Hypermethylation of miR-203 in endometrial carcinomas

Abstract

Objectives: Aberrant expression of SOX4 in endometrial cancer has been identified and partially was contributed to hypermethylation of miR-129-2. Other miRNAs are suspected to influence SOX 4 as well. The current study seeks to identify other hypermethylated miRNAs that regulate SOX4 in endometrial carcinomas.

Methods: Methylation levels of miRNA promoter regions were measured by combined bisulfite restriction analysis (COBRA) and pyrosequencing assays. Gene expression was determined by RT-qPCR. Methylation level of a miRNA locus was corrected with clinicopathologic factors for 252 gynecological specimens.

Results: In silico analysis identified 13 miRNA loci bound on the 3'-UTR of SOX4. Using COBRA assays, increased methylation of miR-203, miR-219-2, miR-596, and miR-618 was detected in endometrial cancer cells relative to those seen in a normal cell line and in normal endometrium. Transfection of a miR-203 mimic decreased SOX4 gene expression. Hypermethylation of miR-203 was detected in 52% of type I endometrioid endometrial carcinomas (n=131) but was not seen in any of 10 uninvolved normal endometria (P<0.001). Methylation status of miR-203 was significantly associated with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.001). Furthermore, hypermethylation of miR-203 was found in endometrioid and clear endometrial subtype tumors, but not in cervical squamous cell and ovarian carcinomas.

Conclusions: Hypermethylation of miR-203 is a frequent event in endometrial carcinomas and is strongly associated with microsatellite instability and MLH1 methylation status. Thus, miR-203 methylation level might represent a marker for patients with endometrioid and clear endometrial sub-cancers.

Keywords: DNA methylation; Endometrial carcinoma; SOX4; miR-203.

Fig. 1

miR-203 is a novel hypermethylated marker in endometrial cancer. (A) The diagram of predicted miRNA binding sites on SOX4 3′-UTR. (B) Summary of the methylation status by COBRA of thirteen miRNA regions in endometrial cancer cell lines (from left to right: AN3CA, Ishikawa, HEC1A, KLE, RL95-2 and SK-UT-1B) and one normal (N) pooled sample derived from two noncancerous endometria as a negative control. m: methylated; and u: unmethylated. (C) Hypermethylation of miR-203 in endometrial cancer cell lines, as revealed by COBRA analysis. E6/E7: normal endometrial cell line; SssI, methylated positive control; N: normal endometrium. +, AciI restriction enzyme added; and −, without AciI.

Fig. 2

Relative expression levels of miR-203 (A), miR-219-2 (B), miR-596 (C), and miR-618 (D) in endometrial cancer cells after treatment with DAC and/or TSA. Gene expression was determined by RT-qPCR analysis and compared to untreated controls. U48 was used as an internal control gene. Error bar: SD; and *: P<0.05 compared with untreated control of the same cell type.

Fig. 3

Methylation of the miR-203 CpG island and clinicopathologic covariates in primary endometrioid endometrial carcinomas (EECs). (A) Methylation profiles of miR-203 in 10 normal endometrial tissues and 131 primary tumors following pyrosequencing analysis. (B) Dot plots demonstrating miR-203 hypermethylation in EEC tumors. (C-D) Dot plots indicating that the methylation level of miR-203 is correlated with MSI status and MLH1 methylation. m: MLH1 methylated; and u: MLH1 unmethylated.

Fig. 4

Hypermethylation of miR-203 in subtypes of primary gynecological tumors. (A) Methylation analysis of 23 paired endometrioid endometrial (EEC) tissues, measured by pyrosequencing analysis. (B) Dot plots demonstrating miR-203 hypermethylation in another set of primary EEC and clear endometrial tumors, but not in serous, mixed Müllerian (MM), or other mixed (mix) tumors. (C) Dot plots indicating methylation levels of miR-203 in cervical squamous cell carcinomas (CvSCC) and ovarian carcinomas (OvCa).

Fig. 5

Down-regulation of SOX4 mRNA expression by miR-203 (A), miR-335 (B) and miR-618 (C). Cancer cells (Ishikawa) underwent transient transfection with miR-203, miR-335, or miR-618 at the indicated concentrations for 24 h. Gene expression was determined by RT-qPCR analysis and compared to untreated controls. GAPDH was used as an internal control gene. Error bar, SD; and *, P<0.05 compared with untreated cells. (D) A proposed model of miR-203 and SOX4 interactions in normal and cancer cells.