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. 2014 Jan 31;9(1):e87767.
doi: 10.1371/journal.pone.0087767. eCollection 2014.

Integrated analysis of differential miRNA and mRNA expression profiles in human radioresistant and radiosensitive nasopharyngeal carcinoma cells

Xin-Hui Li  1 , Jia-Quan Qu  2 , Hong Yi  1 , Peng-Fei Zhang  1 , Hong-Mei Yi  1 , Xun-Xun Wan  1 , Qiu-Yan He  1 , Xu Ye  1 , Li Yuan  1 , Jing-Feng Zhu  1 , Jiao-Yang Li  1 , Zhi-Qiang Xiao  1
Affiliations

Integrated analysis of differential miRNA and mRNA expression profiles in human radioresistant and radiosensitive nasopharyngeal carcinoma cells

Xin-Hui Li et al. PLoS One. .

Abstract

Background: The purpose of this study was to identify miRNAs and genes involved in nasopharyngeal carcinoma (NPC) radioresistance, and explore the underlying mechanisms in the development of radioresistance.

Methods: We used microarrays to compare the differences of both miRNA and mRNA expression profiles in the radioresistant NPC CNE2-IR and radiosensitive NPC CNE2 cells, applied qRT-PCR to confirm the reliability of microarray data, adopted databases prediction and anticorrelated analysis of miRNA and mRNA expression to identify the miRNA target genes, and employed bioinformatics tools to examine the functions and pathways in which miRNA target genes are involved, and construct a miRNA-target gene regulatory network. We further investigated the roles of miRNA-23a and its target gene IL-8 in the NPC radioresistance.

Results: THE MAIN FINDINGS WERE FOURFOLD: (1) fifteen differential miRNAs and 372 differential mRNAs were identified, and the reliability of microarray data was validated for randomly selected eight miRNAs and nine genes; (2) 174 miRNA target were identified, and most of their functions and regulating pathways were related to tumor therapeutic resistance; (3) a posttranscriptional regulatory network including 375 miRNA-target gene pairs was constructed, in which the ten genes were coregulated by the six miRNAs; (4) IL-8 was a direct target of miRNA-23a, the expression levels of IL-8 were elevated in the radioresistant NPC tissues and showed inverse correlation with miRNA-23a expression, and genetic upregulation of miRNA-23a and antibody neutralization of secretory IL-8 could reduce NPC cells radioresistance.

Conclusions: We identified fifteen differential miRNAs and 372 differential mRNAs in the radioresistant NPC cells, constructed a posttranscriptional regulatory network including 375 miRNA-target gene pairs, discovered the ten target genes coregulated by the six miRNAs, and validated that downregulated miRNA-23a was involved in NPC radioresistance through directly targeting IL-8. Our data form a basis for further investigating the mechanisms of NPC radioresistance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Validation of microarray-based detection of differentially expressed miRNAs and mRNAs in the NPC CNE2-IR and CNE2 cells by qRT-PCR.
Nine miRNAs (A) and eight mRNAs (B) selected from micorarry data were detected by qRT-PCR. Fold changes from the microarray were given by log2 values (right y-axis). Fold changes from the qRT-PCR were determined using the 2−ΔΔCt method and normalized to the endogenous control GAPDH or U6 (left y-axis). Error bars represent the standard deviation of the mean (SD). Importantly, the fold changes (y-axis) cannot be directly compared between assays due to differences in calculation methods, but the general trend of upregulation and downregulation can be compared. (C) The nine miRNA-target gene pairs with an inverse correlation of expression identified by microarray analysis and validated by qRT-PCR.
Figure 2
Figure 2. The posttranscriptional regulatory network of miRNAs and target genes in the radioresistant NPC cells.
Eleven miRNAs and 174 target genes with an inverse correlation of expression were built into a bipartite network using Cytoscape v2.6. The diamonds and ellipses represent the miRNAs and genes, respectively. The red and green colors represent the relatively high and low expression, respectively. The larger geometric drawing indicates the more miRNAs or genes interacted with it.
Figure 3
Figure 3. Validation of IL-8 as a target of miRNA-23a.
(A) (top) Diagrammatic representation of binding site of miRNA-23a in the 3′UTR of IL-8; (bottom) miRNA-23a mimic significantly reduced the luciferase activity of a dual luciferase reporter with the 3′UTR of IL-8 compared to the controls. Values are the means ± SD of percent changes over controls after normalization to the Renilla luciferase activity. (B) A representative result of Western blot shows the expression level of IL-8 in the CNE2 and CNE2-IR cells, and CNE2-IR cells transfected with miRNA-23a mimic or mimic control. β-actin was used as an internal control for loading. Three experiments were done; columns, mean; bars, S.D. *P<0.05 and **P<0.01 differ from the controls. DLR-blank, a dual luciferase reporter without the 3′UTR of IL-8; DLR-IL8 3′UTR, a dual luciferase reporter with the 3′UTR of IL-8.
Figure 4
Figure 4. The Expressions of miRNA-23a and IL-8 in the NPC tissues with different radiosensitivity.
(A) A representative immunohistochemical result shows no detectable IL-8 expression in the nasopharyngitis tissue, low IL-8 expression in the radiosensitive NPC tissue, and high IL-8 expression in the radioresistant NPC tissue. Original magnification, ×200. (B) Expression levels of miRNA-23a in the radiosensitive and radioresistant NPC tissues. (C) Correlation analysis between IL-8 and miRNA-23a. Pearson's correlation coefficient and P-value for individual analysis are shown in the inserts.
Figure 5
Figure 5. The roles of miRNA-23a and IL-8 in the radioresistance of NPC cells.
(A) and (B). A representative clonogenic survival assay shows that transfection of miRNA-23a mimic decreased the radioresistance of NPC CNE2-IR cells. CNE2-IR cells and its transfectants were irradiated with a range of 2-10 Gy radiation doses, and colonies that formed after incubation of 12 d were counted to calculate the survival fractions, and dose survival curve was drawn. (C) Hoechst 33258 staining shows that transfection of miRNA-23a mimic increased the apoptosis of irradiation-induced CNE2-IR cells. CNE2-IR cells and its transfectants were exposed to 6 Gy irradiation, incubated for 48 h, and then assessed for cell apoptosis using the cell-permeable DNA dye Hoechst 33258. (D) A histogram shows the apoptotic rate of CNE2-IR cells and its transfectants 48 h after 6 Gy irradiation. (E) and (F) A representative clonogenic survival assay shows that neutralization of secretory IL-8 using anti-human IL-8 antibody decreased the radioresistance of NPC CNE2-IR cells. CNE2-IR cells were cultured with DMEM medium supplemented with 2% FCS and monoclonal mouse anti-human IL-8 antibody (2.5 µg/mL) or mouse control IgG1 (2.5 µg/mL), and irradiated with a range of 2-10 Gy radiation doses, and colonies that formed after incubation of 12 d were counted to calculate the survival fractions, and dose survival curve was drawn.
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Grants and funding

This research program was supported by National Natural Science Foundation of China (81230053, 81172559, 81272959), National Basic Research Program of China (2013CB910502). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.