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MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3 Xiunan Li, Yumei Wu*, Aihui Liu, Xin Tang Department of Breast Surgery, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, 100006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2016 Accepted 26 June 2016 Available online xxx
MiR-27b downregulation is significantly associated with tamoxifen resistance in breast cancer cells. However, how it is downregulated in tamoxifen resistant (TamR) breast cancer cells and its downstream regulation were not clear. By performing MSP assay and QRT-PCR analysis with the use of 5-AZA-dC, a DNA methyltransferase inhibitor, we observed that TamR MCF-7 cells had significantly higher levels of methylation in the miR-27b promoter region than tamoxifen sensitive MCF-7 (TamS) cells and demethylation restored miR-27b expression. Re-expression of miR-27b sensitized TamR MCF-7 cells to tamoxifen, inhibited invasion and reversed epithelial-mesenchymal transition (EMT)-like properties. By using bioinformatics analysis and following dual luciferase and western blot analysis, this study confirmed a direct regulation of miR-27b on HMGB3 expression by binding to the 30 UTR. In addition, this study also found that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b in reducing tamoxifen resistance and cell invasion and in reversing EMT-like properties. Therefore, we infer that HMGB3 is a functional target of miR-27b in modulation of tamoxifen resistance and EMT. © 2016 Elsevier Inc. All rights reserved.
Keywords: MiR-27b Promoter methylation Tamoxifen HMGB3
1. Introduction Breast cancer is one of the most commonly diagnosed female malignancies and the second leading cause of mortality of all cancers in women [1]. A large proportion (about 70%) of the breast cancer cases were estrogen receptor (ER) positive [2,3]. Therefore, Tamoxifen, an antagonist of the ER, was the most commonly prescribed endocrine therapy for ER-positive breast cancer [4]. Although the use of tamoxifen has significantly improved patient survival, there are still a large proportion of the patients develop acquired tamoxifen resistance, which is a major cause of cancer recurrence and mortality [4]. The mechanism underlying endocrine resistance remains poorly understood, despite some important players involved were identified. Previous studies found that dysregulated miRNAs are involved in the development of acquired tamoxifen resistance in breast cancer cells. For example, miR-574-3p downregulation is associated
* Corresponding author. Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, 17#, Qihelou Street, Dongcheng District, Beijing, 100006, China. E-mail address: [email protected] (Y. Wu).
with tamoxifen resistant (TamR) breast cancer and can modulate tamoxifen sensitivity by targeting Clathrin heavy chain (CLTC) [5]. MiR-320 downregulation in breast cancer tissue contributes to acquired tamoxifen resistance by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRgamma) as well as their downstream effectors, c-Myc and Cyclin D1 [6]. In fact, a dozes of miRNAs are dysregulated in TamR breast cancer and affect the sensitivity via multiple signaling pathways [7e9]. Although the role of the dysregulated miRNAs have been gradually revealed, the mechanisms involved in their dysregulation have not been fully understood. Alterations of epigenetic modulation, such as methylation, histone modification and non-coding RNAs are quite common in carcinogenesis [10,11]. This mechanism is also involved in the dysregulated miRNAs in drug resistance of breast cancer. For example, epigenetically downregulated miR-375 in HER2-positive breast cancer can induce trastuzumab resistance by targeting IGF1R [12]. Methylation downregulated miR-149 can modulate chemoresistance by targeting GlcNAc N-deacetylase/N-sulfotransferase-1 in human breast cancer [13]. The transcription of miR-15a/16 cluster is suppressed in TamR breast cancer cells due to an epigenetic mechanism [14]. MiR-27b downregulation is significantly associated with
http://dx.doi.org/10.1016/j.bbrc.2016.06.133 0006-291X/© 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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tamoxifen resistance in breast cancer cells [14]. In addition, its downregulation also contributes to breast cancer stem cell generation [14]. However, how it is downregulated in the cells and its downstream regulation were not clear. In this study, we firstly reported that miR-27b downregulation in TamR breast cancer cells is a result of promoter methylation and HMGB3 is a direct target of miR-27b in modulating tamoxifen resistance and epithelialmesenchymal transition (EMT). 2. Methods 2.1. Cell culture and treatment The tamoxifen sensitive (TamS) human breast cancer cell line MCF-7 was obtained from ATCC (Manassas, VA, USA). The MCF-7 derived tamoxifen resistant cells (TamR) were generated by using a conventional stepwise method as described in previous study [15], which induces drug resistance by increasing concentrations of tamoxifen over 8 months. All of the cancer cells were grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 units of penicillin/ml and 100 mg of streptomycin/ml. MiR-27b mimics, HMGB3 siRNA and the scramble negative controls were all purchased from Ribobio (Shanghai, China). TamS and TamR cells were transfected with 100 nM miR-27b using Lipofectamie 2000 (Invitrogen, Carlsbad, CA, USA) and transfected with 100 nM HMGB3 siRNA using Hiperfect transfection reagent (QIAGEN, GmbH, Hilden, Germany) according to manufacturer’s instruction. For 5-AZA-dC treatment, the cells were cultured with 2.5 mM 5-AZA-dC (Sigma-Aldrich, St. Louis, MO, USA) for 48 h, and then were harvested for the MSP assay and qRT-PCR analysis of miR-27b expression. 2.2. Bioinformatics analysis and prediction The Affymetrix® GeneChip® miRNA Array 3.0 data of miRNA expression in tamoxifen sensitive and tamoxifen resistant MCF7 cells were retrieved in NCBI GEO Datasets (http://www.ncbi.nlm. nih.gov/gds, GSE66607). The possible targets of miR-27b were predicted using TargetScan 7.1 (http://www.targetscan.org/). The CpG island and the possible methylation sites in the promoter regions of miR-27b were predicted by using MethPrimer (http:// www.urogene.org/methprimer/). The primers used for Methylation-Specific PCR were also designed with MethPrimer. 2.3. QRT-PCR analysis Total RNAs in the cell samples were extracted using the TRIzol reagent (Invitrogen) according to manufacturer’s instructions. MiRNAs specific cDNA was synthesized using the stem-loop primers and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). To quantify miR-27b expression, qRT-PCR analysis was performed using TaqMan MicroRNA Assay Kit (Applied Biosystems), with U6 snRNA used as the endogenous control. All PCR reactions were performed using an ABI Prism 7500 (Applied Biosystems). The results were calculated using the 2DDCT methods.
