LncRNA GACAT3 predicts poor prognosis and promotes cell proliferation in breast cancer through regulation of miR-497/CCND2

Abstract

Breast cancer is the most common malignancy in women which increases gradually all over the world. LncRNA GACAT3 has been found to be increased in gastric cancer and associated with tumor malignancy. However, whether GACAT3 plays a role in the regulation of breast cancer is not known. In the present study, we found that GACAT3 expression was increased in breast cancer tissues and cells compared with adjacent normal tissues and normal cells. High GACAT3 expression was correlated with the poor prognosis of breast cancer patients. GACAT3 and cyclin D2 (CCND2) contained a binding site of miR-497. miR-497 was decreased in breast cancer tissues and cells compared with adjacent normal tissues and normal cells. Low miR-497 expression was correlated with the poor prognosis of breast cancer patients. In breast cancer tissues, the expression of miR-497 was negatively correlated with GACAT3. Downregulation of GACAT3 increased miR-497 expression. miR-497 mimic reduced the luciferase of GACAT3 and CCND2. Anti-miR-497 reversed the effects of GACAT3 downregulation. We also found that GACAT3 may act as a ceRNA for miR-497, enhancing the expression of CCND2. In conclusion, GACAT3 promotes breast cancer malignancy by sponging miR-497, leading to the enhancement of its endogenous target CCND2. These results suggest that GACAT3/miR-497/CCND2 is a potential therapeutic target and biomarker for breast cancer.

Keywords

Breast cancer CCND2 lncRNA GACAT3 malignancy miR-497

1. Introduction

The incidence of breast cancer, the most common malignancy in women, increases gradually all over the world [1, 2], which can cause nearly half a million of deaths in a year [3]. In the last decades, breast cancer-associated mortality has declined due to major advances in detection, diagnosis, and treatment [4]. Although the prognosis of breast cancer can be predicted by the conventional tumor node metastasis (TNM) staging system, clinical outcomes are significantly differential among patients with similar TNM stages [5]. The molecular diagnostic tests such as Oncotype Dx and Mammaprint have been developed to obtain additional prognostic information, however, the use of these kits is limited by the high cost and regional availability [6]. Therefore, detailed understanding of the basis for breast cancer and identifying prognostic breast cancer biomarkers are of great importance.

Owing to human transcriptome analysis, it has been found that most of the transcribed RNAs are not translated [7]. Long noncoding RNAs (lncRNAs), containing more than 200 nucleotides, are a type of non-coding RNAs, that play an important role in the regulation of gene expression at transcriptional, post-transcriptional and translational regulation levels [8, 9]. Emerging evidence has indicated a crucial role of lncRNAs in the development and progression of cancer [10, 11]. AC130710 has been named them as gastric cancer associated transcript 3 (GACAT3) [12]. GACAT3 is considered to function as a lncRNA which is located on human Chr 2p24.3. GACAT3 has been found to be increased in gastric cancer and associated with tumor size, tumor-node metastasis stages, and distal metastasis [13]. Moreover, GACAT3 was identified to promote gastric cancer progression by negatively regulating miR-497 expression [14]. However, the potential regulatory roles of GACAT3 in breast cancer are largely unknown.

In this study, we found that GACAT3 expression was increased in breast cancer tissues and cell lines. High levels of GACAT3 expression was associated with clinicopathological characteristics and poor prognosis in breast cancer patients. Downregulation of GACAT3 significantly inhibited proliferation, migration and invasion of breast cancer cells. microRNA-497 was involved in GACAT3-induced regulation of proliferation, migration and invasion in breast cancer cells. We suggest that GACAT3 plays a substantial oncogenic role in the progression and metastasis of breast cancer and considered to be a potential therapeutic target.

