HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells

Accepted Manuscript Title: HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells Authors: Kenji Ohe, Shinsuke Miyajima, Tomoko Tanaka, Yuriko Hamaguchi, Yoshihiro Harada, Yuta Horita, Yuki Beppu, Fumiaki Ito, Takafumi Yamasaki, Hiroki Terai, Masayoshi Mori, Yusuke Murata, Makito Tanabe, Kenji Ashida, Munechika Enjoji, Toshihiko Yanase, Nobuhiro Harada, Toshiaki Utsumi, Akila Mayeda PII: DOI: Reference:

S0960-0760(18)30212-7 https://doi.org/10.1016/j.jsbmb.2018.04.007 SBMB 5137

To appear in:

Journal of Steroid Biochemistry & Molecular Biology

Received date: Revised date: Accepted date:

15-9-2017 23-12-2017 13-4-2018

Please cite this article as: Ohe K, Miyajima S, Tanaka T, Hamaguchi Y, Harada Y, Horita Y, Beppu Y, Ito F, Yamasaki T, Terai H, Mori M, Murata Y, Tanabe M, Ashida K, Enjoji M, Yanase T, Harada N, Utsumi T, Mayeda A, HMGA1a induces alternative splicing of estrogen receptor alpha in MCF-7 human breast cancer cells, Journal of Steroid Biochemistry and Molecular Biology (2010), https://doi.org/10.1016/j.jsbmb.2018.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

HMGA1a induces alternative splicing of estrogen receptor alpha in MCF7 human breast cancer cells

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Kenji Ohe1, Shinsuke Miyajima4, Tomoko Tanaka2, Yuriko Hamaguchi2, Yoshihiro

Harada1, Yuta Horita1, Yuki Beppu1, Fumiaki Ito1, Takafumi Yamasaki1, Hiroki Terai1,

Masayoshi Mori1, Yusuke Murata1, Makito Tanabe2, Kenji Ashida3, Munechika Enjoji1, Toshihiko Yanase2, Nobuhiro Harada5, Toshiaki Utsumi4, Akila Mayeda6

Department of Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, 2Department of

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Endocrinology and Diabetes Mellitus, Faculty of Medicine, Fukuoka University, 8-19-1 Nanakuma,

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Jonan-ku, Fukuoka 814-180, Japan. 3

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Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu

University, Fukuoka, Japan.

Department of Breast Surgery, 5Department of Biochemistry, 6Division of Gene Expression

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Mechanism, Institute for Comprehensive Medical Science (ICMS), Fujita Health University, Toyoake, Aichi 470-1192, Japan

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Running title: HMGA1a induces exon-skipping of ERα

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Corresponding author.

Mailing address: Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1

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Nanakuma, Jonan-ku, Fukuoka 814-180, Japan. Phone:(+81-92-871-6631). Fax:(+81-92863-0389). E-mail: [email protected], or Institute for Comprehensive Medical Science (ICMS), Fujita Health University, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, Japan. Phone: (81-562) 93-9377. Fax: (81-562) 93-8834. E-mail: [email protected]. 1

Highlights

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The RNA-binding activity of HMGA1a regulates alternative splicing of ERα in

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MCF-7 breast cancer cells.

An RNA decoy of HMGA1a inhibits HMGA1a-directed ERα alternative splicing and sensitizes tamoxifen-resistant MCF-7 cells to tamoxifen.

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The HMGA1a RNA decoy increases estrogen-dependent growth of MCF-7 cell

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transplanted nude mice.

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Abstract

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The high-mobility group A protein 1a (HMGA1a) protein is known as an oncogene whose expression level in cancer tissue correlates with the malignant potential, and known as a

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component of senescence-related structures connecting it to tumor suppressor networks in

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fibroblasts. HMGA1 protein binds to DNA, but recent studies have shown it exerts novel functions through RNA-binding. Our previous studies have shown that sequence-specific

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RNA-binding of HMGA1a induces exon-skipping of Presenilin-2 exon 5 in sporadic Alzheimer disease. Here we show that HMGA1a induced exon-skipping of the estrogen

