miR-1236 regulates hypoxia-induced epithelial–mesenchymal transition and cell migration/invasion through repressing SENP1 and HDAC3

Highlights

• MiR-1236 represses SENP1 and HDAC3.

• MiR-1236 is crucial for hypoxia-induced EMT.

• MiR-1236 is repressed by Twist1.

Abstract

Intratumoral hypoxia induces epithelial–mesenchymal transition and promotes cancer metastasis. MicroRNAs (miRNAs) are endogenous, single-strand RNA molecules that regulate gene expression. MiRNAs control cell growth, proliferation, differentiation and cell death and may function as oncogenes or tumor suppressors. HDAC3 and SENP1 are two molecules involved in hypoxia-induced EMT and HIF-1α stability, respectively. In this report, we show that miR-1236 plays a critical role in hypoxia-induced EMT and metastasis. MiRNA prediction programs TargetScan and miRanda show that miR-1236 may target HDAC3 and SENP1. MiR-1236 represses the luciferase activity of reporter constructs containing 3'UTR of HDAC3 and SENP1 as well as the expression levels of HDAC3 and SENP1. MiR-1236 abolishes hypoxia-induced EMT and inhibits migration and invasion activity of tumor cells. Hypoxia represses miR-1236 expression. The promoter region of miR-1236 is identified as the NELFE promoter. Twist1, an EMT regulator activated by hypoxia/HIF-1α, is shown to repress the reporter construct driven by the NELFE promoter. The binding site of Twist1 in the NELFE promoter is identified and chromatin immunoprecipitation assays show the direct binding of Twist1 to this site. Overexpression or knockdown of Twist1 in stable cell lines shows the inverse correlation between Twist1 and miR-1236 expression. These results identify a miRNA that regulates hypoxia-induced EMT and metastasis through repressing HDAC3 and SENP1 expression and present a regulatory network that involves many key players in hypoxia-induced EMT.

Introduction

Intratumoral hypoxia is one of the crucial events that mediate cancer metastasis [1], [2]. Hypoxia induces epithelial–mesenchymal transition (EMT), a critical process involved in development and tumor progression [3], [4]. EMT is a process whereby epithelial cell layers lose polarity and cell–cell contact and undergo a dramatic remodeling of cytoskeleton to increase cell migration and invasion activity [3], [4]. EMT is usually mediated by transcriptional regulators, including Snail, Slug, Twist1, ZEB1, ZEB2, etc [3], [4], [5]. Interestingly, these EMT transcriptional regulators are also regulated by hypoxia/hypoxia-inducible factor 1α (HIF-1α) [5], [6].

Hypoxia-induced EMT can occur through various epigenetic mechanisms [7], [8]. One of the important mechanisms is through the interplay between HDAC3 and WDR5 to regulate EMT marker gene (i.e. E-cadherin, vimentin, etc) expression [9]. HDAC3 removes the acetyl group of histone 3 lysine 4 (H3K4Ac) to facilitate the repression of epithelial gene and activation of mesenchymal gene expression [8], [9]. Knockdown of HDAC3 abolishes hypoxia-induced EMT marker gene regulation [8], [9]. HDAC3 obviously plays an important role in hypoxia-induced EMT. SUMO-specific protease 1 (SENP1) reverses the sumolyation of HIF-1α to prevent its ubiquitination and degradation [10]. SENP1 plays a critical role in the stabilization of HIF-1α [10].

MicroRNAs (miRNAs) are 18–24 nucleotide noncoding RNA molecules that regulate gene expression at various levels [11]. MiRNAs may have oncogene or tumor suppressor property depending on their targets [11], [12]. Various miRNAs have been shown to be involved in EMT [12]. Hypoxia/HIF-1α regulates various miRNAs to mediate tumor progression, induce drug resistance, and regulate tumor cell lysis by cytotoxic T cells [13], [14], [15], [16].

MicroRNA miR-1236 is an intronic miRNA (102 bp in size) that is located in chromosome 6p. MiR-1236-3p is the major form and there is no other family member (GeneCards and miRBase database). MiR-1236 and Negative Elongation Factor E (NELFE) share the same promoter and miR-1236 co-expresses with NELFE [17], [18]. MiR-1236 is found only in human, Pan troglodytes and Pongo pygmaeus (GeneCards and miRBase database). MicroRNA miR-1236 has been shown to repress VEGFR-3, α-fetoprotein, VprBP, RORγ, p21 promoter, and ZEB1 to inhibit inflammatory lymphangiogenesis, PI3K/Akt pathway, HIV-1 infection, ulcerative colitis, cell proliferation, or cell migration/invasion, respectively [19], [20], [21], [22], [23], [24]. MiR-1236 has been shown to be induced by IL-1β [19]. MiRNAs that regulate HDAC3 or SENP1 have been identified [25], [26]. However, whether a single miRNA can simultaneously regulate SENP1 and HDAC3 remains to be discovered.

In this study, we show that miR-1236 regulates EMT and cancer cell migration and invasion by simultaneously repressing HDAC3 and SENP1 expression. Due to the ability of SENP1 to stabilize HIF-1α, repression of miR-1236 during hypoxia causes abolishment of SENP1 inhibition and leads to increased HIF-1α levels. We also show that Twist1 represses the expression of miR-1236 by direct binding to the promoter region of miR-1236. We show that miR-1236 expression is repressed by Twist1 under hypoxia, which activates the levels of HDAC3 and SENP1 and contributes to EMT and cancer metastasis induced by hypoxia.