2. Materials and methods

All experimental protocols were approved by the Ethics Committee of Chinese PLA General Hospital and the First Affiliated Hospital of Xinxiang Medical University. A total of 41 breast cancer tissues and the corresponding adjacent normal tissues were obtained from patients who underwent radical surgery at the Department of Oncology, The First Affiliated Hospital of Xinxiang Medical University, between March 2012 and December 2012. None of the patients received adjuvant chemotherapy, immunotherapy, or radiotherapy before surgery. We obtained informed consent from all the patients. The clinic specimens were later stored in liquid nitrogen at $-$ 80 ${}^{\circ}$ C for RNA examination. Two independent experienced clinical pathologists participated in the pathological examination according to the WHO classification. We performed a 5 year follow-up and overall survival (OS) was defined as the length of time between surgery and death or the last follow-up (if death did not occur).

2.1 Cell lines and culture

Normal breast cell line MCF-10A and breast cancer cell lines, including MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3 cells, were obtained from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). MCF10A was cultured in F12/DMEM 1:1 medium. MCF-7 was cultured in MEM medium with sodium pyruvate 0.11 mg/ml and bovine insulin 0.01 mg/ml. MDA-MB-231, MDA- MB-468 and MDA-MB-453 cells were cultured in L-15 medium.T-47D and SK-BR-3 were cultured in DMEM medium. All the cells were cultured in medium with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Gibco, Grand Island, NY, USA) at 37 ${}^{\circ}$ C and 5% CO ${}_{2}$ . The lentivirus vectors carrying shNC, shGACAT3, NC, and GACAT3, and miR-497 mimic and inhibitor and their respective negative control RNAs were obtained from GeneBio Company (Shanghai, China). The transfection was conducted according to the manufacture’s protocols.

2.2 Measurement of proliferation

Proliferation was evaluated through assessment of DNA synthesis by measurement of 5-Bromo-2-deoxyUridine (BrdU) incorporation. In brief, cells were seeded in 96-well culture plates at a density of 2 $\times$ 10 ${}^{3}$ cells/well, cultured for 24–72 h, then incubated with a final concentration of 10 mM BrdU (BD Pharmingen, CA, USA) for 2 h. After the incubation, the medium was removed and cells were fixed for 30 min at room temperature and incubated with peroxidase-coupled anti-BrdU-antibody (Sigma-Aldrich, USA) for 60 min at room temperature. Then the cells were washed three times with PBS and incubated with peroxidase substrate (tetramethylbenzidine) for 30 min. Finally, the absorbance at 450 nM was measured using a microplate reader (Thermo Fisher Scientific, IL, USA). Triplicate individual experiments were performed.

2.3 Measurement of apoptosis

Apoptotic cell death was evaluated using the TdT-mediated dUTP nick end labeling (TUNEL) fluorescence FITC kit (Roche, Basel, Switzerland). After the treatment, cells were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100. Then, cells were incubated with TUNEL in an incubator at 37 ${}^{\circ}$ C for 1 h. After washing with PBS twice, FITC fluorescence was analyzed using a cytometer (BD Biosciences, USA). Triplicate individual experiments were performed. Results were expressed as percentage of apoptotic cells as compared to control.

2.4 Luciferase activity assay

The HEK293T cells were seeded at 1.5 $\times$ 10 ${}^{4}$ /well in 96-well plates and co-transfected with 200 ng of GACAT3-WT, GACAT3-MUT, CCND2-WT, or CCND2-MUT (Sangon Biotech, China). HEK293T cells were also co-transfected with 10 ng of pRL-TK (Promega, USA) and miR-497 mimic or miRNA NC using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The luciferase assay was assayed using the dual-luciferase reporter assay system (Promega, USA) 48 h after transfection. Triplicate individual experiments were performed.

2.5 Invasion and migration assays

The wound healing assay was performed to determine the ability of migration. For the Wounding healing assay, cells were plated in a 6-well plate. Wounds were created with a 100- $\mu$ L pipette tip on cells grown to 100% confluence. The detached cells were washed away with PBS. Cell migration was photographed under a light microscope (Olympus, Japan) at 0 h and 24 h after wounding. For the transwell invasion assay, 1 $\times$ 10 ${}^{5}$ cells were plated into the upper chambers in transwell inserts in 24-well plate with transwell (8 mm pore size, Corning, USA) coated with Matrigel free with serum (BD Biosciences, USA). Medium containing 10% FBS was added into the lower chambers as a chemoattractan. Cells were incubated in an incubator at 37 ${}^{\circ}$ C with 5% CO ${}_{2}$ for 48 h and then the medium was removed and the upper surfaces were rinsed with PBS for thrice. Cells remained in the upper chamber were removed with cotton buds, while cells that invaded the lower surfaces of the filters were fixed in 4% polyformaldehyde for 30 min, air-dried, and stained with 0.1% crystal violet for 10 min. Stained cells adhering to the lower surface of the membrane were counted by photographing six fields per membrane. Results were expressed as fold-changes as compared to control.

