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PGC-1 alpha interacts with microRNA-217 to functionally regulate breast cancer cell proliferation
Biomedicine & Pharmacotherapy 85 (2017) 541–548
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Original article
PGC-1 alpha interacts with microRNA-217 to functionally regulate breast cancer cell proliferation Shaohui Zhang, Xinguo Liu, Jianming Liu, Heng Guo, Hongfeng Xu, Geng Zhang* Department of Pharmacology, Wuhan No. 1 Hospital, Tongji Medical College, Wuhan, Hebei Province, 430030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2016 Received in revised form 14 November 2016 Accepted 14 November 2016
Background: In this study, we explored the functional mechanism of PPARg co-activator 1-alpha (PGC-1a) in regulating miR-217-mediated breast cancer development in vitro. Methods: Dual-luciferase activity assay was applied to examine the binding of miR-217 on PGC-1a gene. Breast cancer cell lines, MCF-7 and MDA-MB-231 were infected by lentivirus to constitutively downregulate miR-217. Its regulation on PGC-1a expression was investigated by qRT-PCR and western blot. PGC-1a gene was subsequently downregulated by siRNA in miR-217-downregulated breast cancer cells to examine its effect on cancer proliferation and cell-cycle progression. In addition, another downstream target gene of miR-217, DACH1, was further downregulated in breast cancer cells to investigate the functional association of PGC-1a and DACH1 in miR-217-mediated breast cancer regulation. Results: PGC-1a gene was directly bound by human miR-217. Downregulation of miR-217 in MCF-7 and MDA-MB-231 cells increased PGC-1a production at both mRNA and protein levels. SiRNA-mediated PGC1a downregulation reversed the inhibition of miR-217-downregulaiton on breast cancer proliferation and cell-cycle progression. Moreover, siRNA-mediated DACH1 downregulation further reversed miR217-downregulaiton induced inhibition on cancer proliferation and cell-cycle progression in PGC-1a downregulated MCF-7 and MDA-MB-231 cells. Conclusion: MiR-217 is the upstream regulator of PGC-1a in breast cancer regulation in vitro, possibly independent of DACH1 signaling pathway. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Breast cancer miR-217 PGC-1 alpha DACH1 Lentivirus siRNA
1. Introduction During past decade, incidence rate of breast cancer is on the rise globally [1,2]. In developing countries like China, breast cancer is one of the leading causes of cancer-related mortality among female patients [3]. While the mean survival rates are gradually improving for patients with breast cancer [4,5], much is needed to understand the underlying mechanisms of breast cancer in order to develop efficient screening and treatment plans to cure the disease. PPARg co-activator 1-alpha (PGC-1a) belongs to the family of transcriptional coactivators, PPARg coactivators (PGC), which was originally found to be regulating mitochondrial biogenesis and metabolic flux [6]. In various types of human cancer, PGC-1a has
* Corresponding author at: Department of Pharmacology, Wuhan No. 1 Hospital, Tongji Medical College, 215 Hongkong Rd., Wuhan, Hebei Province, 430030, China. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.biopha.2016.11.062 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
been shown to be differentially expressed in carcinoma tissues and play important roles in regulating cancer developments [7–12]. In human breast cancer, PGC-1a was shown to be lowly expressed in carcinoma tissues among patients with breast cancer [13,14]. Functionally, PGC-1a was shown to regulate glutamine metabolism and mitochondrial functions in breast cancer cells [15,16]. Interestingly, not only PGC-1a was found to be downregulated, and potentially acting as tumor suppressor in breast cancer, colon cancer and ovarian cancer [10,11,13,14], but also it was demonstrated that PGC-1a can also be upregulated and functioning as tumor suppressor in human cancer [12,17]. However, in human breast cancer, PGC-1a was shown to have no direct effect on the growth of breast cancer cell line MDA-MB-231, either in vitro or in vivo [18]. MicroRNAs (miRNAs) are groups of evolutionally conserved, noncoding small RNAs that functionally suppress the expression of its downstream target genes by binding to the complimentary DNA sequence located at 30 un-translated region (30 -UTR) of targeted
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genes [19,20]. In human breast cancer, miRNAs have been demonstrated to play critical roles in cancer regulations, such as breast cancer carcinogenesis, proliferation, cell-cycle progression and metastasis [21–23]. In addition, miRNAs induced various downstream signaling pathways in breast cancer, such as p27Kip1 and NF-kappaB [24,25]. In a recent study, Zhang and colleagues discovered that, one of the carcinoma-associated miRNAs, microRNA-217 (miR-217), was highly expressed in breast cancer, and inhibition of miR-217 exerted significant tumor suppressive effect on breast cancer proliferation and cell-cycle progression [26]. In this study, we hypothesized that miR-217 is the upstream regulator of PGC-1a in breast cancer in vitro. We firstly used dualluciferase activity assay to examine the binding of miR-217 on PGC1a gene. We then generated breast cancer cell lines with downregulated miR-217 to examine the effect of miR-217 on PGC-1a gene and protein regulations. In addition, we used siRNA transfection to genetically knock down PGC-1a in miR-217downregulated breast cancer cells to examine the functional role of PGC-1a in miR-217 inhibition-mediated cancer regulation. Moreover, another miR-217 downstream target, DACH1 gene was further knocked down to evaluate its association with PGC-1a in regulating breast cancer development in vitro. 