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The Long Noncoding RNA SPRY4-IT1 Increases the Proliferation of Human Breast Cancer Cells by Upregulating ZNF703 Expression

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The Long Noncoding RNA SPRY4-IT1 Increases the Proliferation of Human Breast Cancer Cells by Upregulating ZNF703 Expression Shi et al. Molecular Cancer (2015) 14:51 DOI 10.1186/s12943-015-0318-0 RESEARCH Open Access The long noncoding RNA SPRY4-IT1 increases the proliferation of human breast cancer cells by upregulating ZNF703 expression Yongguo Shi1,2, Juan Li1, Yangchen Liu2, Jie Ding1, Yingrui Fan1, Yun Tian1, Li Wang1, Yifan Lian1, Keming Wang1* and Yongqian Shu3* Abstract Background: Long noncoding RNAs (lncRNAs) have emerged recently as a new class of genes that regulate cellular processes, such as cell growth and apoptosis. The SPRY4 intronic transcript 1 (SPRY4-IT1) is a 708-bp lncRNA on chromosome 5 with a potential functional role in tumorigenesis. The clinical significance of SPRY4-IT1 and the effect of SPRY4-IT1 on cancer progression are unclear. Methods: Quantitative reverse transcriptase PCR (qRT-PCR) was performed to investigate the expression of SPRY4-IT1 in 48 breast cancer tissues and four breast cancer cell lines. Gain and loss of function approaches were used to investigate the biological role of SPRY4-IT1 in vitro. Microarray bioinformatics analysis was performed to identify the putative targets of SPRY4-IT1, which were further verified by rescue experiments, and by western blotting and qRT-PCR. Results: SPRY4-IT1 expression was significantly upregulated in 48 breast cancer tumor tissues comparedwith normal tissues. Additionally, increased SPRY4-IT1 expression was found to be associated with a larger tumor size and an advanced pathological stage in breast cancer patients. The knockdown of SPRY4-IT1 significantly suppressed proliferation and caused apoptosis of breast cancer cells in vitro. Furthermore, we discovered that ZNF703 was a target of SPRY4-IT1 and was downregulated by SPRY4-IT1 knockdown. Moreover, we provide the first demonstration that ZNF703 plays an oncogenic role in ER (−) breast carcinoma cells. Conclusions: SPRY4-IT1 is a novel prognostic biomarker and a potential therapeutic candidate for breast cancer. Keywords: SPRY4-IT1, ZNF703, Proliferation, Breast cancer Background understanding of the genetic and molecular characteris- Despite the improved prognosis of breast cancer patients tics of breast cancer is urgently needed for early diagno- because of early diagnosis, radical surgery and the devel- sis, choice of the appropriate treatment and an improved opment of adjuvant therapy, breast cancer remains the prognosis for patients with breast cancer. most common type of cancer among women [1,2]. Trad- Recent studies have demonstrated that a class of non- itional prognostic markers, such as the estrogen receptor protein-coding RNAs (ncRNAs), which are known as (ER), progesterone receptor (PR), HER2/NEU, and p53 long non-coding RNAs (lncRNAs), participates in cell [3,4], are insufficient indicators of tumor aggressiveness fate determination and human disease pathogenesis and do not adequately discriminate between different [6-9]. lncRNAs are RNA molecules longer than 200 nu- biological and clinical outcomes [5]. Therefore, a better cleotides that are not translated into proteins [10,11]. An increasing body of evidence has suggested that lncRNAs * Correspondence: [email protected]; [email protected] are key regulators in several biological processes and are 1 Department of Oncology, The Second Affiliated Hospital of Nanjing Medical increasingly recognized as diagnostic or prognostic can- University, Nanjing, Jiangsu, PR China 3Department of Oncology, the First Affiliated Hospital of Nanjing Medical cer biomarkers, including in breast cancer [10,12-14]. University, Nanjing, Jiangsu, PR China For example, GAS5, a well-known lncRNA, is markedly Full list of author information is available at the end of the article © 2015 Shi et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shi et al. Molecular Cancer (2015) 14:51 Page 2 of 13 down-regulated in breast cancer and has been suggested assessed the effects of SPRY4-IT1 expression on breast as a diagnostic cancer biomarker [9,15-17]. Another cancer cell phenotypes in vitro. Our results suggest that characterized lncRNA, HOTAIR, is overexpressed in increased SPRY4-IT1 expression may play a role in breast breast cancer, interacts with Polycomb Repressive Com- cancer carcinogenesis. plex 2 (PRC2) and alters the regulation of genes [18], Zinc finger 703 (ZNF703) has been identified as the resulting in aberrant histone H3K27 methylation and genetic driver of the amplification at 8p12 in luminal B gene expression and further promoting cancer invasive- tumors [22-26]. A recent study demonstrated that en- ness and metastasis [18]. Our previous studies have also hanced ZNF703 expression represses E-cadherin expres- shown that patients with higher lncRNA Loc554202 ex- sion and increases lung metastases in a mouse