bromide. The methylated primers (M) were: left, 50 ATTATGTGTGTTAGGAAAGGGAAAC-30 ; right, 50 CTATATACCGCCAACCCGTC-0 3; while the unmethylated primers (U) were: left, 50 - ATTATGTGTGTTAGGAAAGGGAAAT-30 ; right, 50 - ACAATCTATATACCACCAACCCATC-0 3. 2.5. Cell viability assay TamR cells with enforced miR-27b overexpression or HMGB3 knockdown were seeded in a 96-well plate at a density of 3000 cells/well and were cultured in the presence of 4hydroxytamoxifen (5 mM) or ethanol solvent control. After 72 h, cell viability was measured using Cell Titer Glo Luminescent Cell Viability assay (Promega) following the manufacturer’s instructions. was performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). 2.6. Transwell assay Transwell assay was performed to according to the methods introduced in one previous study [16]. In brief, the tanswell insert chamber coated with Matrigel (BD Biosciences Franklin Lakes, NJ, USA) was used according to manufacturer’s instruction. The invaded cells on the bottom side were fixed with 4% polyoxymethylene and stained with 0.1% crystal violet. Cell counting was performed under a light microscope. 2.7. Western blot analysis Cell samples were lysed using a lysis buffer (Beyotime, Shanghai, China). Then, the protein concentration was quantified using a BCA protein assay kit (Beyotime). Then, a conventional western blot analysis was performed following a method described in one previous study [17]. Primary antibodies used included anti-E-cadherin (1:1000, #3195, Cell Signaling, Danvers, MA, USA), anti-N-cadherin (1:1000, #13116, Cell Signaling), anti-HMGB3 (1:1000, #6893, Cell Signaling) and anti-b-actin (1: 2000, ab8227, Abcam). The blot signals were visualized using the ECL Western blotting substrate (Promega, Madison, WI, USA). The relative gray scale of the bands were analyzed using ImageJ software. 2.8. Fluorescence microscopy TamR cells after transfection were grown on coverslips. Then, the cells were fixed, permeabilized in 0.1% Triton X-100 and blocked in 1% BSA. The coverslips were incubated with primary antibodies against E-cadherin (#3195, Cell Signaling) and N-cadherin (#13116, Cell Signaling) respectively at 4 C overnight. Then, the coverslips were washed and further incubated with secondary Anti-Rabbit IgG (H þ L), F(ab’)2 Fragment (Alexa Fluor® 555 Conjugate) (#4413, Cell Signaling) and Anti-Rabbit IgG (H þ L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (#4412, Cell Signaling) respectively for 1 h at room temperature in the dark. Nuclei were stained using Prolong® Gold Antifade Reagent with DAPI (#8961, Cell Signaling). Immunofluorescent images were obtained using an Eclipse Ti-S inverted phase/fluorescent microscope (Nikon, Tochigi, Japan).
2.4. Methylation-specific PCR (MSP) 2.9. Dual luciferase assay DNA was extracted from cell lines by DNeasy Tissue Kit (Qiagen, Hilden, Germany). MSP was performed under the following cycling conditions: 95 C for 4 min, then 40 cycles of 94 C, 25 s; 51 C, 25 s; 72 C, 30 s, followed by a final 5 min extension at 72 C. MSP products were separated electrophoretically on 2% agarose gels and visualized under ultraviolet light after staining with ethidium
To construct luciferase reporter vectors, the wildtype 30 UTR sequence fragment of HMGB3 with predicted miR-27b binding site was chemically synthesized and inserted into the downstream of the luciferase gene of pmirGLO Dual-Luciferase miRNA Target Expression Vector between Xhol/Xbal sites (Promega) and assigned
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
X. Li et al. / Biochemical and Biophysical Research Communications xxx (2016) 1e6
as pGLO-HMGB3-WT. By using the same method, the mutant sequence without miR-27b binding site was inserted into the vector and the recombinant vector was termed as pGLO-HMGB3-MT. TamS and TamR cells cultured in the 96-well plate were cotransfected with either miR-27b mimics or the scramble negative controls and pGLO-HMGB3-WT or pGLO-HMGB3-MT, using Lipofectamie 2000 (Invitrogen). Cells were harvested 24 h after transfection and then luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega) and a GloMaxTM 96 Microplate Luminometer (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to that of Renilla luciferase. 2.10. Statistical analysis Data were presented in the form of means ± standard deviation (SD) based at least three repeats. Comparison between groups was performed using the unpaired t-test. A two-sided p value of <0.05 was considered statistically significant.
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One previous miRNA array (GSE66607) showed that multiple miRNAs were significantly downregulated in TamR MCF-7 cells than in TamS MCF-7 cells (Fig. 1A). MiR-27b is one of the most downregulated miRNAs (Fig. 1A). By performing qRT-PCR analysis, we also confirmed substantially suppressed miR-27b expression in TamR cells than in TamS cells (Fig. 1B). However, how it is downregulated in TamR cells is not clear. Previous studies reported that miR-195 and miR-497 (two miRNAs downregulated in TamR cells, showed in Fig. 1A), were significantly downregulated in breast cancer due to methylation state of CpG islands upstream of the miR-195/497 gene [18]. Therefore, we hypothesized that hypermethylation in the promoter regions is a cause of miR-27b downregulation in TamR cells. To verify this hypothesis, we firstly analyzed the CpG islands in the promoter region of miR-27b and designed two pairs of primers for MSP assay (Fig. 1C). Following MSP assay showed that TamR cells had significantly higher levels of methylation in the miR-27b promoter region than TamS cells (Fig. 1D). 5-AZA-dC treatment significantly decreased the methylation (Fig. 1D) and also restored miR-27b expression in TamR cells (Fig. 1E).
