Estradiol induces HOTAIR levels via GPER-mediated miR-148a inhibition in breast cancer
© Tao et al.; licensee BioMed Central. 2015
Received: 24 October 2014
Accepted: 14 April 2015
Published: 25 April 2015
HOTAIR plays an important role in the regulation of cancer cell proliferation and cancer invasion in breast cancer. The up-regulation of HOTAIR has been reported in both estrogen receptor (ER) positive and triple-negative (TN) breast cancer. It has been reported that HOTAIR is regulated by estrogen (E2) via ERs in ER-positive breast cancer. However, it is unknown how HOTAIR is regulated in TN breast cancer. In this study, we found that HOTAIR was increased in the peripheral blood mononuclear cells and cancer tissues from breast cancer patients, and was especially higher in patients with metastatic breast cancer. In addition, we found that estrogen promoted HOTAIR through its receptor GPER and estrogen-induced breast cancer cell migration was reversed by deleting HOTAIR in TN breast cancer cells MDA-MB-231and BT549. Furthermore, we identified that E2-GPER induces the level of HOTAIR through the suppression of miR-148a. miR-148a level was negatively correlated with HOTAIR level in breast cancer patients. After the mutation of the predicted miR-148a binding sites in HOTAIR, miR-148a had no effect on HOTAIR. In conclusion, our findings offer important new insights into the ability of estrogenic GPER signaling to increase the HOTAIR level by inhibiting miR-148a in breast cancer.
Breast cancer is one of the most common malignant diseases in women. However, the molecular pathogenesis of breast cancer remains poorly defined due to its heterogeneity . Despite advances in the treatment of breast cancer, the effective control of metastasis remains a complex problem. It has been reported that over 90% of the deaths of cancer patients are caused by metastasis, which is formed by the spread of disseminated primary tumor cells to distant anatomic sites . Finding new modalities to treat patients who do not respond to conventional treatments has become increasingly important.
Non-coding RNA has become the focus of “next generation” biology. Non-coding RNA includes microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). Roles for miRNAs have been demonstrated in the regulation of a broad range of biological activities and diseases [3,4]. More recently, thousands of lncRNAs, which are transcribed non-coding RNAs that have more than 200 nucleotides, were discovered and implicated in a variety of biological processes [5,6]. In these thousands of lncRNAs, HOTAIR is a star that is highly expressed in primary breast tumors , hepatocellular carcinoma , colorectal cancer  and gastrointestinal stromal tumors . HOTAIR expression is augmented in primary breast tumors and metastases, and HOTAIR expression level in primary tumors is a powerful predictor of metastases and death [7,11].Therefore, HOTAIR may be a potential therapy target in breast cancer. HOTAIR promotes cancer progression in various ways, including dependents EZH2 to promote cell cycle progression , regulating PTEN methylation  and maintaining the stemness of cancer cells . However, the mechanism by which HOTAIR increases in breast cancer is unknown.
The hormone estrogen (17β-estradiol, E2) has a key role in cell prolife[ration and differentiation through receptor binding and activation [15-17]. The effects of E2 have been widely analyzed in the human mammary gland, where it is responsible for normal epithelial growth and for the development of 70–80% of human breast cancer tumors . Approximately 70% of human breast cancer is estrogen receptor-α positive (ER+) and up to 20% of breast cancer is triple-negative breast cancer (TNBC) .
In ER-positive breast cancer, HOTAIR is transcriptionally induced by E2 through multiple functional estrogen response elements (EREs) in the promoter region . However, as a highly aggressive breast cancer subtype, TNBC lacks a known signaling pathway amenable to targeted therapy. G-protein-coupled estrogen receptor-1 (GPER, formerly known as GPR30) has attracted increasing interest, considering its ability to mediate estrogenic signaling in breast cancer . GPER has also been proposed as a candidate biomarker in triple-negative breast cancer . In addition, in our previous study, we found that E2 can regulate miR-148a expression through GPER . Since HOTAIR increases in both ER-positive and TN breast cancer [24,25], we supposed that estrogen may regulate HOTAIR expression through GPER.
