Original Article Epigenetic Silencing of Mir137 in Gastric Cancer

Int J Clin Exp Med 2016;9(9):17926-17932 www.ijcem.com /ISSN:1940-5901/IJCEM0029440

Original Article Epigenetic silencing of miR137 in gastric cancer

Wei Jin, Tingting Jiang, Jie Sun, Wenxia Xu, Lifeng Feng, Hongchuan Jin, Xian Wang

Department of Medical Oncology, Key Laboratory of Biotherapy in Zhejiang, Sir Runrun Shaw Hospital, Medical School of Zhejiang University, Hangzhou, China Received March 3, 2016; Accepted April 7, 2016; Epub September 15, 2016; Published September 30, 2016

Abstract: The small non-coding RNAs termed microRNAs can induce post-transcriptional silencing of genes critical to the pathogenesis of multiple cancers. Such microRNAs are deregulated and play important roles in carcinogenesis. In this study, we explored the regulation and function of miR137 in gastric cancer. We found that miR137 was downregulated in a variety of gastric cancer cell lines and primary gastric carcinoma tissues. Its downregulation was resulted from DNA methylation of its promoter. Pharmaceutical demethylation restored its expression and its promoter was highly methylated in gastric cancer cells. FMNL2 (formin like 2) was found as a new target regulated by miR137. Both miR137 mimics and FMNL2 siRNA inhibited the growth of gastric cancer cells in vitro. Therefore, we concluded that promoter methylation-mediated downregulation of miR137 can stimulate FMNL2 expression to promote gastric carcinogenesis.

Keywords: Gastric cancer, miR137, methylation

Introduction like Ras and HMGA2 [5-8]. In contrast, the miR- 17-92 cluster, which is located on chromosome Carcinogenesis is believed to be a stepwise 13q31, are upregulated and functions as onco- process with the accumulation of multiple genes in multiple cancers including colorectal genetic and epigenetic changes that often cancer [9, 10]. Therefore, the importance of results in the aberrant activation of oncogenes miRNAs acting as a new layer of gene regulation and/or inactivation of tumor suppressor genes in tumorigenesis is emerging. (TSGs). TSGs need to be inactivated to lose their tumor inhibitory functions during carcino- These cancer-related microRNAs are often genesis, whereas oncogenes, once activated, deregulated in many cancers including gastro- can promote the transformation of normal cells intestinal cancers [11-14]. Similar to protein- into tumor cells. Both TSGs and oncogenes are coding cancer-related genes, these deregulat- usually protein coding genes. Recently an ed microRNAs in cancer development are unusual class of small RNAs have been found subjected to genetic and epigenetic regula- to play an important role in cancer develop- tions. In this study, we explored the regulation ment, although they are not protein-coding and function of miR137 in gastric cancer. It was downregulated in a variety of gastric cancer cell RNAs [1]. These small RNAs are termed microR- lines and primary gastric carcinoma tissues NAs since their mature form are at a length of due to DNA methylation of its promoter. only 19-25 nucleotides. MicroRNAs can induce Moreover, it inhibited the growth of gastric can- post-transcriptional gene silencing through cer cells by target the expression of FMNL2 (for- base-pairing with partially complementary sites min like 2). in the 3’-nontranslated region (3’-UTR) of their target mRNAs. During cancer development, Materials and methods microRNAs can function as either TSGs or oncogenes, depending on the target gene they Cell culture and patients samples regulated [2-4]. For example, Let-7 is down-regulated in many cancers and functions as a TSG All cancer cell lines were grown at 37°C in 10% by suppressing the expression of oncogenes CO2 atmosphere and maintained routinely in miR137 in gastric cancer