model of pression present an advanced pathological breast cancer breast cancer [27]. In this study, we found that ZNF703 stage [19]. is a downstream target gene of SPRY4-IT1 and demon- lncRNA-SPRY4-IT1 (GenBank Accession ID AK024556) strated that ZNF703 promotes ER(−) breast carcinoma isderivedfromanintronoftheSPRY4gene.Khaitanetal. cell proliferation and suppresses apoptosis in vivo. [20] observed that SPRY4-IT1 is highly expressed in mel- anoma cells compared with melanocytes. The knockdown Results of SPRY4-IT1 expression results in defects in cell growth, Expression of SPRY4-IT1 in breast cancer tissues and decreased invasion, and increased rates of apoptosis in breast cancer cell lines melanoma cells [20]. A study conducted by Zou et al. [21] qRT-PCR analysis was used to examine the SPRY4-IT1 showed that aberrant expression of lncRNA SPRY4-IT1 levels in 48 breast cancer tissues and 48 matched normal may contribute to the generation of abnormal HTR-8/ breast tissues. SPRY4-IT1 expression was upregulated SVneo trophoblast cells. However, the expression profile (P < 0.05) in cancerous tissues compared with normal tis- and biological role of SPRY4-IT1 as well as its mechanism sues (Figure 1A). We then evaluated the correlation of in breast cancer remain largely unknown. In this study, we SPRY4-IT1 expression with clinicopathological parameters Figure 1 Relative expression of SPRY4-IT1 in breast cancer tissues and cells compared with adjacent normal tissues and normal breast epithelial cells. (A) Relative expression of SPRY4-IT1 in breast cancer tissues (T) (n = 48) compared with corresponding non-tumor tissues (N) (n = 48). SPRY4-IT1 expression was examined by qPCR and normalized to GAPDH expression and the levels in the cells of interest were compared with the expression levels inMCF-10A and other cells. The results are presented as the -ΔΔCT changes in tumor tissues relative to normal tissues. (B and C) The data are presented as the relative expression levels in tumor tissues. SPRY4-IT1 expression was significantly higher in patients with a higher pathological stage and a larger tumor size (shown as -ΔΔCT). (D) SPRY4-IT1 expression was assessed by qRT-PCR in ER(−) breast cancer tissues and ER(+) breast cancer tissues(shown as -ΔΔCT). (E) The relative SPRY4-IT1 expression levels in breast cancer cell lines (MD-MB-231, MDA-MB-435S and MCF-7) were compared with human breast epithelial cells (MCF-10A), as assessed by qRT-PCR. * P < 0.05 and **P < 0.01. Shi et al. Molecular Cancer (2015) 14:51 Page 3 of 13 (i.e., stage, maximum diameter) to assess its clinical signifi- cycle in MDA-MB-231 and MDA-MB-435S cells cance. As presented in Figures 1B and 1C, both larger tu- through flow cytometry. After treatment with si-SPRY4- mors, which represent a higher tumor burden, and more IT1 or si-NC for 48 h, SPRY4-IT1 knockdown led to a advanced tumors are associated with increased SPRY4-IT1 significant accumulation of cells at the G0/G1-phase expression. Furthermore, we found ER(−)breast cancer tis- (p < 0.05) and a significant decrease in cells in the S-phase sues exhibit increased SPRY4-IT1 expression than ER(+) (p < 0.05; Figure 3A). Consistently, cyclin D1 protein ex- breast cancer tissues (p < 0.01; Figure 1D). These analyses pression was also disrupted in the si-SPRY4-IT1-trans- demonstrate that SPRY4-IT1 may be a potential prognostic fected breast cancer cells compared with the control cells biomarker for breast cancer patients. We then examined (Figure 3B). the expression of SPRY4-IT1 in three human breast We then, investigated the effects of SPRY4-IT1 knock- cancer cell lines, namely MD-MB-231, MDA-MB-435S down on apoptosis. The percentages of apoptotic cells and MCF-7 cells, and in the normal breast epithelium cell were significantly increased in the treated group com- line MCF-10A. Significantly higher expression of SPRY4- pared with the control group (p < 0.05; Figure 3C). Con- IT1 was found in MDA-MB-231 and MD-MB-435S cells, sistently, microscopic analysis of TUNEL staining in compared with MCF-10A cells (p < 0.01; Figure 1E), but MDA-MB-231 and MDA-MB-435S cells also showed MCF-7 cells expressed relatively lower levels of SPRY4-IT1 that the knockdown of SPRY40-IT1 resulted in a higher compared with MCF-10A cells (p <0.01; Figure 1E). These number of apoptotic cells compared with the controls results are consistent with our previous findings. There- (Figure 4D). Additionally, the western blot analysis show fore,SPRY4-IT1wasdepletedinMD-MB-231andMD- that Bax protein was significantly increased in si-SPRY4- MB-435S cells, which exhibit a higher expression of IT1-treated cells, whereas the Bcl-2 protein level was de- SPRY4-IT1. In addition, SPRY4-IT1 was over-expressed in creased (Figure 4E). Taken together, the flow cytometric the MCF-7 cell line after transfection of pcDNA3.1- analysis, TUNEL staining and protein analysis results SPRY4-IT1, which generates a relatively lower level of suggested that SPRY4-IT1 has exerts a critical effect on SPRY4-IT1 expression.
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