2. Materials and methods 2.1. Ethic statement The approval to perform scientific research in our study was granted by the Clinical Research & Ethic Committee at Wuhan No. 1 Hospital, Tongji Medical College, in Wuhan, China. 2.2. Dual-luciferase activity assay To perform co-transfection with luciferase plasmid, mirVana1 miRNA mimics of mature human hsa-miR-217 (miR-217-mimics) and negative control of mirVanaTM miRNA Mimic (miR-mC) were purchased from ThermoFisher (ThermoFisher Scientific, USA). The 30 -UTR of human gene encoding PGC-1a was inserted into a psiCHECK2 (Promega, USA), to generate a firefly luciferase vector, PGC-1&30 -UTR. Putative miR-217 binding site on PGC-1a 30 -UTR was then mutated, and also inserted into psiCHECK2 plasmid to generate a mutant firefly luciferase vector, PGC-1a 30 -UTR (m). In in vitro culture of human HEK-293T cells, miR-mC or miR-217-mimics was co-transfected with PGC-1a 30 -UTR or PGC-1a 30 -UTR(m) for 48 h. A dual-luciferase reporter assay (Promega, USA) was carried out to compare relative luciferase activities between co-transfected HEK-293T cells. 2.3. Breast cancer cell lines with miR-217 downregulation Breast cancer cell lines, MCF-7 and MDA-MB-231, were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Wuhan, China). Cells were cultured in 6-well plates in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, USA), penicillin (100 U/mL) & streptomycin (100 mg/mL) (Pen-Strep, ThermoFisher Scientific, USA) in a tissue-culturing environment with 5% CO2 at 37 C. Lentivirusus containing antagomiR against human mature miR217 (miR-217-inhibitor), and negative control antagomiR (miR-iC) were purchased from Ribobio (Ribobio, Guangzhou, China). In 6well plate, MCF-7 and MDA-MB-231 cells were then transduced with miR-217-inhibitor or miR-iC, along with 6 mg/mL polybrene at multiplicity of infection between 15 and 20. 72 h after transduction, cells were replenished with fresh culture medium (without lentiviruses), and passaged for another week or two to stabilize the
effect of lentiviral transduction. Quantitative RT-PCR was used to evaluate the transduction efficiency. 2.4. Clinical breast cancer samples Clinical breast carcinoma tissues were obtained, through surgery, from 16 patients diagnosed with breast cancer between June 2015 and October 2016. Once obtained, all samples were immediately frozen in liquid nitrogen and stored at 80 C until RNA extraction. All participating patients signed consent forms. All medical and experimental procedures were performed in accordance with the approved protocol by Ethic Committee and the guideline of World Medical Association Declaration of Helsinki. 2.5. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from MCF-7 and MDA-MB-231 cells using a Trizol kit (ThermoFisher Scientific, USA), and quantified by a NanoDro ND-1100 spectrophotometer (ThermoFisher Scientific, USA). Quantitative real-time PCR (qRT-PCR) was carried out on an Applied Biosystems Sequence Detection System 7900 (ABI 7900, ThermoFisher Scientific, USA). For human mature miR-217, gene detection was performed using a Taqman MicroRNA Assays Kit (Applied Biosystems, USA). For human genes for PGC-1a, GACH-1, an SYBR qRT-PCR Assay (ThermoFisher Scientific, USA) used instead. In both cases, single snRNA RNU6 was used as qRT-PCR template. Gene expression levels were then quantified using the 2DDCt method. 2.6. Western blot assay Total protein was extracted from MCF-7 and MDA-MB-231 cells using a Tris-based RIPA lysis buffer (ThermoFisher Scientific, USA), and quantified using a Protein Assay (Bio-Rad, USA). For each experimental sample, 30 mg diluted protein was separated o 8% NuPage gels (Novex, USA) and later electro-transferred to PVDF membranes. After blocking with 5% dry-milk in TBS-T buffer (ThermoFisher Scientific, USA) for 1 h at room temperature (RT), protein-contained membranes were treated with primary antibodies against PGC-1a (1:100, Santa Cruz Biotechnology, USA) and DACH-1 (1:500, Abcam, USA) over night at 4C. After 3 washes (PBST, 10 min each), membranes were further treated with horseradishperoxidase secondary antibodies for 2 h at RT. Blotting was visualized using an enhanced chemiluminescence system (ECL, Pierce, USA). 2.7. PGC-1a downregulation assay ON-TARGETplus siRNA against human PGC-1a (Si-PGC-1a), &&&&&man DACH1 (Si-DACH1), &&d a negative TARGETplus control siRNA (Si-C) were purchased from GE Dharmacon (GE health Care, USA). Transfection of Si-PGC-1a, Si-DACH1 or Si-C was performed in MCF-7 and MDA-MB-231 cells using DharmaFECT Transfection Reagent (GE health Care, USA) for 24 h, followed by qRT-PCR to confirm transfection efficiency. 2.8. Proliferation assay MCF-7 and MDA-MB-231 cells were plated in 96-well plate (5 103 cells/well). They were allowed to proliferate for 5 days. Daily proliferation rates were then measured using a Vybrant MTT Cell Proliferation Assay (ThermoFisher Scientific, USA) at optical density (O.D.) of 570 nm.