To study whether HOTAIR is regulated by E2 via GPER in breast cancer cells, we measured the mRNA levels of HOTAIR in breast cancer cells after treatment with E2. Furthermore, we investigated the regulation mechanism of E2 on HOTAIR expression. We found that E2 up-regulated HOTAIR in breast cancer cells through GPER via the suppression of miR-148a. Taken together, we are reporting a new mechanism of E2 regulating HOTAIR expression in breast cancer.
Materials and methods
Patients and sample collection
Breast cancer variables
Age (years) median (range)
< 2 cm
Estrogen receptor status
Invasive ductal carcinomas
Months surviving (mean)
The blood was collected from a cubital vein with an anti-coagulant (heparin sodium) and processed immediately. The controls and patients were matched for age and gender where possible. Peripheral blood mononuclear cells (PBMCs) were separated by centrifugation on Ficoll gradient.
MDA-MB-231and BT549 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All cells were maintained in a humidified incubator at 37°C and 5% CO2. For the E2 (Sigma-Aldrich, USA) stimulation experiments, the cells were cultured for at least 3 days in phenol red-free RPMI1640 with 5% dextran-coated charcoal-treated serum before the E2 treatment.
RT-PCR and real-time PCR
RNA was extracted using TRIzol. Total RNA (1 μg) was reverse-transcribed using a RevertAid First Strand cDNA Synthesis Kit (Fermentas). HOTAIR and miR-148a were measured using qRT-PCR (Roche). The expression of HOTAIR was determined in triplicate in three to six separate experiments and normalized using GAPDH, and miR-148a was normalized using U6. Real-time PCR was performed in the ABI PRISM 7300 Sequence Detection System 2.1 (PE Applied Biosystems) using relative quantification. Analysis and fold differences were determined using the comparative cycle threshold (CT) method. Fold change was calculated from the △△CT values with the formula 2-△△CT.
The primers are miR-148a -F:5′-ACACTCCAGCTGGGACAAAGTTCTG-3′; miR-148a -R:5′- CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGTCAGTGCAC -3′; U6 -F:5′-CTCGCTTCGGCAGCACA-3′; U6- R:5′-AACGCTTCACGAATTTGCGT-3′; HOTAIR -F:5′-TTTGGACTGTAAAATATGGC-3′; HOTAIR -R:5′-TTCTGACACTGAACGGACT-3′; GAPDH-F:5′- GTGAAGCAGGCGTCGGA -3′ GAPDH-R:5′- AGCCCCAGCGTCAAAGG -3′.
RNA oligonucleotides and transfection
The siRNA sequences targeting human HOTAIR (siHOTAIR-1 UAACAAGACCAGAGAGCUGUU; siHOTAIR-2 CCACAUGAACGCCCAGAGAUU; siHOTAIR 3 GAACGGGAGUACAGAGAGAUU) or negative control RNA (NC CUACAACAGCCACAACGUCdTdT) were designed and produced by Genepharma (Shanghai, China). siRNA transfection was performed using Lipo2000 (QIAGEN). siRNAs with 20 nmol/L were used for transfection in a serum-free medium. The total RNA was prepared 24 ~ 48 hours after transfection and used for quantity RT-PCR analysis.
Migration and invasion assays
For the transwell migration assay, the breast cancer cells were trypsinized and placed in the upper chamber of each insert (Corning, Cambridge, USA) containing the non-coated membrane. Then, a medium supplemented with 20% fetal bovine serum (600 μl) was added to the lower chambers. After 24, 36 and 48 hours of incubation at 37°C, the upper surface of the membrane was wiped with a cotton tip, and the cells attached to the lower surface were stained for 10 min with crystal violet. The cells in five random fields of view at × 100 magnification were counted and expressed as the average number of cells per field of view. All assays were performed in triplicate.