3. Results 3.1. MiR-27b downregulation in TamR cells is a result of promoter methylation To identify dysregulated miRNAs in tamoxifen resistant MCF7 cells, we firstly retrieved miRNA Array data in NCBI GEO Datasets.
3.2. Enforced miR-27b expression sensitizes TamR cells to tamoxifen, inhibits invasion and reverses EMT-like properties Then, we investigated the regulative effect of miR-27b on tamoxifen sensitivity of the breast cancer cells. TamR cells transfected with miR-NC had no response to 5 mM 4-hydroxytamoxifen.
Fig. 1. MiR-27b downregulation in TamR cells is a result of promoter methylation. A. MicroRNA array data of dysregulated miRNAs in TamR MCF-7 cells (data retrieved from NCBI GEO Datasets, with the number: GSE66607). The bars represent fold change of miRNAs (TamR vs. TamS). B. QRT-PCR analysis of miR-27b expression in TamS and TamR MCF-7 cells. C. Bioinformatics analysis of CpG island in promoter regions of miR-27b and the location of methylated and unmethylated primers used for MSP analysis. D. TamS and TamR MCF7 cells with or without the treatment of 5-AZA-dC were used for MSP assay and the products were separated electrophoretically on 2% agarose gels. M, amplification with methylated primers; U, amplification with unmethylated primers. E. QRT-PCR analysis of miR-27b expression in TamR MCF-7 cells with or without the treatment of 5-AZA-dC. **p < 0.01.
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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Fig. 2. Enforced miR-27b expression sensitizes TamR cells to tamoxifen, inhibits invasion and reverses EMT-like properties. A. The relative growth inhibiting effect of 5 mM 4hydroxytamoxifen treatment on TamR MCF-7 cells with or without transfection of miR-27b. B. Representative images of transwell assay of invaded TamS and TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. C. Quantitation of invaded cells showed in figure B. D and E. Immunofluorescent staining (D, red: E-cadherin, green: N-cadherin, Blue: DAPI) and western blot analysis (E) of E-cadherin and N-cadherin in TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. F. Quantitation of relative gray scale of E-cadherin and N-cadherin showed in figure E. *p < 0.05, **p < 0.01.
However, the cells with enforced miR-27b expression had approximately 15% loss of cell viability under the treatment (Fig. 2A), suggesting that miR-27b re-expression was capable of sensitizing TamR cells to tamoxifen. Next, we studied whether miR27b could modulate the invasive capacity of the TamR cells. Transwell assay showed that TamR cells had a significantly higher level of invasive capacity than TamS cells and transfection of miR27b significantly reduced the capacity (Fig. 2BeC). This reduction was accompanied with decreased expression of mesenchymal marker N-cadherin and increased expression of epithelial marker E-Cadherin (Fig. 2DeF). 3.3. MiR-27b directly targets HMGB3 in breast cancer cells Since we identified miRNA-27b as a regulator of both tamoxifen resistance and EMT, we decided to further explore the downstream genes involved in these modulations. Our preliminary studies showed that HMGB3, which is relevant to drug resistance and breast cancer cell proliferation and metastasis [19,20], is a highly possible target of miR-27b (Fig. 3A). Therefore, we hypothesized that HMGB3 upregulation might be a result of miR-27b downregulation and HMGB3 might contribute tamoxifen resistance and accompanying EMT-like properties. To test this hypothesis, we first detected the expression of HMGB3 in TamS and TamR cells and found that it was significantly upregulated in TamR cells (Fig. 3 B and E). In addition, transfection of miR-27b mimics significantly decreased HMGB3 levels in both TamS and TamR cells (Fig. 3 B and E). To further verify the direct regulation of miR-27b on HMGB3, we cloned luciferase reporter constructs carrying either wildtype or mutant 30 -UTR of HMGB3 downstream of the luciferase gene.
Transfection with miR-27b mimics significantly reduced relative luciferase activity of the reporter containing wildtype sequence in both TamS and TamR cells (Fig. 3CeD). However, miR-27b mimics had no suppressive effect on luciferase activity of the reporter with mutant miR-27b targeting sites (Fig. 3CeD). 3.4. Silencing of HMGB3 phenocopies the effects of miR-27b in TamR cells Since we confirmed that HMGB3 is a direct target of miR-27b, we further investigated the functional role of HMGB3 in breast cancer cells. TamR cells were firstly transfected with HMGB3 siRNA (Fig. 4AeB). Knockdown of HMGB3 sensitized TamR cells to tamoxifen treatment (Fig. 4C) and reduced the invasive capacity (Fig. 4D). In addition, HMGB3 knockdown also increased E-cadherin and decreased N-cadherin in TamR cells (Fig. 4EeG). These results suggest that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b and it is a functional target of miR27b involved in both tamoxifen resistance and EMT. 4. Discussion One recent study found that multiple miRNAs are significantly downregulated in TamR breast cancer cells, such as miR-497, miR221/222, miR-195 and miR-27b [7]. Although miR-27b is one of the most downregualted miRNAs in TamR cells, how it is dysregulated and its functional role in breast cancer has not been fully understood. Previous studies reported that the methylation state of CpG islands upstream of the miR-195/497 gene is responsible for the downregulation of miR-497, miR-195 and miR-221/222 in breast
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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Fig. 3. MiR-27b directly targets HMGB3 in breast cancer cells. A Bioinformatics analysis of putative binding site between miR-27b and 30 UTR of HMGB3 (positive: 487e493). The sequence of mutant HMGB3 30 UTR used for dual luciferase was also given. B and E. Western blot analysis images (B) and Quantitation of relative gray scale (E) of HMGB3 in TamS and TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. C and D. Dual luciferase assay of the inhibiting effect of miR-27b mimics on Renilla luciferase expression in TamS (C) and TamR (D) MCF-7 cells co-transfected with pGLO-HMGB3-WT or pGLO-HMGB3-MT. **p < 0.01.
