Upregulation of miR-31* is negatively associated with recurrent/newly formed oral leukoplakia

Abstract

Background: Oral leukoplakia (OLK) is a potentially malignant disorder of the oral cavity. However, the underlying mechanism of OLK is still unclear. In this study, we explore possible miRNAs involved in OLK.

Methodology/principal findings: Using miRNA microarrays, we profiled miRNA expression in OLK and malignantly transformed OLK (mtOLK) tissue samples. The upregulation of miR-31*, miR-142-5p, miR-33a, miR-1259, miR-146b-5p, miR-886-3p, miR-886-5p, miR-519d, and miR-301a along with the downregulation of miR-572, miR-611, miR-602, miR-675, miR-585, miR-623, miR-637, and miR-1184 in mtOLK were new observations. Fluorescence in situ hybridization (FISH) analyses confirmed that miR-31* is highly expressed in mtOLK. There was a significant difference between the FISH score (p<0.05) in patients with or without recurrent/newly formed OLK. Functional analyses demonstrated that a miR-31* inhibitor decreased apoptosis in the Leuk-1, which is an immortalized oral epithelial cell line spontaneously derived from an oral leukoplakia lesion. miR-31* regulated apoptosis, cell proliferation, migration, and invasion in the HOIEC, which is a HPV E6/E7-immortalized oral epithelial cell line. Furthermore, miR-31* modulated the biological functions of apoptosis, cell proliferation, cell cycle, migration, and invasion in the oral squamous cell carcinoma cell line, Cal-27. Using bioinformatic analyses and dual luciferase reporter assays, we determined that the 3' untranslated region of fibroblast growth factor 3 (FGF3) is the target of miR-31*. Expression of FGF3 was downregulated or upregulated in the presence of a miR-31* mimic or inhibitor, respectively.

Conclusions/significance: Upregulation of miR-31* is negatively associated with recurrent/newly formed OLK. MiR-31* may exert similar but distinguishable effects on biological function in oral cells with different malignant potential. FGF3 is the target of miR-31*. miR-31* may play an important role during OLK progression through regulating FGF3. MiRNA* strands may also have prominent roles in oral carcinogenesis.

Figure 1. Unsupervised hierarchical cluster analysis of miRNA expression in OLK and mtOLK.

The marked separation of OLK and mtOLK based on the miRNAs profiling is exhibited. Scale is shown in the upper right. Red represents a high expression and green represents a low expression. OLK, oral leukoplakia; mtOLK, malignant transformed oral leukoplakia.

Figure 2. FISH detection of miR-31* expression in OLK and mtOLK.

(A) ∼ (C), no positive signal is detected in OLK. (D) ∼ (L), the representative positive signals are showed in mtOLK. (D) ∼ (F), tumor nest; (G) ∼ (I), vascular area; (J) ∼ (L), inflammation. FISH signals are visualized in green, while blue depicts nuclear DAPI stain. OLK, oral leukoplakia; mtOLK, malignant transformed oral leukoplakia.

Figure 3. Representative results of miR-31* effects on biological functions.

(A) the MTT assay in HIOEC; (B) the Annexin V assay in Leuk-1; (C) flow cytometry analysis for cell cycle in Cal-27; (D) representative field of view of HIOEC invasion inserts at a 100× magnification, (Da) miR-31* mimic, (Db) miR-31* inhibitor, (Dc) negative control, (Dd) Quantification of relative numbers of invading cells representing average counts from 6 fields-of-view per insert per sample ±SD, **p<0.05, ***p<0.001; (E) representative field of view of HIOEC migration inserts at a 100× magnification, (Ea) miR-31* mimic, (Eb) miR-31* inhibitor, (Ec) negative control, (Ed) quantification of relative numbers of invading cells representing average counts from 6 fields-of-view per insert per sample ±SD, **p<0.05, ***p<0.001.

Figure 4. Dual luciferase reporter assay for miR-31* and targeted genes.

(A) Psicheck-FGF3-wt/mt and miR-31*; (B) Psicheck-IL5Rα-wt/mt and miR-31*. NC  =  negative control; mimic  =  miR-31* mimic; wt  =  wild type; mt  =  mutant type.

Figure 5. miR-31* mimics/inhibitors regulating mRNA and protein expression of FGF3 in oral cells.

(A) QRT-PCR analysis of FGF3 expression in Leuk-1, HIOEC and Cal-27 cell lines, **p<0.05; (B) Western blot analysis of of FGF3 expression in Leuk-1, HIOEC and Cal-27 cell lines transfected with miR-31* mimic, miR-31* inhibitor and negative control. The representative blot is shown (upper for FGF3). The histogram shows the average volume density normalized by the loading control, GAPDH (lower).