2.6 Quantitative real-time PCR

Total RNA was extracted from cultured cells or tissues, using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. First-strand cDNA was synthesized 500 ng RNA using a PrimeScript ${}^{\@setsize{\scriptsize}{8pt}{\viipt}{\@viipt}\textregistered}$ RT reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) according to the manufacturer’s protocol. Quantitative RT-PCR was performed using SYBR premix real-time PCR Reagent (Takara, China) on a Biorad CFX96 real-time PCR system (Biorad, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were used as the endogenous control for detection of mRNA and miRNA expression, respectively. Relative quantification was analyzed by the comparative threshold cycle (2 ${}^{-\Delta\Delta\text{Ct}}$ ) method. Triplicate individual experiments were performed.

2.7 Statistical analysis

Data were shown as mean $\pm$ SEM and were analyzed statistically using GraphPad Prism 5.0. One-way Analysis of Variance (ANOVA) and student’s t-tests were conducted for statistical analysis of either multiple or two groups. Pearson Correlation Coefficient and Kaplan-Meier methods were used to analyze correlation and OS, respectively. $P<$ 0.05 was considered to be statistically significant.

Figure 1.

Upregulation of lncRNA GACAT3 expression is correlated with poor prognosis in breast cancer patients. (A) Flowchart of experiments in the present study. (B) GACAT3 expression in adjacent normal tissues and breast cancer tissues were measured by qRT-PCR. (C) GACAT3 expression in normal breast cells and breast cancer cell lines were measured by qRT-PCR. (D) The breast cancer patients were divided into GACAT3 high-expression and low-expression group and the relationship between GACAT3 expression level and prognosis of breast cancer patients was analyzed using Kaplan–Meier analysis. # $p<$ 0.05, compared with control.

Figure 2.

GACAT3 is a target of miR-497. (A) Cell proliferation in MCF-7 cells transfected with LV-GACAT3. (B) Cell proliferation in MCF-7 cells transfected with LV-shGACAT3. (C) The predicted binding sites of miR-497 in GACAT3 (GACAT3-WT) and GACAT3 mutant (GACAT3-MUT) sequences. (D) miR-497 expression in MCF-7 cells transfected with LV-GACAT3. (E) miR-497 expression in MCF-7 cells transfected with LV-shGACAT3. (F) Luciferase activity was determined in cells co-transfected with miR-497 mimic or NC-mimic and pGL3 luciferase reporters containing GACAT3-WT or GACAT3-MUT sequences. # $p<$ 0.05, compared with control.

Figure 3.

Downregulation of miR-497 expression is correlated with poor prognosis in breast cancer patients. (A) miR-497 expression in adjacent normal tissues and breast cancer tissues were measured by qRT-PCR. (B) miR-497 expression in normal breast cells and breast cancer cell lines were measured by qRT-PCR. (C) The breast cancer patients were divided into miR-497 high-expression and low-expression group and the relationship between miR-497 expression level and prognosis of breast cancer patients was analyzed using Kaplan-Meier analysis. (D) Pearson’s correlation analysis of the relationship between GACAT3 and miR-497. # $p<$ 0.05, compared with control.

Figure 4.

Downregulation of GACAT3 decreases proliferation and increases apoptosis by negative regulation of miR-497. MCF-7 cells were co-transfected with LV-shGACAT3 and anti-miR-497. (A) Cell proliferation was detected by BrdU incorporation. (B) Flow cytometry was used to detect the apoptosis by TUNEL staining. Caspase 3 and 9 activities were measured using commercial kits (C and D). (E and F) Bax and Bcl-2 expression ws measured by qRT-PCR. # $p<$ 0.05, compared with shNC. ## $p<$ 0.05, compared with shGACAT3.

