Evidence for MicroRNA-Mediated Regulation of Steroidogenesis by

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Evidence for microRNA-mediated regulation of steroidogenesis by hypoxia Man Kit Richard YU, Gayathri Chaturvedi, Steve Kwan Hok Tong, Suraia Nusrin, John Paul Giesy, Rudolf S.S. Wu, and Richard Kong Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es504676s • Publication Date (Web): 12 Dec 2014 Downloaded from http://pubs.acs.org on December 16, 2014

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Environmental Science & Technology

ORIGINAL RESEARCH ARTICLE

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Evidence for microRNA-mediated regulation of steroidogenesis by hypoxia

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Richard Man Kit Yu1, Gayathri Chaturvedi2, Steve Kwan Hok Tong2, Suraia Nusrin2,

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John Paul Giesy2,3,4, Rudolf Shiu Sun Wu4,5, Richard Yuen Chong Kong*,2,4

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School of Environmental and Life Sciences, The University of Newcastle, Callaghan, New

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South Wales, Australia; 2

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Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,

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Kowloon, Hong Kong Special Administrative Region, China; 3

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Saskatchewan, Saskatoon, Saskatchewan, Canada; 4

State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue,

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Department of Veterinary Biomedical Sciences and Toxicology Centre, University of

Kowloon, Hong Kong Special Administrative Region, China; 5

School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong

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Special Administrative Region, China.

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*To whom correspondence should be addressed: Richard Y. C. Kong, Department of Biology

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and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong

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Special Administrative Region, China. Tel.: 852-3442-7794; Fax: 852-3442-0522; E-mail:

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[email protected] .

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ACS Paragon Plus Environment


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ABSTRACT

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Environmental hypoxia can occur in both natural and occupational environments. Over the

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recent years, the ability of hypoxia to cause endocrine disruption via perturbations in steroid

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synthesis (steroidogenesis) has become increasingly clear. To further understand the

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molecular mechanism underlying hypoxia-induced endocrine disruption, the steroid-

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producing human cell line H295R was used to identify microRNAs (miRNAs) affecting

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steroidogenic gene expression under hypoxia. Hypoxic treatment of H295R cells resulted in

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the downregulation of seven steroidogenic genes and one of these, CYP19A1 (aromatase),

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was shown to be regulated by the transcription factor hypoxia-inducible factor-1 (HIF-1).

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Using bioinformatic and luciferase reporter analyses, miR-98 was identified to be a

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CYP19A1-targeting miRNA from a subset of HIF-1-inducible miRNAs. Gain- and loss-of-

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function analysis suggested that under hypoxia, the increased expression of miR-98 led to the

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downregulation of CYP19A1 mRNA and protein expression and that it may have contributed

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to a reduction in estradiol (E2) production. Intriguingly, luciferase reporter assays using

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deletion constructs of a proximal 5′-flanking region of miR-98 did not reveal a hypoxia-

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responsive element (HRE)-containing promoter. Overall, this study provided evidence for the

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role of miRNAs in regulating steroidogenesis and novel insights into the molecular

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mechanisms of hypoxia-induced endocrine disruption.

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ABSTRACT ART

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INTRODUCTION

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Environmental hypoxia is a state of reduced oxygen availability. Typically, it occurs in

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eutrophic waters (aquatic hypoxia), at high altitudes (hypobaric hypoxia) or in low-oxygen

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workplace environments, such as fire-protected rooms (normobaric hypoxia). Modulation of

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steroid synthesis (steroidogenesis) by hypoxia has been widely observed in various tissues,

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such as the gonads,1‒6 placenta7‒9 and adrenal glands.10,11 Because the steroidogenesis

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pathway is catalyzed by a number of oxygen-sensitive cytochrome P450 enzymes (CYPs),

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hypoxia is often thought to hinder steroidogenesis through enzymatic inhibition. However,

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recent evidence has suggested that it can also modulate the expression of steroidogenic genes,

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leading to either stimulation or inhibition of steroid production.3‒9,12‒14

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From invertebrates to mammals, the transcriptional response to hypoxia is primarily

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mediated by the transcription factor hypoxia-inducible factor-1 (HIF-1). HIF-1 is a

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heterodimeric protein that consists of an oxygen-sensitive HIF-1α subunit and a constitutively

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expressed HIF-1β subunit.15 Under hypoxic conditions, HIF-1α accumulates and dimerizes