Fig. 4. Silencing of HMGB3 phenocopies the effects of miR-27b in TamR cells. A-B. Western blot analysis images (A) and quantitation of relative gray scale (B) of HMGB3 in TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. C. The relative growth inhibiting effect of 5 mM 4-hydroxytamoxifen treatment on TamR MCF-7 cells with or without transfection of HMGB3 siRNA or the negative controls. D. Quantitation of transwell assay of invaded TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. E and F. Immunofluorescent staining (E, red: E-cadherin, green: N-cadherin, Blue: DAPI) and western blot analysis (F) of E-cadherin and N-cadherin in TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. G. Quantitation of relative gray scale of E-cadherin and N-cadherin showed in figure F. **p < 0.01.
cancer cells [18,21]. Another study found that E2F7 can compete with E2F1 at miR-15a/16 promoter and decrease the transcription of miR-15a/16 cluster, leading to acquired tamoxifen resistance [7]. These findings suggest that epigenetic regulation might be an important mechanism of dysregulated miRNAs in TamR cells. One previous study found that miR-27b functioned as an inhibitor of tumor progression in colorectal cancer and DNA hypermethylation of miR-27b CpG islands decreased miR-27b expression [22]. Therefore, we hypothesized that hypermethylation might also be a mechanism of miR-27b downregulation in TamR cells. By
performing MSP assay and qRT-PCR analysis with the use of 5-AZAdC, a DNA methyltransferase inhibitor, we observed that TamR cells had significantly higher levels of methylation in miR-27b promoter region than TamS cells and demethylation restored miR-27b expression in TamR cells. In breast cancer, the effect of miR-27b dysregulation has gradually been revealed. One recent study observed that miR-27b was downregulated in mammary cell transformation and its downregulation was associated with ErbB2-dependent invasion in threedimensional (3D) cultures of human mammary epithelial cells [23].
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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In addition, its loss also contributes to breast cancer stem cell generation by activating ENPP1 [14]. In multiple types of cancer, miR-27b dysregulation is involved in modulation of chemosensitivity. For example, miR-27b can sensitize cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo via activating p53-dependent apoptosis and reducing CYP1B1-mediated drug detoxification [24]. In breast cancer cells, miR-27b can decrease acquired drug resistance by attenuating the formation of the side-population cells of breast cancer cells [14]. However, the role of miR-27b in tamoxifen resistance has not been reported. Multiple previous studies reported that enhanced EMT is a possible mechanism of acquired tamoxifen resistance in breast cancer cells [25,26]. Interestingly, these studies also observed that reexpression of the downregulated miRNAs in TamR cells, such as miR-375 and miR-200 can sensitize the cells to tamoxifen and also partially reverse the EMT like properties [25e27]. In TamR cells, we found that re-expression of miR-27b could sensitize cells to tamoxifen, inhibit invasion and reverse EMT-like properties. Therefore, we decided to investigate its downstream effector. HMGB3 protein belongs to the high mobility group box (HMGbox) subfamily and has 99% homology with HMGB1 and HMGB2 [28]. They all play important roles in DNA replication, transcription, recombination, and repair in cancer cells [29]. HMGB3 has been reported as an oncogene modulating drug resistance and breast cancer cell proliferation and metastasis [19,20]. Our bioinformatics analysis predicted that the 30 UTR of HMGB3 has a highly conserved targeting site of miR-27b and the following dual luciferase and western blot analysis confirmed the direct regulation of miR-27b on HMGB3 expression. More importantly, our study found that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b in reducing tamoxifen resistance and cell invasion and in reversing EMT-like properties. Therefore, we infer that HMGB3 is a functional target of miR-27b in both tamoxifen resistance and EMT. Conflict of interest None. Acknowledgment This study was supported by Beijing Natural Science Foundation (7142055). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.133 References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9e29. [2] C.K. Osborne, V. Bardou, T.A. Hopp, G.C. Chamness, S.G. Hilsenbeck, S.A. Fuqua, J. Wong, D.C. Allred, G.M. Clark, R. Schiff, Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer, J. Natl. Cancer Inst. 95 (2003) 353e361. [3] O. Gojis, B. Rudraraju, M. Gudi, K. Hogben, S. Sousha, R.C. Coombes, S. Cleator, C. Palmieri, The role of SRC-3 in human breast cancer, Nat. Rev. Clin. Oncol. 7 (2010) 83e89. [4] G. Early Breast Cancer Trialists’ Collaborative, Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials, Lancet 365 (2005) 1687e1717. [5] T. Ujihira, K. Ikeda, T. Suzuki, R. Yamaga, W. Sato, K. Horie-Inoue, T. Shigekawa, A. Osaki, T. Saeki, K. Okamoto, S. Takeda, S. Inoue, MicroRNA-574-3p, identified by microRNA library-based functional screening, modulates tamoxifen response in breast cancer, Sci. Rep. 5 (2015) 7641. [6] M. Lu, K. Ding, G. Zhang, M. Yin, G. Yao, H. Tian, J. Lian, L. Liu, M. Liang, T. Zhu, F. Sun, MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to
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Huang, miRNA-27b targets vascular endothelial growth factor C to inhibit tumor progression and angiogenesis in colorectal cancer, PloS one 8 (2013) e60687. [23] A. Pincini, G. Tornillo, F. Orso, M. Sciortino, B. Bisaro, P. Leal Mdel, A. Lembo, M.F. Brizzi, E. Turco, C. De Pitta, P. Provero, E. Medico, P. Defilippi, D. Taverna, S. Cabodi, Identification of p130Cas/ErbB2-dependent invasive signatures in transformed mammary epithelial cells, Cell cycle 12 (2013) 2409e2422. [24] Y. Shang, B. Feng, L. Zhou, G. Ren, Z. Zhang, X. Fan, Y. Sun, G. Luo, J. Liang, K. Wu, Y. Nie, D. Fan, The miR27b-CCNG1-P53-miR-508-5p axis regulates multidrug resistance of gastric cancer, Oncotarget 7 (2016) 538e549. [25] A. Ward, A. Balwierz, J.D. Zhang, M. Kublbeck, Y. Pawitan, T. Hielscher, S. Wiemann, O. Sahin, Re-expression of microRNA-375 reverses both tamoxifen resistance and accompanying EMT-like properties in breast cancer, Oncogene 32 (2013) 1173e1182. [26] T.T. Manavalan, Y. Teng, L.M. Litchfield, P. 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Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3 Xiunan Li, Yumei Wu*, Aihui Liu, Xin Tang Department of Breast Surgery, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, Beijing, 100006, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 June 2016 Accepted 26 June 2016 Available online xxx
MiR-27b downregulation is significantly associated with tamoxifen resistance in breast cancer cells. However, how it is downregulated in tamoxifen resistant (TamR) breast cancer cells and its downstream regulation were not clear. By performing MSP assay and QRT-PCR analysis with the use of 5-AZA-dC, a DNA methyltransferase inhibitor, we observed that TamR MCF-7 cells had significantly higher levels of methylation in the miR-27b promoter region than tamoxifen sensitive MCF-7 (TamS) cells and demethylation restored miR-27b expression. Re-expression of miR-27b sensitized TamR MCF-7 cells to tamoxifen, inhibited invasion and reversed epithelial-mesenchymal transition (EMT)-like properties. By using bioinformatics analysis and following dual luciferase and western blot analysis, this study confirmed a direct regulation of miR-27b on HMGB3 expression by binding to the 30 UTR. In addition, this study also found that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b in reducing tamoxifen resistance and cell invasion and in reversing EMT-like properties. Therefore, we infer that HMGB3 is a functional target of miR-27b in modulation of tamoxifen resistance and EMT. © 2016 Elsevier Inc. All rights reserved.