Figure 5.

Downregulation of GACAT3 decreases malignancy in glioma by negative regulation of miR-497. MCF-7 cells were co-transfected with LV-shGACAT3 and anti-miR-497. (A) The invasion of transfected cells was determined by Transwell assay. (B) The migration of transfected cells was determined by Wounding healing assay. mRNA expression of E-cadherin (C), N-cadherin (D) and Vimentin (E) was determined. # $p<$ 0.05, compared with shNC. ## $p<$ 0.05, compared with shGACAT3.

Figure 6.

GACAT3 acts as a ceRNA for miR-497 to enhance CCND2 expression. (A) Bioinformatics analysis reveals the putative binding sequences of miR-497 on CCND2 and the mutant sequences. (B) miR-497 mimic reduced the mRNA expression of CCND2. (C) Luciferase activity was determined in cells co-transfected with miR-497 mimic or NC-mimic and pGL3 luciferase reporters containing CCND2-WT or CCND2-MUT sequences. (D) RIP assay of the enrichment of Ago2 on GACAT3 and CCND2 transcripts relative to IgG in stable shGACAT3 cells. (E) The mRNA expression of CCND2 was assayed in stable stable shGACAT3 cells co-transfected with LV-CCND2. Cell proliferation (F) and apoptosis (G) were determined in stable stable shGACAT3 cells co-transfected with LV-CCND2. # $p<$ 0.05, compared with shNC. ## $p<$ 0.05, compared with shGACAT3.

3. Results

3.1 Upregulation of LncRNA GACAT3 is associated with poor prognosis of breast cancer patients

To investigate the role of GACAT3 in the development and prognosis of breast cancer, we firstly examined the expression level of GACAT3 in breast cancer tissues (Fig. 1A). Compared with adjacent normal tissues, GACAT3 expression was significantly increased in breast cancer tissues (Fig. 1B). Moreover, the expression of GACAT3 was significantly higher in breast cancer cell lines, including MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3 cells, as compared to normal breast cell line MCF-10A (Fig. 1C). To detect the clinical relevance of GACAT3 level in breast cancer, we evaluated the association of GACAT3 expression level with OS. We showed that patients with higher GACAT3 expression had shorter OS time than those cases with lower GACAT3 expression (Fig. 1D).

3.2 miR-497 is a target of GACAT3 in the regulation of breast cancer cell proliferation

To investigate the role of GACAT3 in breast cancer cell proliferation, MCF-7 cells were transfected with either LV-shGACAT3 or LV-GACAT3. The results showed that downregulation of GACAT3 markedly inhibited MCF-7 cell proliferation (Fig. 2A), but upregulation of GACAT3 showed a promotive effect on MCF-7 cell proliferation (Fig. 2B). miR-497 has been reported to be a target of GACAT3 in the regulation of gastric cancer (Fig. 2C) [14]. In the current study, we tested the possible interaction between GACAT3 and miR-497 in the context of breast cancer. We showed that upregulation of GACAT3 markedly inhibited miR-497 expression in MCF-7 cells (Fig. 2D), while downregulation of GACAT3 markedly increased miR-497 expression (Fig. 2E). Moreover, transfection of miR-497 mimics significantly decreased GACAT3 WT luciferase activity but not the GACAT3 MUT luciferase. The results indicated that miR-497 is a target of GACAT3 in the regulation of breast cancer cell proliferation.

3.3 Downregulation of miR-497 is associated with poor prognosis of breast cancer patients

To investigate the role of miR-497 in the development and prognosis of breast cancer, we examined the expression level of miR-497 in breast cancer tissues. Compared with adjacent normal tissues, miR-497 expression was significantly decreased in breast cancer tissues (Fig. 3A). Moreover, the expression of miR-497 was significantly lower in breast cancer cell lines, including MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3 cells, as compared to normal breast cell line MCF-10A (Fig. 3B). To detect the clinical relevance of miR-497 level in breast cancer, we evaluated the association of miR-497 expression level with OS. We showed that patients with lower miR-497 expression had shorter OS time than those cases with higher miR-497 expression (Fig. 3C). Moreover, the expression level of miR-497 was negatively correlated with GACAT3 in tumor tissues (Fig. 3D). Furthermore, we tested the role of miR-497 in MCF-7 cell proliferation. As shown in Fig. 3E and F, miR-497 mimic significantly inhibited the proliferation of MCF-7 cells, while miR-497 inhibitor remarkably increased MCF-7 cell proliferation.

