An estrogen receptor a suppressor, microRNA-22, is downregulated in estrogen receptor a-positive human breast cancer cell lines and clinical samples Jianhua Xiong1,*, Dianke Yu2,*, Na Wei1, Hanjiang Fu3, Tianjing Cai1, Yuanyu Huang1, Chen Wu2, Xiaofei Zheng3, Quan Du1, Dongxin Lin2 and Zicai Liang1 Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing, China Department of Etiology and Carcinogenesis, State Key Laboratory of Molecular Oncology, Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China Beijing Institute of Radiation Medicine, China Keywords breast carcinoma; estrogen receptor a; microRNA-22; proliferation Correspondence Z Liang, Laboratory of Nucleic Acid Technology, Institute of Molecular Medicine, Peking University, Beijing 100871, China Fax: +86 10 62769862 Tel: +86 10 62769862 E-mail: liangz@pku.edu.cn Dongxin Lin, Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, China Fax: +86 10 67722460 Tel: +86 10 87788491 E-mail: lindx72@cicams.ac.cn *These authors contributed equally to this work (Received 30 September 2009, revised January 2010, accepted 25 January 2010) doi:10.1111/j.1742-4658.2010.07594.x Previous studies have suggested that microRNAs (miRNAs) may play important roles in tumorigenesis, but little is known about the functions of most miRNAs in cancer development In the present study, we set up a cell-based screen using a luciferase reporter plasmid carrying the whole  4.7 kb 3¢-UTR of estrogen receptor a (ERa) mRNA cotransfected with a synthetic miRNA expression library to identify potential ERa-targeting miRNAs Among all the miRNAs, miR-22 was found to repress robustly the luciferase signal in both HEK-293T and ERa-positive MCF-7 cells Mutation of the target site was found to abrogate this repression effect of miR-22, whereas antagonism of endogenous miR-22 in MDA-MB-231 cells resulted in elevated reporter signals We assessed the miR-22 expression patterns in five breast cancer cell lines and 23 clinical biopsies and revealed that there is a significant inverse association between the miR-22 levels and ERa protein expression To evaluate the potential of miR-22 as a potential therapeutic intervention, we found that reduction of endogenous ERa protein levels and suppression of cancer cell growth could be achieved in MCF-7 cells by miR-22 overexpression in a way that can be recapitulated by the introduction of specific small interfering RNA against ERa The phenomena can be rescued by the reintroduction of ERa Taken together, our data indicate that miR-22 was frequently downregulated in ERa-positive human breast cancer cell lines and clinical samples Direct involvement in the regulation of ERa may be one of the mechanisms through which miR-22 could play a pivotal role in the pathogenesis of breast cancer Introduction MicroRNAs (miRNAs), a class of endogenous short ( 22 nucleotides) noncoding RNAs, have been reported to be capable of suppressing the expression of protein-coding genes at the post-transcriptional level by cleaving target mRNAs and ⁄ or repressing their translation [1] Aberrant expression of miRNAs is known to be involved in various human diseases, including cancer [2,3] In two recent studies, more than 50% of human miRNA genes have been mapped to the cancer-related chromosomal regions with high Abbreviations DMEM, Dulbecco’s modified Eagle’s medium; ERa, estrogen receptor a; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miRNA, microRNA; siRNA, small interfering RNA 1684 FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS J Xiong et al frequencies of amplification or deletion, and frequent genomic alterations of miRNAs were observed in cancers [4,5] Emerging evidence shows that miRNAs function as oncogenes or tumor suppressors to modulate multiple oncogenic cellular processes, including cell proliferation, apoptosis, invasion and migration [6–8] For example, it has been shown that p53-dependent miR-34b and miR-34c cooperate to inhibit the proliferation of neoplastic epithelial ovarian cells [6], and miR-15 and miR-16 simulate apoptosis