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receptor alpha (ERα) gene and increased ERα46 mRNA expression in MCF-7 breast cancer cells. An RNA-decoy of HMGA1a efficiently blocked this event and reduced

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ERα46 protein expression. Blockage of HMGA1a RNA-binding property consequently induced cell growth through reduced ERα46 expression in MCF-7 cells and increased sensitivity to tamoxifen in the tamoxifen-resistant cell line, MCF-7/TAMR1. Stable expression of an HMGA1a RNA-decoy in MCF-7 cells exhibited decreased ERα46 protein expression and increased estrogen-dependent tumor growth when these cells were 2

implanted in nude mice. These results show HMGA1a is involved in alternative splicing of the ERα gene and related to estrogen-related growth as well as tamoxifen sensitivity in

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MCF-7 breast cancer cells.

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Keywords: Estrogen receptor,Breast Cancer

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Introduction High-mobility group A (HMGA) gene products are known to be oncogenic and show

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increased expression in tumors with stronger malignancy or metastatic potential [1-3]. They cause neoplastic transformation [4, 5] and are known to affect chromatin structure through their DNA-binding function [6]. On the other hand, HMGA proteins have also been found to be involved in cellular senescence and heterochromatin formation through tumor suppressor networks in diploid fibroblast cells [7].

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Estrogen receptor alpha (ERα) gene is known to express various isoforms through

alternative splicing in a tissue-specific, disease-specific manner [8]. One of these isoforms,

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ERα46, antagonizes the function of full-length ERα66 in mammary carcinoma cells [9]. It

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is expressed through a promoter upstream of non-coding exon E/F in nonreproductive

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tissue and in MCF-7 mammary carcinoma cells [10]. This isoform lacks the N terminal A/B

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domain (173 amino acids) found in full-length ERα66, but retains the DNA-binding domain [10]. The B domain is known to possess one of the two transactivation functions (AFs), the

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ligand-independent AF-1. Therefore, ERα46 functions as a potent functional competitor of the mitogenic full-length ERα66 AF-1 activity on estrogen-responsive elements of target

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gene promoters [9]. Indeed, reduced ERα46 protein expression has been found in

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antiestrogen-resistant breast cancer cell lines correlating with dominant full-length ERα66 transcriptional activity [11].

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We provide here evidence that the RNA-binding activity of HMGA1a is involved in the expression of the ERα46 protein by inducing alternative splicing of the ERα gene in

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regulating estrogen-dependent tumor growth and tamoxifen sensitivity of MCF-7 cells.

Materials and methods

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Plasmids

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HMGA1a expression plasmid was constructed by PCR of human genomic DNA and

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inserted into EcoRI-BamHI site of pSG5. The plasmid for expressing nucleus-directed

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RNA-decoy of HMGA1aBSmutNLS (HMGA1a binding sequence mutant_nuclear localization signal) and HMGA1aBSwtNLS (HMGA1a binding sequence wild

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type_nuclear localization signal) was constructed by annealing the two oligonucleotides

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(Table 1, see decoy plasmid construction) HMGA1aBSmut_NLSf and HMGA1aBSmut_NLSr for HMGA1aBSmutNLS,

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HMGA1aBSwt_NLSf and HMGA1aBSwt_NLSr for HMGA1aBSwtNLS. These annealed oligonucleotides were subcloned into pcDNA6.2-GW/miR (Invitrogen) according to the

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manufacturer’s instructions to generate pcDNA6.2-HMGA1aBSmutNLS and pcDNA6.2-

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HMGA1aBSwtNLS, respectively.

Tamoxifen treatment

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MCF-7 cells, HMGA1aBSmut RNA decoy and HMGA1aBSwt RNA decoy MCF-7 stable transfectants were treated with 10 μM of tamoxifen for three days after 24h starvation in

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phenol-red free DMEM (Sigma, D5921) with 10% charcoal/dextran treated FBS (Hyclone, SH30068.02).