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2.9. Cell cycle assay MCF-7 and MDA-MB-231 cells were fixed by ice-cold 70% Ethanol over night at 4 C, and immunostained with 50 mg/mL propidium iodide (Sigma-Aldrich, USA) for 1 h at 37 C. DNA content was measured using a FACSCalibur flow cytometer (BD Biosciences, CA). Cell population at G0/G1, S or G2/M stages was then determined using hisograms on a ModFit software (Verity Software House, USA). 2.10. Statistical analysis Each assay was carried out for more than 3 times. Data was presented as means S.E.M. Statistical analysis was conducted using student’s t-test on a windows-based SPSS software (SPSS, version 13.0, USA). Statistical difference was declared if P < 0.05. 3. Results 3.1. Identifying PGC-1a upstream miRNA regulators We used online microRNA binding algorithm, TargetScan (www.targetscan.org) to search possible upstream miRNA regulators of PGC-1a. The criteria for selecting upstream miRNA candidate is that Context++ score has to be >90%. Based on this, we found 10 miRNA candidates (Table 1). Then, as PGC-1a was shown to be lowly expressed in breast cancer, we were looking for miRNAs with high expression levels in carcinoma tissues, and proven oncogenic roles in regulating breast cancer development. Thus, miR-217 is very likely the candidate upstream regulator of PGC-1a, as miR-217 was shown to be upregulated in breast cancer, and inhibition of miR-217 exerted inhibitory effect on proliferation and cell-cycle in breast cancer cell line, MDA-MB-231 [26]. 3.2. MiR-217 is the upstream regulator of PGC-1a in breast cancer We then examined, through functional assays, whether miR217 was indeed an upstream regulator of PGC-1a in breast cancer. Firstly, as revealed on the website of TargetScan (www.targetscan. org), 30 -UTR of PGC-1a gene has a putative binding sequence complimentary to the sequence of human mature miR-217 (Fig. 1A). Secondly, a dual-luciferase activity assay demonstrated that human mature miR-217 did bind to wild-type 30 -UTR of PGC1a gene, but not mutant PGC-1a 30 -UTR without putative binding sequence (Fig. 1B, *P < 0.05). Thirdly, we compared endogenous expression levels of miR-217 and PGC-1a gene in 16 clinical samples of breast cancer carcinoma, and found that miR-217 and PGC-1a gene was inversely expressed in breast cancer (Fig. 2C, P < 0.05, R2 = 0.271). Fourthly, we used lentiviral transduction to suppress endogenous miR-217 expression in two breast cancer cell
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lines, MCF-7 and MDA-MB-231. Analysis of qRT-PCR showed that, miR-217 was downregulated, whereas PGC-1a gene upregulated in MCF-7 and MDA-MB-231 cells (Fig. 2D, * P < 0.05). Fifthly, analysis western blot demonstrated that, PGC-1a protein expression levels were also significantly upregulated in MCF-7 and MDA-MB-231 cells (Fig. 2E). Therefore, our data clearly demonstrated that miR-217 was the upstream regulator of PGC-1a in human breast cancer. 3.3. Inhibition of PGC-1a reverses miR-217-downregulation induced suppression on breast cancer MiR-217 downregulation was shown to suppress breast cancer proliferation and cell-cycle transition [26]. We thus investigated whether PGC-1a&has functional role in this regulation. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, we transfected them with a PGC-1a-targeted siRNA (Si-PGC-1a) or a nonspecific siRNA (Si-C) for 24 h. QRT-PCR showed that mRNA levels of PGC-1a&were significantly suppressed by Si-PGC-1a transfection (Fig. 2A, *P < 0.05). In addition, western blot analysis confirmed that, PGC-1a proteins were also downregulated by Si-PGC-1a transfection (Fig. 2B). SiRNA-transfected MCF-7 and MDA-MB-231 were then evaluated by a 5-day MTT proliferation assay. The result demonstrated that, in miR-217-downregualted breast cancer cells, inhibition of PGC-1a significantly promoted cancer proliferations (Fig. 2C, *P < 0.05). Moreover, a flow cytometry assay showed that, in miR217-downregualted breast cancer cells, inhibition of PGC-1a also significantly progressed cell cycle transition from G0/G1 to S stage (Fig. 2D, *P < 0.05). 3.4. PGC-1a inhibition is independent of DACH1 expression in breast cancer It was demonstrated that DACH1 was involved in the functional regulation of miR-217 in beast cancer [26]. We then asked whether there is an interaction between PGC-1a and DACH1. In siRNA transfected miR-217-downregulated MCF-7 and MDA-MB-231 cells, we used qRT-PCR to evaluate the gene expression of DACH1. It showed that inhibition of PGC-1a had no effect on mRNA level of PGC-1a in breast cancer cells (Fig. 3A, *P < 0.05). We also examined DACH1 proteins in siRNA-transfected breast cancer cells. Analysis of western blot demonstrated that, PGC-1a had no effect on protein level of PGC-1a in breast cancer cells either (Fig. 3B). After transfecting MCF-7 or MDA-MB-231 cells with PGC-1atargeted siRNA, we transfected breast cancer cells with 2nd set of siRNAs, Si-C or DACH-1-targeted siRNA (si-DACH1). 24 h after transfection, qRT-PCR and western blot assay showed that siDACH1 significantly downregulated endogenous DACH1 at both mRNA (Fig. 3C, *P < 0.05) and protein levels (Fig. 3D, *P < 0.05),
Table 1 Possible upstream microRNAs of PGC-1a. Data were obtained from TargetScan (www.targetscan.rog). MicroRNA
Context++ score (%)
Function in breast cancer
Functions in other human cancers
miR-137 miR-138-5p miR-204-5p miR-211-5p miR-219-5p miR-140-5p miR-152-3p miR-148a-3p miR-148b-3p miR-217
97 98 96 96 94 92 91 91 91 90
Tumor suppressor [27] Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Oncogene [26]
Tumor suppressor [28] Tumor suppressor [29,30] Tumor suppressor [31,32] Oncogene or Tumor suppressor [33,34] Tumor suppressor [35,36] Tumor suppressor [37,38] Unknown Unknown Unknown Oncogene or Tumor suppressor [39–41]
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Fig. 1. PGC-1a is the downstream target of miR-217 in breast cancer. (A) Diagram were shown for a putative binding sequence of wild type human PGC-1a 30 -UTR by miR-217. A mutated PGC-1a 30 -UTR did not including the binding sequence. (B) In a dual-luciferase activity assay, HEK-293T cells were co-transfected with miR-mC or miR-217-mimics, and firefly luciferase plasmid of PGC-1a 30 -UTR or PGC-1a 30 -UTR(m) for 48 h. Relative luciferase activities were then measured and normalized to the value in cells cotransfected with miR-mC and PGC-1a 30 -UTR luciferase plasmid. (*P < 0.05). (C) In breast carcinoma samples obtained from 16 patients, endogenous gene expression levels of miR-217 and PGC-1a were compared (P < 0.05, R2 = 0.271). (D) MCF-7 and MDA-MB-231 cells were transduced with lentiviruses of miR-iC or miR-217-inhibitor. QRT-PCR was carried out to measure endogenous expression levels of miR-217 and PGC-1a gene (*P < 0.05). (E) Western blot analysis was also used to evaluate PGC-1a protein levels in MCF-7 and MDA-MB-231 cells.
further confirming that regulation on DACH1 expression is independent of PGC-1a inhibition. 3.5. PGC-1a regulation in miR-217-dwonregulated breast cancer is independent of DACH1 Finally, we asked whether PGC-1a and DACH1 might have functional interaction in miR-217-dwonregulated breast cancer. In
double-transfected miR-217-dwonregulated MCF-7 and MDA-MB231 cells, we conducted a 5-day MTT proliferation assay. It showed that inhibition of DACH1 could further promote breast cancer proliferation, even after PGC-1a was downregulated (Fig. 4A, *P < 0.05). Moreover, analysis of flow cytometry assay showed that inhibition of DACH1 also further progressed cell cycle transition from G0/G1 to S stage, even after PGC-1a was downregulated, in miR-217-dwonregulated breast cancer cells (Fig. 4B, *P < 0.05).