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with HIF-1β to activate expression of HIF-1 target genes through binding to their hypoxia-

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responsive elements (HREs). To date, there are over 100 genes that are known to be regulated

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by HIF-1, including those involved in oxygen delivery (erythropoietin, EPO), angiogenesis

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(vascular endothelial growth factor, VEGF) and glucose uptake (glucose transporter 1,

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GLUT-1).16 The involvement of HIF-1 in the regulation of steroidogenic gene expression has

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been previously suggested by a number of studies. For instance, exposure to cobalt chloride

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(CoCl2, a chemical inducer of HIF-1) decreases CYP11A1 mRNA expression and hence

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progesterone production in testicular Leydig cells.17 Other studies have demonstrated that

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HIF-1 can bind to and activate the gene promoters of 3β-HSD1 and CYP19A1 in Leydig

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cells3 and breast adenocarcinoma cells,14 respectively. Despite these new insights, it remains

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to be determined whether hypoxia or HIF-1 regulates steroidogenic expression via any post-

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transcriptional mechanisms, such as microRNA (miRNA) regulation.

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MiRNAs have emerged as a novel class of post-transcriptional regulators of gene

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expression. They are small non-coding RNAs (20‒22 nucleotides) that bind imperfectly to

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the 3′-untranslated regions (3′-UTRs) of their target mRNAs, causing mRNA degradation

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and/or translational repression. It has been estimated that over one-third of human genes are

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regulated by miRNAs.18 Previously, a genome-wide screen has implicated approximately 50

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miRNAs in human ovarian steroidogenesis.19 Notably, most of the miRNAs have been shown

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to inhibit, and not to stimulate, the release of steroids. This raises the possibility that under

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hypoxia, some of these or other miRNAs may be induced by HIF-1 and be responsible for the

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hypoxic repression of certain steroidogenic genes.

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The main goals of this study were to examine the effects of hypoxia on the expression of a

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subset of steroidogenic genes essential for estradiol (E2) synthesis, including HMGR, StAR,

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CYP11A1, CYP17A1, 3β-HSD2, 17β-HSD1 and CYP19A1 (Figure 1), and to identify HIF-1-

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mediated miRNAs that contribute to the observed differential gene expression. To

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accomplish these goals, we employed the human adrenocortical carcinoma cell line H295R as

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an in vitro steroidogenesis model. This cell line expresses all of the key enzymes involved in

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adrenal and gonadal steroidogenesis, overcoming the limitation associated with the frequent

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tissue- and developmental stage-specific expression of these genes in vivo. Its versatility is

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exemplified by its use in the recently OECD validated H295R Steroidogenesis Assay for the

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screening and detection of endocrine disrupting chemicals.20 Our results have indicated that

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miR-98 is an HIF-1-regulated miRNA that attenuates CYP19A1 expression and E2

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production in hypoxic H295R cells.

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MATERIALS AND METHODS

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Cell Culture. H295R cells were obtained from the ATCC and cultured as described

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previously.21 Exposure of H295R cells (1.2 × 106) to normoxia (20% O2) or hypoxia (1% O2)

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was carried out at 37°C for 24 h. The normoxic or hypoxic conditions were created in a

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CO2 incubator by mixing 20% O2, 75% N2 and 5% CO2 or 1% O2, 94% N2 and 5% CO2,

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respectively, using a gas mixer (WITT). The O2 levels were monitored using a

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GasAlertMax XT II gas detector (BW Technologies) throughout the experiment.

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qRT-PCR. qRT-PCR for quantifying expression of steroidogenic genes was performed as

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described previously.21 miR-98 expression was quantified using a TaqMan MiRNA Reverse

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Transcription Kit and a TaqMan Gene Expression Assay (both from Applied Biosystems).

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U6 snRNA was used as a normalization control. The relative expression levels of

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steroidogenic genes and miR-98 were analyzed using the comparative threshold cycle (CT)

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method.22

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Western Blot Analysis—Western blot analysis was performed as previously described23

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using anti-HIF1α (BD Biosciences, #610959), anti-CYP19A1 (Santa Cruz, #sc-30086) and

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anti-β-actin (Sigma, #A2228) antibodies.