in chronic lymphocytic leukemia by targeting BCL2 [7] On the other hand, miR-10b initiates breast cancer invasion and metastasis by repressing homeobox D10 [8] Moreover, miRNA expression profiling has been used as a signature to distinguish different cancer types and to provide an accurate classification of poorly differentiated tumors [9] In view of the roles that miRNAs play in human diseases, including cancer, miRNAs have been considered to be potential drug candidates or therapeutic targets [10] Breast cancer is one of the most common and prevalent cancers in women and a leading cause of cancerrelated death [11] As in other common cancers, the formation and progression of breast cancer is a multistep process involving genetic and epigenetic alterations that drive unrestrained cell proliferation and growth [12,13] Several aberrantly expressed miRNAs have been identified in breast cancer, such as miR-21, miR-205 and miR-17-5p, which were shown to be involved in the formation of breast cancer by targeting the TPM1, HER3 and AIB1 genes, respectively [14– 16] However, the functional roles of most miRNAs in the development of breast cancer remain unknown In this study, we identified miR-22 as a potent regulator of ESR1 encoding estrogen receptor a (ERa) and demonstrated that miR-22 is frequently downregulated in ERa-positive human breast cancer cell lines and clinical samples In addition, further functional studies showed that ERa plays an important role in miR-22-mediated growth retardation of tumor cells Results Identification of miRNAs that might target ERa 3¢-UTR To identify human miRNAs that might target ERa 3¢-UTR, we used the targetscan program (http:// www.targetscan.org/) to predict miRNAs that have the interaction with 3¢-UTR of ESR1 mRNA Along the  4.3 kb full length of 3¢-UTR of ESR1 mRNA, 59 miRNAs had conserved target sites and partial miRNA families broadly conserved among vertebrates MicroRNA-22 and estrogen receptor a were enumerated according to their conserved target positions (Fig 1A) In addition, miR-206, which has two target sites on 3¢-UTR of ESR1 mRNA, was previously reported as a negative regulator of ERa [17] miR-9 and miR-1 were implicated in crucial cancerrelated cell signaling regulation [18,19] The 62 miRNAs were chosen as our preferred candidates for ERa regulators To evaluate comprehensively miRNAs–ESR1 mRNA interactions, we used the screening system based on a luciferase reporter plasmid carrying the full-length 3¢-UTR of ESR1 mRNA As a result, nine miRNAs were found to suppress the expression of the reporter by more than 40%, and 25 miRNAs were found to suppress the expression of the reporter by more than 20% (Fig 1B) This might constitute the major category of miRNAs that play regulatory roles on ERa through interactions with 3¢-UTR of ESR1 mRNA As indicated in Fig 1B, miR-22 could induce an 40% reduction in the luciferase signal Direct regulation of ERa expression by miR-22 We compared three popular miRNA target prediction programs and found that miR-22 was highly scored in all three algorithms [targetscan, miranda (http:// www.microrna.org/microrna/home.do) and pictar (http://pictar.mdc-berlin.de/)] for targeting ESR1 (Table S1) [20,21] The targetscan prediction suggested that ESR1 has an extremely conserved miR-22 target site (position 2292–2298 of human ESR1 3¢-UTR) in human and other mammalian species, including the chimpanzee, rhesus monkey, mouse, rat, dog and rabbit (Fig 2A) The predicted DG of 70 bp 5¢- and 3¢-flanking regions of neighboring potential conserved miR-22 target site was determined by mfold and the resulting DG values ()10.70 and )8.40 kcalỈmol)1, respectively) suggested that miR-22 may have access to its conserved target ESR1 mRNA site [22,23] We therefore constructed a reporter plasmid (pGL3m–ESR1–3¢-UTR–WT) with the 4.3 kb ESR1 3¢-UTR cloned downstream to a firefly luciferase reporter gene and used both vector-expressed miR-22 and synthetic miR-22 to evaluate the suppression effects of the miRNAs on the reporter gene expression