DMEM or RPMI1640 medium supplemented bolically active cells to convert 3-(4,5-dimethy- with 10% fetal bovine serum. All primary gastric lthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4- carcinoma tissues as well as their adjacent sulfophenyl)-2H-tetrazolium (MTS) into forma- ® non-tumor tissues were collected from patients zan using CellTiter 96 AQueous Assay kit (Pro- who underwent surgical resection of tumors. All mega, Madison, WI, USA). Briefly, cells are patients provided written informed consent for seeded in 12-well plates and transfected with the use of their tissues. All tissues had been siRNA or microRNA mimic for 48 h. Cells were pathologically confirmed. Tissue samples were then harvested and reseeded into 96-well immediately snap-frozen in liquid nitrogen, and plates. After another 24 h, the quantity of stored at -80°C until further analysis. formazan was measured as 490 nm absor- bance after 1 hour incubation with CellTiter 96 Total RNA extraction AQueous One Solution Reagent following Cell pellets or tissues were homogenized in instructions provided. Trizol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA containing small RNA was extracted Bisulfite treatment of DNA and methylation using miRNeasy Mini Kit (Qiagen, Hilden, analysis Germany) according to the manufacturer’s protocol. DNase treatment was carried out to Methylation status of miR137 was determined remove any contaminating DNA. The concen- by BGS (bisulfite genome sequencing) using tration of all RNA samples were quantified by bisulfite modified genomic DNA as the tem- NanoDrop 1000 (Nanodrop, Wilmington, DE, plate. Genomic DNA was bisulphite-treated USA). with Zymo DNA Modification Kit (Zymo Rese- arch, Orange, CA, USA) according to the proto- Real-time PCR for microRNA quantification col provided by the manufacturer. MSP was carried out for 40 cycles with annealing tempera- For microRNA quantitative PCR (qPCR), reverse ture at 62°C, as previously described [15]. transcription was performed using the miScript microRNA quantification system (Qiagen). In For BGS, PCR products amplified with BGS brief, total RNA was polyadenylated and reverse primers were purified with Illustra GFXTM PCR transcribed to cDNA using miScript Reverse and gel band purification kit (GE Healthcare life Transcription kit (Qiagen). Real-time PCR was science, Uppsala, Sweden) and cloned into performed using miScript SYBR Green PCR kit pCR4-TOPO vector for sequencing (Invitrogen). (Qiagen) in ABI PRISM 7900 Real-time PCR sys- At least 6 colonies were randomly chosen for tem (Applied Biosystems). The expression lev- els of microRNAs were normalized to U6. The plasmid extraction and sequencing analysis amplification was performed at 95°C for 15 using ABI PRISM BigDye Terminator Cycle min, followed by 40 cycles of 94°C for 15 s, Sequencing Kit in ABI 3100 sequencer (Applied 55°C for 30 s and 70°C for 30 s. Biosystems).

SiRNA and microRNA mimic transfection Western blot analysis

FMNL2 depletion and microRNA re-expression Cells were lyzed in Lammeli’s lysis buffer, were achieved by transfection with siRNAs and resolved in SDS-PAGE minigel and transferred microRNA mimic (Qiagen, Hilden, Germany), res- onto Nitrocellulose membrane (Millipore, Bill- pectively. Cells were seeded in 12-well plates erica, MA). Membranes were probed overnight (1x105/well) and transfected with siRNA duplx- at 4°C with primary antibodies against FMNL2 es or miRNA mimic (10 nM) using Lipofec- (Abnova, Taipei, Taiwan) or actin (Santa Cruz TM tamine 2000 reagent (Invitrogen) according Biotechnology, Santa Cruz, CA, USA) at 1:1000 to the manufacture’s instructions. Cells were dilution, washed extensively with 0.1% Tween- harvested for RNA and protein extraction after 20 in PBS and incubated with secondary anti- 72 h. bodies conjugated with horse-radish peroxi- Cell growth assay (MTS assay) dase at 1:10000 dilution. The signals were visualized with enhanced chemiluminescence Cell growth was measured by a non-radioactive (Amersham Life Science Inc., Buckinghamshire, proliferation assay based on the ability of meta- UK).

17927 Int J Clin Exp Med 2016;9(9):17926-17932 miR137 in gastric cancer

Figure 1. MiR137 was downregulated in gastric cancer. A. MiR137 expressions in gastric cancer cell lines and non-tumor stomach epithelial cell line (GES-1). B. MiR137 expressions in primary gastric carcinoma tissues as well as adjacent non-tumor tissues were quantified by real-time RT-PCR using U6 for the normalization. The Wilcoxon matched pairs test was used to calculate the p value (n=58, p<0.05).

Figure 2. Promoter hypermethylation mediates the downregulation of miR137. A. Methylation of miR137 promoter in gastric cancer cells were determined by BGS. B. MiR137 expression in gastric cancer cells before and after Aza treatment were determined by real time RT-PCR.