Keywords: MiR-27b Promoter methylation Tamoxifen HMGB3
1. Introduction Breast cancer is one of the most commonly diagnosed female malignancies and the second leading cause of mortality of all cancers in women [1]. A large proportion (about 70%) of the breast cancer cases were estrogen receptor (ER) positive [2,3]. Therefore, Tamoxifen, an antagonist of the ER, was the most commonly prescribed endocrine therapy for ER-positive breast cancer [4]. Although the use of tamoxifen has significantly improved patient survival, there are still a large proportion of the patients develop acquired tamoxifen resistance, which is a major cause of cancer recurrence and mortality [4]. The mechanism underlying endocrine resistance remains poorly understood, despite some important players involved were identified. Previous studies found that dysregulated miRNAs are involved in the development of acquired tamoxifen resistance in breast cancer cells. For example, miR-574-3p downregulation is associated
* Corresponding author. Department of Gynecologic Oncology, Beijing Obstetrics and Gynecology Hospital, Capital Medical University, 17#, Qihelou Street, Dongcheng District, Beijing, 100006, China. E-mail address: [email protected] (Y. Wu).
with tamoxifen resistant (TamR) breast cancer and can modulate tamoxifen sensitivity by targeting Clathrin heavy chain (CLTC) [5]. MiR-320 downregulation in breast cancer tissue contributes to acquired tamoxifen resistance by targeting cAMP-regulated phosphoprotein (ARPP-19) and estrogen-related receptor gamma (ERRgamma) as well as their downstream effectors, c-Myc and Cyclin D1 [6]. In fact, a dozes of miRNAs are dysregulated in TamR breast cancer and affect the sensitivity via multiple signaling pathways [7e9]. Although the role of the dysregulated miRNAs have been gradually revealed, the mechanisms involved in their dysregulation have not been fully understood. Alterations of epigenetic modulation, such as methylation, histone modification and non-coding RNAs are quite common in carcinogenesis [10,11]. This mechanism is also involved in the dysregulated miRNAs in drug resistance of breast cancer. For example, epigenetically downregulated miR-375 in HER2-positive breast cancer can induce trastuzumab resistance by targeting IGF1R [12]. Methylation downregulated miR-149 can modulate chemoresistance by targeting GlcNAc N-deacetylase/N-sulfotransferase-1 in human breast cancer [13]. The transcription of miR-15a/16 cluster is suppressed in TamR breast cancer cells due to an epigenetic mechanism [14]. MiR-27b downregulation is significantly associated with
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Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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tamoxifen resistance in breast cancer cells [14]. In addition, its downregulation also contributes to breast cancer stem cell generation [14]. However, how it is downregulated in the cells and its downstream regulation were not clear. In this study, we firstly reported that miR-27b downregulation in TamR breast cancer cells is a result of promoter methylation and HMGB3 is a direct target of miR-27b in modulating tamoxifen resistance and epithelialmesenchymal transition (EMT). 2. Methods 2.1. Cell culture and treatment The tamoxifen sensitive (TamS) human breast cancer cell line MCF-7 was obtained from ATCC (Manassas, VA, USA). The MCF-7 derived tamoxifen resistant cells (TamR) were generated by using a conventional stepwise method as described in previous study [15], which induces drug resistance by increasing concentrations of tamoxifen over 8 months. All of the cancer cells were grown in RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, 100 units of penicillin/ml and 100 mg of streptomycin/ml. MiR-27b mimics, HMGB3 siRNA and the scramble negative controls were all purchased from Ribobio (Shanghai, China). TamS and TamR cells were transfected with 100 nM miR-27b using Lipofectamie 2000 (Invitrogen, Carlsbad, CA, USA) and transfected with 100 nM HMGB3 siRNA using Hiperfect transfection reagent (QIAGEN, GmbH, Hilden, Germany) according to manufacturer’s instruction. For 5-AZA-dC treatment, the cells were cultured with 2.5 mM 5-AZA-dC (Sigma-Aldrich, St. Louis, MO, USA) for 48 h, and then were harvested for the MSP assay and qRT-PCR analysis of miR-27b expression. 2.2. Bioinformatics analysis and prediction The Affymetrix® GeneChip® miRNA Array 3.0 data of miRNA expression in tamoxifen sensitive and tamoxifen resistant MCF7 cells were retrieved in NCBI GEO Datasets (http://www.ncbi.nlm. nih.gov/gds, GSE66607). The possible targets of miR-27b were predicted using TargetScan 7.1 (http://www.targetscan.org/). The CpG island and the possible methylation sites in the promoter regions of miR-27b were predicted by using MethPrimer (http:// www.urogene.org/methprimer/). The primers used for Methylation-Specific PCR were also designed with MethPrimer. 2.3. QRT-PCR analysis Total RNAs in the cell samples were extracted using the TRIzol reagent (Invitrogen) according to manufacturer’s instructions. MiRNAs specific cDNA was synthesized using the stem-loop primers and the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). To quantify miR-27b expression, qRT-PCR analysis was performed using TaqMan MicroRNA Assay Kit (Applied Biosystems), with U6 snRNA used as the endogenous control. All PCR reactions were performed using an ABI Prism 7500 (Applied Biosystems). The results were calculated using the 2DDCT methods.