It was found that in HEK293T and ERa-positive MCF-7 cells, miR-22 had a potent inhibitory effect on the expression of the reporter gene with the ESR1 3¢-UTR tag (Fig 2B, C) To examine whether the ERa silencing is mediated by specific and direct interaction of miR-22 with the ESR1 target site, the complementary site for the miR-22 seed region was mutated to FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1685 MicroRNA-22 and estrogen receptor a J Xiong et al A B Fig Identification of miRNAs that might target ERa 3¢-UTR (A) The 59 miRNAs predicted as having broadly conserved sites by the TARGETSCAN program Partial miRNA families broadly conserved among vertebrates were enumerated according to their conserved target positions (B) The effects of the predicted 59 miRNAs as well as three interested miRNAs (miR-206, miR-9 and miR-1) on reporter gene expression of pGL3m–ESR1–3¢-UTR–WT Relative luciferase activity was measured 48 h after transfection and normalized by Renilla luciferase activity generated by cotransfected pRL-TK vector The normalized luciferase activity for the controls was set as 100% Data are presented as mean ± standard deviation from at least three independent experiments form pGL3m–ESR1–3¢-UTR–MUT (Fig 2A) Both pcDNA3.0–miR-22 and miR-22 duplex reduced luciferase activities expressed in pGL3m–ESR1–3¢-UTR– WT by  50%, but such a reduction was completely abolished in pGL3m–ESR1-3¢–UTR–MUT (Fig 2B, C) Moreover, knockdown of endogenous miR-22 in MDA-MB-231 cells that express a relatively high level miR-22 could elevate the luciferase signal of pGL3m– ESR1–3¢-UTR–WT (Fig 2D), further suggesting that silencing of ERa was indeed by the interaction of miR-22 with the 3¢-UTR of ESR1 The effect of miR-22 on endogenous ERa protein levels was also examined in MCF-7 and MDA-MB- 1686 231 cells The results showed that an ectopic increase in either synthesized or vector-expressed miR-22 in MCF-7 led to an 50% reduction in ERa protein levels (Fig 2E) Conversely, ERa expression was significantly elevated by inhibiting endogenous miR-22 in MDA-MB-231 cells (Fig 2F) Interestingly, the reduction in ERa protein levels was markedly greater than the reduction in ESR1 mRNA levels determined by quantitative RT-PCR (Fig 2G) These results demonstrated that miR-22 could regulate ERa expression by directly binding to ERa 3¢-UTR, and inhibited ERa expression through both destabilizing mRNA and inhibiting translation FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS J Xiong et al MicroRNA-22 and estrogen receptor a A B C E D F G Fig Direct regulation of ERa expression by miR-22 (A) A putative miR-22-binding target region in the 3¢-UTR of ESR1 mRNA among mammalian species (upper panel, shown in red); site-direct mutations in the sequence complimentary to the seed region for miR-22 (lower panel, shown in red) (B–D) Relative luciferase activity of pGL3m–ESR1–3¢-UTR–WT (ESR1–3¢-UTR–WT) and pGL3m–ESR1–3¢-UTR–MUT (ESR1–3¢-UTR–MUT) in HEK293T and MCF-7 cells cotransfected with pcDNA-3.0–miR-22 or pcDNA-3.0 and synthetic miR-22 duplex or control RNA duplex,and in MDA-MB-231 cells with anti-miR-22 or control anti-miR Relative luciferase activity was measured 48 h after transfection and normalized by Renilla luciferase activity generated by cotransfected pRL-TK vector The normalized luciferase activity for the controls was set as Data are presented as mean ± standard deviation from at least three independent experiments (**P < 0.01) (E) Suppression of ERa expression in MCF-7 cells by pcDNA-3.0–miR-22 or synthetic miR-22 duplex MCF-7 cells were harvested 48 h after transfection and cell lysate was applied to a western blot b-actin was used as a loading control and the relative density of bands was densitometrically quantified (F) Upregulation of ERa expression in MDA-MB-231 cells by anti-miR-22 MDA-MB-231 cells were harvested 48 h after transfection and cell lysate was applied to a western blot b-actin was used as a loading control (G) Relative level of ERa mRNA was detected using quantitative RT-PCR