Transfection and RT-PCR

Transfection of MCF-7 cells was done by using Lipofectamine 2000 and Plus Reagent

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(Invitrogen). Cells were harvested 48 h after transfection. Total RNA of transfected MCF-7

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cells was extracted by TRIzol reagent (Invitrogen). After digesting with RQ1 DNase

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(Promega), one microgram of total RNA was reverse transcribed using M-Mulv reverse

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transcriptase (New England Biolabs) and random primer (Promega). Nested PCR of E/F ERα mRNA was performed using two sets of primers, step 1: E/F1f and E/F1r, step 2:

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and ERα46 (180 bp).

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E/F2f and E/F2r [10]. The two step PCR (20 cycles + 25 cycles) amplifies ERα (702 bp)

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Immunoblot assays

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MCF-7 cells were washed twice with PBS and resuspended in 1 ml TRIzol reagent (Invitrogen). After passing through a 25G needle five times, protein was purified from the

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interphase and organic phase by precipitating with 6V of a solution for precipitating protein (50% Ethanol, 24.5% Acetone, 24.5% Methanol, 1% distilled water). 20 μg of protein was boiled in sample buffer and separated by 10% sodium dodecyl sulfate-PAGE (SDS-PAGE), transferred to nitrocellulose membrane and analyzed by an ERα antibody (HC-20) (SantaCruz, sc-543) which recognizes the C-terminus of the protein and by HMGA1a antibody 6

(FL95; Santa Cruz) or phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (CST Japan, 9101S). Anti-rabbit immunoglobulin G conjugated to alkaline phophatase (Promega)

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was used as secondary antibody and detected by BCIP (5-bromo-4-chloro-3indolylphosphate) (Promega).

Generation of HMGA1a-RNA-decoy stable cell line and MTS assay

pcDNA6.2-HMGA1aBSmutNLS and pcDNA6.2-HMGA1aBSwtNLS were transfected into

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MCF-7 cells and cells were split and growth media containing DMEM supplemented with

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10% FBS and 10 μg/ml blasticidin (Invitrogen) was added after forty-eight hours. After 4

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days, blasticidin-resistant colonies were identified and further propagated. Cell viability

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was checked by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-

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sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 Aqueous One Solution Cell

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Proliferation Assay kit (Promega) in 96-well plates at a density of 500 cells/well in 50 μL medium. Prior the measurement cells were left to attach for 24 h at 37°C. At time 0 and

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each 24 h thereafter, 100 μl of MTS reagent was added to the cells and incubated at 37°C

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for 2 h. Relative colorimetric changes were measured at 492 nm.

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Tumor growth in vivo BALB/c nude mice (Balb/c-nu/nuSlc) (female, 4-8W) were obtained from Chubu Scientific Materials, Nagoya, Japan. The mice were kept under conditions of a 12-h light, 12-h dark cycle (lights on from 8:00 to 20:00), constant temperature (23–25 °C), and free access to 7

food and water. Mice were implanted with 17β-Estradiol pellets subcutaneously (0.25 mg/pellet, 60 days slow release; Innovative Research of America) one week prior to

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implanting 5×106 MCF-7 mammary carcinoma cells in phenol red free Matrigel (BD, Japan) (0.1 ml). The use of animals was kept to the minimum necessary to validate the data, and all animal protocols were carried out according to the National Institutes of Health

guidelines for the care and use of laboratory animals and approved by the Fujita Health University Committee for Animal Care. Tumor widths and lengths were recorded and

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tumor volumes were calculated using the formula (l × w2/2) [12]. After four weeks,

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animals were killed using an approved procedure. The use of animals was kept to the

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minimum necessary to validate the data, and all animal protocols were carried out

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according to the National Institutes of Health guidelines for the care and use of laboratory animals and approved by the Fujita Health University Committee for Animal Care

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(Protocol No. I0831 accepted in April 2011).

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Statistical Analysis

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Data are presented as mean±SD. Group differences were analyzed by Students t-test using

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Microsoft Excel.

