Upregulation of CCAT2 promotes cell proliferation by repressing the P15 in breast cancer

Biomedicine & Pharmacotherapy 91 (2017) 1160–1166

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Upregulation of CCAT2 promotes cell proliferation by repressing the P15 in breast cancer Xin Deng, Yi Zhao* , Xin Wu, Guoqing Song Department of Pancreatic and Breast Surgery, Shengjing Hospital of China Medical University, Shenyang, Liaoning, PR China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 15 March 2017 Received in revised form 5 May 2017 Accepted 5 May 2017

Background: Long non-coding RNAs (lncRNAs) are demonstrated to function as modulators of both transcriptional and post-transcriptional regulation in various types of tumors progression. The objective of the study is to investigate the clinical significance and underlying mechanism of Colon cancer associated transcript 2 (CCAT2) involved in breast cancer. Methods: QT-PCR was performed to examine the relative expression levels of CCAT2 in breast cancer tissues and adjacent normal tissues. Kaplan-Meier survival curves and log rank test were applied to assess the correlation between CCAT2 expression and the overall survival (OS) time in patients. MTT cell proliferation assay, transwell invasion assay and cell cycle analysis were conducted to detect the cell proliferation and invasion. Western blot analysis, RNA immunoprecipitation (RIP) and Chromatin immunoprecipitation (ChIP) assays were performed to detect the association between CCAT2 and P15. The tumor xenograft in nude mice was performed to evaluate the effect of CCAT2 expression on tumor growth in vivo. Results: Our results confirmed that CCAT2 expression levels in tumor tissues were markedly increased than that in adjacent normal tissues. Higher CCAT2 expression was found to show a significantly correlation with advanced TNM stage and lymph node metastasis in patients. Kaplan-Meier survival curves and log-rank test showed that higher CCAT2 expression was closely correlated with shorter over survival (OS) time in patients. In vitro, knockdown of CCAT2 showed that cell proliferation and invasion capabilities were suppressed and increased G0-G1 phase cell proportion but reduced S phase cell proportion in MCF-7 and MDA-MB-231 cells. Moreover, when CCAT2 silencing, the cell cycle relative protein CyclinD1, CyclinE1 and CDK4 expression were downregulated, but p15 was up-regulated in MCF7 and MDA-MB-231 cells. Besides, we confirmed that CCAT2 suppressed the p15 expression level via interacting with EZH2 in breast cancer cells. In vivo, the tumor growth was inhibited after knockdown of CCAT2. Conclusion: Our results indicated that CCAT2 may be a potential prognostic marker and therapeutic target for breast cancer. © 2017 Elsevier Masson SAS. All rights reserved.

Keyword: Colon cancer associated transcript 2 Breast cancer EZH2 p15

1. Introduction Breast cancer is the most common cancer afflicting female worldwide [1]. Although larger progress has been advanced for novel therapeutics of breast cancer patients and decreased the incidences of mortality, metastatic breast cancer still is a deadly disease. Tumor metastasis occurs at about 10% of breast cancers patients at diagnosis and up to 70% of breast cancer involving in lymph nodes will recur [2,3]. Therefore, to explore the molecular

* Corresponding author at: Department of Pancreatic and Breast Surgery, Shengjing Hospital of China Medical University, No.36, San Hao street, Shenyang, Liaoning, 110004, PR China. E-mail address: [email protected] (Y. Zhao). http://dx.doi.org/10.1016/j.biopha.2017.05.030 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.

basis of breast cancer development and investigate how to inhibit the malignant behavior may be important for breast cancer treatment. Long nocoding RNA (LncRNAs) is identified to be non-protein coding transcripts longer than 200 nucleotides. Studies have revealed that lncRNAs could regulate most of biological processes including cell proliferation, cell differentiation, cell migration and cell apoptosis [4,5]. LncRNAs play crucial roles in regulation of many cancer types including breast cancer, serving as oncogenes or tumor suppressors [6]. The long non-coding RNA SPRY4-IT1 was reported to promote cell proliferation in human breast cancer by upregulating ZNF703 expression [7]. LncRNA-ATB could dramatically enhance trastuzumab resistance and invasion-metastasis cascade by competitively biding miR-200c, up-regulating the ZEB1