with GAPDH as an internal control FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1687 MicroRNA-22 and estrogen receptor a J Xiong et al Frequent downregulation of miR-22 expression in ERa-positive breast cancer cell lines and clinical samples To evaluate the therapeutic potential and to extend the mechanistic insight of miR-22 as an ERa suppressor, we measured its expression levels using quantitative RT-PCR in five breast cancer cell lines that are either ERa positive (MCF-7, T-47D and BT-474) or ERa negative (MDA-MB-231 and SK-BR-3), and 23 breast tumor specimens, of which 10 are ERa positive and 13 are ERa negative Among all breast cancer cell lines examined, miR-22 expression was found to be significantly lower in ERapositive lines, such as MCF-7 (2.290 ± 0.499), T-47D (1.573 ± 0.325) and BT474 (1.152 ± 0.318), than in ERa-negative lines, such as MDA-MB-231 (10.732 ± 1.923) and SK-BR-3 (4.269 ± 1.027) The differences were determined by Student’s t-test as P = 0.0018 for the comparison between MCF-7 and MDA-MB-231, P = 0.0012 for the comparison between T-47D and MDA-MB-231 and P = 0.0010 for the comparison between BT-474 and MDA-MB231, but P = 0.0399 for the comparison between MCF-7 and SK-BR-3, P = 0.0123 for the comparison between T-47D and SK-BR-3 and P = 0.0074 for the comparison between BT-474 and SK-BR-3 (Fig 3A) The ERa expression status of breast cancer cell lines was confirmed using immunoblotting (Fig 3B) For breast cancer clinical samples, ERa-positive breast tumor specimens had significantly lower miR-22 levels (0.913 ± 0.807, range 0.112–2.10) than ERanegative specimens (2.410 ± 2.550, range 0.615–9.64; P = 0.044; Fig 3C) As indicated, the P value was 0.044; the Kruskal–Wallis one-way analysis of variance test indicated that the levels of miR-22 were inversely associated with ERa expression status in tumor specimens, which is in good agreement with the inverse correlation between the expression of miR-22 and ERa in breast cancer cell lines ERa is potentially involved in miR-22-mediated repression of ERa-positive breast cancer cell growth To investigate the role of ERa in miR-22-mediated repression of human cancer cell growth of ERa-positive breast cancer cells we used two specific small interfering RNAs (siRNA) against ERa MCF-7 cells were transfected with ERa siRNAs or control RNA duplex After incubation for 48 h, the expression of ERa was subjected to quantitative RT-PCR detection or 1688 A B C Fig Frequent downregulation of miR-22 expression in ERa-positive breast cancer cell lines and tumor specimens (A) For breast cancer cell lines, the expression levels of mature miR-22 were determined by quantitative RT-PCR with U6 as an internal standard miR-22 expression levels are presented as mean ± standard deviation from at least three independent experiments The P values of comparisons between two groups are as follows: P = 0.0018, comparison between MCF-7 and MDA-MB-231; P = 0.0012, comparison between T-47D and MDA-MB-231; P = 0.0010, comparison between BT-474 and MDA-MB-231; P = 0.0399, comparison between MCF-7 and SK-BR-3; P = 0.0123, comparison between T-47D and SK-BR-3; P = 0.0074, comparison between BT-474 and SK-BR-3 (B) The ERa expression status of breast cancer cell lines was examined by western blotting (C) For breast cancer specimens, miR-22 expression data are illustrated using a box plot The line inside each box is the median; the upper and lower limits of the box are the 75th and 25th percentiles, respectively, and the vertical bars above and below the box indicate the maximum and minimum values The solid circles are outlier values The expression levels of mature miR-22 were determined by quantitative RT-PCR with U6 as an internal standard (*P = 0.044) FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS J Xiong et al immunoblot analysis The result showed that the two siRNAs could reduce ERa mRNA and protein level significantly (Fig 4A,B) Further functional studies showed that knockdown of ERa by the two siRNAs could mimic the inhibitory effect of