesion. KEGG analysis showed that target genes were significantly involved in adherens junction, cell adhesion molecules, p53 signalling pathway et al. Comprehensive analysis of the coordinate expression of miRNAs and mRNAs reveals that miR-29a/c, miR-34b, miR-34c-3p, miR-34c-5p, miR-429, miR-203, miR-222, miR-1/206, miR-141, miR-18a/b, miR-544, miR-205 and miR-149 may play important roles on the development of NPC. We proposed an integrative strategy for identifying the miRNA-mRNA regulatory modules and TF-miRNA regulatory networks. TF including ETS2, MYB, Sp1, KLF6, NFE2, PCBP1 and TMEM54 exert regulatory functions on the miRNA expression.

Conclusions: This study provides perspective on the microRNA expression during the development of NPC. It revealed the global trends in miRNA interactome in NPC. It concluded that miRNAs might play important regulatory roles through the target genes and transcription factors in the stepwise development of NPC.

Figure 1

miRNA expression profile in the stepwise development of NPC. (A) The microdissection was performed with Methyl Green staining to separate tumor cells to non-tumor cells; (B)Hierarchical clustering result of 48 differentially-expressed miRNAs. Each row represents the expression profile of a miRNA across 22 samples and each column represents a sample. The sample IDs and clinical stages information are listed above the 'heatmap'. Red and green colors respectively indicate either higher and lower expression levels of the miRNA. Samples are well separated into control and NPC patient groups except three. Using the dendrogram-based methods for clustering, the samples can be further separated into five subgroups on hierarchical clustering, which were correlated with the NPC clinic stages. (C) Dynamic miRNA expressions were classified into 6 different patterns.

Figure 2

Target gene prediction of miRNAs. miRNA-gene interactions were built into a bipartite network (the miRNAome). The red rectangles indicate the up-regulated miRNAs, the blue rectangles indicate the down-regulated miRNAs. The red ellipses indicate the up-regulated target genes, the blue ellipses indicate the down-regulated target genes.

Figure 3

The expression miRNAs were verified by real-time PCR in 38 NPC cases and 10 normal. (A) the up-regulated miRNAs; (B) the down-regulated miRNAs.

Figure 4

The expression of corresponding target genes of miRNAs were verified by real-time PCR in 38 NPC cases and 10 normal. (A) representative target genes of upregulated miRNAs; (B) representative target genes of downregulated miRNAs.

Figure 5

The miRNA transcriptional network for NPC development. These transcription factor-miRNA interactions were built into a network. The triangles represent transcription factors which regulate the miRNA expression; the squares represent the miRNAs which were regulated by TFs. The larger triangles represent the more miRNAs which are regulated.