bromide. The methylated primers (M) were: left, 50 ATTATGTGTGTTAGGAAAGGGAAAC-30 ; right, 50 CTATATACCGCCAACCCGTC-0 3; while the unmethylated primers (U) were: left, 50 - ATTATGTGTGTTAGGAAAGGGAAAT-30 ; right, 50 - ACAATCTATATACCACCAACCCATC-0 3. 2.5. Cell viability assay TamR cells with enforced miR-27b overexpression or HMGB3 knockdown were seeded in a 96-well plate at a density of 3000 cells/well and were cultured in the presence of 4hydroxytamoxifen (5 mM) or ethanol solvent control. After 72 h, cell viability was measured using Cell Titer Glo Luminescent Cell Viability assay (Promega) following the manufacturer’s instructions. was performed according to the manufacturer’s protocol (BD Pharmingen, San Diego, CA, USA). 2.6. Transwell assay Transwell assay was performed to according to the methods introduced in one previous study [16]. In brief, the tanswell insert chamber coated with Matrigel (BD Biosciences Franklin Lakes, NJ, USA) was used according to manufacturer’s instruction. The invaded cells on the bottom side were fixed with 4% polyoxymethylene and stained with 0.1% crystal violet. Cell counting was performed under a light microscope. 2.7. Western blot analysis Cell samples were lysed using a lysis buffer (Beyotime, Shanghai, China). Then, the protein concentration was quantified using a BCA protein assay kit (Beyotime). Then, a conventional western blot analysis was performed following a method described in one previous study [17]. Primary antibodies used included anti-E-cadherin (1:1000, #3195, Cell Signaling, Danvers, MA, USA), anti-N-cadherin (1:1000, #13116, Cell Signaling), anti-HMGB3 (1:1000, #6893, Cell Signaling) and anti-b-actin (1: 2000, ab8227, Abcam). The blot signals were visualized using the ECL Western blotting substrate (Promega, Madison, WI, USA). The relative gray scale of the bands were analyzed using ImageJ software. 2.8. Fluorescence microscopy TamR cells after transfection were grown on coverslips. Then, the cells were fixed, permeabilized in 0.1% Triton X-100 and blocked in 1% BSA. The coverslips were incubated with primary antibodies against E-cadherin (#3195, Cell Signaling) and N-cadherin (#13116, Cell Signaling) respectively at 4 C overnight. Then, the coverslips were washed and further incubated with secondary Anti-Rabbit IgG (H þ L), F(ab’)2 Fragment (Alexa Fluor® 555 Conjugate) (#4413, Cell Signaling) and Anti-Rabbit IgG (H þ L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) (#4412, Cell Signaling) respectively for 1 h at room temperature in the dark. Nuclei were stained using Prolong® Gold Antifade Reagent with DAPI (#8961, Cell Signaling). Immunofluorescent images were obtained using an Eclipse Ti-S inverted phase/fluorescent microscope (Nikon, Tochigi, Japan).
2.4. Methylation-specific PCR (MSP) 2.9. Dual luciferase assay DNA was extracted from cell lines by DNeasy Tissue Kit (Qiagen, Hilden, Germany). MSP was performed under the following cycling conditions: 95 C for 4 min, then 40 cycles of 94 C, 25 s; 51 C, 25 s; 72 C, 30 s, followed by a final 5 min extension at 72 C. MSP products were separated electrophoretically on 2% agarose gels and visualized under ultraviolet light after staining with ethidium
To construct luciferase reporter vectors, the wildtype 30 UTR sequence fragment of HMGB3 with predicted miR-27b binding site was chemically synthesized and inserted into the downstream of the luciferase gene of pmirGLO Dual-Luciferase miRNA Target Expression Vector between Xhol/Xbal sites (Promega) and assigned
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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as pGLO-HMGB3-WT. By using the same method, the mutant sequence without miR-27b binding site was inserted into the vector and the recombinant vector was termed as pGLO-HMGB3-MT. TamS and TamR cells cultured in the 96-well plate were cotransfected with either miR-27b mimics or the scramble negative controls and pGLO-HMGB3-WT or pGLO-HMGB3-MT, using Lipofectamie 2000 (Invitrogen). Cells were harvested 24 h after transfection and then luciferase activity was measured using the Dual-Luciferase reporter assay system (Promega) and a GloMaxTM 96 Microplate Luminometer (Promega) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to that of Renilla luciferase. 2.10. Statistical analysis Data were presented in the form of means ± standard deviation (SD) based at least three repeats. Comparison between groups was performed using the unpaired t-test. A two-sided p value of <0.05 was considered statistically significant.
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One previous miRNA array (GSE66607) showed that multiple miRNAs were significantly downregulated in TamR MCF-7 cells than in TamS MCF-7 cells (Fig. 1A). MiR-27b is one of the most downregulated miRNAs (Fig. 1A). By performing qRT-PCR analysis, we also confirmed substantially suppressed miR-27b expression in TamR cells than in TamS cells (Fig. 1B). However, how it is downregulated in TamR cells is not clear. Previous studies reported that miR-195 and miR-497 (two miRNAs downregulated in TamR cells, showed in Fig. 1A), were significantly downregulated in breast cancer due to methylation state of CpG islands upstream of the miR-195/497 gene [18]. Therefore, we hypothesized that hypermethylation in the promoter regions is a cause of miR-27b downregulation in TamR cells. To verify this hypothesis, we firstly analyzed the CpG islands in the promoter region of miR-27b and designed two pairs of primers for MSP assay (Fig. 1C). Following MSP assay showed that TamR cells had significantly higher levels of methylation in the miR-27b promoter region than TamS cells (Fig. 1D). 5-AZA-dC treatment significantly decreased the methylation (Fig. 1D) and also restored miR-27b expression in TamR cells (Fig. 1E).