MDA-MB-231 cells were stimulated with 1 μM G1 (Sigma-Aldrich, USA) with or without 100 nM G15 (Sigma-Aldrich, USA) for 6 h. Then cell lysates were harvested in a cell lysis buffer (Boster, Wuhan, China), dissolved in 9% SDS–PAGE buffer, and subjected to western blotting using primary detection antibodies against total or phosphorylated ERK1/2 (diluted 1:1000; BioWorld, St Louis Park, MN, USA). Membranes were incubated overnight at 4°C before incubation with the appropriate HRP-conjugated secondary antibodies. Immunodetection was conducted using the enhanced chemiluminescence system (Amersham Pharmacia Biotech).
Luciferase reporter assay
The full length of HOTAIR was amplified and cloned into downstream of PGL3-control vector (Promega). Cells plated on 24-well plates were transfected with 100 ng plasmid and 200 nmol/L of miR-148a mimics (RiboBio Co., Ltd., Guangzhou, China), miR-148a inhibitors (RiboBio Co., Ltd., Guangzhou, China) or their negative control (RiboBio Co., Ltd., Guangzhou, China). The miRNA mimics were a sequence of synthetic mature miRNAs used for functional studies of miRNAs. The miRNA inhibitors were synthetic antisense oligonucleotides, which are complementary to the mature miRNA sequence and used for loss-of-function studies of miRNAs. After 48 hours, the cells were lysed and assayed with a dual luciferase assay (Promega) according to the manufacturer’s instructions. For HOTAIR promoter analysis, the HOTAIR promoter (−35 to −2286) was amplified and cloned into a PGL3-basic vector (Promega).Transfection efficiency was estimated by co-transfecting the cells with SV-40 Renilla luciferase. Luciferase activity was measured using the dual luciferase assay system (Promega) and a 96-well luminometer (Fluoroskan Ascent Fl, Labsystems). Three independent experiments were performed in triplicate.
A statistical analysis was performed using Prism 5.0. One-way analysis of variance (ANOVA) and Tukey post hoc tests were used for comparisons within a group. The student t test was used for comparing two different treatments for one cell. All tests were two-sided and p < 0.05 was considered significant. The association analysis was evaluated with Fisher’s exact test.
The HOTAIR level was increased in the PBMCs and tumor tissues from the breast cancer patients
E2-induced HOTAIR increases the migration of breast cancer cells
GPER mediates the promotion effect of E2 on HOTAIR levels
miR-148a targets HOTAIR in breast cancer cells
LncRNAs are of high interest as potential breast cancer therapeutics. However, their expression and function in breast cancer still need to be elucidated. Estrogen signaling is important in the development and progression of breast cancer . HOTAIR, one of the important lncRNAs in the promotion of breast cancer migration, increases in both ER-positive and TN breast cancer [24,25], indicating that estrogen may regulate HOTAIR in a different way other than through ER. In the present study, we found that HOTAIR was increased in breast cancer patients, and was especially higher in migrated breast cancer. In addition, we found that estrogen promoted HOTAIR through its receptor GPER and estrogen-induced breast cancer cell migration was reversed by deleting HOTAIR. Furthermore, we identified that E2-GPER increases the level of HOTAIR through the suppression of miR-148a.
HOTAIR can regulate gene expression through changes in chromatin states and epigenetic modifications [27,28]. Recently, the up-regulation of HOTAIR was observed in several cancers, including breast cancer [7,28-30], hepatocellular carcinoma [11,31], colorectal cancer (CRC) [9,32], pancreatic cancer , non-small cell lung cancer (NSCLC)  and esophageal squamous cell carcinoma (ESCC) [32,33]. Furthermore, HOTAIR has promoted the migration and invasion of breast carcinoma cells , CRC cells , pancreatic cancer cells , NSCLC cells  and ESCC cells [32,33]. Therefore, to investigate the way HOTAIR regulates cells is very important for cancer clinical therapy.