Results

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HMGA1a induces and an HMGA1a RNA decoy inhibits ERα46 expression From our previous studies, we have shown sequence-specific RNA-binding of HMGA1a

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induces aberrant splicing of the presenilin-2 gene in sporadic Alzheimer’s disease [13]. HMGA1a is an oncogenic product, the expression level of which correlates with the

malignant potential of cancerous mammary cells [14]. Thus, we searched for an HMGA1a RNA-binding sequence [13] (5’-GCUGCUACAAG-3’) in the ERα gene. Since HMGA1a

functions only when it is adjacent a 5’ splice site sequence [15], the search was performed

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by attaching a GU dinucleotide to the 3’ end of the HMGA1a RNA-binding sequence.

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Using a web-based program, Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/),

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we aligned genomic sequence of the ERα gene and 5’-GCUGCUACAAGGU-3’ (note that

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the bold underlined GU dinucleotide is attached). In this way, a candidate was located 33

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nucleotides upstream the authentic 5’ splice site of ERα exon 1 (Fig. 1A). The 5’ splice site

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score of the adjacent pseudo-5’ splice site (MaxEnt 5’ splice site score: 8.67 [16]) is comparable to the authentic 5’ splice site (MaxEnt 5’ splice site score: 8.63) of this exon.

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The HMGA1a RNA-binding candidate sequence in ERα exon 1 is 5’- GCGGCUACACG 3’, a two-base mismatch of the original one we found previously, 5’-GCUGCUACAAG-3’

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[13] (mismatch underlined).

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Since an HMGA1a RNA-binding candidate sequence was in exon 1 of the ERα gene,

we tested whether it alters the expression of ERα46, an exon 1-skipped isoform of ERα which antagonizes proliferative action of full-length ERα66 in MCF-7 mammary carcinoma cells [9]. Transient expression of HMGA1a-induced endogenous ERα46 mRNA expression accompanied with reduced expression of ERα66 full-length non-reproductive 9

form [17] of ERα mRNA (Fig. 1B, lanes 1 and 2). Knockdown strategies were considered for confirming this result, but since HMGA1a is also a transcription factor [3], it was

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necessary to knockdown only its RNA-binding function. Transfection of the HMGA1a RNA-binding sequence using modified RNA oligonucleotides had the risk of modifying

off-target microRNA function, with similar antisense sequences in other mRNAs. Thus, we reinforced a novel strategy utilizing HMGA1a RNA-binding sequence combined with

nuclear localization signal (5’-AGUGUU-3’) found in certain microRNAs [18] (Fig. 1C).

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Transient expression of this HMGA1a RNA decoy inhibited ERα46 mRNA expression

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(Fig. 1C, lane 4), which was restored by co-expression of HMGA1a (Fig. 1C, lane 5).

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HMGA1a RNA decoy presumably saturated RNA-binding of the relatively low level of

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endogenous HMGA1a in MCF-7 cells [3]. This inhibition and reconstitution of HMGA1a

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RNA-binding activity showed that HMGA1a is responsible for ERα46 mRNA expression.

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HMGA1aBSwt RNA decoy decreased ERα46 protein level and increased cell viability To test the influence of the RNA-binding function of HMGA1a in MCF-7 cells,

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HMGA1aBSwt RNA decoy along with a mutant (HMGA1aBSmut RNA decoy) as control were stably expressed in MCF-7 cells and checked for the expression of full-length ERα66

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and ERα46 protein by immunoblot analysis using an ERα antibody which recognizes the Cterminus of the protein. As expected, MCF-7 cell stable transfectants of HMGA1a RNA decoy showed decreased expression of ERα46 protein compared with control RNA (HMGA1a RNA decoy mutant) (Fig. 2A). When assessed by MTS assay, HMGA1aBSwt

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RNA decoy exhibited an increase of cell viability compared to the control HMGA1aBSmut RNA decoy (Fig. 2B). This indicated that an RNA decoy of HMGA1a reduced ERα46

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protein level; a result of its ability to reduce ERα46 mRNA (Fig. 1). The increased cell viability by HMGA1aBSwt RNA decoy implied that HMGA1a RNA binding activity may

be related to the anti-oncogenic properties of HMGA1a [7]. When we tested the expression of phosphor-MAPK, we found it coincided with induced HMGA1a expression (Fig. 2C,

compare lanes 1 and 2, 3 and 4). However, the RNA decoy of HMGA1a did not attenuate

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phospho-MAPK expression (Fig. 2C, compare lanes 1 and 3 or 2 and 4). Thus, the RNA-

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binding activity of HMGA1a influenced alternative splicing of ERα and possibly its DNA-

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binding activity induced growth factor signaling. Since blocking RNA-binding activity of

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HMGA1a decreased ERα46, we hypothesized that this HMGA1a RNA decoy would increase estrogen-dependency in tamoxifen-resistant MCF-7 (MCF7/TAMR1) cells.