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Fig. 2. PGC-1a had opposite regulatory effect as miR-217 in breast cancer. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, siRNA transfection with Si-C or SiPGC-1a was performed for 24 h. (A) QRT-PCR was carried out to measure endogenous expression levels of PGC-1a gene (*P < 0.05). (B) Western blot analysis was also used to evaluate PGC-1a protein levels in MCF-7 and MDA-MB-231 cells. (C) After transfections, MCF-7 and MDA-MB-231 cells were re-plated and evaluated through a 5-day MTT proliferation assay (*P < 0.05). (D) MCF-7 and MDA-MB-231 cells were also evaluated through a flow cytometry assay to assess their cell-cycle progression (*P < 0.05).
Therefore, our data clearly demonstrated that signaling pathways of PGC-1a and DACH1 were independent during their regulations in miR-217 downregulation-mediated breast cancer. 4. Discussions In human breast cancer, though PGC-1a was found to be differentially expressed in carcinoma tissues than in normal breast epithelia tissues [13,14], it has no direct functional role in regulating breast cancer regulation [18]. In this study, we sought after the upstream regulator, or the epigenetic signaling pathway
of miRNA, in association with PGC-1a in human breast cancer. With the criterial of have clear oncogenic role in breast cancer, and having Content++ Score >90%, we showed that miR-217 might be the candidate, as its binding Content++ Score with PGC-1a is 90%, and more importantly it functionally acts of promoter on breast cancer proliferation and cell-cycle progression [26]. The results of several biochemical assays confirmed our hypothesis of miR-217 being the upstream regulator of PGC-1a in human breast cancer. Firstly, a co-transfection dual-luciferase activity assay showed that human mature mir-217 did bind the 30 UTR of PGC-1a gene. Secondly, in lentivirus-mediated,
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Fig. 3. PGC-1a inhibition has no effect on DACH1 expression in breast cancer. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, siRNA transfection with Si-C or Si- PGC-1a was performed for 24 h. (A) QRT-PCR was carried out to measure endogenous gene expression of DACH1 (*P < 0.05). (B) Western blot analysis was also used to evaluate DACH1 protein levels in MCF-7 and MDA-MB-231 cells. (C) In MCF-7 and MDA-MB-231 cells transfected with Si-PGC-1a, 2nd siRNA transfection with Si-C or SiDACH1 was performed for 24 h. QRT-PCR was carried out to measure endogenous gene expression of DACH1 (*P < 0.05). (D) Western blot analysis was then also used to evaluate DACH1 protein levels.
endogenously miR-217-downregulated breast cancer cell lines MCF-7 and MDA-MB-231 cells, both PGC-1a mRNA and protein levels were significantly upregulated. Most importantly, while PGC-1a gene was downregulated, through siRNA transfection, cancer proliferation and cycle–cycle progression were significantly promoted in miR-217-downregulated MCF-7 and MDA-MB-231 cells. This is a novel, and quite interesting property of PGC-1a in vitro regulation discovered by our study in breast cancer. In previous studies, although PGC-1a was shown to be lowly expressed in breast cancer carcinoma tissues [13,14], no direct functional role of PGC-1a was found in breast cancer regulation [18]. Thus, as we revealed in this study, it seems like the general expression level of PGC-1a was ineffective to trigger any functional
in vitro regulation in breast cancer. However, at high expression level, induced by miR-217 downregulation, the inhibition of PGC1a may exert oncogenic modulation on breast cancer. In previous report, DACH1 was shown to be the downstream target gene of miR-217, as it reversed breast cancer regulation induced by miR-217 [26]. As we demonstrated in this study that miR-217 was the upstream regulator of PGC-1a gene, it would be interesting to investigate what the functional relationship between PGC-1a and DACH1. Thus, in this study, we performed double transfection of both PGC-1a and DACH1-targeted siRNAs in miR217-downregulated breast cancer cells. Interestingly, we found that, after the application of PGC-1a-targeted siRNA, downregulation of DACH1 showed further oncogenic effect by
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Fig. 4. Synergic effect of PGC-1a and DACH1 in breast cancer regulation. In miR-217-donwregulated MCF-7 and MDA-MB-231 cells, they were transfected with Si- PGC-1a for 24 h, followed by 2nd siRNA transfection with Si-C or Si-DACH1 for another 24 h. (A) MCF-7 and MDA-MB-231 cells were re-plated and evaluated through a 5-day MTT proliferation assay (*P < 0.05). (C) MCF-7 and MDA-MB-231 cells were also evaluated through a flow cytometry assay to assess their cell-cycle progression (* P < 0.05).
promoting cancer proliferation and inducing cancer cell-cycle progression. These observations thus suggested that the regulatory signaling pathways of PGC-1a and DACH1 are independent to each other, as downregulations of PGC-1a and DACH1 exerted additive effects to reverse the tumor suppression of miR-217 downregulation on breast cancer proliferation and cell-cycle progression in vitro. Further investigations, including those in vivo ones on downstream signaling pathways of PGC-1a and DACH1 would certainly help to elucidate the complex regulatory network associated with miRNA in breast cancer. 5. Conclusion Overall, our study demonstrated that miR-217 was the upstream epigenetic regulator of PGC-1a in human breast cancer. The functional pathway of PGC-1a in regulating miR-217mediated breast cancer development may be independent of known miR-217 target gene of DACH1.
