SP1-Induced Upregulation of the Long Noncoding RNA TINCR Regulates Cell Proliferation and Apoptosis by Affecting KLF2 Mrna Stability in Gastric Cancer

ORIGINAL ARTICLE SP1-induced upregulation of the long noncoding RNA TINCR regulates cell proliferation and apoptosis by affecting KLF2 mRNA stability in gastric cancer

T-p Xu1, X-x Liu2, R Xia1, L Yin1, R Kong3, W-m Chen1, M-d Huang4 and Y-q Shu1

The long noncoding RNA TINCR shows aberrant expression in human squamous carcinomas. However, its expression and function in gastric cancer remain unclear. We report that TINCR is strongly upregulated in human gastric carcinoma (GC), where it was found to contribute to oncogenesis and cancer progression. We also revealed that TINCR overexpression is induced by nuclear transcription factor SP1. Silencing TINCR expression inhibited cell proliferation, colony formation, tumorigenicity and apoptosis promotion, whereas TINCR overexpression promoted cell growth, as documented in the SGC7901 and BGC823 cell lines. Mechanistic analyses indicated that TINCR could bind to STAU1 (staufen1) protein, and inﬂuence KLF2 mRNA stability and expression, then KLF2 regulated cyclin-dependent kinase genes CDKN1A/P21 and CDKN2B/P15 transcription and expression, thereby affecting the proliferation and apoptosis of GC cells. Together, our ﬁndings suggest that TINCR contributes to the oncogenic potential of GC and may constitute a potential therapeutic target in this disease.

Oncogene (2015) 34, 5648–5661; doi:10.1038/onc.2015.18; published online 2 March 2015

INTRODUCTION lncRNA, eventually triggering rapid mRNA degradation.16,17 For Increased cell proliferation is a crucial characteristic of malignancy example, studies have showen that ARF1, KLF2 and SERPINE1 15,18,19 and a hallmark cancer capability.1 Malignant proliferation is the mRNAs are bona fide SMD targets. main reason for dismal outcomes in cancer patients. Transcription The Kruppel-like factor (KLF) family, also known as SP1-like generates many long noncoding RNAs (abbreviated as ncRNA or proteins, consists of a set of transcription factors that are lncRNA) that are capable of influencing diverse cellular processes present in diverse organisms, in which they function in cell 20,21 like proliferation, apoptosis or metastasis.2 An increasing number differentiation and proliferation. KLF2 is one of the of studies have revealed that deregulated lncRNA expression has a prominent members due to its tumor suppressor function in functional role in a variety of disease states,3,4 and many lncRNAs malignancies like prostate cancer, ovarian cancer, leukemia and 22–25 exhibit tissue-specific expression.5–7 In addition, recent reports breast cancer. Its growth-inhibitory, pro-apoptotic function showed that some lncRNAs exhibit multiple gene expression is mediated via the regulation of the expression of the cell cycle- models and have significant roles during cellular development in inhibiting cyclin-dependent kinase genes CDKN2B/P15 and multiple carcinomas.8–12 CDKN1A/P21.24,25 Both these genes are crucial CDK inhibitors TINCR, an lncRNA producing a 3.7-kb transcript, controls human and lead to a transition from the G1 phase to the S phase of the epidermal differentiation and is downregulated in human squa- cell cycle.26 mous cell carcinoma.13,14 Kretz et al.14 first confirmed that TINCR Here we report a novel role for TINCR in tumor development could bind to staufen1 (STAU1) protein and mediate differentiated and GC cell growth. We show that: (a) the level of TINCR is mRNA stabilization. The double-stranded RNA-binding protein higher in tumor cells and tumor tissues than in normal gastric STAU1 has various roles in gene expression. In addition to its role epithelial cells and adjacent normal tissues; (b) the upregulation in mRNA localization, STAU1 binds to an STAU1-binding site in the of TINCR is induced by the transcription factor SP1; (c) patients 3’-untranslated region (3’UTR) of its target mRNAs. STAU1 can with high TINCR expression in their gastric cancer cells have a induce mRNA degradation, which is termed STAU1-mediated poor prognosis; (d) decreased TINCR expression abolishes mRNA decay (SMD).15 SMD is a translation-dependent mechanism tumorigenicity in nude mice; (e) TINCR regulates cell growth, that occurs when STAU1, together with the nonsense-mediated cell cycle progression and apoptosis; (f) TINCR alters KLF2 mRNA decay factor UPF1, is bound sufficiently downstream of a expression by affecting mRNA stability via STAU1 -mediated termination codon.15 In addition to the STAU1-binding site in the mRNA decay; (g) CDKN2B/P15 and CDKN1A/P21 are the direct 3’UTR of the target mRNA, STAU1 can recognize an RNA duplex targets of the transcription factor KLF2; and (h) forced KLF2 formed by intermolecular base pairing between an Alu sequence in expression inhibits proliferation and pro-apoptosis in gastric the 3’UTR of the target mRNA and another Alu sequence in an cancer cells.