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and ZNF-217 expression, and then promoting cell EMT process in breast cancer [8]. H19-derived miR-675 enhanced tumorigenesis and metastasis in breast cancer by downregulating c-Cbl and Cbl-b expression levels [9]. Overexpression of HOTAIR promotes cell proliferation, whereas HOTAIR silencing significantly inhibits cell survival and abolished tamoxifen-resistant cell growth in breast cancer [10]. Downregulation of NBAT1 in breast cancer is associated with tumor metastasis nature and poor patient prognosis [11]. GAS5 suppresses cell proliferation by acting as a molecular sponge for miR-21, leading to the de-repression of phosphatase and tensin homologs (PTEN) in breast cancer [12]. The above strong evidence showed that LncRNAs present important biological function in breast cancer progression. LncRNA CCAT2, a novel long non-coding RNA transcript encompassing the rs6983267 SNP, which is over-expressed in some tumors, such as, cervical squamous cell cancer [13], gastric cancer [14], non-small cell lung cancer [15],and so on. In breast cancer, CCAT2 is found to promote cell migration and downregulate chemosensitivity to 50 FU in an rs6983267-independent manner in breast cancer [16]. CCAT2 promotes cell growth in vitro and tumor formation in vivo and regulated the WNT signaling pathway in breast cancer [17]. However, the contributions of CCAT2 to breast cancer still need to be investigated. In present study, we confirmed that CCAT2 was markedly upregulated in breast cancer and CCAT2 silencing suppressed the cell proliferation, cell invasion and cell cycle progression in breast cancer in vitro and in vivo inhibited tumor growth. Besides, CCAT2 inhibited the expression of p15 by interacting with EZH2 in breast cancer cells. Thus, our study indicated that CCAT2 may be a potential therapeutic target to suppress breast cancer malignant behavior.

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CCAT2-1, 50 -GUGCAACUCUGCAAUUUAAUU-30 , si-CCAT2-2, 50 UUAAAUUGCAGAGUUGCACUU-30 , si-EZH2, sense: 50 -GAGGUUCAGACGAGCUGAUUU-30 , antisense: 50 -AUCAGCUCGUCUGAACCUCUU-30 .si-p15, sense: 50 -CGGAGUCAACCGUUUCGGGUU-30 , 0 antisense: 5 -CCCGAAACGGUUGACUCCGUU-30 . Cells cultured on a 6-well plate and were transfected using Lipofectamine 2000 (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Breast cancer cells were harvested after transfection at 48 h for further analysis. 2.4. Cell proliferation assay The 2  103 MCF-7 and MDA-MB-231 cells were seeded in a 96well plate per well. 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Kit (Beyotime, China) was conducted to assess the cell proliferation ability in accordance with the guidelines. After transfection at indicated time of 1–5 days, the cells were used to detect the cell proliferation ability. The absorbance of each well was determined on a microplate reader at 490 nm. All of the experiments were repeated at least three times. 2.5. Transwell invasion assay MCF-7 and MDA-MB-231 cell invasion ability was assessed using transwell invasion assays by Matrigel-coated (BD Biosciences) transwell chambers (Corning Costar, Corning, USA) 0.1.5  105 cells in serum-free medium were placed on the upper chambers and the lower chambers were added with medium containing 10% FBS. After incubation in a humidified incubator with 5% CO2 at 37  C for 24 h, the invasive cells on the lower chambers were fixed and stained with crystal violet. The cells were counted under the microscope.

2. Materials and methods 2.6. Cell cycle analysis 2.1. Patients and tissue samples The 120 cases paired of breast tissues and adjacent normal tissues were collected from patients who underwent surgical resection between February 2008 and March 2013 at Department of Pancreatic and Breast Surgery, Shengjing Hospital of China Medical University. All of tissue specimens were snap-frozen and immediately stored in liquid nitrogen after resection until RNA analysis. The breast tissues and adjacent normal tissues were confirmed by two experienced pathologists. The clinicopathological feathers were collected from patients. Informed written consents were obtained from all patients involving in this study. The ethics committee of Sheng jing Hospital of China Medical University approved the study. 2.2. Cell lines culture The human breast cancer cell lines MCF-7 and MDA-MB-231 and a human normal breast epithelial cell lines MCF10A were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). MCF10A was cultured in Dulbecco’s Modified Essential Medium (DMEM)/ F12 medium supplemented with 10% FBS. The others two cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 mg/ml streptomycin in a humidified incubator with 5% CO2 at 37  C.