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Original article
PGC-1 alpha interacts with microRNA-217 to functionally regulate breast cancer cell proliferation Shaohui Zhang, Xinguo Liu, Jianming Liu, Heng Guo, Hongfeng Xu, Geng Zhang* Department of Pharmacology, Wuhan No. 1 Hospital, Tongji Medical College, Wuhan, Hebei Province, 430030, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 August 2016 Received in revised form 14 November 2016 Accepted 14 November 2016
Background: In this study, we explored the functional mechanism of PPARg co-activator 1-alpha (PGC-1a) in regulating miR-217-mediated breast cancer development in vitro. Methods: Dual-luciferase activity assay was applied to examine the binding of miR-217 on PGC-1a gene. Breast cancer cell lines, MCF-7 and MDA-MB-231 were infected by lentivirus to constitutively downregulate miR-217. Its regulation on PGC-1a expression was investigated by qRT-PCR and western blot. PGC-1a gene was subsequently downregulated by siRNA in miR-217-downregulated breast cancer cells to examine its effect on cancer proliferation and cell-cycle progression. In addition, another downstream target gene of miR-217, DACH1, was further downregulated in breast cancer cells to investigate the functional association of PGC-1a and DACH1 in miR-217-mediated breast cancer regulation. Results: PGC-1a gene was directly bound by human miR-217. Downregulation of miR-217 in MCF-7 and MDA-MB-231 cells increased PGC-1a production at both mRNA and protein levels. SiRNA-mediated PGC1a downregulation reversed the inhibition of miR-217-downregulaiton on breast cancer proliferation and cell-cycle progression. Moreover, siRNA-mediated DACH1 downregulation further reversed miR217-downregulaiton induced inhibition on cancer proliferation and cell-cycle progression in PGC-1a downregulated MCF-7 and MDA-MB-231 cells. Conclusion: MiR-217 is the upstream regulator of PGC-1a in breast cancer regulation in vitro, possibly independent of DACH1 signaling pathway. © 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Breast cancer miR-217 PGC-1 alpha DACH1 Lentivirus siRNA
1. Introduction During past decade, incidence rate of breast cancer is on the rise globally [1,2]. In developing countries like China, breast cancer is one of the leading causes of cancer-related mortality among female patients [3]. While the mean survival rates are gradually improving for patients with breast cancer [4,5], much is needed to understand the underlying mechanisms of breast cancer in order to develop efficient screening and treatment plans to cure the disease. PPARg co-activator 1-alpha (PGC-1a) belongs to the family of transcriptional coactivators, PPARg coactivators (PGC), which was originally found to be regulating mitochondrial biogenesis and metabolic flux [6]. In various types of human cancer, PGC-1a has
* Corresponding author at: Department of Pharmacology, Wuhan No. 1 Hospital, Tongji Medical College, 215 Hongkong Rd., Wuhan, Hebei Province, 430030, China. E-mail address: [email protected] (G. Zhang). http://dx.doi.org/10.1016/j.biopha.2016.11.062 0753-3322/© 2016 Elsevier Masson SAS. All rights reserved.
been shown to be differentially expressed in carcinoma tissues and play important roles in regulating cancer developments [7–12]. In human breast cancer, PGC-1a was shown to be lowly expressed in carcinoma tissues among patients with breast cancer [13,14]. Functionally, PGC-1a was shown to regulate glutamine metabolism and mitochondrial functions in breast cancer cells [15,16]. Interestingly, not only PGC-1a was found to be downregulated, and potentially acting as tumor suppressor in breast cancer, colon cancer and ovarian cancer [10,11,13,14], but also it was demonstrated that PGC-1a can also be upregulated and functioning as tumor suppressor in human cancer [12,17]. However, in human breast cancer, PGC-1a was shown to have no direct effect on the growth of breast cancer cell line MDA-MB-231, either in vitro or in vivo [18]. MicroRNAs (miRNAs) are groups of evolutionally conserved, noncoding small RNAs that functionally suppress the expression of its downstream target genes by binding to the complimentary DNA sequence located at 30 un-translated region (30 -UTR) of targeted
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genes [19,20]. In human breast cancer, miRNAs have been demonstrated to play critical roles in cancer regulations, such as breast cancer carcinogenesis, proliferation, cell-cycle progression and metastasis [21–23]. In addition, miRNAs induced various downstream signaling pathways in breast cancer, such as p27Kip1 and NF-kappaB [24,25]. In a recent study, Zhang and colleagues discovered that, one of the carcinoma-associated miRNAs, microRNA-217 (miR-217), was highly expressed in breast cancer, and inhibition of miR-217 exerted significant tumor suppressive effect on breast cancer proliferation and cell-cycle progression [26]. In this study, we hypothesized that miR-217 is the upstream regulator of PGC-1a in breast cancer in vitro. We firstly used dualluciferase activity assay to examine the binding of miR-217 on PGC1a gene. We then generated breast cancer cell lines with downregulated miR-217 to examine the effect of miR-217 on PGC-1a gene and protein regulations. In addition, we used siRNA transfection to genetically knock down PGC-1a in miR-217downregulated breast cancer cells to examine the functional role of PGC-1a in miR-217 inhibition-mediated cancer regulation. Moreover, another miR-217 downstream target, DACH1 gene was further knocked down to evaluate its association with PGC-1a in regulating breast cancer development in vitro. 2. Materials and methods 2.1. Ethic statement The approval to perform scientific research in our study was granted by the Clinical Research & Ethic Committee at Wuhan No. 1 Hospital, Tongji Medical College, in Wuhan, China. 