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Quantification of Hormones. Extraction and quantification of E2 and testosterone (T) in the cell culture medium were performed as described by Hecker et al.24

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Overexpression and Knockdown of miR-98. Overexpression and knockdown of miR-98

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were conducted using precursor (pre-miR-98) and inhibitor (anti-miR-98) molecules

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(Ambion), respectively. These molecules were transfected into H295R cells using a Neon

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electroporation system (Invitrogen; 3 × 1600 V, 10 ms) at final concentrations of 100 and 150

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nM for the pre-miR-98 and anti-miR-98, respectively. After 24 h, fresh medium was added to

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the electroporated cells, and they were incubated under hypoxic conditions for an additional

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24 h. The cells were then harvested for qRT-PCR and Western blot analyses. 6


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Preparation of Lentiviral Constructs for Overexpression and Knockdown of HIF-1α.

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Under normoxia, the HIF-1α protein is rapidly degraded via the ubiquitin-proteasome

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pathway that is initiated by the hydroxylation of two conserved proline residues (P402 and P564)

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present in the oxygen-dependent degradation (ODD) domain of HIF-1α.25,26 To express the

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normoxia-resistant HIF-1α protein, we generated an HIF-1α mutant construct (HIF1α:∆ODD),

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in which P402 and P564 were substituted with alanine residues using a GeneArt Site-Directed

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Mutagenesis System (Invitrogen). The mutated HIF-1α ORF was then cloned into a

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pENTR/D-TOPO entry vector (Invitrogen) and subsequently transferred to a pLenti6/V5-

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DEST lentiviral destination vector (Invitrogen) to produce an expression construct, pLenti-

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HIF1α. To accomplish the siRNA-mediated knockdown of HIF-1α, two complementary

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ssDNA oligonucleotides targeting HIF-1α were designed using BLOCK-iT RNAi Express

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software (Invitrogen). The ssDNA oligonucleotides were annealed to generate dsDNA

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oligonucleotides (HIF-1αi 956), which were then cloned into a pcDNA 6.2-GW/EmGFP

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vector (Invitrogen). Subsequently, an entry clone was generated via a pDONR221 donor

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vector (Invitrogen). The target sequence was finally transferred to a pLenti6/V5-DEST vector

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(Invitrogen) to generate an expression construct, pLenti-HIF1αi956, which was used for HIF-

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1α knockdown.

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Lentivirus Production and Transduction. Lentiviruses for overexpression and

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knockdown of HIF-1α were produced using a ViraPower Lentiviral Gateway Expression Kit

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(Invitrogen). Briefly, 3 x 106 293FT cells were seeded 1 day before transfection. A total of 3

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μg of lentiviral constructs and 9 μg of packing mix were cotransfected into the 293FT cells

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using Lipofectamine 2000 (Invitrogen). The transfected cells were then incubated at 37°C for

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48 h. Next, the cells were incubated with Lentivirus Concentrator (Clontech) at 4°C for 4 h to

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concentrate the viruses. Viral particles were recovered by centrifuging the medium at 3,000 

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g for 5 min at 4°C, and the pellet was resuspended in ice-cold DMEM/F12 complete medium 7



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and stored at ‒80°C until use. H295R cells (1 × 106) were seeded 1 day prior to transduction.

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A viral suspension containing either pLenti-HIF1α, pLenti-HIF1αi956 or empty pLenti6/V5-

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DEST (control) was applied to the H295R cells in the presence of polybrene (6 μg/ml;

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Sigma). The transduced cells were incubated at 37°C for 24 h. After incubation, the cells

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were fed with fresh medium and kept under normoxia or hypoxia at 37°C for another 24 h

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before harvesting.

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Preparation of Luciferase Reporter Constructs. To assess the interaction between miR-

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98 and CYP19A1 mRNA, a 174-bp fragment of the CYP19A1 3′-UTR was amplified by PCR

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using

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GCAAAGCTTCATGACCCCAAAGCCAAG-3′

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CAGACTAGTGCGAATTCCAAGGTGTGC-3′ (reverse), which contained HindIII and SpeI

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sites (underlined), respectively. The PCR product was double-digested with HindIII and SpeI

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and cloned into the HindIII-SpeI site of the pMIR-REPORT luciferase vector (Ambion)

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immediately downstream of the CMV-driven firefly luciferase gene. A construct possessing

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mutations disrupting the putative miR-98 binding sites in the 3′-UTR of CYP19A1 was

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prepared using a GeneArt Site-Directed Mutagenesis System (Invitrogen) with the following