1Department of Oncology, The First Afﬁliated Hospital of Nanjing Medical University, Nanjing, People’s Republic of China; 2Department of Gastrointestinal Surgery, Northern Jiangsu People's Hospital, Clinical Medical School, Yangzhou University, Yangzhou, People’s Republic of China; 3Clinical Medical Examination Center, Northern Jiangsu People's Hospital, Clinical Medical School, Yangzhou University, Yangzhou, People’s Republic of China and 4Department of Medical Oncology, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an, People’s Republic of China. Correspondence: Professor Y-q Shu, Department of Oncology, The First Afﬁliated Hospital of Nanjing Medical University, No. 300 Guangzhou Road, Nanjing 210029, Jiangsu Province, People's Republic of China. E-mail: [email protected] or [email protected] Received 3 September 2014; revised 14 January 2015; accepted 16 January 2015; published online 2 March 2015 TINCR contributes to the oncogenic potential of GC T-p Xu et al 5649

Figure 1. Increased TINCR expression in GC; the transcription factor SP1 is involved in TINCR upregulation; and the clinical signiﬁcance of TINCR. (a) TINCR expression was examined by quantitative reverse transcription PCR in 80 paired human GC tissues and adjacent noncancerous tissues (Wilcoxon's signed-rank test, Po0.01). Data are represented as log2 fold changes (cancer/normal), and deﬁned as ‘o − 1’ for underexpression and ‘41’ for overexpression. The patients were divided into a low-TINCR expression group (n = 25) and a high TINCR expression group (n = 55) according to whether TINCR was up or downregulated in their tumor tissue samples compared with the corresponding adjacent noncancerous tissue samples. (b) Real-time PCR analysis of TINCR expression in the normal gastric epithelium cell line (GES1) and GC cells. Bars: s.d.; *Po0.05, **Po0.01. (c) The predicted positions of putative SP1-binding sites in − 1 kb human TINCR promoter by gene sequence analysis (above) and the primers designed for ChIP assays have been provided in Supplementary Table S1. Quantitative ChIP assays were used to show direct binding of SP1 to endogenous TINCR promoter regions (below). Bars: s.d.; **Po0.01. (d) A dual-luciferase reporter assay was performed by co-transfecting the TINCR promoter fragment (TINCR-pGL3-F) or deleted TINCR promoter fragment (TINCR- pGL3-D) with SP1 expression vector or an empty vector in SGC7901 cells. Luciferase activity was expressed as relative to that of the pGL3 vector (a promoter-less vector). Bars: s.d.; **Po0.01. (e) Quantitative PCR analysis of TINCR expression levels following the treatment of SGC7901 and BGC823 cells with siRNAs-SP1. Bars: s.d.; *Po0.05, **Po0.01. (f) The receiver operating characteristic curve for prediction of gastric cancer based on TINCR expression level, using corresponding adjacent non-tumorous tissues as a control. (g) Kaplan–Meier analysis of disease-free survival based on TINCR expression in all the 80 patients.