2.2. Dual-luciferase activity assay To perform co-transfection with luciferase plasmid, mirVana1 miRNA mimics of mature human hsa-miR-217 (miR-217-mimics) and negative control of mirVanaTM miRNA Mimic (miR-mC) were purchased from ThermoFisher (ThermoFisher Scientific, USA). The 30 -UTR of human gene encoding PGC-1a was inserted into a psiCHECK2 (Promega, USA), to generate a firefly luciferase vector, PGC-1&30 -UTR. Putative miR-217 binding site on PGC-1a 30 -UTR was then mutated, and also inserted into psiCHECK2 plasmid to generate a mutant firefly luciferase vector, PGC-1a 30 -UTR (m). In in vitro culture of human HEK-293T cells, miR-mC or miR-217-mimics was co-transfected with PGC-1a 30 -UTR or PGC-1a 30 -UTR(m) for 48 h. A dual-luciferase reporter assay (Promega, USA) was carried out to compare relative luciferase activities between co-transfected HEK-293T cells. 2.3. Breast cancer cell lines with miR-217 downregulation Breast cancer cell lines, MCF-7 and MDA-MB-231, were purchased from Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Wuhan, China). Cells were cultured in 6-well plates in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher Scientific, USA), supplemented with 10% fetal bovine serum (FBS, ThermoFisher Scientific, USA), penicillin (100 U/mL) & streptomycin (100 mg/mL) (Pen-Strep, ThermoFisher Scientific, USA) in a tissue-culturing environment with 5% CO2 at 37 C. Lentivirusus containing antagomiR against human mature miR217 (miR-217-inhibitor), and negative control antagomiR (miR-iC) were purchased from Ribobio (Ribobio, Guangzhou, China). In 6well plate, MCF-7 and MDA-MB-231 cells were then transduced with miR-217-inhibitor or miR-iC, along with 6 mg/mL polybrene at multiplicity of infection between 15 and 20. 72 h after transduction, cells were replenished with fresh culture medium (without lentiviruses), and passaged for another week or two to stabilize the
effect of lentiviral transduction. Quantitative RT-PCR was used to evaluate the transduction efficiency. 2.4. Clinical breast cancer samples Clinical breast carcinoma tissues were obtained, through surgery, from 16 patients diagnosed with breast cancer between June 2015 and October 2016. Once obtained, all samples were immediately frozen in liquid nitrogen and stored at 80 C until RNA extraction. All participating patients signed consent forms. All medical and experimental procedures were performed in accordance with the approved protocol by Ethic Committee and the guideline of World Medical Association Declaration of Helsinki. 2.5. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from MCF-7 and MDA-MB-231 cells using a Trizol kit (ThermoFisher Scientific, USA), and quantified by a NanoDro ND-1100 spectrophotometer (ThermoFisher Scientific, USA). Quantitative real-time PCR (qRT-PCR) was carried out on an Applied Biosystems Sequence Detection System 7900 (ABI 7900, ThermoFisher Scientific, USA). For human mature miR-217, gene detection was performed using a Taqman MicroRNA Assays Kit (Applied Biosystems, USA). For human genes for PGC-1a, GACH-1, an SYBR qRT-PCR Assay (ThermoFisher Scientific, USA) used instead. In both cases, single snRNA RNU6 was used as qRT-PCR template. Gene expression levels were then quantified using the 2DDCt method. 2.6. Western blot assay Total protein was extracted from MCF-7 and MDA-MB-231 cells using a Tris-based RIPA lysis buffer (ThermoFisher Scientific, USA), and quantified using a Protein Assay (Bio-Rad, USA). For each experimental sample, 30 mg diluted protein was separated o 8% NuPage gels (Novex, USA) and later electro-transferred to PVDF membranes. After blocking with 5% dry-milk in TBS-T buffer (ThermoFisher Scientific, USA) for 1 h at room temperature (RT), protein-contained membranes were treated with primary antibodies against PGC-1a (1:100, Santa Cruz Biotechnology, USA) and DACH-1 (1:500, Abcam, USA) over night at 4C. After 3 washes (PBST, 10 min each), membranes were further treated with horseradishperoxidase secondary antibodies for 2 h at RT. Blotting was visualized using an enhanced chemiluminescence system (ECL, Pierce, USA). 2.7. PGC-1a downregulation assay ON-TARGETplus siRNA against human PGC-1a (Si-PGC-1a), &&&&&man DACH1 (Si-DACH1), &&d a negative TARGETplus control siRNA (Si-C) were purchased from GE Dharmacon (GE health Care, USA). Transfection of Si-PGC-1a, Si-DACH1 or Si-C was performed in MCF-7 and MDA-MB-231 cells using DharmaFECT Transfection Reagent (GE health Care, USA) for 24 h, followed by qRT-PCR to confirm transfection efficiency. 2.8. Proliferation assay MCF-7 and MDA-MB-231 cells were plated in 96-well plate (5 103 cells/well). They were allowed to proliferate for 5 days. Daily proliferation rates were then measured using a Vybrant MTT Cell Proliferation Assay (ThermoFisher Scientific, USA) at optical density (O.D.) of 570 nm.
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2.9. Cell cycle assay MCF-7 and MDA-MB-231 cells were fixed by ice-cold 70% Ethanol over night at 4 C, and immunostained with 50 mg/mL propidium iodide (Sigma-Aldrich, USA) for 1 h at 37 C. DNA content was measured using a FACSCalibur flow cytometer (BD Biosciences, CA). Cell population at G0/G1, S or G2/M stages was then determined using hisograms on a ModFit software (Verity Software House, USA). 2.10. Statistical analysis Each assay was carried out for more than 3 times. Data was presented as means S.E.M. Statistical analysis was conducted using student’s t-test on a windows-based SPSS software (SPSS, version 13.0, USA). Statistical difference was declared if P < 0.05. 3. Results 3.1. Identifying PGC-1a upstream miRNA regulators We used online microRNA binding algorithm, TargetScan (www.targetscan.org) to search possible upstream miRNA regulators of PGC-1a. The criteria for selecting upstream miRNA candidate is that Context++ score has to be >90%. Based on this, we found 10 miRNA candidates (Table 1). Then, as PGC-1a was shown to be lowly expressed in breast cancer, we were looking for miRNAs with high expression levels in carcinoma tissues, and proven oncogenic roles in regulating breast cancer development. Thus, miR-217 is very likely the candidate upstream regulator of PGC-1a, as miR-217 was shown to be upregulated in breast cancer, and inhibition of miR-217 exerted inhibitory effect on proliferation and cell-cycle in breast cancer cell line, MDA-MB-231 [26]. 3.2. MiR-217 is the upstream regulator of PGC-1a in breast cancer We then examined, through functional assays, whether miR217 was indeed an upstream regulator of PGC-1a in breast cancer. Firstly, as revealed on the website of TargetScan (www.targetscan. org), 30 -UTR of PGC-1a gene has a putative binding sequence complimentary to the sequence of human mature miR-217 (Fig. 1A). Secondly, a dual-luciferase activity assay demonstrated that human mature miR-217 did bind to wild-type 30 -UTR of PGC1a gene, but not mutant PGC-1a 30 -UTR without putative binding sequence (Fig. 1B, *P < 0.05). Thirdly, we compared endogenous expression levels of miR-217 and PGC-1a gene in 16 clinical samples of breast cancer carcinoma, and found that miR-217 and PGC-1a gene was inversely expressed in breast cancer (Fig. 2C, P < 0.05, R2 = 0.271). Fourthly, we used lentiviral transduction to suppress endogenous miR-217 expression in two breast cancer cell
