Development of a novel microRNA promoter microarray for ChIP-on-chip assay to identify epigenetically regulated microRNAs

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Abstract

To gain a global view of epigenetic alterations around microRNA (miRNA) promoter regions, and to identify epigenetically regulated miRNAs, we developed a novel miRNA promoter microarray for chromatin immunoprecipitation (ChIP)-on-chip assay. We designed a custom oligo microarray covering regions spanning −10 to +2.5 kb of precursor miRNAs in the human genome. This microarray covers 541 miRNAs, each of which is covered by approximately 100 probes (60-mer) over its 12.5-kb genomic position, that includes predicted transcription start sites. Using this custom-made miRNA promoter microarray, we successfully performed ChIP-on-chip assay to identify miRNAs regulated by histone modification. Fifty-three miRNAs (9.8%) showed increased levels of both histone H3 acetylation and histone H3-K4 methylation in AGS gastric cancer cells treated with the DNA-methylation inhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitor 4-phenylbutyric acid. One of these miRNAs, miR-9, is downregulated in gastric cancer tissues and is activated by chromatin-modifying drugs, suggesting that it may be a potential target for epigenetic therapy of gastric cancer.

Highlights

► We developed a novel miRNA promoter microarray for ChIP-on-chip assay. ► Using this miRNA array, we successfully identified epigenetically regulated miRNAs. ► Tumor suppressor miRNAs such as miR-9 and miR-34a were identified by this assay. ► miR-9 is a potential target for epigenetic therapy of gastric cancer.

Introduction

MicroRNAs (miRNAs) are ∼22 nucleotide (nt) non-coding RNAs that can post-transcriptionally downregulate the expression of various target genes. Currently, ∼1500 human miRNAs have been identified in the human genome, each of which potentially controls hundreds of target genes. In animals, miRNA genes are generally transcribed by RNA polymerase II (pol II) to form primary transcripts (pri-miRNAs). Pol II-transcribed pri-miRNAs are capped with 7-methylguanosine and are polyadenylated. The nuclear RNase III enzyme Drosha and its co-factor DGCR8 process pri-miRNAs into ∼60-nt precursor miRNAs (pre-miRNAs), which form an imperfect stem-loop structure. Pre-miRNAs are transported into the cytoplasm by exportin 5 and are subsequently cleaved by Dicer into mature miRNAs, which are then loaded into the RNA-induced silencing complex (RISC). The miRNA/RISC complex downregulates specific gene products by translational repression via binding to partially complementary sequences in the 3′-untranslated regions of the target mRNAs or by directing mRNA degradation via binding to perfectly complementary sequences.

MircoRNAs are expressed in a tissue-specific manner and play important roles in cell proliferation, apoptosis, and differentiation during mammalian development [1]. Links between miRNAs and the development and progression of human malignancies, including gastric cancer, are becoming increasingly apparent [2], [3]. Because miRNAs can have large-scale effects through regulation of a variety of target genes during carcinogenesis, understanding the regulatory mechanisms controlling miRNA expression is important. Epigenetic alterations such as DNA methylation and histone modification play critical roles in chromatin remodeling and regulation of gene expression in mammalian development and human diseases, including cancer. We have recently reported that some miRNAs are regulated by epigenetic alterations at their CpG island promoters. Epigenetic treatment with chromatin-modifying drugs such as the DNA-demethylating agent 5-aza-2′-deoxycytidine (5-Aza-CdR) and the histone deacetylase (HDAC) inhibitor 4-phenylbutyric acid (PBA) can reactivate some important tumor suppressor miRNAs, and this may be a novel therapeutic approach for human cancers [4], [5], [6], [7]. To gain a global view of epigenetic alterations around miRNA promoter regions and to identify epigenetically regulated miRNAs, we developed a novel miRNA promoter microarray for chromatin immunoprecipitation (ChIP)-on-chip assay and used it to identify candidate miRNAs regulated by epigenetic mechanisms in human gastric cancer cells.

Section snippets

MicroRNA promoter microarray

As shown in Fig. 1, we designed a custom oligo microarray covering regions −10 to +2.5 kb surrounding the genomic positions of pre-miRNAs in the human genome (NCBI36/hg18). Briefly, we first downloaded genomic coordinates of pre-miRNAs from the Manchester (previously Sanger) miRBase v10.1. The set of genomic coordinates at the 5′ end of the pre-miRNAs was positioned at zero, and in silico pre-designed probes were searched to fit a 4 × 44 K microarray from the high-definition ChIP probe database in

Identification of candidates of epigenetically regulated miRNAs by ChIP-on-chip assay with a novel miRNA promoter array

We designed a custom oligo microarray covering from −10 to +2.5 kb surrounding the genomic positions of pre-miRNAs in the human genome (Fig. 1). This microarray covers 541 miRNAs with 125 base spacing between the probes on average, and each miRNA is covered with approximate 100 probes (60 mer) over its 12.5-kb genomic position, that includes predicted TSSs.

To investigate miRNAs, which are regulated by epigenetic alterations, we treated AGS cells with the DNA-methylation inhibitor 5-Aza-CdR and

Discussion

Using a custom miRNA promoter microarray, we performed comprehensive ChIP-on-chip analysis of histone modifications in predicted human miRNA promoter regions. Because recent studies have shown that the majority of TSSs occur within 10 kb upstream of the 5′-end of pre-miRNAs [10], [11], we designed microarray probes from 10 kb upstream to 2.5 kb downstream (relative to the TSS) of each pre-miRNAs to comprehensively analyze histone modifications. We identified miRNAs as candidates for epigenetic

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientists B (21790327 to Y.S.), Grant-in-Aid for Young Scientists A (23680090 to Y.S.), Grant-in-Aid for Scientific Research B (22300169, to H.S.), Grant-in-Aid for challenging Exploratory Research (24659103, to H.S.) from the Japan Society for the Promotion of Science, a Grant from Takeda Science Foundation (to Y.S.), a Research Fund of Mitsukoshi Health and Welfare Foundation (to H.S.), and a Grant from the Smoking Research Foundation (to

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