miR-22 on the proliferation and colony formation of breast cancer cells, whereas a control siRNA duplex did not show an effect (Fig 4C,D) We then went further to determine whether overexpression of ERa could counterbalance the antigrowth effect of miR-22 on MCF-7 cells We forced MCF-7 cells to express ERa constitutively using a construct encoding the entire encoding region of ERa mRNA, but lacking the ERa 3¢-UTR, thus yielding an mRNA that is resistant to miR-22-mediated inhibition of translation Indeed, we found that the miR-22-induced cell growth repression phenotype was partially rescued by the introduction of this vector expressing an miRNA-resistant ERa transcript (Fig 4C, D) These findings suggest that ERa plays an important role in miR-22-retarded growth of ERa-positive breast cancer cells Discussion To date, more than 700 human miRNAs have been identified using experiment-driven methods and computation-driven approaches [24,25] miRNAs have diverse expression patterns in different cell types and it is well accepted that miRNAs regulate numerous physiological and pathological processes [1,26] The biological function of most miRNAs is, however, largely unknown miRNAs have been relatively better investigated in tumor cells and it has already been shown that miRNAs can function as both tumor suppressors and oncogenes by directly regulating genes involved in related pathways Unrestrained cell proliferation and deregulated cell death underlie neoplastic progression in almost all cancer types [13,27] An increasing number of miRNAs have been implicated in tumorigenesis via the regulation of cancer cell proliferation and growth For instance, let-7 can inhibit proliferation of lung and liver cancer cells by targeting multiple cell cycle oncogenes [28] and miR-34b and miR-34c have a cooperative negative effect on proliferation and colony formation of ovarian cancer cells [6], whereas overexpression of the miR-17-92 cluster miRNAs enhance lung cancer cell proliferation and growth as oncogenes [29] The highly conserved human miR-22 gene is located at a fragile cancer-relevant genomic region in chromosome 17 (17p13.3), and mapped to an exon of the C17orf91 gene [4,30] To date, several genes, including HOXA6, HOXA4, HSPG2, GPNMB, CLIC4 and SP1, have been predicted as targets of miR-22 [31–33], MicroRNA-22 and estrogen receptor a whereas ERa has been suggested as a direct target of this miRNA in a recent work [34] miRNA expression profiling data revealed that miR-22 had a great reduction in acute myeloid leukemia with mutations in NPM1 compared with acute myeloid leukemia without NPM1 mutations, and HOXA6–HOXA4 were predicted as targets of miR-22 [31] miR-22 has been detected with a distinct expression pattern in human Duchenne muscular dystrophy, where HSPG2, GPNMB and CLIC4 were predicted as its potential targets [32] HSPG2 has been reported to contribute to tumor growth and angiogenesis in vivo [35], and GPNMB was identified as a pathological and diagnostic marker in melanocyte tumor progression [36] Moreover, CLIC4 was found to participate in stressinduced apoptosis in human osteosarcoma cells [37] Flow cytometry analysis showed that overexpression of miR-22 could reduce ERa and SP1 protein levels in pancreatic cancer cells [33] Estrogen receptors (mainly ERa and ERb) constitute a group of ligand-activated nuclear receptors that are activated by estrogen Human ERa is a transcription factor that regulates diverse gene expression, and is implicated in cancers by stimulating cell proliferation and tumor growth [38,39] An miRNA library-based screening with miR-206, miR-18a and miR-221 ⁄ 222 as putative positive controls [17,40,41] demonstrated that miR-22 could robustly suppress the luciferase signal of ERa 3¢-UTR tethered vector By mutating the complementary site for the miR-22 seed region, we showed that repression of ERa by miR-22 was almost completely abolished Conversely, the ERa signal intensities were significantly elevated by knockdown of endogenous miR-22 in MDA-MB-231 cells that