3.4 Upregulation of miR-497 is involved in GACAT3 knockdown-induced inhibition of cell proliferation

To test whether regulation of miR-497 was involved in GACAT3-exhibited regulation of breast cancer cell proliferation, we co-transfected MCF-7 cells with LV-shGACAT3 and miR-497 inhibitor. As shown in Fig. 4A, LV-shGACAT3-induced decrease of cell proliferation was significantly inhibited by miR-497 inhibitor. Downregulation of GACAT3 also promoted apoptosis in MCF-7 cells, which effect was inhibited by miR-497 inhibitor (Fig. 4B). Moreover, Downregulation of GACAT3 increased the activities of caspase 3 and 9 and miR-497 inhibitor inhibited this effect (Fig. 4C and D). The mRNA expression of Bax was increased by LV-shGACAT3, while Bcl-2 expression was decreased by LV-shGACAT3 (Fig. 4E and F). The effect of LV-shGACAT3 on Bax and Bcl-2 expression was inhibited by miR-497 inhibitor (Fig. 4E and F).

3.5 Upregulation of miR-497 is involved in GACAT3 knockdown-induced inhibition of epithelial-mesenchymal transition

In the next step, we explored the effect of GACAT3 and miR-497 on epithelial-mesenchymal transition (EMT) in breast cancer cells. In Fig. 5A, we showed that GACAT3 knockdown significantly decreased the invasion ability, which effect was prohibited by miR-497 inhibitor. The results of Wounding healing assay showed that GACAT3 knockdown remarkably decreased the ability of migration in MCF-7 cells. The inhibitory effect of GACAT3 knockdown on MCF-7 cell migration was suppressed by miR-497 inhibitor (Fig. 5B). We further determined the effect of GACAT3 and miR-497 on the expression of EMT biomarkers. As shown in Fig 5C, expression of E-Cadherin was increased by GACAT3 knockdown, which effect was suppressed by miR-497 inhibitor. GACAT3 knockdown also decreased the expression of N-Cadherin and Vimentin and 497 inhibitor suppressed this effect (Fig. 5D and E). These results indicated that upregulation of miR-497 was involved in GACAT3 knockdown-induced inhibition of EMT.

3.6 GACAT3 is predicted to be a ceRNA of Cyclin D2 by directly binding miR-497

Using bioinformatics analysis, we found a predicted binding site of miR-497 in Cyclin D2 (CCND2) (Fig. 6A). Transfection of miR-497 mimic significantly reduced the expression of CCND2 in MCF-7 cells (Fig. 6B). Transfection of miR-497 mimic markedly decreased the luciferase of CCND2-WT (Fig. 6C), whereas miR-497 mimic did not affect the luciferase activity of CCND2 when the binding site was mutational (Fig. 6C). To investigate whether GACAT3 can act as a ceRNA for CCND2 by sponging miR-497 in breast cancer, we performed an RIP assay on Ago2. Downregulation of GACAT3 decreased the enrichment of Ago2 on GACAT3 but remarkably increased the enrichment on CCND2 (Fig. 6D). Downregulation of GACAT3 significantly decreased the expression of CCND2, which effect was inhibited by miR-497 inhibitor (Fig. 6E). Moreover, the effect of GACAT3 knockdown on cell proliferation and apoptosis was inhibited by upregulation of CCND2 expression (Fig. 6F and G). Overexpression of CCND2 significantly increased cell proliferation but did not significantly affect apoptosis in MCF-7 cells (Fig. 6F and G). In general, these data indicated that GACAT3 functioned as a ceRNA to regulate CCND2 expression by sponging miR-497.