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lines, MCF-7 and MDA-MB-231. Analysis of qRT-PCR showed that, miR-217 was downregulated, whereas PGC-1a gene upregulated in MCF-7 and MDA-MB-231 cells (Fig. 2D, * P < 0.05). Fifthly, analysis western blot demonstrated that, PGC-1a protein expression levels were also significantly upregulated in MCF-7 and MDA-MB-231 cells (Fig. 2E). Therefore, our data clearly demonstrated that miR-217 was the upstream regulator of PGC-1a in human breast cancer. 3.3. Inhibition of PGC-1a reverses miR-217-downregulation induced suppression on breast cancer MiR-217 downregulation was shown to suppress breast cancer proliferation and cell-cycle transition [26]. We thus investigated whether PGC-1a&has functional role in this regulation. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, we transfected them with a PGC-1a-targeted siRNA (Si-PGC-1a) or a nonspecific siRNA (Si-C) for 24 h. QRT-PCR showed that mRNA levels of PGC-1a&were significantly suppressed by Si-PGC-1a transfection (Fig. 2A, *P < 0.05). In addition, western blot analysis confirmed that, PGC-1a proteins were also downregulated by Si-PGC-1a transfection (Fig. 2B). SiRNA-transfected MCF-7 and MDA-MB-231 were then evaluated by a 5-day MTT proliferation assay. The result demonstrated that, in miR-217-downregualted breast cancer cells, inhibition of PGC-1a significantly promoted cancer proliferations (Fig. 2C, *P < 0.05). Moreover, a flow cytometry assay showed that, in miR217-downregualted breast cancer cells, inhibition of PGC-1a also significantly progressed cell cycle transition from G0/G1 to S stage (Fig. 2D, *P < 0.05). 3.4. PGC-1a inhibition is independent of DACH1 expression in breast cancer It was demonstrated that DACH1 was involved in the functional regulation of miR-217 in beast cancer [26]. We then asked whether there is an interaction between PGC-1a and DACH1. In siRNA transfected miR-217-downregulated MCF-7 and MDA-MB-231 cells, we used qRT-PCR to evaluate the gene expression of DACH1. It showed that inhibition of PGC-1a had no effect on mRNA level of PGC-1a in breast cancer cells (Fig. 3A, *P < 0.05). We also examined DACH1 proteins in siRNA-transfected breast cancer cells. Analysis of western blot demonstrated that, PGC-1a had no effect on protein level of PGC-1a in breast cancer cells either (Fig. 3B). After transfecting MCF-7 or MDA-MB-231 cells with PGC-1atargeted siRNA, we transfected breast cancer cells with 2nd set of siRNAs, Si-C or DACH-1-targeted siRNA (si-DACH1). 24 h after transfection, qRT-PCR and western blot assay showed that siDACH1 significantly downregulated endogenous DACH1 at both mRNA (Fig. 3C, *P < 0.05) and protein levels (Fig. 3D, *P < 0.05),
Table 1 Possible upstream microRNAs of PGC-1a. Data were obtained from TargetScan (www.targetscan.rog). MicroRNA
Context++ score (%)
Function in breast cancer
Functions in other human cancers
miR-137 miR-138-5p miR-204-5p miR-211-5p miR-219-5p miR-140-5p miR-152-3p miR-148a-3p miR-148b-3p miR-217
97 98 96 96 94 92 91 91 91 90
Tumor suppressor [27] Unknown Unknown Unknown Unknown Unknown Unknown Unknown Unknown Oncogene [26]
Tumor suppressor [28] Tumor suppressor [29,30] Tumor suppressor [31,32] Oncogene or Tumor suppressor [33,34] Tumor suppressor [35,36] Tumor suppressor [37,38] Unknown Unknown Unknown Oncogene or Tumor suppressor [39–41]
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Fig. 1. PGC-1a is the downstream target of miR-217 in breast cancer. (A) Diagram were shown for a putative binding sequence of wild type human PGC-1a 30 -UTR by miR-217. A mutated PGC-1a 30 -UTR did not including the binding sequence. (B) In a dual-luciferase activity assay, HEK-293T cells were co-transfected with miR-mC or miR-217-mimics, and firefly luciferase plasmid of PGC-1a 30 -UTR or PGC-1a 30 -UTR(m) for 48 h. Relative luciferase activities were then measured and normalized to the value in cells cotransfected with miR-mC and PGC-1a 30 -UTR luciferase plasmid. (*P < 0.05). (C) In breast carcinoma samples obtained from 16 patients, endogenous gene expression levels of miR-217 and PGC-1a were compared (P < 0.05, R2 = 0.271). (D) MCF-7 and MDA-MB-231 cells were transduced with lentiviruses of miR-iC or miR-217-inhibitor. QRT-PCR was carried out to measure endogenous expression levels of miR-217 and PGC-1a gene (*P < 0.05). (E) Western blot analysis was also used to evaluate PGC-1a protein levels in MCF-7 and MDA-MB-231 cells.
further confirming that regulation on DACH1 expression is independent of PGC-1a inhibition. 3.5. PGC-1a regulation in miR-217-dwonregulated breast cancer is independent of DACH1 Finally, we asked whether PGC-1a and DACH1 might have functional interaction in miR-217-dwonregulated breast cancer. In
double-transfected miR-217-dwonregulated MCF-7 and MDA-MB231 cells, we conducted a 5-day MTT proliferation assay. It showed that inhibition of DACH1 could further promote breast cancer proliferation, even after PGC-1a was downregulated (Fig. 4A, *P < 0.05). Moreover, analysis of flow cytometry assay showed that inhibition of DACH1 also further progressed cell cycle transition from G0/G1 to S stage, even after PGC-1a was downregulated, in miR-217-dwonregulated breast cancer cells (Fig. 4B, *P < 0.05).