expressed a relatively high level of miR-22 These results demonstrate that ERa is a direct target of miR-22 miR-22 treatment was found to dramatically reduce the endogenous translational yield of ERa, and knockdown of endogenous miR-22 could elevate ERa protein expression Because ERa expression is routinely monitored in breast cancer samples as a prognostic marker, we went further to assess the correlation between miR-22 expression and ERa protein levels in breast cancer cell lines and surgical specimens It was interesting to find that downregulation of miR-22 expression occurs frequently, not only in ERa-positive human breast cancer cell lines, but also in surgical specimens compared with ERa-negative counterparts This result made it appealing to examine whether miR-22 could also be used as a marker for the identification of breast cancer subtypes in addition to ERa itself, as miR-22 probably regulates a different set of genes in comparison with the regulatory profile of ERa FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1689 MicroRNA-22 and estrogen receptor a A J Xiong et al B C D Fig ERa is potentially involved in miR-22-mediated repression of human ERa-positive breast cancer cell growth (A) Regulation of ERa expression by two ERa siRNAs MCF-7 cells were transfected with control RNA duplex or ERa siRNAs, total RNAs were prepared and analyzed for ERa mRNA expression by quantitative RT-PCR at 48 h after transcription The data were normalized against the expression of GAPDH mRNA Data are presented as mean ± standard deviation from at least three independent experiments (B) Regulation of ERa expression by two ERa siRNAs MCF-7 cells were transfected with control RNA duplex or ERa siRNAs, total cellular proteins were prepared and analyzed for ERa protein expression by western blotting at 48 h after transfection b-actin was used as a loading control and the relative density of bands was densitometrically quantified (C, D) Knockdown of ERa could recapitulate the phenotype of repressed cell growth induced by miR-22 overexpression MCF-7 cells were transfected with control RNA duplex or miR-22 duplex or ERa siRNAs for 24 h incubation In the other two groups, reintroduction of ERa abrogates the antigrowth effect of miR-22 MCF-7 cells were first transfected with miR-22 duplex, and at 24 h after transfection, sequentially transfected with ERa-expressing vector pcDNA3.1–ESR1 (indicated as ERa) or empty vector pcDNA3.1 (empty vector) for 24 h incubation (C) Single-cell suspensions containing 20 000 cells treated respectively were seeded in each well of 24-well plates at 37 °C as attached monolayers in DMEM containing 10% fetal bovine serum Cells were harvested by treatment with trypsin and counted every 24 h in triplicate (D) Single-cell suspensions containing 10 000 cells treated respectively were seeded in each well of six-well plates coated with soft agar The plates were photographed after incubation at 37 °C for weeks Data are presented as mean ± standard deviation from at least three independent experiments *P < 0.05; **P < 0.01; ***P < 0.001, compared with control-RNA-duplex-transfected cells or comparison between two groups as indicated 1690 FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS J Xiong et al In summary, we showed that frequent downregulation of miR-22 expression is associated with ERa-positive human breast cancer cells, and miR-22 can directly regulate ERa expression We further showed that ERa is potentially involved in miR-22-mediated repression of human cancer cell growth of ERa-positive breast cancer cells It would then be interesting to explore whether miR-22 could serve as a potential therapeutic reagent in the treatment of cancer in which ERa plays an important role Materials and methods MicroRNA-22 and estrogen receptor a RNA isolation and quantitative RT-PCR detection Total RNAs from cultured cells were isolated using TRI Reagent (Sigma, St Louis, MO, USA) and converted into cDNA using ImPro-II reverse transcriptase (Promega) Detection of the mature form of miR-22 