4. Discussion

It has been demonstrated that the expression of lncRNA GACAT3 is increased in gastric cancer and associated with tumor size, tumor-node metastasis stages, and distal metastasis [13]. Moreover, the interaction between GACAT3 and miR-497 is involved in the regulation of gastric cancer progression [14]. However, the role of GACAT3 and miR-497 is not known in breast cancer. Competing endogenous RNAs (ceRNAs) are RNA transcripts which can communicate with each other at post-transcription level by competing for shared miRNAs [15, 16]. ceRNA networks link the function of protein-coding mRNAs with that of non-coding RNAs such as microRNA, lncRNA, pseudogenic RNA and circular RNA [15, 16]. Large amount of evidence has shown that lncRNA can act as a ceRNA for target mRNA that may represents a widespread form of post-transcriptional regulation of gene expression in both physiology and pathology [15, 16]. Whether GACAT3 acts as a ceRNA is not clear.

In our study, we found that GACAT3 expression was increased in breast cancer tissues and cells. High expression of GACAT3 in breast cancer tissues predicted poor prognosis. miR-497 expression was decreased in breast cancer tissues and cells. Low expression of miR-497 in breast cancer tissues predicted poor prognosis. miR-497 expression was negatively associated with GACAT3 expression. Furthermore, we investigated the role of GACAT3 and miR-497 in breast cancer malignancy. Metastasis remains the major cause of death in breast cancer patients. At the molecular level, cancer metastasis is thought to be initiated by the process of EMT [17, 18]. E-Cadherin, N-Cadherin, and Vimentin were critical regulators of EMT [19]. Downregulation of GACAT3 inhibited cell proliferation, increased apoptosis, decreased invasion and migration ability, and decreased the ability of EMT. The effect of GACAT3 knockdown on cell proliferation, apoptosis, metastasis, and EMT was suppressed by miR-497 inhibitor. These results indicated that GACAT3 played an oncogenic role in breast cancer through inhibition of miR-497.

Numerous studies have reported the role of miR-497 in the development of several types of cancers. Generally speaking, miR-497 plays a tumor suppressive role. miR-497 overexpression decreases proliferation, migration and invasion of human retinoblastoma cells via targeting vascular endothelial growth factor A [20]. In renal cell carcinoma, miR-497 plays an inhibitory role by targeting VEGFR-2 [21]. In bladder cancer cells, miR-497 upregulation inhibits cell invasion and metastasis [22]. miR-497 inhibits tumor growth through targeting insulin receptor substrate 1 in colorectal cancer [23]. miR-497 inhibits thyroid cancer tumor growth and invasion by suppressing BDNF [24]. In breast cancer, it has been found that miR-497 acted as a tumor suppressor [25, 26, 27]. In the present study, we confirmed the inhibitory role of miR-497 in breast cancer.

Moreover, we investigated the mechanism of GAC AT3/miR-497 interaction-induced regulation of breast cancer. We showed that CCND2 was a target of miR-497. Activation of CCND2 has been shown to promote the development and progression of thyroid cancer [28]. In our study, we confirmed that upregulation of CCND2 inhibited the tumor suppressive role of miR-497 and increased cell proliferation in breast cancer cells. GACAT3 functioned as a ceRNA to regulate CCND2 expression by sponging miR-497. More and more studies have suggest that lncRNA and mRNA integration network reconstruction plays important roles in the development and malignancy of cancers [29]. miRNAs also plays a vital role in the regulation of cancer development and functions as crucial biomarkers for the diagnosis or prognosis of cancers [30]. Based on these findings, we propose that GACAT3 and miR-497 may be useful biomarkers for prediction of progression or prognosis of breast cancer. Large scale clinical investigations should be conducted to test the role of GACAT3 and miR-497 in the prediction of the progression and prognosis of breast cancer.

In conclusion, the findings demonstrate that GAC AT3 plays an oncogenic role in breast cancer. GACAT3 acts as a ceRNA for miR-497 to boost breast cancer progression by upregulation of CCND2 expression. Our findings identify the role of GACAT3/miR-497/CCND2 in the development of breast cancer. These results suggest that GACAT3/miR-497/CCND2 is a potential therapeutic target and biomarker for breast cancer.

Footnotes

Conflict of interest

The authors declare none conflict of interest.

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