After MCF-7 or MDA-MB-231 cells transfection with si-NC or siCCAT2, cells were harvested and fixed in 70% ice-cold ethanol and then the cells were added with bovine pancreatic 5 mg/ml RNase and 10 mg/ml propidium iodide (Beyotime, China) for 30 min. FACS-Calibur System (BD Biosciences, NJ, USA) was used to detect the cell cycle distribution. The cell cycle distribution is presented as the percentage of cells in the G0/G1, S, and G2/M populations. The experiments were repeated at least three times. 2.7. RNA isolation and quantitative real-time PCR (qRT-PCR) Total RNA was extracted from breast cancer samples and adjacent normal tissues or cells by Trizol reagent (TAKARA, Dalian, China) according to the manufacturer’s instructions. The relative cDNA was reversed from 1 mg total RNA using the Prime Script RT Master Mix (TAKARA, Dalian, China). The RT-PCR assay was performed using SYBR Premix Ex TaqTMII (TAKARA) on the ABI 7500HT fast real-time PCR System (Applied Biosystems). Relative quantification of RNA expression levels was evaluated using the 2DDCt method. All of experiment was performed in three times. The primer sequences were: for CCAT2-forward was 50 -CCACATCGCTCAGACACCAT-30 and the CCAT2-reverse was 50 ACCAGGCGCCCAATACG-30 . ForGAPDH-forward was 50 0 CGCTCTCTGCTCCTCCTGTTC-3 and GAPDH-reverse was 50 ATCCGTTGACTCCGACCTTCAC-30 .p15-forward, 50 -GGACTAGTGGAGAAGGTGCG-30 and p15-reverse, 50 GGGC GCTGCCCATCATCATG 30 .

2.3. Cell transfection 2.8. Western blot analysis The siRNA oligonucleotide including si-NC, si-CCAT2-1, siCCAT2-2, si-EZH2, si-p15 were designed and synthesized by Ribobio, Guangzhou, China. The silencing sequences as follow: si-

Cells were lysed by RIPA buffer containing protease inhibitor cocktail (Sigma).Total protein was separated by SDS-PAGE on 10%

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polyacrylamide gels and then transferred to Hybond-P polyvinylidene difluoride (PVDF) membrane (Millipore). The membrane was incubated with anti-Cyclin D1 (1:1500; Santa Cruzy, CA, USA), Cyclin E1 (1:2000; CST, USA), CDK4 (1:2000; Santa Cruzy, CA, USA), GAPDH (1:1000; CST, USA) and p15 (1:1500; Santa Cruzy, CA, USA) overnight at 4  C. The secondary antibodies were conjugated to horseradish peroxidase, and western bands were visualized using the ECL Western Blot Detection System (Billerica, MA, USA). The relative protein expression was analyzed using Image-Pro Plus software (version 6.0, Media Cybernetics, Inc., USA). All of experiment was performed at least in three times.

2.9. RNA immunoprecipitation (RIP) assay RIP experiments were performed according to previous study [18], and used by a Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) according to the manufacturer’s instructions in MDA-MB-231 cells. Antibody for RIP assays of EZH2 was from Cell signaling Technology, USA. 2.10. Chromatin immunoprecipitation (ChIP) assay ChIP assay was carried out as described previously using the Pierce Agarose ChIP Kit according to the manufacturer’s protocol (Invitrogen, USA) [19]. The MDA-MB-231 cells were processed with formaldehyde and incubated for 20 min to generate DNA-protein cross-links. Cell lysates were sonicated to generate chromatin fragments of 200–300 bp and immunoprecipitated with EZH2 or H3K27me3-specific antibody (CST, USA). The IgG was used as the control. Precipitated chromatin DNA was recovered and detected by qRT-PCR assays. 2.11. Tumor xenograft in nude mice The nude mice were subcutaneously injected into the left inguinal with 4  106 MDA-MB-231 cells with either control lentivirus or a lentivirus-shRNA-CCAT2, respectively. The tumors volumes were measured every 7 days after implantation. The tumor volume was calculated by the modified ellipse formula (volume = length  width2/2). The mice were sacrificed after 4 weeks. 2.12. Statistical analysis The SPSS 13.0 software was used to analyze the data in the study. The Student’s t-test and Kaplan-Meier curve and log-rank test were used to analyze the CCAT2 expression and the association between CCAT2 expression and the overall survival time. The P values less than 0.05 were considered statistically significant.