Construction of microRNA reporter vector FMNL2 interaction. The sequences of the oligo- nucleotides used were. The 3’-UTR of FMNL2 mRNA containing intact miR137 binding sequences was inserted imme- Luciferase activity assay diately downstream of a CMV promoter-driven firefly luciferase cassette in a pMIR-reporter 1x105 of cells were co-transfected with pMIR- vector according to the protocol provided Luciferase-FMNL2-3’UTR and pRL-CMV-Renilla (Ambion, Austin, TX, USA). Restriction enzymes (Promega) constructs in the presence of 30 Spe I and Hind III were used. To make control pmol of miR-137 mimic or control oligonucle- vectors, the mutant construct contains point otides. At 48 h post-transfection, activities of mutations of the seed sequence of miR-137- firefly luciferase and renilla luciferase were

17928 Int J Clin Exp Med 2016;9(9):17926-17932 miR137 in gastric cancer

Figure 3. MiR137 mimic inhibited the growth of gastric cancer cells. A. Growth of gastric cancer cells after miR137 mimic transfection were determined by MTS assay. Representative experiments in triplicate were shown as mean ± SD. The asterisks indicate the statistical difference (Student’s t-test, p<0.05). B. Apoptosis of gastric cancer cells after miR137 mimic transfection were determined by flowcytometry after FITC-Annexin V and PI double staining. Representative experiments in triplicate were shown as mean ± SD. The asterisks indicate the statistical difference (Student’s t-test, p<0.05).

Figure 4. FMNL2 is a direct target of miR137. A. Effect of miR137 mimic on FMNL2 expression was determined by western blotting. Actin was used as the control. B. Expression of luciferase in the presence of miR137 or control oligos was determined by luciferase reporter assay. Representative experiments in triplicate were shown as mean ± SD. The asterisk indicates significant difference (p<0.05, Student’s t test). C. The effect of FMNL2 depletion on cell growth was evaluated by MTS assay. Representative experiments in triplicate were shown as mean ± SD. The asterisks indicate the statistical difference (Student’s t-test, p<0.05). measured by using the Dual-GloTM luciferase cinoma tissues were determined by real-time assay system (Promega) as described by the RT-PCR. MiR137 was significantly downregulat- manufacturer. Relative luciferase activity was ed in a variety of gastric cancer cell lines (Figure normalized with renilla luciferase activity and 1A). In addition, its expression was also signifi- then compared with the pMIR-reporter vector cantly reduced in gastric carcinoma tissues control. (n=58, p<0.05, Wilcoxon matched pairs test) (Figure 1B). Results Promoter methylation medicates miR137 MiR137 is downregulated in gastric cancer downregulation

The expression of miR137 in in vitro cultured We then set to understand how miR137 was gastric cancer cell lines and primary gastric car- downregulated in gastric cancer. It has been