3. Results 3.1. MiR-27b downregulation in TamR cells is a result of promoter methylation To identify dysregulated miRNAs in tamoxifen resistant MCF7 cells, we firstly retrieved miRNA Array data in NCBI GEO Datasets.
3.2. Enforced miR-27b expression sensitizes TamR cells to tamoxifen, inhibits invasion and reverses EMT-like properties Then, we investigated the regulative effect of miR-27b on tamoxifen sensitivity of the breast cancer cells. TamR cells transfected with miR-NC had no response to 5 mM 4-hydroxytamoxifen.
Fig. 1. MiR-27b downregulation in TamR cells is a result of promoter methylation. A. MicroRNA array data of dysregulated miRNAs in TamR MCF-7 cells (data retrieved from NCBI GEO Datasets, with the number: GSE66607). The bars represent fold change of miRNAs (TamR vs. TamS). B. QRT-PCR analysis of miR-27b expression in TamS and TamR MCF-7 cells. C. Bioinformatics analysis of CpG island in promoter regions of miR-27b and the location of methylated and unmethylated primers used for MSP analysis. D. TamS and TamR MCF7 cells with or without the treatment of 5-AZA-dC were used for MSP assay and the products were separated electrophoretically on 2% agarose gels. M, amplification with methylated primers; U, amplification with unmethylated primers. E. QRT-PCR analysis of miR-27b expression in TamR MCF-7 cells with or without the treatment of 5-AZA-dC. **p < 0.01.
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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Fig. 2. Enforced miR-27b expression sensitizes TamR cells to tamoxifen, inhibits invasion and reverses EMT-like properties. A. The relative growth inhibiting effect of 5 mM 4hydroxytamoxifen treatment on TamR MCF-7 cells with or without transfection of miR-27b. B. Representative images of transwell assay of invaded TamS and TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. C. Quantitation of invaded cells showed in figure B. D and E. Immunofluorescent staining (D, red: E-cadherin, green: N-cadherin, Blue: DAPI) and western blot analysis (E) of E-cadherin and N-cadherin in TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. F. Quantitation of relative gray scale of E-cadherin and N-cadherin showed in figure E. *p < 0.05, **p < 0.01.
However, the cells with enforced miR-27b expression had approximately 15% loss of cell viability under the treatment (Fig. 2A), suggesting that miR-27b re-expression was capable of sensitizing TamR cells to tamoxifen. Next, we studied whether miR27b could modulate the invasive capacity of the TamR cells. Transwell assay showed that TamR cells had a significantly higher level of invasive capacity than TamS cells and transfection of miR27b significantly reduced the capacity (Fig. 2BeC). This reduction was accompanied with decreased expression of mesenchymal marker N-cadherin and increased expression of epithelial marker E-Cadherin (Fig. 2DeF). 3.3. MiR-27b directly targets HMGB3 in breast cancer cells Since we identified miRNA-27b as a regulator of both tamoxifen resistance and EMT, we decided to further explore the downstream genes involved in these modulations. Our preliminary studies showed that HMGB3, which is relevant to drug resistance and breast cancer cell proliferation and metastasis [19,20], is a highly possible target of miR-27b (Fig. 3A). Therefore, we hypothesized that HMGB3 upregulation might be a result of miR-27b downregulation and HMGB3 might contribute tamoxifen resistance and accompanying EMT-like properties. To test this hypothesis, we first detected the expression of HMGB3 in TamS and TamR cells and found that it was significantly upregulated in TamR cells (Fig. 3 B and E). In addition, transfection of miR-27b mimics significantly decreased HMGB3 levels in both TamS and TamR cells (Fig. 3 B and E). To further verify the direct regulation of miR-27b on HMGB3, we cloned luciferase reporter constructs carrying either wildtype or mutant 30 -UTR of HMGB3 downstream of the luciferase gene.
Transfection with miR-27b mimics significantly reduced relative luciferase activity of the reporter containing wildtype sequence in both TamS and TamR cells (Fig. 3CeD). However, miR-27b mimics had no suppressive effect on luciferase activity of the reporter with mutant miR-27b targeting sites (Fig. 3CeD). 3.4. Silencing of HMGB3 phenocopies the effects of miR-27b in TamR cells Since we confirmed that HMGB3 is a direct target of miR-27b, we further investigated the functional role of HMGB3 in breast cancer cells. TamR cells were firstly transfected with HMGB3 siRNA (Fig. 4AeB). Knockdown of HMGB3 sensitized TamR cells to tamoxifen treatment (Fig. 4C) and reduced the invasive capacity (Fig. 4D). In addition, HMGB3 knockdown also increased E-cadherin and decreased N-cadherin in TamR cells (Fig. 4EeG). These results suggest that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b and it is a functional target of miR27b involved in both tamoxifen resistance and EMT. 4. Discussion One recent study found that multiple miRNAs are significantly downregulated in TamR breast cancer cells, such as miR-497, miR221/222, miR-195 and miR-27b [7]. Although miR-27b is one of the most downregualted miRNAs in TamR cells, how it is dysregulated and its functional role in breast cancer has not been fully understood. Previous studies reported that the methylation state of CpG islands upstream of the miR-195/497 gene is responsible for the downregulation of miR-497, miR-195 and miR-221/222 in breast
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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Fig. 3. MiR-27b directly targets HMGB3 in breast cancer cells. A Bioinformatics analysis of putative binding site between miR-27b and 30 UTR of HMGB3 (positive: 487e493). The sequence of mutant HMGB3 30 UTR used for dual luciferase was also given. B and E. Western blot analysis images (B) and Quantitation of relative gray scale (E) of HMGB3 in TamS and TamR MCF-7 cells after transfection of miR-27b mimics or the negative controls. C and D. Dual luciferase assay of the inhibiting effect of miR-27b mimics on Renilla luciferase expression in TamS (C) and TamR (D) MCF-7 cells co-transfected with pGLO-HMGB3-WT or pGLO-HMGB3-MT. **p < 0.01.