© 2015 Macmillan Publishers Limited Oncogene (2015) 5648 – 5661 TINCR contributes to the oncogenic potential of GC T-p Xu et al 5650 RESULTS GES1. Significantly high TINCR expression was found in SGC7901 TINCR was upregulated in tissues and cell lines, and was regulated (Po0.01), BGC823 (Po0.01), MGC803 (P = 0.015) and MKN45 by SP1 (P = 0.031) compared with that in GES1 (Figure 1b). fi TINCR expression was significantly higher in the tumor tissues than Next we investigated the mechanisms controlling tissue-speci c TINCR expression. We performed a computational screen and in the adjacent normal tissues, as investigated using quantitative found that three tandem putative SP1-binding sites at the regions PCR assays (Po0.01; Figure 1a). TINCR expression was also − 163 to − 153 bp (E1), − 88 to − 78 bp (E2) and − 16 to − 6 bp (E3) detected in the GC cell lines, including MGC803, BGC823, in the TINCR promoter (Figure 1c). We used chromatin immuno- MKN45 and SGC7901, and the normal gastric epithelium cell line precipitation (ChIP) assays to determine which region in the TINCR promoter mediated SP1-binding to the endogenous TINCR promoter. An obvious SP1-binding activity on the endogenous Table 1. Correlation between TINCR expression and clinicopathological TINCR promoter was observed at the region around − 0.2 kb (E1; characteristics of gastric cancer Figure 1c). Consistent with this finding, the − 201 to +163-bp fi fl Clinical parameter TINCR χ2-test region of the TINCR promoter-driven re y luciferase expression expression P-value vector induced TINCR transcriptional activity via SP1 overexpression, as indicated by a luciferase reporter assay (Figure 1d). To validate this finding, we deleted the E1 binding site and repeated High no. cases Low no. cases the reporter assay. The results showed that the deletion of the (n = 55) (n = 25) SP1-binding motif E1 significantly impaired the effect of SP1 on Age (years) 0.544 TINCR transcription activation, suggesting that SP1 binds to their o50 26 10 special binding motifs to regulate TINCR transcription (Figure 1d). 450 29 15 We next determined whether the overexpression of TINCR is mediated by SP1. We knocked down endogenous SP1 expression in GC cells by transfection with small-interfering RNAs (siRNAs) Gender 0.755 targeting the SP1 gene (Supplementary Figure S1a). TINCR levels Male 35 15 were significantly reduced in cells transfected with siRNAs Female 20 10 (Figure 1e). These results indicate the possibility that TINCR upregulation in GC is mediated by the transcription factor SP1. Location 0.249 Distal 27 10 TINCR expression and clinicopathological factors in GC Middle 21 8 Proximal 7 7 TINCR expression levels in tumor tissues were categorized as low or high depending on whether TINCR expression was up or downregulated compared with the corresponding adjacent Size 0.131 noncancerous tissue samples. Clinicopathologic factors were 45cm 32 10 analyzed in the high- and low-TINCR expression groups. As shown o5cm 23 15 in Table 1, the high TINCR group (n = 55) showed a greater depth of invasion (P = 0.005) and higher tumor stage (P = 0.002) than the low-TINCR expression group (n = 25). However, there was no Histologic 0.211 significant correlation between TINCR expression and other differentiation 4 Well 4 1 clinicopathological features (P 0.05). The clinical data of all Moderately 15 10 patients are shown in Supplementary Table S2. Poorly 33 10 Undifferentiated 3 4 Table 2. Univariate and multivariate Cox regression analyses TINCR for DFS of patients in study cohort (n = 80) Invasion depth 0.005a T1 10 7 Variables DFS T2 16 15 T3 18 3 HR 95% CI P-value T4 11 0 Univariate analysis Age (o50 vs 450 years) 0.685 0.352–1.333 0.265 – a Gender (male vs female) 0.651 0.312 1.360 0.254 TNM stages 0.002 Location (distal vs middle+ proximal) 0.733 0.377–1.427 0.361 I1115 Tumor size ( 45vso5 cm ) 1.118 0.576–2.172 0.742 II 13 5 Histologic differentiation 1.413 0.702–2.846 0.333 III 23 5 (well+ moderately vs poorly+ IV 8 0 undifferentiated) Invasion depth (T3+T4 vs T1+T2) 1.845 0.943–3.612 0.074 TNM stage (III+IV vs I+II) 4.628 2.071–10.342 o0.001a Lymphatic 0.175 Lymphatic metastasis (no vs yes) 0.308 0.140–0.678 0.003a metastasis Distant metastasis (no vs yes) 0.461 0.190–1.115 0.086 Yes 24 15 Expression of TINCR (high vs low) 2.242 1.010–4.976 0.047a No 31 10 Multivariate analysis TNM stage (III+IV vs I+II) 3.222 1.268–8.184 0.014a Lymphatic metastasis (no vs yes) 0.570 0.233–1.391 0.517 Distant metastasis 0.052 – Yes 8 0 Expression of TINCR (high vs low) 1.373 0.589 3.200 0.463 No 47 25 Abbreviations: CI, confidence interval; DFS, disease-free survival; HR, hazard a a ratio. Overall Po0.05. Overall Po0.05.

