MicroRNA expression profiling and Notch1 and Notch2 expression in minimal deviation adenocarcinoma of uterine cervix

Abstract

Background: MicroRNA (miRNA) expression is known to be deregulated in cervical carcinomas. However, no data is available about the miRNA expression pattern for the minimal deviation adenocarcinoma (MDA) of uterine cervix. We sought to detect deregulated miRNAs in MDA in an attempt to find the most dependable miRNA or their combinations to understand their tumorigenesis pathway and to identify diagnostic or prognostic biomarkers. We also investigated the association between those miRNAs and their target genes, especially Notch1 and Notch2.

Methods: We evaluated miRNA expression profiles via miRNA microarray and validated them using.real-time PCR assays with 24 formalin-fixed, paraffin-embedded tissue blocks of MDA and 11 normal proliferative endocervical tissues as control. Expression for Notch1 and 2 was assessed by immunohistochemistry.

Results: MiRNA-135a-3p, 192-5p, 194-5p, and 494 were up-regulated, whereas miR-34b-5p, 204-5p, 299-5p, 424-5p, and 136-3p were down-regulated in MDA compared with normal proliferative endocervical tissues (all P<0.05). Considering the second-order Akaike Information Criterion consisting of likelihood ratio and number of parameters, miR-34b-5p showed the best discrimination power among the nine candidate miRNAs. A combined panel of miR-34b-5p and 194-5p was the best fit model to discriminate between MDA and control, revealing 100% sensitivity and specificity. Notch1 and Notch2, respective target genes of miR-34b-5p and miR-204-5p, were more frequently expressed in MDA than in control (63% vs. 18%; 52% vs. 18%, respectively, P<0.05). MiR-34b-5p expression level was higher in Notch1-negative samples compared with Notch1-positive ones (P<0.05). Down-regulated miR-494 was associated with poor patient survival (P=0.036).

Conclusions: MDA showed distinctive expression profiles of miRNAs, Notch1, and Notch2 from normal proliferative endocervical tissues. In particular, miR-34b-5p and 194-5p might be used as diagnostic biomarkers and miR-494 as a prognostic predictor for MDA. The miR-34b-5p/Notch1 pathway as well as Notch2 might be important oncogenic contributors to MDA.

Figure 1

Unsupervised hierarchical clustering analysis based on miRNA array data. Array data from 8 minimal deviation adenocarcinomas (MDA) (C1–C8) and 8 normal proliferative endocervical tissues (NE) (N1–N8) shows 47 overexpressed miRNAs (red) and 49 underexpressed miRNAs (green) in MDA compared to NE.

Figure 2

Validation of the differentially-expressed miRNAs from the microarray data with real-time quantitative PCR. Expression of each miRNA in a validation set composed of normal proliferative endocervical tissues (NE) and minimal deviation adenocarcinoma (MDA) of uterine cervix is shown by an individual scatter plot.

Figure 3

Immunohistochemical staining of Notch1 and Notch2 in minimal deviation adenocarcinoma (MDA) of uterine cervix and their correlation with miRNAs. (A) Notch1 is predominantly localized at the apical membranes and (B) Notch2 is expressed in the cytoplasm of the tumor cells. High magnification view (inset, ×400). Differential expression of (C) Notch1 and (D) Notch2 in MDA compared to normal proliferative endocervical tissue (NE). Expression levels of (E) miR-34b-5p and (F) miR-204-5p according to Notch1 and Notch2 status, respectively.

Figure 4

Kaplan-Meier survival curve and log-rank test for miR-494 level. The survival rate of patients with down-regulated miR-494 levels was significantly lower than that of patients with up-regulated miR-494 levels (log-rank test P =0.036).