piR-31470 epigenetically suppresses the expression of glutathione S-transferase pi 1 in prostate cancer via DNA methylation

Highlights

• piR-31470 was highly expressed in prostate cancer cells.

• piR-31470 could bind to PIWIL4 to form the PIWIL4/piR-31470 complex.

• The PIWIL4/piR-31470 complex could recruit DNMT1, DNMT3α and MBD2 to initiate and maintain the hypermethylation of GSTP1.

• The inactivation of GSTP1 increased the vulnerability to oxidative stress and promoted proliferation in prostate cells.

Abstract

Inactivation of glutathione S-transferase pi 1 (GSTP1) via hypermethylation is an early and common event in prostate carcinogenesis. Functional inactivation of GSTP1 increases the susceptibility to oxidative stress and enhance progression risk of the prostatic carcinoma. In this study, we hypothesized that the Piwi-interacting RNA (piRNA) could be a sequence-recognition and guidance molecule for induction of promoter methylation of GSTP1 facilitating prostate carcinogenesis. We found that piR-31470 was highly expressed in prostate cancer cells, and piR-31470 could bind to piwi-like RNA-mediated gene silencing 4 (PIWIL4) to form the PIWIL4/piR-31470 complex. This complex could bind to the nascent RNA transcripts of GSTP1, and recruit DNA methyltransferase 1, DNA methyltransferase 3 alpha and methyl-CpG binding domain protein 2 to initiate and maintain the hypermethylation and inactivation of GSTP1. Our data demonstrated that the overexpression of piR-31470 inhibited the levels of GSTP1 and increased vulnerability to oxidative stress and DNA damage in human prostate epithelial RWPE1 cells. In conclusion, this study characterized the roles of the PIWIL4/piR-31470 complex in the regulation of the transcription of GSTP1 by methylating the CpG island of GSTP1. This discovery may provide a novel therapeutic strategy by targeting piRNAs for the epigenetic treatment of prostate cancer.

Introduction

DNA methylation has critical roles in the control of gene activity. Hypermethylation of the CpG islands in the promoter regions of tumor-suppressor genes is a major event in the origin of many cancers [1]. Hypermethylation and inactivation of these genes can affect several cellular processes such as cell cycle, DNA repair, cell-to-cell interaction, metabolism of carcinogens, angiogenesis and apoptosis, all of which can be involved in the development of cancer [2,3]. The methylation of genomic DNA is catalyzed by DNA methyltransferases (DNMTs) which are essential for the cytosine methylation and for the maintenance of methylation throughout cell replication [4]. Enhanced expression of DNMTs is associated with hypermethylation of tumor-suppressor genes in several cancers, for example, prostate cancer [5,6]. However, the DNMTs do not recognize specific DNA sequences and the profiles of hypermethylation of tumor-suppressor genes are specific to the cancer type [7,8]. The molecular mechanism that guides the DNMTs to their target sites yet remains largely unexplored.

Previous studies indicated that Piwi-interacting RNAs (piRNAs) are logical candidates for sequence-specific targeting in the genome [9,10]. piRNAs are small non-coding RNAs of 26–31 nucleotides in length. piRNAs are first found to be enriched in animal gonads and they are thought to be critical for silencing transposon loci, via DNA methylation, in order to maintain genomic integrity in germline cells [11,12]. However, only about 20% of piRNAs map to transposon and other repeat genomic regions [13], suggesting that there may be a diverse set of functions for piRNAs. Recent studies have shown that aberrant expression of piRNA is a signature feature across multiple cancer types. Remarkably, an increasing number of functional evidence concerning piRNAs is involved in the regulation of epigenetic mechanisms in tumorigenesis [[14], [15], [16]]. Further supporting the hypothesis, piRNAs may be critical for silencing tumor-suppressor genes, via DNA methylation, resulting in tumor progression.

Glutathione S-transferase pi 1 (GSTP1) methylation, an early event in prostate cancer, can be detected in an estimated up to 90% of prostate cancers [17]. Still, it is presently unknown which mechanisms are involved in GSTP1 methylation, apart from a general observation of increased methylation owing to elevated expression of DNMTs in cancer. The loss of GSTP1 can increase the susceptibility to somatic genomic alterations, caused by oxidative or electrophilic carcinogens, and enhance progression risk in prostatic carcinoma [[18], [19], [20], [21], [22]]. Consequently, repression of GSTP1 hypermethylation is beneficial for antioxidation and cancer prevention.

In the present study, we have provided compelling evidence suggesting a novel mechanism of piRNA as a guidance molecule for sequence-recognition and recruitment of DNMTs to the specific sequence to induce the methylation of GSTP1 in prostate cancer.