17929 Int J Clin Exp Med 2016;9(9):17926-17932 miR137 in gastric cancer reported that miR137 downregulation in hu- miR137 was also reported in human gastric man cancers was attributed to promoter hyper- carcinoma tissues [17]. In this study, we found methylation [16-19]. Therefore, we asked whe- promoter methylation of miR137 was indeed ther promoter hypermethylation was responsi- responsible for its epigenetic silencing in gas- ble for miR137 downregulation in gastric can- tric cancer cells. Furthermore, we have previ- cer cells. Indeed, miR137 promoter was highly ously reported that miR137 seemed to be one methylated in gastric cancer cell lines such as of microRNAs suppressed by active wnt signal- AGS, MKN28 and NCI-N87 but not non-tumor ing in colorectal cancer [23]. Therefore, it would stomach epithelial cell line GES-1 (Figure 2A). be interesting to know whether and how the In consistent with this, miR137 expressions in active wnt signaling contributes to the promot- gastric cancer cell lines were significantly er methylation of miR137. restored after pharmacological demethylation (Figure 2B). MicroRNAs function to regulate the expression of target genes mainly in the post-transcription- FMNL2 is the direct target of miR-137 al manner. More than one target genes could be targeted by one single microRNA, as long as We then investigated whether downregulation they contain the microRNA-binding sites in the of miR-137 was relevant to the development of 3’-UTR. MiR137 has been reported to affect gastric cancer. After restoration of miR137 the expression of several target genes such as expression by transfection of miR137 mimic DCLK1, BMP7, cdc42 and MCL-1 [24-27]. In into gastric cancer cell lines, cell growth was this study, we identified FMNL2 a new target of significantly inhibited accompanied with the miR137. Transfection of miR137 mimic reduced induction of cell apoptosis (Figure 3A and 3B). protein expression of FMNL2 (Figure 3). Moreover, miR137 significantly inhibited the FMNL2 is the direct target of miR137 activity of luciferase driven by wild-type but not Using algorithms such as PicTar (http://pictar. miR137-interaction deficient 3’-UTR Figure( 3). bio.nyu.edu/), TargetScan (http://www.targets- Similar to miR137 transfection, knock-down of can.org/) and miRanda (http://www.microrna. FMNL2 results in the growth inhibition of gas- org/), FMNL2 (formin-like 2) was identified as tric cancer cells (Figure 3). one possible target of miR137. FMNL2 expres- As named, FMNL2 belongs to formin superfam- sion was indeed reduced after transfection of ily, encoding a formin-related protein that are a miR137 mimic (Figure 4A). Moreover, miR137 group of proteins involved in the polymerization can significantly suppress the expression of of actin and associate with the fast-growing luciferase controlled by wild-type FMNL2 3’- endof actin filaments. Bioinformatic analysis UTR but not mutated FMNL2 3’-UTR (Figure revealed it was evolutionally conserved with 4B). Importantly, similar to miR137 mimic orthologs in fish, Drosophila, and nematode. trasnfection, siRNA mediated-FMNL2 knock- The gene contains at least 27 exons and spans down led to significant growth inhibition of gas- around 314 kb in the genome. In human, its tric cancer cells (Figure 4C). expression was high in many fetal and adult Discussion organs such as brain, liver, spleen, kindey and ovary [28]. Formin-related proteins have been Like many other microRNAs with tumor sup- implicated in morphogenesis, cytokinesis, and pressor function, miR137 was found to be cell polarity. However, their relevance to human downregulated in various type of human can- cancer pathogenesis remain controversial. For cers such as colorectal cancer, glioblastoma, example, FMNL2 was found to promote metas- and oral squamous cell carcinoma [20-22]. tasis in colorectal cancer while down-regulation Such microRNAs with tumor suppressor func- of formin-like 2 predicted poor prognosis in tion could be downregulated in human cancer hepatocellular carcinoma [29, 30]. In colorectal either genetically or epigenetically. Indeed, epi- cancer, miR137 was down-regulated to pro- genetic silencing of miR137 have been report- mote cell invasion and metastasis by directly ed in human cancers including lung cancer and targeting FMNL2 [31]. Nevertheless, FMNL2 squamous cell carcinoma of the head and neck may have formin-independent functions in the [18, 19]. Interestingly, promoter methylation of development of cancers. The biological rele-