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Intriguingly, this was the case, expressing the RNA decoy sensitized the cells to tamoxifen

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in these cells (Fig. 2D, column “HMGA1a BSwt RNA decoy”).

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HMGA1aBSwt RNA decoy induce tumor formation in vivo When the MCF-7 cell stable transfectants of HMGA1aBSwt RNA decoy and control

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(HMGA1aBSmut RNA decoy) were implanted in ovariectomized nude mice along with an estrogen pellet (0.25 mg/pellet, 60 days slow release), tumors stably expressing HMGA1a RNA decoy exhibited increased growth compared to control MCF-7 cells and HMGA1a RNA decoy mutant stable cells after four weeks (Fig. 3). Taken together, we have shown

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HMGA1a blocked estrogen-response in MCF-7 through alternative splicing, and a decoy RNA of HMGA1a RNA-binding attenuated this event and promoted tumor-growth of

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MCF-7 cell stable transfectants of the HMGA1a RNA decoy in nude-mice (Fig. 3). Taken together, a working model of HMGA1a RNA-binding activity on estrogen-

dependent tumor growth is shown in Fig. 4. The RNA decoy of HMGA1a induced tumor growth, but shows the exciting possibilities of hyperstable binding of U1 snRNP to the

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upstream 5’ splice site in ERα exon 1 may suppress tumor growth in breast cancer cells.

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Discussion

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The expression of estrogen receptor alpha (ERα) is a hallmark in breast cancer, in

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that it is the main target for hormonal therapy. Seventy percent of breast tumors are ERαpositive [19]. Estrogen deprivation therapies such as the use of selective estrogen receptor

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modulators (SERM) [20] or aromatase inhibitors [21] are effective for these ERα-positive

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breast tumors. However, it is known that 50% of patients with advanced disease do not respond to the representative SERM, tamoxifen (de novo resistance) and that most patients

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experience tumor relapse (acquired resistance). Crosstalk between ERα and growth factor signaling is known to be the underlying mechanism for this resistance to therapy [22, 23].

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We propose here a mechanism of ERα gene expression through regulation of alternative splicing. The isoform we analyzed, ERα46, is regulated through alternative splicing of ERα exon 1 and is known to antagonize the function of full-length ERα66 [9]. According to our results of differential phospho-MAPK expression that shows no attenuation by the 12

HMGA1a RNA decoy, and the induced phospho-MAPK expression by HMGA1a, we believe the DNA-binding activity of HMGA1a is mainly stimulating the growth factor

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signaling as previously reported in MCF-7 cells [24]. Future studies are needed to delineate the proportional involvement of RNA- and DNA-binding activity in different contexts of hormone-resistant breast cancer.

During extensive analyses of the RNA-binding function of HMGA1a [13, 15, 25], we found an RNA element in the ERα gene where HMGA1a may function by regulating

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alternative splicing. The HMGA1a-binding sequence needs to be adjacently upstream a 5’

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splice site to trap and inhibit normal dissociation of U1 snRNP during spliceosome

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assembly [15]. We found an HMGA1a-binding sequence that fulfills these requirements in

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ERα exon 1 located 33 nucleotides upstream the authentic-5’ splice site and also adjacently

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upstream a pseudo-5’ splice site (Fig.1A). Hyperstable U1 snRNP / 5’ splice site-induced

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dysfunction [26] has been gathering attention, recently. hnRNPA1 [27], HMGA1a [15], TIA-1 [28] and hnRNPL [29] have been shown to inhibit splicing when the binding-site is

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in the exon and juxtaposed to a pseudo- or an authentic-5’ splice site. Previous reports in D. melanogaster have shown P-element somatic inhibitor protein (PSI) binds to a short