In ER-positive breast cancer cells, it has been demonstrated that HOTAIR is transcriptionally induced by E2 through multiple functional EREs in its promoter . Estrogen receptors (ERs), along with various ER coregulators such as histone methylases mixed-lineage leukemia 1 (MLL1) and MLL3 and CREB-binding protein/p300, bind to the promoter of HOTAIR in an E2-dependent manner. The level of histone H3 lysine-4 trimethylation, histone acetylation and RNA polymerase II recruitment is enriched at the HOTAIR promoter in the presence of E2. The knockdown of ERs and MLLs downregulated the E2-induced HOTAIR expression . However, in TN breast cancer, HOTAIR is also up-regulated, indicating that HOTAIR may be regulated in a different way. Here, we found that E2 could up-regulate HOTAIR levels through GPER in TN breast cancer cells. Our findings further confirm the important role of GPER in cancer development.
E2 regulates the HOTAIR level through the down-regulation of miR-148a. Several miRNAs have been reported to regulate HOTAIR levels. miR-141 suppresses HOTAIR in an Ago2-dependent manner . In addition, HOTAIR can also regulate miRNA levels . miR-148a functions as a tumor suppressor in cancer cells. It has been reported that miR-148a inhibits tumor metastasis by targeting IGF-IR and IRS1 . Moreover, miR-148a suppresses the epithelial-mesenchymal transition and metastasis of hepatoma cells by targeting Met/Snail signaling . We found that the miR-148a level was negatively correlated with the HOTAIR level in breast cancer patients. In addition, we predicted and proved that there are miR-148a binding sequences in HOTAIR. Tumor suppressor miRNA inhibits tumor promoting lncRNA, which may be an important regulation method in cancer.
HOTAIR is becoming a potential therapy target in many cancers. However, its transcription regulation method is unknown. Our findings offer important new insights into the ability of estrogenic GPER signaling to increase the HOTAIR level by inhibiting miR-148a. These findings provide new targets for breast cancer therapy.
Authors are grateful to patients for their important contribution to this study. We also thank all the members of this team for their hard work and invaluable assistance.
- Vargo-Gogola T, Rosen JM. Modelling breast cancer: one size does not fit all. Nat Rev Cancer. 2007;7:659–72.View ArticlePubMedGoogle Scholar
- Fidler IJ. The pathogenesis of cancer metastasis: the ‘seed and soil’ hypothesis revisited. Nat Rev Cancer. 2003;3:453–8.View ArticlePubMedGoogle Scholar
- Espinoza-Lewis RA, Wang DZ. MicroRNAs in heart development. Cur Top Dev Biol. 2012;100:279–317.View ArticleGoogle Scholar
- Zhao G, Zhou X, Chen S, Miao H, Fan H, Wang Z, et al. Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia. J Biomed Sci. 2014;21:81.View ArticlePubMed CentralPubMedGoogle Scholar
- Ulitsky I, Bartel DP. LincRNAs: genomics, evolution, and mechanisms. Cell. 2013;154:26–46.View ArticlePubMed CentralPubMedGoogle Scholar
- Morris KV, Mattick JS. The rise of regulatory RNA. Nat Rev Genet. 2014;15:423–37.View ArticlePubMed CentralPubMedGoogle Scholar
- Gupta RA, Shah N, Wang KC, Kim J, Horlings HM, Wong DJ. Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature. 2010;464:1071–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Geng YJ, Xie SL, Li Q, Ma J, Wang GY. Large intervening non-coding RNA HOTAIR is associated with hepatocellular carcinoma progression. J Int Med Res. 2011;39:2119–28.View ArticlePubMedGoogle Scholar