was performed using Quantitect SYBR Green PCR Kit (Qiagen, Hilden, Germany) and quantitative RT-PCR Primer Sets (Ribobio, Guangzhou, China) with the U6 small nuclear RNA as an internal control Detection of the ERa mRNA was performed using Quantitect SYBR Green PCR Kit (Qiagen), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA as an internal control The detection primers for ESR1 and GAPDH are included in Table S2 Cell lines and cultures Five breast carcinoma cell lines were obtained from the Cell Resource Center of Peking Union Medical College (Beijing, China) and maintained in our laboratory HEK293T (American Type Culture Collection, Manassas, VA, USA) and MCF-7 cells were maintained in 10% fetal bovine serum-supplemented Dulbecco’s modified Eagle’s medium (DMEM) (Hyclone, Logan, UT, USA); MDA-MB-231 cells were maintained in 10% fetal bovine serum-supplemented L-15 (Gibco, Grand Island, NY, USA); SK-BR-3 cells were maintained in 10% fetal bovine serum-supplemented RPMI-1640 (Hyclone); T-47D and BT-474 cells were maintained in RPMI-1640 (Hyclone) plus 10% fetal bovine serum and 0.2 mL)1 insulin Vector construction A DNA segment encompassing the mature miR-22 sequence and its 5¢- and 3¢-flanking regions (130 and 80 bp, respectively) was cloned into the BamHI and XhoI sites in pcDNA3.0 (Invitrogen, Carlsbad, CA, USA) to create the miR-22 expression vector pcDNA3.0–miR-22 pGL3m was modified from a firefly luciferase-expressing vector pGL3-control (Promega, Madison, WI, USA) by inserting a multiple cloning sequence downstream of the XbaI site, including EcoRV, ApaI, SacII, NdeI, PstI, EcoRI and NruI sites The insertion site is immediately downstream of the stop codon of the firefly luciferase reporter gene A 4.3 kb fragment encoding the full-length 3¢-UTR of human ESR1 mRNA (Genbank accession no NM_000125) was cloned between the SacII and EcoRI sites in pGL3m, forming pGL3m–ESR1–3¢-UTR–WT, in which, site-specific mutations were performed to disrupt the binding site of miR-22, forming pGL3m–ESR1–3¢UTR–MUT The ERa-expressing vector (pcDNA3.1– ESR1) was created by cloning the ESR1 coding sequence into the EcoRI and NheI sites of pcDNA3.1 (Invitrogen) The primers used in the subcloning experiments are included in Table S2; all the construct products were validated by sequencing RNA oligoribonucleotides and cell transfections The miRNA mimic library was obtained from Ribobio (Guangzhou, China); miR-22 duplex and the negative control RNA duplex (indicated as control RNA duplex) were obtained from GenePharma (Shanghai, China) (Fig S1) The control RNA duplex was used to eliminate the potential nonsequence-specific effects and its sequences were nonhomologous to any human genome sequences MCF-7 cells were transfected with 50 nm RNA duplex using lipofectamine 2000 (Invitrogen) and counted 24 h after transfection for plating wells to observe proliferation and colony formation The anti-miR-22 was a 2¢-O-methyl-modified oligoribonucleotide designed as an inhibitor of miR-22, and its sequence is 5¢-ACAGUCUUCAACUGGCAGCUU-3¢ The negative control for anti-miR-22 in the antagonism experiments was control anti-miR, with a sequence of 5¢-GUG GAUAUUGUUGCCAUCA-3¢ The sequences of two siRNAs for ESR1 are as follows: ERa siRNA #2 sense strand 5¢-UCAUCGCAUUCC UUGCAAAdTdT-3¢, antisense strand 5¢- UUUGCAAGGAAUGCGAUGAdTdT-3¢; ERa siRNA #3 sense strand 5¢- GGAGAAUGUUGAAACA CAAdTdT-3¢, antisense strand 5¢- UUGUGUUUCAA CAUUCUCCdTdT-3¢ The transfection efficiency was monitored by fluorescence-activated cell sorting, using a carboxyfluorescein (FAM)-labeled siRNA Western blot Forty-eight hours after transfection, the cells were lysed using cell lysis buffer (Cell Signaling Technology, Beverly, MA, USA) Isolated proteins were separated in 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA, USA), and detected with antibodies for human ERa (Cell Signaling Technology), b-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ECL kit (Santa Cruz Biotechnology) The intensity of protein bands was quantified using image