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pyrimidine-rich sequence [30] at the upstream pseudo-5’ splice site, interacts with hrp48 (homolog of mammalian hnRNPA1) [31], and with U1-70K protein to increase U1 snRNP-

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binding to the pseudo-5’ splice site and reduce U1 snRNP binding to the authentic-5’ splice site [32]. This induces intron retention of IVS3 of P-transposable element in somatic and germ line specific manner [33]. HMGA1a is highly reminiscent to PSI in the sense that it

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also binds to a sequence at the upstream pseudo 5’ splice site, interacts with hnRNPA1 [34] and U1-70K [13].

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We have shown here for the first time that an oncogene to be involved in alternative splicing of its target gene; HMGA1a regulates alternative splicing of the ERα gene. The

isoform we analyzed, ERα46, is known to lose expression in hormone-resistant cell lines [11]. Our HMGA1a RNA decoy suppressed HMGA1a-mediated alternative splicing and showed a decrease of ERα46 protein in MCF-7 cells and resulted in increased estrogen-

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mediated tumor growth in nude mice.

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Transgenic studies of HMGA1 have shown that MCF-7 cells increase sensitivity to

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the activated Ras/ERK mitogenic signaling pathway [24], and transgenic mice develop pituitary adenomas and NK cell lymphomas [5]. There have been reports about the

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correlation of HMGA1 and ERα expression in breast cancer, showing negative correlation

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[35] [36], and increased expression [37] and promotion of metastasis [38] in basal-like breast cancer. HMGA1a is not expressed in normal breast tissue, but found in hyperplastic

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regions with cellular atypia and ductal carcinoma correlating with c-erbB-2 (Her2)

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expression [39]. It has been shown that knockdown of HMGA1a in MCF-7 cells inhibits both proliferation and metastatic progression [3]. MCF-7 breast cancer cells are estrogen

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receptor positive and are a benign type of cell line with characteristics of normal mammary epithelial cells [40]. Thus, we believe HMGA1a functions anti-oncogenic through its sequence-specific RNA-binding properties in MCF-7 cells. The context-dependent HMGA1a-mediated alternative splicing of the ERα gene is involved in growth of ERαpositive breast tumors. This not only extends our previous findings that HMGA1a regulates 14

alternative splicing through its sequence-specific RNA-binding potential [15], but also the possibility of its involvement in other HMGA1a-related cancer through regulation of

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alternative splicing of its target gene. It must be taken of note that we have blocked the RNA-binding of HMGA1a, and that transcriptional regulation by HMGA1a is not

precluded. Splicing regulation through the HMGA1a binding site of ERα exon 1 may be a

promising therapeutic target, but simple overexpression of HMGA1a in estrogen receptorpositive breast cancer have to avoided due to its effects in promoting tumor progression.

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Moreover, the capability of HMGA1a RNA decoy to sensitize tamoxifen-resistant MCF-7

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cells to tamoxifen must be tested in vivo, as well as other cell lines or patient derived

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xenografts models.

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Author contributions

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K.O. conceived of the presented idea, developed the theory, carried out the experiments,

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and wrote the manuscript. S.M. helped the in vivo experiments. T.T., Yo.H., Yu.H., T.Y., F.I., Y.B., H.T., M.M., Y.M., M.E., T.Y. helped providing experimental environment.

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N.H., T.U., A.M. helped supervise the project. All authors discussed the results and

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contributed to the final manuscript.

Funding

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This work was supported by a Grant-in-Aid for Scientific Research (C) to K.O. (grant 24591920 and 15K10050) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and a Grants from Fujita Health University to A.M. and T.U.

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Declaration of interest No potential conflict of interest was disclosed.

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Acknowledgements

We are grateful to Gert Weber and Professor Reinhard Lührmann (Max Planck Institute of Biophysical Chemistry, Göttingen, Germany) for providing the purified U1 snRNP. We also thank Professor Raymond Reeves (Washington State University, WA, USA) and

Professor Masashi Narita (University of Cambridge, Cancer Research UK Cambridge

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Centre, UK) for helpful discussion.