3. Results 3.1. CCAT2 is increased in breast cancer tissues and predicts a poor prognosis in breast cancer patients To validate the role of CCAT2 expression in breast cancer, we firstly detected the expression of CCAT2 in 120 pairs of breast cancer tissues and paired adjacent normal tissues in patients. The results were shown in Fig. 1A, we found that the CCAT2 expression levels were significantly upregulated in breast cancer tissues compared with the adjacent normal tissues in patients (Fig. 1A, P < 0.001). Next, we assessed the correlation between CCAT2 expression level and clinicopathological factors. As shown in

Table 1, higher CCAT2 was closely correlated with TNM stage (P = 0.009, Table 1) and lymph node metastasis (P = 0.001, Table 1). Kaplan-Meier survival analysis and log rank test confirmed that higher CCAT2 expression was significantly correlated with shorter patient survival time (Log rank = 15.43, P < 0.001, Fig. 1B). Taken together, our results indicated that upregulation of CCAT2 expressions in breast cancer was correlated with the poor prognosis for the breast cancer patients. 3.2. The association between CCAT2 expression and cell proliferation, invasion and cell cycle in the breast cancer cells To further investigate the biological role of CCAT2 in breast cancer, we detected the expression of CCAT2 in human breast cancer cell lines MCF-7 and MDA-MB-231 and a human normal breast epithelial cell lines MCF10A. The qRT-PCR analysis results showed that CCAT2 was higher expression in breast cancer cells than that in the MCF10A cells (Fig. 1C). Furthermore, the CCAT2 was knocked down in MCF-7 and MDA-MB-231 cells, the si-CCAT22 was selected to study the function of CCAT-2 based on its higher efficiency of knockdown in the following experiment (Fig. 1D and E). The proliferation ability was examined in MCF-7 and MDA-MB231 cell after CCAT2 silencing by MTT cell proliferation assays. We confirmed that knockdown of CCAT2 in MCF-7 and MDA-MB-231 cells significantly inhibited cell proliferation ability compared with that in the si-NC group (Fig. 2A and B). In addition, knockdown of CCAT2 displayed a decrease in S phase cell proportion by flow cytometry analysis compared with that in the si-NC group in MCF7 and MDA-MB-231 cells (Fig. 2C and D). The transwell cell invasive assays in MCF-7 and MDA-MB-231 cells showed that cell invasive number was significantly decreased in CCAT2 silencing group, compared with that in the si-NC group (Fig. 2E and F).Thus, these results indicated that reduced CCAT2 suppressed cell proliferation and invasion in breast cancer. 3.3. Knockdown of CCAT2 upregulates p15 expression by interacting with EZH2 in breast cancer cells To explore the molecular mechanism of CCAT2 involved in breast cancer, we detected the cell cycle related protein expression after knockdown of CCAT2 in MCF-7 and MDA-MB-231 cells. The results demonstrated that CCAT2 silencing inhibited the expression of CyclinD1, Cyclin E1 and CDK4 and upregulated the p15 expression in MCF-7 and MDA-MB-231 cells (Fig. 3A and B). P15 played an important role in tumor growth in breast cancer [20]. EZH2, a core subunit of polycomb repressive complex2, is a histone methyltransferase that specifically catalyze the trimethylation of lysine residue 27 of histone 3 (H3K27me3) of target gene [21,22]. The further results confirmed p15 was up-regulation in transcriptional level by qRT-PCR analysis after transfection of siRNAs against CCAT2 in MDA-MB-231 cells (Fig. 4A). Next, we sought to detect whether CCAT2 regulated p15 expression via interacting with EZH2. An RNA immunoprecipitation assay using EZH2 or H3k27me3 antibodies confirmed that CCAT2 could bind to EZH2 or H3k27me3 in MDA-MB-231 cells (Fig. 4B). Moreover, we knocked down EZH2 by si-EZH2 oligos and the results showed an increased mRNA and protein expression levels of p15 in MDA-MB231 cells (Fig. 4C and D). Furthermore, we performed chromatin immunoprecipitation (ChIP)-quantitative real-time PCR (ChIPqPCR) to evaluate EZH2 or trimethylation of histone H3 lysine 27 (H3K27me3) statues at p15 gene, which is a marker of suppressed chromatin. We confirmed that knockdown of CCAT2 by siRNA oligos decreased EZH2 or H3K27me3 level of p15 promoter region in MDA-MB-231 cells (Fig. 4E). Therefore, these data indicated that CCAT2 repressed the P15 expression via interacting with EZH2 in breast cancer cells.