j software (National Institutes of Health, Bethesda, MD, USA) FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS 1691 MicroRNA-22 and estrogen receptor a J Xiong et al Table Clinical characteristics of patients with breast carcinoma Soft-agar colony assay ERa positive ERa negative Variable n (%) n (%) Total no Age < 50 years ‡ 50 years Tumor size £ cm > cm Lymph node metatasis No Yes Histology Invasive ductal carcinoma Medullary carcinoma 10 (43.5) 13 (56.5) (30.0) (70.0) (46.2) (53.8) Anchorage-independent growth was carried out in six-well plates coated with 0.6% soft agar in DMEM plus 10% fetal bovine serum Twenty-four hours after transfection, · 104 transfected cells were plated into each well of six-well plates and maintained in DMEM plus 10% fetal bovine serum for weeks Colonies were stained with 1.25 mgỈmL)1 nitroblue tetrazolium for 16 h before imaging (50.0) (50.0) (53.8) (46.2) Statistical analysis (40.0) (60.0) (61.5) (38.5) 10 (100) (0) 12 (92.3) (7.7) Luciferase reporter assay For miRNA duplex library analysis, HEK293T and MCF-7 cells per well in 24-well plates were cotransfected with 50 nm miRNA duplex, 120 ng pGL3m–ESR1–3¢-UTR–WT and ng pRL-TK (Promega) in duplicate For miR-22 analysis, cells were cotransfected with plasmids (300 ng pcDNA3– miR-22 or pcDNA3.0) or duplexes (33 nm miR-22 duplex or control RNA duplex), 120 ng pGL3m–ESR1–3¢-UTR–WT or pGL3m–ESR1–3¢-UTR–MUT and ng pRL-TK in triplicate In MDA-MB-231 cells, cells were cotransfected with 200 nm anti-miR-22 or control anti-miR, 120 ng pGL3m– ESR1–3¢-UTR–WT or pGL3m–ESR1–3¢-UTR–MUT and ng pRL-TK in triplicate For the above analyses, cell lysates were analyzed 48 h after transfection using the DualLuciferase Reporter Assay System (Promega) and the experiments were independently repeated at least three times Luciferase activity was detected using the Synergy HT microplate fluorescence reader (Bio-Tek Instruments, Winooski, VT, USA) The pRL-TK vector constitutively expressing Renilla luciferase was cotransfected as an internal control to minimize experimental variability caused by the differences in cell viability or transfection efficiencies Tissue specimens and RNA extraction Fresh breast cancer tissues of 23 individual patients were procured from surgical resection specimens collected in the Cancer Hospital, Chinese Academy of Medical Sciences (Beijing, China) in 2009 The clinical characteristics of patients with breast carcinoma are shown in Table No patients received treatment before surgery and they signed informed consent forms for sample collection ERa protein expression status was confirmed at diagnosis using standard immunohistochemistry procedures Total RNA was isolated and then converted to cDNA using miR-22 RT primer (Ribobio) and ImPro-II reverse transcriptase (Promega) 1692 Data are presented as mean ± standard deviation from at least three independent experiments and differences were assessed using Student’s t test The Kruskal–Wallis one-way analysis of variance test was used to test the significance of 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Table S1 Partial predicted miRNAs that target ESR1 3¢-UTR by targetscan, miranda or pictar Table S2 Primers for cloning miR-22, ESR1 3¢-UTR and ESR1 coding sequence and for the detection of ESR1 and GAPDH mRNA by quantitative RT-PCR This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 1684–1694 ª 2010 The Authors Journal compilation ª 2010 FEBS ... 5¢-UCAUCGCAUUCC UUGCAAAdTdT-3¢, antisense strand 5¢- UUUGCAAGGAAUGCGAUGAdTdT-3¢; ERa siRNA #3 sense strand 5¢- GGAGAAUGUUGAAACA CAAdTdT-3¢, antisense strand 5¢- UUGUGUUUCAA CAUUCUCCdTdT-3¢ The transfection... that miR-22 is frequently downregulated in ERa-positive human breast cancer cell lines and clinical samples In addition, further functional studies showed that ERa plays an important role in. .. candidates or therapeutic targets [10] Breast cancer is one of the most common and prevalent cancers in women and a leading cause of cancerrelated death [11] As in other common cancers, the formation