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Figure legends Figure 1. HMGA1a regulates alternative splicing of the ERα gene in MCF-7 cells. (A)

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HMGA1a RNA-binding site and surrounding sequences in ERα exon 1 is indicated

(Estrogen receptor alpha exon 1). Arrows show the pseudo-5’ splice site and authentic 5’

splice site with 5’ splice site MAXENT scores. The HMGA1a binding site in ERα exon 1 is compared with the previously reported one in presenilin-2 (PS2) exon 5. Mutated bases are underlined (HMGA1a_BSmut). (B) Transient expression of HMGA1a induces increase of

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ERα46 mRNA. Right graph is quantification of RT-PCR in left panel. (C) Top shows the

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sequence of decoy-RNA of HMGA1aBSwt and control RNA (cont RNA) HMGA1aBSmut

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with nuclear localization signal (NLS) 5’-AGUGUU-3’ at 3’ end. These RNAs were

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expressed by a micro RNA expression vector. 5’-GCUGCUACAAG-3’ is the HMGA1a

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RNA-binding sequence found in Presenilin-2 exon 5. HMGA1a RNA-binding sequence is

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indicated and mutant with underlined nucleotides is where it is different from wild type. Nested RT-PCR of endogenous ERα and ERα46 mRNA in MCF-7 cells. Compared to cont

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RNA, decoy RNA had a strong exon-inclusion activity (decrease of ERα46/(ERα46+ERα66) mRNA ratio). Transient expression of HMGA1a to decoy RNA

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restored the decreased ERα46/(ERα46+ERα66) mRNA ratio. ( is a band resulting from

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unconventional splicing)

Figure 2. HMGA1a RNA-decoy suppresses ER46 protein and increases viability of MCF-7 breast cancer cells. (A) Western blot analysis of MCF-7 stable transfectants of

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HMGA1aBS RNA-decoys using ERα antibody which recognizes the C-terminus of ERα protein. Right graph is quantification of ERα66/(ERα46+ERα66) protein. (b) MTS assay of

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MCF-7 stable transfectants of HMGA1aBS RNA-decoys. indicates p<0.05, ** indicates p<0.01.

Figure 3. HMGA1a RNA-decoy promotes tumor growth in vivo. (A) Left picture is

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representative appearance of tumor growth, the tumor volume of which was quantified as

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the right graph for MCF-7 stable transfectants of HMGA1aBSwt RNA-decoy implanted in

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ovariectomized nude mice along with an estrogen pellet (0.25 mg/pellet, 60 days slow

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release). indicates p<0.05, ** indicates p<0.01.

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Figure 4. Model and mechanism of HMGA1a-targeted ER alternative splicing resulting

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in altered estrogen-response in MCF-7 breast cancer cells.

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Tables Table 1 Oligonucleotides used in this study Nucleotides corresponding to HMGA1a RNA-binding site are underlined and micro-RNA nucleus localization signal is in bold 5’HMGA1aBSmut_NLSf TGCTGTACCGCCGAGATCAGAGTGTTGTTTTGGCCACT GACTGACAACACTCTGATCTCGGCGGTA-3’ 5’HMGA1aBSmut_NLSr CCTGTACCGCCGAGATCAGAGTGTTGTCAGTCAGTGGC CAAAACAACACTCTGATCTCGGCGGTAC-3’ 5’HMGA1aBSwt_NLSf TGCTGTACCGCTGCTACAAGAGTGTTGTTTTGGCCACT GACTGACAACACTCTTGTAGCAGCGGTA-3’ 5’HMGA1aBSwt_NLSr CCTGTACCGCTGCTACAAGAGTGTTGTCAGTCAGTGGC CAAAACAACACTCTTGTAGCAGCGGTAC-3’ primers used for RTPCR E/F1f 5’-AAGGAGTAAGCACAAAGATCTC-3’ E/F1r 5’-CTCACAGGACCAGACTCCATAATGG-3’ E/F2f 5’-CAGCACTTCTTCAAAAAGGATGTAGA-3’ E/F2r 5’-AGCATAGTCATTGCACACTGC-3’

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decoy plasmid construction

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