17930 Int J Clin Exp Med 2016;9(9):17926-17932 miR137 in gastric cancer vance and underlying mechanisms warrants [8] Roush S and Slack FJ. The let-7 family of mi- further investigations. croRNAs. Trends Cell Biol 2008; 18: 505-516. [9] He L, Thomson JM, Hemann MT, Hernando- In summary, we found that miR137 was down- Monge E, Mu D, Goodson S, Powers S, Cordon- regulated through promoter methylation in Cardo C, Lowe SW, Hannon GJ and Hammond human gastric cancer. It can inhibit the in vitro SM. A microRNA polycistron as a potential human oncogene. Nature 2005; 435: 828-833. growth of gastric cancer cells probably through [10] Diosdado B, van de Wiel MA, Terhaar Sive direct targeting of FMNL2. Therefore, FMNL2 Droste JS, Mongera S, Postma C, Meijerink WJ, could be a novel target for the diagnosis and Carvalho B and Meijer GA. MiR-17-92 cluster is therapy of human gastric cancer. associated with 13q gain and c-myc expression during colorectal adenoma to adenocarci- Acknowledgements noma progression. Br J Cancer 2009; 101: 707-714. This work was supported by High Level Talents [11] Volinia S, Calin GA, Liu CG, Ambs S, Cimmino A, Program from the Department of Health and Petrocca F, Visone R, Iorio M, Roldo C, Ferracin 151 talents program in Zhejiang. M, Prueitt RL, Yanaihara N, Lanza G, Scarpa A, Vecchione A, Negrini M, Harris CC and Croce Disclosure of conflict of interest CM. A microRNA expression signature of human solid tumors defines cancer gene targets. None. Proc Natl Acad Sci U S A 2006; 103: 2257- 2261. [12] Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Address correspondence to: Drs. Wei Jin and Xian Poon TC, Ng SS and Sung JJ. Differential ex- Wang, Department of Medical Oncology, Key Labo- pression of microRNAs in plasma of colorectal ratory of Biotherapy in Zhejiang, Sir Runrun Shaw cancer patients: A potential marker for colorec- Hospital, Medical School of Zhejiang University, tal cancer screening. Gut 2009; 58: 1375-81. Hangzhou, China. E-mail: [email protected] (WJ); [13] Ng EK, Tsang WP, Ng SS, Jin HC, Yu J, Li JJ, [email protected] (XW) Rocken C, Ebert MP, Kwok TT and Sung JJ. Mi- croRNA-143 targets DNA methyltransferases References 3A in colorectal cancer. Br J Cancer 2009; 101: 699-706. [1] Ambros V. The functions of animal microRNAs. [14] Shin VY, Jin H, Ng EK, Cheng AS, Chong WW, Nature 2004; 431: 350-355. Wong CY, Leung WK, Sung JJ and Chu KM. NF- [2] Esquela-Kerscher A and Slack FJ. Oncomirs - kappaB targets miR-16 and miR-21 in gastric microRNAs with a role in cancer. Nat Rev Can- cancer: involvement of prostaglandin E recep- cer 2006; 6: 259-269. tors. Carcinogenesis 2011; 32: 240-245. [3] Lam EK, Wang X, Shin VY, Zhang S, Morrison H, [15] Wang LJ, Jin HC, Wang X, Lam EK, Zhang JB, Sun J, Ng EK, Yu J and Jin H. A microRNA con- Liu X, Chan FK, Si JM and Sung JJ. ZIC1 is tribution to aberrant Ras activation in gastric downregulated through promoter hypermethyl- cancer. Am J Transl Res 2011; 3: 209-218. ation in gastric cancer. Biochem Biophys Res [4] Feng L, Ma Y, Sun J, Shen Q, Liu L, Lu H, Wang Commun 2009; 379: 959-963. F, Yue Y, Li J, Zhang S, Lin X, Chu J, Han W, [16] Kozaki K, Imoto I, Mogi S, Omura K and Inaza- Wang X and Jin H. YY1-MIR372-SQSTM1 regu- wa J. Exploration of tumor-suppressive microR- latory axis in autophagy. Autophagy 2014; 10: NAs silenced by DNA hypermethylation in oral 1442-1453. cancer. Cancer Res 2008; 68: 2094-2105. [5] Johnson SM, Grosshans H, Shingara J, Byrom [17] Steponaitiene R, Kupcinskas J, Langner C, M, Jarvis R, Cheng A, Labourier E, Reinert KL, Balaguer F, Venclauskas L, Pauzas H, Tamelis Brown D and Slack FJ. RAS is regulated by the A, Skieceviciene J, Kupcinskas L, Malferthein- let-7 microRNA family. Cell 2005; 120: 635- er P and Link A. Epigenetic silencing of miR- 647. 137 is a frequent event in gastric carcinogen- [6] Lee YS and Dutta A. The tumor suppressor mi- esis. Mol Carcinog 2016; 55: 376-386. croRNA let-7 represses the HMGA2 oncogene. [18] Langevin SM, Stone RA, Bunker CH, Lyons-Wei- Genes Dev 2007; 21: 1025-1030. ler MA, LaFramboise WA, Kelly L, Seethala RR, [7] Kumar MS, Erkeland SJ, Pester RE, Chen CY, Grandis JR, Sobol RW and Taioli E. MicroR- Ebert MS, Sharp PA and Jacks T. Suppression NA-137 promoter methylation is associated of non-small cell lung tumor development by with poorer overall survival in patients with the let-7 microRNA family. Proc Natl Acad Sci U squamous cell carcinoma of the head and S A 2008; 105: 3903-3908. neck. Cancer 2011; 117: 1454-1462.

