miR-137 is a tumor suppressor in endometrial cancer and is repressed by DNA hypermethylation

Abstract

Endometrial cancer is the most common gynecological cancer in the United States. We wanted to identify epigenetic aberrations involving microRNAs (miRNAs), whose genes become hypermethylated in endometrial primary tumors. By integrating known miRNA sequences from the miRNA database (miRBase) with DNA methylation data from methyl-CpG-capture sequencing, we identified 111 differentially methylated regions (DMRs) associated with CpG islands (CGIs) and miRNAs. Among them, 22 DMRs related to 29 miRNAs and within 8 kb of CGIs were hypermethylated in endometrial tumors but not in normal endometrium. miR-137 was further validated in additional endometrial primary tumors. Hypermethylation of miR-137 was found in both endometrioid and serous endometrial cancer (P < 0.01), and it led to the loss of miR-137 expression. Treating hypermethylated endometrial cancer cells with epigenetic inhibitors reactivated miR-137. Moreover, genetic overexpression of miR-137 suppressed cancer cell proliferation and colony formation in vitro. When transfected cancer cells were implanted into nude mice, the cells that overexpressed miR-137 grew more slowly and formed smaller tumors (P < 0.05) than vector transfectants. Histologically, xenograft tumors from cancer cells expressing miR-137 were less proliferative (P < 0.05), partly due to inhibition of EZH2 and LSD1 expression (P < 0.01) in both the transfected cancer cells and tumors. Reporter assays indicated that miR-137 targets EZH2 and LSD1. These results suggest that miR-137 is a tumor suppressor that is repressed in endometrial cancer because the promoter of its gene becomes hypermethylated.

Figure 1

DNA methylation of miR-137 in primary endometrial tissues. (a) Outline for identifying hypermethylated miRNA in endometrial cancer. (b) Methylation profiles of 10 normal endometrial tissues (N) and 67 primary tumors (Ca) created after methyl-capture sequencing analysis. Red and white dashed-line squares represent methylated and unmethylated regions, respectively. Green bar: the nearby CpG island.

Figure 2

miR-137 is hypermethylated and shows loss of expression in human primary endometrial tumors. (a) Genomic map of the CpG island nearby miR-137 (black bar), COBRA and bisulfite pyrosequencing (Pyro) amplicons and TCGA methylation probes. CpG sites are lines under bars. (b) Dot plot showing miR-137 methylation levels in 10 pairs of endometrioid endometrial cancer (EEC) and normal adjacent tissues (NAT) quantified by bisulfite pyrosequencing. (c) DNA methylation was determined by bisulfite pyrosequencing in human endometrial tissues. NAT: normal adjacent tissue; Serous: serous endometrial tumor. Each dot represents one specimen. Each horizontal line indicates the mean of methylation within each group. DNA methylation of miR-137 by probes in paired samples (d) and subtypes of endometrial tumors (e) in TCGA cohort. Pair (n=33); N: normal endometrium (n=13); EEC (n=312); serous (n=98). (f) Expression of miR-137 in TCGA endometrial cohort. Gray portions: miR-137 not expressed; white portions: miR-137 expressed. Number in each bar indicates sample size. *: P<0.05; **: P<0.01; ***: P<0.001.

Figure 3

miR-137 is hypermethylated and reactivated by epigenetic inhibitors in endometrial cancer cells. (a) DNA methylation levels determined by bisulfite pyrosequencing in a normal endometrial cell line (EM-E6/E7/TERT) and 11 endometrial cancer cell lines. (b–c) Reactivation of pre-/pri- and mature miR-137 after epigenetic inhibitors in endometrial cancer cells after endometrial cancer cells were treated with epigenetic inhibitors. Cancer cells (HEC1A, Ishikawa H, and Hec50co) were treated with 5-aza-2′-deoxycytidine (DAC) and/or trichostatin A (TSA). Gene expression was determined by RT-qPCR and compared to untreated controls (CTR). U6 served as an internal control. Bars: means±SD; *: P<0.05.

Figure 4

Functional analysis of miR-137 in endometrial cancer cells. (a) Relative expression of miR-137 in stably transfected clones of HEC1A and Ishikawa H cancer cells by RT-qPCR. pCMV-MIR: mock transfection with vector; miR-137: miR-137 transfection. U6 served as an internal control. Bars: means±SD. (b) Cellular proliferation in transfected HEC1A and Ishikawa H cells at different time points, as determined by MTS assays. (c–d) Colony number and size in miR-137-transfected HEC1A and Ishikawa H endometrial cancer cells. (e–f) Gene expression by RT-qPCR and Western blotting in transfected cells. Bars: means±SD. *: P<0.05; **: P<0.01; ***: P<0.001.

Figure 5

miR-137 impairs xenograft tumor formation. (a) Xenograft tumor growth curves in miR-137 stably transfected clones of HEC1A cells. pCMV-MIR: mock transfection with vector; miR-137: miR-137 transfection. (b) Tumor weights of miR-137- or mock-transfected HEC1A cells. (c) Protein expression of EZH2 and LSD1 in transfected cells by Western blotting. β-actin serves as a loading control. (d–e) Representative sections and summary of H&E and IHC staining of xenograft tumors. Bars: means±SE. *: P<0.05; **: P<0.01.

Figure 6

miR-137 targets EZH2 amd LSD1. (a) 3′-UTR reporter assays were conducted in cells co-transfected with reporter plasmids, negative contol miRNA, miR-137, or anti-miR-137. Fold changes were normalized to plamsid and negative control miRNA transfections. Gene expression of EZH2 (b) and LSD1 (c) in miR-137-transfected HEC1A cancer cells by RT-qPCR. The stably transfected cells were subsequently transfected with EZH2 and control vector pCMV-HA (HA), or with LSD1 and its empty vector pCMV6 (PS). GAPDH served as an internal control. (d) Cellular proliferation in transfected cells at different time points was determined by MTS assays. *: P<0.05; ***: P<0.001.