MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma - PubMed (original) (raw)

doi: 10.1371/journal.pone.0027840. Epub 2011 Nov 22.

Shan Gao, Toby Hulf, Tennille Sibbritt, Shalima Nair, Daniela Elena Costea, Sune B Villadsen, Vivi Bakholdt, Jesper B Bramsen, Jens A Sørensen, Annelise Krogdahl, Susan J Clark, Jørgen Kjems

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MicroRNA alterations and associated aberrant DNA methylation patterns across multiple sample types in oral squamous cell carcinoma

Erik D Wiklund et al. PLoS One. 2011.

Abstract

Background: MicroRNA (miRNA) expression is broadly altered in cancer, but few studies have investigated miRNA deregulation in oral squamous cell carcinoma (OSCC). Epigenetic mechanisms are involved in the regulation of >30 miRNA genes in a range of tissues, and we aimed to investigate this further in OSCC.

Methods: TaqMan® qRT-PCR arrays and individual assays were used to profile miRNA expression in a panel of 25 tumors with matched adjacent tissues from patients with OSCC, and 8 control paired oral stroma and epithelium from healthy volunteers. Associated DNA methylation changes of candidate epigenetically deregulated miRNA genes were measured in the same samples using the MassArray® mass spectrometry platform. MiRNA expression and DNA methylation changes were also investigated in FACS sorted CD44(high) oral cancer stem cells from primary tumor samples (CSCs), and in oral rinse and saliva from 15 OSCC patients and 7 healthy volunteers.

Results: MiRNA expression patterns were consistent in healthy oral epithelium and stroma, but broadly altered in both tumor and adjacent tissue from OSCC patients. MiR-375 is repressed and miR-127 activated in OSCC, and we confirm previous reports of miR-137 hypermethylation in oral cancer. The miR-200 s/miR-205 were epigenetically activated in tumors vs normal tissues, but repressed in the absence of DNA hypermethylation specifically in CD44(high) oral CSCs. Aberrant miR-375 and miR-200a expression and miR-200c-141 methylation could be detected in and distinguish OSCC patient oral rinse and saliva from healthy volunteers, suggesting a potential clinical application for OSCC specific miRNA signatures in oral fluids.

Conclusions: MiRNA expression and DNA methylation changes are a common event in OSCC, and we suggest miR-375, miR-127, miR-137, the miR-200 family and miR-205 as promising candidates for future investigations. Although overall activated in OSCC, miR-200/miR-205 suppression in oral CSCs indicate that cell specific silencing of these miRNAs may drive tumor expansion and progression.

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Conflict of interest statement

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

Figures

Figure 1

Figure 1. Hierarchical clustering of OSCC and healthy oral tissue miRNA expression profiles.

MiRNA expression profiling was performed using TaqMan® TLDA low density miRNA arrays for primary tumor/matched normal adjacent pairs from 4 OSCC patients, as well as normal epithelium and stroma from 2 healthy controls. The samples encompass 2 non-metastatic tumors (18110T & 11063T), 2 metastatic tumors (17093T & 28169T), 4 matched adjacent normal tissues (18110N, 11063N, 17093N & 28169N), 2 healthy connective tissue (stroma) (H14C & H5C) and 2 healthy epithelium (H14E & H5E), respectively. Expression was normalized to RNU48 (2−dCt) and hierarchical clustering was performed using Cluster 3.0. for relative miRNA levels, and is color coded from bright green (undetected) to red (highest expression).

Figure 2

Figure 2. DNA methylation and expression patterns for candidate epigenetically deregulated miRNAs.

MiRNA CpG methylation (a–f) and expression (g–k) patterns in OSCC in and normal oral tissues. DNA methylation levels were measured using the MassArray® mass spectrometry platform for CpG enriched regions in the miR-375 (a), miR-127 (b), miR-200ab-429 (c), miR-200c-141 (d), miR-205 (e) and miR-137 (f) loci in a panel of 25 OSCC tumor/matched adjacent pairs and 8 healthy control epithelium/stroma pairs. Values are presented as average percentage CpG methylation across the full regions analyzed for each locus. Relative expression of miR-375 (g), miR-127 (h), miR-200abc (average) (i), miR-141 (j) and miR-205 (k) was evaluated in the same samples by individual TaqMan® miRNA assays performed in triplicates and normalized to RNU48 (2−dCt). Box plots indicate the median value (horizontal line) and the 25th–75th percentile range (box) with whiskers at 1.5×IQR. Values outside this range are shown as outliers (points). P-values were determined by the paired two-tailed Student's t-test for matched patient tumor/adjacent and healthy epithelium/stroma pairs overall, and the unpaired two-tailed Student's t-test for tumor vs. healthy epithelium.

Figure 3

Figure 3. miR-200 and miR-205 expression and methylation differences in individual tumor vs. matched adjacent pairs.

Expression and DNA methylation was determined as explained in the legend to Figures 2, and represents the same data visualized as the difference between individual pairs of tumor and matched adjacent epithelium. (a) miR-200a, miR-200b, miR-200c, miR-141 and miR-205 −fold change expression (+/− SD), and (b) average % CpG methylation change of the miR-200ab-429, miR-200c-141 and miR-205 loci (+/− SE). X-axis numbers are OSCC patient identifiers.

Figure 4

Figure 4. miRNA expression and DNA methylation in oral cancer stem cells.

MiR-200 family and miR-205 expression (a) and DNA methylation (b), and miR-137 expression (c) and DNA methylation (d) in OSCC cancer stem cells (CSCs) purified by FACS sorting the 5% most CD44 staining cells from 3 primary samples (CD44high), compared to the 5% least staining cells (CD44low). Similar control non-CSC cells were sorted from 3 healthy individuals. Relative miRNA expression (+/− SD) was determined by individual TaqMan® miRNA assays normalized to RNU48 (2−dCt), and percentage CpG methylation levels (+/− SE) across the indicated regions were measured using the MassArray® mass spectrometry platform. Statistical significance was determined by the paired one-tailed Student's t-test for CD44high vs. CD44low cells from individual patients.

Figure 5

Figure 5. miRNA expression and DNA methylation in oral rinse and saliva.

(a) miR-375 (b) miR-200a and (c) miR-200c relative to miR-191 expression ratio, and (d) absolute miR-200c-141 DNA methylation level in oral rinse and saliva from 15 OSCC patients and 7 healthy controls. The box plots represent the median value (horizontal line) and the 25th–75th percentile range (box) with whiskers at 1.5×IQR. Values outside this range are indicated as outliers (points). MiRNA ratio (+/− SD) was determined by individual TaqMan® miRNA assays, normalizing miR-375 to miR-191 Ct values (2−dCt). Percentage CpG methylation (+/− SE) was measured using the MassArray® mass spectrometry platform. P-values were determined by the unpaired two-tailed Student's t-test for OSCC patients vs. healthy controls.

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