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primers: 5-AAGTATTTTTTAATCCTAATTCAAAATTTAACAGTTAC-3′ (forward) and

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5-GTAACTGTTAAATTTTGAATTAGGATTAAAAAATACTT-3′ (reverse). Both the

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wild-type and mutated plasmid clones (designated as pMIR-19-WT and pMIR-19-MUT,

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respectively) were verified by DNA sequencing.

genomic

DNA

from

H295R

cells

and

the

following

(forward)

primers: and

5′5′-

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To assess the transactivation potential of HIF-1 on the miR-98 promoter, five deletion

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constructs of the miR-98 5′-flanking region (with or without putative HREs) were generated

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from genomic DNA by PCR using the following primers (restriction sites are underlined):

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forward primers, 5′-ACGTTGCTAGCCGCTGGACAGAAGAATGCAA-3′ for construct

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pGL-miR98p1 (‒2687/+17), 5′-AGGATGCTAGCTTGACTGTGTCTGCCGTAATGTG-3′ 8


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5′-

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for

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TGAACCTCGAGAGTGAACCTAGCCTGTGACTGA-3′ for construct pGL-miR98p3 (‒

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1473/+17), 5′-ATCCAGCTAGCAACAGAATCTCTCAGGTAAC-3′ for construct pGL-

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miR98p4 (‒1055/+17), 5′-GTCTCGCTAGCTCCTAATTCTGTGGTACC-3′ for construct

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pGL-miR98p5

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GCATCAAGCTTGGCATGAGCAGAATCCTCTAA-3′. The PCR products were double-

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digested with NheI (or XhoI) and HindIII and cloned into the NheI-HindIII or XhoI-HindIII

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site of a pGL4.10 vector (Promega).

construct

pGL-miR98p2

(‒522/+17),

and

a

common

(‒2283/+17),

reverse

primer,

5′-

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Transient Transfection and Luciferase Reporter Assay. Transfection into HeLa cells

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was carried out using Lipofectamine 2000 (Invitrogen) in a 24-well plate (1 × 105 cells/well).

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For the miRNA-mRNA interaction assay, cells were co-transfected with pMIR-REPORT

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constructs with or without pre-miR-98 (100 nM) or scrambled oligos (Ambion). For the miR-

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98 promoter assay, cells were co-transfected with pGL-miR98p1‒5 deletion constructs and

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HIF-1α expression vector pCMV-HIF1α, which contained the full-length HIF1α:∆ODD

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sequence inserted in pCMV-TNT (Promega). EPO-HRE-luciferase reporter plasmid (pGL-

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Epo-HRE), which contained four copies of the HRE sequence from the human EPO promoter,

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was used as a positive control. Cells were harvested at 24 h after transfection, and reporter

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assays were performed using a Dual-Luciferase Reporter Assay Kit (Promega). Luciferase

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expression was measured with a FLUOstar Optima plate reader (BMG Labtech) and

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normalized using β-galactosidase expression as an internal control. Data are expressed as the

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mean of the relative luciferase units (RLUs) measured in three independent experiments.

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Statistical Analysis. Student’s t-test or ANOVA followed by Student-Newman-Keuls

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post hoc test was used to test the null hypothesis that there was no significant difference

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between the values in the control and treatment groups. The results are presented as the mean

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± SD. Differences were considered to be statistically significant at a p-value of ≤0.05. 9



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RESULTS

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Hypoxia Downregulates CYP19A1 mRNA Expression via HIF-1. Exposure of H295R

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cells to hypoxia (1% O2) significantly suppressed mRNA expression of the majority of the

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steroidogenic genes examined with the exception of CYP11A1, which was not significantly

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affected by hypoxia (Figure 2A). All other genes, including HMGR, StAR, CYP17A1, 3β-

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HSD2, 17β-HSD1 and CYP19A1, showed 34-fold reductions in expression in the hypoxic

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cells. The activation of HIF-1 in hypoxic cells was supported by an increase in the expression

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of GLUT-1 (a HIF-1 target gene). To investigate the role of HIF-1 in regulating steroidogenic

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gene expression, gain- and loss-of-function experiments were carried out. Overexpression of

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HIF-1α under normoxia significantly decreased mRNA expression of StAR, CYP11A1 and

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CYP19A1 and increased that of CYP17A1; however, all of the changes in expression were

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present at low levels (

Evidence for MicroRNA-Mediated Regulation of Steroidogenesis by

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