- Kogo R, Shimamura T, Mimori K, Kawahara K, Imoto S, Sudo T. Long noncoding RNA HOTAIR regulates polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers. Cancer Res. 2011;71:6320–6.View ArticlePubMedGoogle Scholar
- Niinuma T, Suzuki H, Nojima M, Nosho K, Yamamoto H, Takamaru H. Upregulation of miR-196a and HOTAIR drive malignant character in gastrointestinal stromal tumors. Cancer Res. 2012;72:1126–36.View ArticlePubMedGoogle Scholar
- Yang Z, Zhou L, Wu LM, Lai MC, Xie HY, Zhang F, et al. Overexpression of long non-coding RNA HOTAIR predicts tumor recurrence in hepatocellular carcinoma patients following liver transplantation. Ann Surg Oncol. 2011;18:1243–50.View ArticlePubMedGoogle Scholar
- Zhang K, Sun X, Zhou X, Han L, Chen L, Shi Z, et al. Long non-coding RNA HOTAIR promotes glioblastoma cell cycle progression in an EZH2 dependent manner. Oncotarget. 2014 Nov 4. [Epub ahead of print]Google Scholar
- Li D, Feng J, Wu T, Wang Y, Sun Y, Ren J, et al. Long intergenic noncoding RNA HOTAIR is overexpressed and regulates PTEN methylation in laryngeal squamous cell carcinoma. Am J Pathol. 2013;182:64–70.View ArticlePubMedGoogle Scholar
- Pádua Alves C, Fonseca AS, Muys BR, de Barros E, Lima Bueno R, Bürger MC, et al. Brief report: The lincRNA Hotair is required for epithelial-to-mesenchymal transition and stemness maintenance of cancer cell lines. Stem Cells. 2013;31:2827–32.View ArticlePubMedGoogle Scholar
- Deng H, Zhang XT, Wang ML, Zheng HY, Liu LJ, Wang ZY. ER-α36-mediated rapid estrogen signaling positively regulates ER-positive breast cancer stem/progenitor cells. PLoS One. 2014;9, e88034.View ArticlePubMed CentralPubMedGoogle Scholar
- Scaling AL, Prossnitz ER, Hathaway HJ. GPER mediates estrogen-induced signaling and proliferation in human breast epithelial cells and normal and malignant breast. Horm Cancer. 2014;5:146–60.View ArticlePubMedGoogle Scholar
- Zhao G, Nie Y, Lv M, He L, Wang T, Hou Y. ERβ-mediated estradiol enhances epithelial mesenchymal transition of lung adenocarcinoma through increasing transcription of midkine. Mol Endocrinol. 2012;26:1304–15.View ArticlePubMedGoogle Scholar
- Meneses-Morales I, Tecalco-Cruz AC, Barrios-García T, Gómez-Romero V, Trujillo-González I, Reyes-Carmona S, et al. SIP1/NHERF2 enhances estrogen receptor alpha transactivation in breast cancer cells. Nucleic Acids Res. 2014;42:6885–900.View ArticlePubMed CentralPubMedGoogle Scholar
- Yager JD, Davidson NE. Estrogen carcinogenesis in breast cancer. N Engl J Med. 2006;354:270–82.View ArticlePubMedGoogle Scholar
- Bhan A, Hussain I, Ansari KI, Kasiri S, Bashyal A, Mandal SS. Antisense transcript long noncoding RNA (lncRNA) HOTAIR is transcriptionally induced by estradiol. J Mol Bio. 2013;425:3707–22.View ArticleGoogle Scholar
- Tamaki M, Konno Y, Kobayashi Y, Takeda M, Itoga M, Moritoki Y, et al. Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils. Immunol Lett. 2014;160:72–8.View ArticlePubMedGoogle Scholar
- Lappano R, Pisano A, Maggiolini M. GPER function in breast cancer: an overview. Front Endocrinol (Lausanne). 2014;5:66.Google Scholar
- Tao S, He H, Chen Q, Yue W. GPER mediated estradiol reduces miR-148a to promote HLA-G expression in breast cancer. Biochem Biophys Res Commun. 2014;451:74–8.View ArticlePubMedGoogle Scholar