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Fig. 1. CCAT2 is dramatically increased in breast cancer tissues and predicts a poor prognosis in breast cancer patients. (A) QRT-PCR was performed to examine the relative expression of CCAT2 in the 120 cases of breast cancer tissues and the normal adjacent tissue. (B) Kaplan-Meier curve and log rank test were applied to analyze the association between CCAT2 expression and the overall survival time. (C) QRT-PCR was performed to examine the relative expression of CCAT2 in human breast cancer cell lines MCF-7 and MDA-MB-231 and a human normal breast epithelial cell lines MCF10A. (E)-(F) QRT-PCR was performed to examine the relative expression of CCAT2 after transfecting si-NC, si-CCAT2-1 or si-CCAT2-2 into MCF-7 and MDA-MB-231 cells, respectively. *** P < 0.001, **P < 0.05.

3.4. CCAT2 inhibits the tumor growth in vivo To evaluate CCAT2 whether affected the tumor cell growth in vivo, the mice were subcutaneously injected into the left inguinal using MDA-MB-231 cells with either control lentivirus or a lentivirus-shRNA-CCAT2, respectively. Every 7 days, the tumor volume was measured, after 28 days, the results showed that the tumor volume was smaller in lv-shRNA-CCAT2 group, compared with that in the control group (Fig. 5A). Moreover, the results also demonstrated that tumor growth was significantly inhibited in lvshRNA-CCAT2 group, compared with the control group (Fig. 5B). Thus, these data revealed that CCAT2 inhibited the tumor growth in vivo in breast cancer. 4. Discussion Previously reported in the literatures that higher CCAT2 play important role in tumor progression, for example, CCAT2 expression and MYC amplification are significantly associated with poorer overall survival in ESCC patients [23]. CCAT2 expression levels shows higher diagnostic performance than conventional serum biomarkers, like AFP, CA153, and NSE in esophageal squamous cell carcinoma [24]. Knockdown of CCAT2 could decrease cell proliferation and migration as well as induce apoptosis in bladder cancer cells [25]. In our study, qRT-PCR analysis demonstrated that CCAT2 was upregulated in tumor tissues than in the adjacent normal tissues. Higher CCAT2 was significantly correlated with TNM stage and lymph node

