MicroRNA deregulation and pathway alterations in nasopharyngeal carcinoma

Abstract

MicroRNAs (miRNAs) are a family of small non-coding RNA molecules of about 20-23 nucleotides in length, which negatively regulate protein-coding genes at post-transcriptional level. Using a stem-loop real-time-PCR method, we quantified the expression levels of 270 human miRNAs in 13 nasopharyngeal carcinoma (NPC) samples and 9 adjacent normal tissues, and identified 35 miRNAs whose expression levels were significantly altered in NPC samples. Several known oncogenic miRNAs, including miR-17-92 cluster and miR-155, are among the miRNAs upregulated in NPC. Tumour suppressive miRNAs, including miR-34 family, miR-143, and miR-145, are significantly downregulated in NPC. To explore the roles of these dysregulated miRNAs in the pathogenesis of NPC, a computational analysis was performed to predict the pathways collectively targeted by the 22 significantly downregulated miRNAs. Several biological pathways that are well characterised in cancer are significantly targeted by the downregulated miRNAs. These pathways include TGF-Wnt pathways, G1-S cell cycle progression, VEGF signalling pathway, apoptosis and survival pathways, and IP3 signalling pathways. Expression levels of several predicted target genes in G1-S progression and VEGF signalling pathways were elevated in NPC tissues and showed inverse correlation with the down-modulated miRNAs. These results indicate that these downregulated miRNAs coordinately regulate several oncogenic pathways in NPC.

Figure 1

MicroRNA (miRNA) expression patterns distinguish normal from NPC tissues. (A) Unsupervised hierarchical clustering of 223 miRNAs in seven normal (blue)–NPC (red) paired tissues. The hierarchical clustering was performed using squared Euclidean as distance measure and Ward's method for linkage analysis. MicroRNA levels were expressed as 39 – C_t after global median normalisation. (B) Selection of miRNAs differentially expressed in seven paired normal–NPC tissues. Differentially expressed miRNAs were selected based on t-test (P<0.01) and median fold change (⩾3-fold). (C) Principle component analysis using the expression levels of 35 miRNAs in 9 normal (blue) and 13 NPC (red) samples. (D) Unsupervised hierarchical clustering of 35 differentially expressed miRNAs in normal (blue) and NPC (red) samples. The hierarchical clustering was performed using Pearson's dissimilarity as distance measure and Ward's method for linkage analysis. MicroRNA levels were expressed after standardisation.

Figure 2

Significantly modulated MicroRNAs (miRNAs) in 9 normal and 13 NPC tissues. (A) Expression levels of four miRNAs significantly upregulated in NPC tissues. (B) Expression levels of four miRNAs significantly downregulated in NPC tissues. Expression levels of miRNAs were expressed as 39 – C_t after normalisation. P-value for each miRNA was calculated using two-tailed t-test.

Figure 3

Analysis of collective regulated pathways. (A) Flow diagram depicting the process to identify the coregulated targets and specifically enriched pathways. (B) Distribution of predicted targets regulated by down-miR and ctrl-miR. (C) Distribution of cumulative target number coregulated by multiple microRNAs. (D) Pathways significantly and specifically enriched by down-miR targets.

Figure 4

The upregulation of microRNA (miRNA) coregulated targets in NPC. (A) Quantification of coregulated targets in growth factor-regulated G1-S cell cycle pathway : cyclin D2 (CCND2), cyclin E2 (CCNE2), and CDC25A. (B) Quantification of coregulated targets involved in VEGF pathway: VEGFA, PLCG1 (phospholipase C-γ1), and AKT. Expression level of each gene is expressed as 39 – C_t after normalised to three internal controls. (C) Specimens of NPC tissues were stained with the anti-CCNE2 antibody. Shown here are two representative cases containing tumour cells with positive CCNE2 staining (left panels), as well as non-tumour epithelial cells with negative CCNE2 staining (right panels). Original magnification: × 400; Bar, 100 μm.

Figure 5

Inverse correlation between cyclin E2 expression with miR-34c, miR-200a, and miR-9 in NPC. (A) Predicted binding sites for miR-34c-5p, miR-200a, and miR-9 on the 3′UTR of cyclin E2. (B) Expression levels of miR-34c-5p, miR-200a, and miR-9 in normal and NPC tissues. (C) Correlation analysis between cyclin E2 and three microRNAs. Pearson's correlation coefficient and P-value for individual analysis are shown in the inserts. (D) Quantification of miR-9 in HK1 cells after transduction with either negative control lentivirus (HK1/neg) or lentivirus encoding miR-9 (HK1/miR-9). Levels of miR-9 and miR-16 were expressed as 39 – C_t. (E) Quantification of CCNE2 and β-2-microglobulin (B2M) transcripts in HK1/neg and HK1/miR-9 cells. Results are mean±s.d. of three independent experiments. A significant reduction in CCNE2 level was observed after miR-9 expression (HK1/neg vs HK1/miR-9, CCNE2: P=0.014, B2M: P=0.258). (F) Reduced expression of CCNE2 protein in HK1/miR-9 cells.