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Fig. 2. PGC-1a had opposite regulatory effect as miR-217 in breast cancer. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, siRNA transfection with Si-C or SiPGC-1a was performed for 24 h. (A) QRT-PCR was carried out to measure endogenous expression levels of PGC-1a gene (*P < 0.05). (B) Western blot analysis was also used to evaluate PGC-1a protein levels in MCF-7 and MDA-MB-231 cells. (C) After transfections, MCF-7 and MDA-MB-231 cells were re-plated and evaluated through a 5-day MTT proliferation assay (*P < 0.05). (D) MCF-7 and MDA-MB-231 cells were also evaluated through a flow cytometry assay to assess their cell-cycle progression (*P < 0.05).
Therefore, our data clearly demonstrated that signaling pathways of PGC-1a and DACH1 were independent during their regulations in miR-217 downregulation-mediated breast cancer. 4. Discussions In human breast cancer, though PGC-1a was found to be differentially expressed in carcinoma tissues than in normal breast epithelia tissues [13,14], it has no direct functional role in regulating breast cancer regulation [18]. In this study, we sought after the upstream regulator, or the epigenetic signaling pathway
of miRNA, in association with PGC-1a in human breast cancer. With the criterial of have clear oncogenic role in breast cancer, and having Content++ Score >90%, we showed that miR-217 might be the candidate, as its binding Content++ Score with PGC-1a is 90%, and more importantly it functionally acts of promoter on breast cancer proliferation and cell-cycle progression [26]. The results of several biochemical assays confirmed our hypothesis of miR-217 being the upstream regulator of PGC-1a in human breast cancer. Firstly, a co-transfection dual-luciferase activity assay showed that human mature mir-217 did bind the 30 UTR of PGC-1a gene. Secondly, in lentivirus-mediated,
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Fig. 3. PGC-1a inhibition has no effect on DACH1 expression in breast cancer. In MCF-7 and MDA-MB-231 cells with miR-217 downregulation, siRNA transfection with Si-C or Si- PGC-1a was performed for 24 h. (A) QRT-PCR was carried out to measure endogenous gene expression of DACH1 (*P < 0.05). (B) Western blot analysis was also used to evaluate DACH1 protein levels in MCF-7 and MDA-MB-231 cells. (C) In MCF-7 and MDA-MB-231 cells transfected with Si-PGC-1a, 2nd siRNA transfection with Si-C or SiDACH1 was performed for 24 h. QRT-PCR was carried out to measure endogenous gene expression of DACH1 (*P < 0.05). (D) Western blot analysis was then also used to evaluate DACH1 protein levels.
endogenously miR-217-downregulated breast cancer cell lines MCF-7 and MDA-MB-231 cells, both PGC-1a mRNA and protein levels were significantly upregulated. Most importantly, while PGC-1a gene was downregulated, through siRNA transfection, cancer proliferation and cycle–cycle progression were significantly promoted in miR-217-downregulated MCF-7 and MDA-MB-231 cells. This is a novel, and quite interesting property of PGC-1a in vitro regulation discovered by our study in breast cancer. In previous studies, although PGC-1a was shown to be lowly expressed in breast cancer carcinoma tissues [13,14], no direct functional role of PGC-1a was found in breast cancer regulation [18]. Thus, as we revealed in this study, it seems like the general expression level of PGC-1a was ineffective to trigger any functional
in vitro regulation in breast cancer. However, at high expression level, induced by miR-217 downregulation, the inhibition of PGC1a may exert oncogenic modulation on breast cancer. In previous report, DACH1 was shown to be the downstream target gene of miR-217, as it reversed breast cancer regulation induced by miR-217 [26]. As we demonstrated in this study that miR-217 was the upstream regulator of PGC-1a gene, it would be interesting to investigate what the functional relationship between PGC-1a and DACH1. Thus, in this study, we performed double transfection of both PGC-1a and DACH1-targeted siRNAs in miR217-downregulated breast cancer cells. Interestingly, we found that, after the application of PGC-1a-targeted siRNA, downregulation of DACH1 showed further oncogenic effect by
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Fig. 4. Synergic effect of PGC-1a and DACH1 in breast cancer regulation. In miR-217-donwregulated MCF-7 and MDA-MB-231 cells, they were transfected with Si- PGC-1a for 24 h, followed by 2nd siRNA transfection with Si-C or Si-DACH1 for another 24 h. (A) MCF-7 and MDA-MB-231 cells were re-plated and evaluated through a 5-day MTT proliferation assay (*P < 0.05). (C) MCF-7 and MDA-MB-231 cells were also evaluated through a flow cytometry assay to assess their cell-cycle progression (* P < 0.05).
promoting cancer proliferation and inducing cancer cell-cycle progression. These observations thus suggested that the regulatory signaling pathways of PGC-1a and DACH1 are independent to each other, as downregulations of PGC-1a and DACH1 exerted additive effects to reverse the tumor suppression of miR-217 downregulation on breast cancer proliferation and cell-cycle progression in vitro. Further investigations, including those in vivo ones on downstream signaling pathways of PGC-1a and DACH1 would certainly help to elucidate the complex regulatory network associated with miRNA in breast cancer. 5. Conclusion Overall, our study demonstrated that miR-217 was the upstream epigenetic regulator of PGC-1a in human breast cancer. The functional pathway of PGC-1a in regulating miR-217mediated breast cancer development may be independent of known miR-217 target gene of DACH1.
Conflict of interest None. Funding This work is supported by Natural Science Foundation of Hubei Province, China (2013CFC120). References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) E359–386. [2] E.M. Ward, C.E. DeSantis, C.C. Lin, J.L. Kramer, A. Jemal, B. Kohler, O.W. Brawley, T. Gansler, Cancer statistics: breast cancer in situ, CA Cancer J. Clin. 65 (2015) 481–495. [3] W. Chen, R. Zheng, H. Zeng, S. Zhang, J. He, Annual report on status of cancer in China, 2011, Chin. J. Cancer Res. 27 (2015) 2–12. [4] D.N. Lo-Fo-Wong, K. Sitnikova, M.A. Sprangers, H.C. de Haes, Predictors of health care use of women with Breast cancer: a systematic review, Breast J. 21 (2015) 508–513.
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