metastasis. Kaplan-Meier survival curves and log rank test showed that increased CCAT2 expression was significantly correlated with shorter OS in patients. Furthermore, knockdown of CCAT2 showed that cell proliferation, cell invasion and cell cycle progression were inhibited in breast cancer in vitro and tumor volume was inhibited after CCAT2 silencing in vivo. These results showed that CCAT2 played a key role in breast cancer progression. Recently, studies have revealed that lncRNAs may be an important class of pervasive genes involved in regulating downstream targets via EZH2 [26]. GIHCG physically associates with EZH2 and upregulated H3K27me3 and DNA methylation levels on the miR-200b/a/429 promoter [27]. HOXA-AS2 could be an oncogene for GC partly through suppressing P21, PLK3, and DDIT3 expression by interacting with EZH2 could directly bind to the promoter of P21, PLK3 and DDIT3, inducing H3K27 trimethylated [28]. Long non-coding RNA-EBIC promotes tumor cell invasion by binding to EZH2 and inhibiting E-cadherin in cervical cancer [29]. NBAT1 is associated with PRC2 member EZH2 and regulates DKK1 (dickkopf WNT signaling pathway inhibitor 1) in a PRC2 dependent manner in breast cancer [11]. RIP assays showed that CCAT2 could bind to EZH2 and H3k27me3 in breast cancer cells. ChIP assays demonstrated that CCAT2 inhibited p15 expression by EZH2 and H3k27me3 in breast cancer cells. P15, a member of the INK4 family of cyclin-dependent kinase inhibitors, is capable of inducing cell cycle arrest in G1 phase and has been identified as a tumor suppressor [20].These results demonstrated that CCAT2 promoted cell proliferation via regulating p15 expression in breast cancer.

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Fig. 2. The association between CCAT2 expression and cell proliferation and invasion ability in the breast cancer. (B) MTT assays were performed to determine cell proliferation capacity in MCF-7 and MDA-MB-231 cells after transfected with si-NC or si-CCAT2, respectively. (C)- (D) Flow cytometry analysis was performed to detect the cell cycle distribution in MCF-7 and MDA-MB-231 cells after transfected with si-NC or si-CCAT2, respectively. (E)-(F) Transwell cell invasive assay was used to detect the cell invasive ability in MCF-7 and MDA-MB-231 cells after transfected with si-NC or si-CCAT2, respectively. All of the experiments were independently repeated at least three times, ** P < 0.05.

Table 1 The relationship between clinicopathological factors and CCAT2 expression in 120 primary breast cancer. Factors

Age(years) 50 >50 Tumor size <2 cm >2 cm TNM stage I/II III HER-2 status Negative Positive PR status Negative Positive lymph node metastasis Negative Positive ER Negative Positive Histological grade G1/2 G3

Number of Patients

CCAT2 expression

p-value

Lower(n = 55)

Higher (n = 65)

92 28

39 16

53 12

90 30

40 15

50 15

79 41

43 12

36 29

55 65

20 35

35 30

39 81

20 35

19 46

80 40

45 10

35 30

43 77

23 32

20 45

82 38

42 13

40 15

0.170

0.597

0.009**

0.055

0.318

0.001**

0.209

0.662

ER = estrogen receptor; PR = progesterone receptor; HER–2 = c-erb B-2. ** P < 0.05.

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Fig. 3. The association between CCAT2 expression and cell cycle protein. (A) Western-blot analysis was used to detect the protein expression of CyclinD1, CyclinE1, p15 and CDK4 in MCF-7 cells transfected with si-NC or si-CCAT2. (B)Western-blot analysis was performed to detect the protein expression of CyclinD1, CyclinE1, p15 and CDK4 in MDA-MB-231 cells transfected with si-NC or si-CCAT2. All of the experiments were independently repeated at least three times.

5. Conclusions In conclusion, our study demonstrated that CCAT2 was upregulated in breast cancer tissues and increased CCAT2 predicted a shorter OS for breast cancer patients. Furthermore, we revealed that CCAT2 suppressed P15 expression by binding with EZH2. These findings may provide a novel therapeutic target of breast cancer.

Competing interests The authors declare that they have no competing interests. Ethics approval and consent to participate All patients had written the informed consent before surgery. Clinicopathological follow-up infomation from breast cancer

Fig. 4. Knockdown of CCAT2 upregulated p15 expression through interacting with EZH2. (A) QRT-PCR was performed to test the mRNA expression of p15 in MDA-MB-231 cells after transfected with si-NC or si-CCAT2, respectively. (B) RNA immunoprecipitation (RIP) showed that CCAT2 physically associates with EZH2 and H3k27me3 using control IgG antibody in MDA-MB-231 cells. (C)-(D) QRT-PCR and western-blot analysis were performed to examine the relative expression of p15 after transfecting si-NC, siEZH2 into MDA-MB-231 cells, respectively. (E) Chromatin immunoprecipitation(ChIP) assay using EZH2 and H3K27me3 antibodies confirmed that CCAT2 knockdown decreased EZH2 and H3K27me3 recruitment to the promoter region of p15 in the MDA-MB-231 cells. All of the experiments were independently repeated at least three times, ** P < 0.05.