17931 Int J Clin Exp Med 2016;9(9):17926-17932 miR137 in gastric cancer

[19] Kang N, Choi SY, Kim YK, Yoo Ie R, Han DH, Lee [26] Deng J, Lei W, Xiang X, Zhang L, Lei J, Gong Y, DS, Kim YS, Hong SH, Kang JH, Lee KY, Park Song M, Wang Y, Fang Z, Yu F, Feng M, Sun Z, JK, Sung SW, Park MS, Yim HW, Kim SJ and Chen J, Zhan Z and Xiong J. Cullin 4A (CUL4A), Park JY. Silencing of miR-137 by aberrant pro- a direct target of miR-9 and miR-137, promoter hypermethylation in surgically resected motes gastric cancer proliferation and invasion lung cancer. Lung Cancer 2015; 89: 99-103. by regulating the Hippo signaling pathway. On- [20] Silber J, Lim DA, Petritsch C, Persson AI, Mau- cotarget 2016; 7: 10037-50. nakea AK, Yu M, Vandenberg SR, Ginzinger [27] Sakaguchi M, Hisamori S, Oshima N, Sato F, DG, James CD, Costello JF, Bergers G, Weiss Shimono Y and Sakai Y. miR-137 Regulates WA, Alvarez-Buylla A and Hodgson JG. miR-124 the Tumorigenicity of Colon Cancer Stem Cells and miR-137 inhibit proliferation of glioblasto- Through the Inhibition of DCLK1. Mol Cancer ma multiforme cells and induce differentiation Res 2016; 14: 354-62. of brain tumor stem cells. BMC Med 2008; 6: [28] Katoh M and Katoh M. Identification and char- 14. acterization of human FMNL1, FMNL2 and [21] Dang J, Bian YQ, Sun JY, Chen F, Dong GY, Liu FMNL3 genes in silico. Int J Oncol 2003; 22: Q, Wang XW, Kjems J, Gao S and Wang QT. Mi- 1161-1168. croRNA-137 promoter methylation in oral li- [29] Zhu XL, Zeng YF, Guan J, Li YF, Deng YJ, Bian chen planus and oral squamous cell carcino- XW, Ding YQ and Liang L. FMNL2 is a positive ma. J Oral Pathol Med 2013; 42: 315-321. regulator of cell motility and metastasis in [22] Balaguer F, Link A, Lozano JJ, Cuatrecasas M, colorectal carcinoma. J Pathol 2011; 224: Nagasaka T, Boland CR and Goel A. Epigenetic 377-388. silencing of miR-137 is an early event in [30] Liang L, Guan J, Zeng Y, Wang J, Li X, Zhang X colorectal carcinogenesis. Cancer Res 2010; and Ding Y. Down-regulation of formin-like 2 70: 6609-6618. predicts poor prognosis in hepatocellular carci- [23] Wang X, Lam EK, Zhang J, Jin H and Sung JJ. noma. Hum Pathol 2011; 42: 1603-1612. MicroRNA-122a functions as a novel tumor [31] Liang L, Li X, Zhang X, Lv Z, He G, Zhao W, Ren suppressor downstream of adenomatous pol- X, Li Y, Bian X, Liao W, Liu W, Yang G and Ding yposis coli in gastrointestinal cancers. Bio- Y. MicroRNA-137, an HMGA1 target, suppress- chem Biophys Res Commun 2009; 387: 376- es colorectal cancer cell invasion and metasta- 380. sis in mice by directly targeting FMNL2. Gastro- [24] Yang Y, Li F, Saha MN, Abdi J, Qiu L and Chang enterology 2013; 144: 624-635 e624. H. miR-137 and miR-197 Induce Apoptosis and Suppress Tumorigenicity by Targeting MCL-1 in Multiple Myeloma. Clin Cancer Res 2015; 21: 2399-2411. [25] Yang YR, Li YX, Gao XY, Zhao SS, Zang SZ and Zhang ZQ. MicroRNA-137 inhibits cell migra- tion and invasion by targeting bone morphoge- netic protein-7 (BMP7) in non-small cell lung cancer cells. Int J Clin Exp Pathol 2015; 8: 10847-10853.

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