- Chisholm KM, Wan Y, Li R, Montgomery KD, Chang HY, West RB. Detection of long non-coding RNA in archival tissue: correlation with polycomb protein expression in primary and metastatic breast carcinoma. PLoS One. 2012;7, e47998.View ArticlePubMed CentralPubMedGoogle Scholar
- Sørensen KP, Thomassen M, Tan Q, Bak M, Cold S, Burton M, et al. Long non-coding RNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer. Breast Cancer Res Trea. 2013;142:529–36.View ArticleGoogle Scholar
- Luo H, Yang G, Yu T, Luo S, Wu C, Sun Y, et al. GPER-mediated proliferation and estradiol production in breast cancer-associated fibroblasts. Endocr Relat Cancer. 2014;21:355–69.View ArticlePubMed CentralPubMedGoogle Scholar
- Tsai MC, Manor O, Wan Y, Mosammaparast N, Wang JK, Lan F, et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science. 2010;329:689–93.View ArticlePubMed CentralPubMedGoogle Scholar
- Lu L, Zhu G, Zhang C, Deng Q, Katsaros D, Mayne ST, et al. Association of large noncoding RNA HOTAIR expression and its downstream intergenic CpG island methylation with survival in breast cancer. Breast Cancer Res Trea. 2012;136:875–83.View ArticleGoogle Scholar
- Sorensen KP, Thomassen M, Tan Q, Bak M, Cold S, Burton M, et al. Long non-coding RNA HOTAIR is an independent prognostic marker of metastasis in estrogen receptor-positive primary breast cancer. Breast Cancer Res Treat. 2013;142:529–36.View ArticlePubMedGoogle Scholar
- Kim K, Jutooru I, Chadalapaka G, Johnson G, Frank J, Burghardt R, et al. HOTAIR is a negative prognostic factor and exhibits pro-oncogenic activity in pancreatic cancer. Oncogene. 2013;32:1616–25.View ArticlePubMed CentralPubMedGoogle Scholar
- Liu XH, Liu ZL, Sun M, Liu J, Wang ZX, De W. The long non-coding RNA HOTAIR indicates a poor prognosis and promotes metastasis in non-small cell lung cancer. BMC Cancer. 2013;13:464.View ArticlePubMed CentralPubMedGoogle Scholar
- Ge XS, Ma HJ, Zheng XH, Ruan HL, Liao XY, Xue WQ, et al. HOTAIR a prognostic factor in esophageal squamous cell carcinoma inhibits WIF-1 expression and activates Wnt pathway. Cancer Sci. 2013;104:1675–82.View ArticlePubMedGoogle Scholar
- Li X, Wu Z, Mei Q, Guo M, Fu X, Han W. Long non-coding RNA HOTAIR a driver of malignancy predicts negative prognosis and exhibits oncogenic activity in oesophageal squamous cell carcinoma. Br J Cancer. 2013;109:2266–78.View ArticlePubMed CentralPubMedGoogle Scholar
- Chiyomaru T, Fukuhara S, Saini S, Majid S, Deng G, Shahryari V, et al. Long non-coding RNA HOTAIR is targeted and regulated by miR-141 in human cancer cells. J Biol Chem. 2014;289:12550–65.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang H, Cai K, Wang J, Wang X, Cheng K, Shi F, et al. MiR-7, inhibited indirectly by LincRNA HOTAIR, directly inhibits SETDB1 and reverses the EMT of breast cancer stem cells by downregulating the STAT3 pathway. 2014;32:2858–68.Google Scholar
- Xu Q, Jiang Y, Yin Y, Li Q, He J, Jing Y, et al. A regulatory circuit of miR-148a/152 and DNMT1 in modulating cell transformation and tumor angiogenesis through IGF-IR and IRS1. J Mol Cell Biol. 2013;5:3–13.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang JP, Zeng C, Xu L, Gong J, Fang JH, Zhuang SM. MicroRNA-148a suppresses the epithelial-mesenchymal transition and metastasis of hepatoma cells by targeting Met/Snail signaling. Oncogene. 2014;33:4069–76.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.