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Fig. 5. CCAT2 promoted breast cancer cell proliferation in vivo. (A) Representation of nude mice tumor formation in lentivirus-control or lentivirus-shRNA-CCAT2 group. (B) Tumor volume of mice were evaluated every 7 days in lentivirus-control and lentivirus-shRNA-CCAT2 group, respectively, ** P < 0.05.

patients was available. This study was approved by the Human Ethics Committee of Department of Pancreatic and Breast Surgery, Shengjing Hospital of China Medical University and all clinical investigations have been conducted according to the principles expressed in the Declaration of Helsinki. Consent for publication All authors read and approved the final manuscript for publication. Authors’ contributions Xin Deng and Yi Zhao design the experiments, Xin Wu and Guoqing Song perform data collection, Xin Deng and Yi Zhao performed the data analysis. Xin Deng and Yi Zhao wrote the manuscript. Acknowledgments This work was supported by Liaoning Provincial Natural Science Funds (CB65 and D239). References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA Cancer J. Clin. 61 (2011) 69–90. [2] F. Cardoso, N. Harbeck, L. Fallowfield, S. Kyriakides, E. Senkus, Locally recurrent or metastatic breast cancer: ESMO clinical practice guidelines for diagnosis, treatment and follow-up, Ann. Oncol. 23 (Suppl. 7) (2012) vii11–vii19. [3] F.C. Geyer, M.A. Lopez-Garcia, M.B. Lambros, J.S. Reis-Filho, Genetic characterization of breast cancer and implications for clinical management, J. Cell Mol. Med. 13 (2009) 4090–4103. [4] X. Shi, M. Sun, H. Liu, Y. Yao, Y. Song, Long non-coding RNAs: a new frontier in the study of human diseases, Cancer Lett. 339 (2013) 159–166. [5] J.S. Mattick, A new paradigm for developmental biology, J. Exp. Biol. 210 (2007) 1526–1547. [6] Q. Wu, L. Guo, F. Jiang, L. Li, Z. Li, F. Chen, Analysis of the miRNA-mRNA-lncRNA networks in ER+ and ER- breast cancer cell lines, J. Cell Mol. Med. 19 (2015) 2874–2887. [7] Y. Shi, J. Li, Y. Liu, J. Ding, Y. Fan, Y. Tian, L. Wang, Y. Lian, K. Wang, Y. Shu, The long noncoding RNA SPRY4-IT1 increases the proliferation of human breast cancer cells by upregulating ZNF703 expression, Mol. Cancer. 14 (2015) 51. [8] S.J. Shi, L.J. Wang, B. Yu, Y.H. Li, Y. Jin, X.Z. Bai, LncRNA-ATB promotes trastuzumab resistance and invasion-metastasis cascade in breast cancer, Oncotarget 6 (2015) 11652–11663. [9] C. Vennin, N. Spruyt, F. Dahmani, S. Julien, F. Bertucci, P. Finetti, T. Chassat, R.P. Bourette, X. Le Bourhis, E. Adriaenssens, H19 non coding RNA-derived miR-675 enhances tumorigenesis and metastasis of breast cancer cells by down regulating c-Cbl and Cbl-b, Oncotarget 6 (2015) 29209–29223. [10] X. Xue, Y.A. Yang, A. Zhang, K.W. Fong, J. Kim, B. Song, S. Li, J.C. Zhao, J. Yu, ncRNA HOTAIR enhances ER signaling and confers tamoxifen resistance in breast cancer, Oncogene 35 (2016) 2746–2755. [11] P. Hu, J. Chu, Y. Wu, L. Sun, X. Lv, Y. Zhu, J. Li, Q. Guo, C. Gong, B. Liu, S. Su, NBAT1 suppresses breast cancer metastasis by regulating DKK1 via PRC2, Oncotarget 6 (2015) 32410–32425.

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