Fig. 4. Silencing of HMGB3 phenocopies the effects of miR-27b in TamR cells. A-B. Western blot analysis images (A) and quantitation of relative gray scale (B) of HMGB3 in TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. C. The relative growth inhibiting effect of 5 mM 4-hydroxytamoxifen treatment on TamR MCF-7 cells with or without transfection of HMGB3 siRNA or the negative controls. D. Quantitation of transwell assay of invaded TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. E and F. Immunofluorescent staining (E, red: E-cadherin, green: N-cadherin, Blue: DAPI) and western blot analysis (F) of E-cadherin and N-cadherin in TamR MCF-7 cells transfected with HMGB3 siRNA or the negative controls. G. Quantitation of relative gray scale of E-cadherin and N-cadherin showed in figure F. **p < 0.01.
cancer cells [18,21]. Another study found that E2F7 can compete with E2F1 at miR-15a/16 promoter and decrease the transcription of miR-15a/16 cluster, leading to acquired tamoxifen resistance [7]. These findings suggest that epigenetic regulation might be an important mechanism of dysregulated miRNAs in TamR cells. One previous study found that miR-27b functioned as an inhibitor of tumor progression in colorectal cancer and DNA hypermethylation of miR-27b CpG islands decreased miR-27b expression [22]. Therefore, we hypothesized that hypermethylation might also be a mechanism of miR-27b downregulation in TamR cells. By
performing MSP assay and qRT-PCR analysis with the use of 5-AZAdC, a DNA methyltransferase inhibitor, we observed that TamR cells had significantly higher levels of methylation in miR-27b promoter region than TamS cells and demethylation restored miR-27b expression in TamR cells. In breast cancer, the effect of miR-27b dysregulation has gradually been revealed. One recent study observed that miR-27b was downregulated in mammary cell transformation and its downregulation was associated with ErbB2-dependent invasion in threedimensional (3D) cultures of human mammary epithelial cells [23].
Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133
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In addition, its loss also contributes to breast cancer stem cell generation by activating ENPP1 [14]. In multiple types of cancer, miR-27b dysregulation is involved in modulation of chemosensitivity. For example, miR-27b can sensitize cancer cells to a broad spectrum of anticancer drugs in vitro and in vivo via activating p53-dependent apoptosis and reducing CYP1B1-mediated drug detoxification [24]. In breast cancer cells, miR-27b can decrease acquired drug resistance by attenuating the formation of the side-population cells of breast cancer cells [14]. However, the role of miR-27b in tamoxifen resistance has not been reported. Multiple previous studies reported that enhanced EMT is a possible mechanism of acquired tamoxifen resistance in breast cancer cells [25,26]. Interestingly, these studies also observed that reexpression of the downregulated miRNAs in TamR cells, such as miR-375 and miR-200 can sensitize the cells to tamoxifen and also partially reverse the EMT like properties [25e27]. In TamR cells, we found that re-expression of miR-27b could sensitize cells to tamoxifen, inhibit invasion and reverse EMT-like properties. Therefore, we decided to investigate its downstream effector. HMGB3 protein belongs to the high mobility group box (HMGbox) subfamily and has 99% homology with HMGB1 and HMGB2 [28]. They all play important roles in DNA replication, transcription, recombination, and repair in cancer cells [29]. HMGB3 has been reported as an oncogene modulating drug resistance and breast cancer cell proliferation and metastasis [19,20]. Our bioinformatics analysis predicted that the 30 UTR of HMGB3 has a highly conserved targeting site of miR-27b and the following dual luciferase and western blot analysis confirmed the direct regulation of miR-27b on HMGB3 expression. More importantly, our study found that silencing of HMGB3 indeed partially phenocopied the effects of miR-27b in reducing tamoxifen resistance and cell invasion and in reversing EMT-like properties. Therefore, we infer that HMGB3 is a functional target of miR-27b in both tamoxifen resistance and EMT. Conflict of interest None. Acknowledgment This study was supported by Beijing Natural Science Foundation (7142055). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.133 References [1] R. Siegel, J. Ma, Z. Zou, A. Jemal, Cancer statistics, 2014, CA Cancer J. Clin. 64 (2014) 9e29. [2] C.K. Osborne, V. Bardou, T.A. Hopp, G.C. Chamness, S.G. Hilsenbeck, S.A. Fuqua, J. Wong, D.C. Allred, G.M. Clark, R. Schiff, Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer, J. Natl. Cancer Inst. 95 (2003) 353e361. [3] O. Gojis, B. Rudraraju, M. Gudi, K. Hogben, S. Sousha, R.C. Coombes, S. Cleator, C. Palmieri, The role of SRC-3 in human breast cancer, Nat. Rev. Clin. Oncol. 7 (2010) 83e89. [4] G. Early Breast Cancer Trialists’ Collaborative, Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials, Lancet 365 (2005) 1687e1717. [5] T. Ujihira, K. Ikeda, T. Suzuki, R. Yamaga, W. Sato, K. Horie-Inoue, T. Shigekawa, A. Osaki, T. Saeki, K. Okamoto, S. Takeda, S. Inoue, MicroRNA-574-3p, identified by microRNA library-based functional screening, modulates tamoxifen response in breast cancer, Sci. Rep. 5 (2015) 7641. [6] M. Lu, K. Ding, G. Zhang, M. Yin, G. Yao, H. Tian, J. Lian, L. Liu, M. Liang, T. Zhu, F. Sun, MicroRNA-320a sensitizes tamoxifen-resistant breast cancer cells to
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Please cite this article in press as: X. Li, et al., MiR-27b is epigenetically downregulated in tamoxifen resistant breast cancer cells due to promoter methylation and regulates tamoxifen sensitivity by targeting HMGB3, Biochemical and Biophysical Research Communications (2016), http:// dx.doi.org/10.1016/j.bbrc.2016.06.133