Oncogene (2015) 5648 – 5661 © 2015 Macmillan Publishers Limited TINCR contributes to the oncogenic potential of GC T-p Xu et al 5651 TINCR is a diagnostic marker and high TINCR expression is the high TINCR patients had higher recurrence rates (median DFS: associated with poor prognosis in GC patients 21 months) than the low-TINCR patients (median DFS: 29.7 months, We used the non-tumorous tissues adjacent to the tumor tissues as a P = 0.035; Figure 1g). The 3-year DFS was 31.6% for high TINCR control to produce a receiver operating characteristic curve. The expression patients, whereas the low-TINCR expressing patients had cutoff value for predicting gastric cancer tissues from normal tissues a 3-year DFS of 57.6%. The results of univariate analyses of the clinical was 9.05 (Δ Ct value). The area under the receiver operating variables that were considered to be potential survival predictors characteristic curve was 0.701 (95% CI (confidence interval) = 0.619– are shown in Table 2. Further analysis in a multivariate Cox 0.782, Po0.001; Figure 1f). The sensitivity and specificity were 0.65 proportional hazards model showed that the TNM stage was strongly and 0.71. Kaplan–Meier analysis and log-rank test were used to associated with DFS. The results revealed that TINCR expression was evaluate the effects of TINCR expression and clinicopathological a significant prognostic indicator of DFS (hazard ratio = 2.242; characteristics on disease-free survival (DFS). The results showed that 95% CI, 1.010–4.976; P = 0.047) in patients with GC (Table 2).

Figure 2. Effects of TINCR on GC proliferation and apoptosis in vitro. TINCR knockdown in GC cells transfected with siRNA or shRNA against TINCR or TINCR upregulation by pcDNA3.1-TINCR vector. TINCR depletion inhibits GC cell growth, as detected by the (a) MTT assay and (c) colony-formation assay, whereas ectopic expression of TINCR promotes GC cell growth, as examined by the (b) MTT assay and (d) colony- formation assay. Bars: s.d.; *Po0.05, **Po0.01. (e,f) Cell cycle analyses in the SGC7901 and BGC823 cell lines. Relative to scrambled siRNA- transfected cells, TINCR knockdown induced signiﬁcantly increased number of cells in the G0/G1 phase and reduced the number of cells in the S phase. Relative to empty vector-transfected cells, TINCR upregulation promotes cell cycle progression. Representative ﬂuorescence activated cell sorting images and statistics based on three independent experiments are shown in (e) and (f). Bars: s.d.; *Po0.05, **Po0.01. (g,h) Downregulating TINCR promoted apoptosis in GC cells. Representative FACS images and statistics based on three independent experiments are shown in (g). Bars: s.d.; *Po0.05, **Po0.01. (h) Apoptosis-related proteins were detected by immunoblotting, using extracts from the control and TINCR-depleted GC cells. GAPDH is used as loading control.

Figure 2. Continued

Effects of TINCR on the proliferation and apoptosis of GC cells and shRNA2 significantly inhibited the colony formation of GC cell To determine whether TINCR is required for the maintenance of lines compared with the control Scrambled RNA (Po0.001; malignant phenotypes of GC cells, we used chemically synthesized Figure 2c), whereas TINCR overexpression promoted the cells siRNAs to knock down endogenous TINCR in GC cell lines. To proliferation (Figure 2d). To further explore whether the effects of exclude off-target effects, we designed two different siRNAs, both TINCR on GC cell proliferation reflected cell cycle arrest, we were considered appropriate for TINCR knockdown (Supplementary examined cell cycle progression by using flow cytometric analysis. Figure S1b, upper panel). MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5- The results revealed that GC cells transfected with siRNA1 or siRNA2 diphenyltetrazolium bromide) tetrazolium) assay revealed that cells had an obvious cell cycle arrest in the G0–G1 phase and the transiently transfected with siRNA1 and siRNA2 and not Scrambled population of cells in the S phase was decreased (Figure 2e). In RNA, had significantly inhibited growth and proliferation of GC cells contrast, TINCR overexpression promoted cell cycle progression (Figure 2a). Meanwhile, we induced ectopic overexpression of TINCR (Figure 2f). by transfecting the GC cell lines with the pcDNA3.1-TINCR To determine whether GC cell proliferation was influenced by expression vector. The transfected cells were selected by the apoptosis, we performed flow cytometric analysis. The results addition of G418 (Figure S1b, middle panel). The overexpression of showed that the fraction of apoptotic cells was significantly TINCR significantly increased the growth of both the cell lines increased among the siRNA1- and siRNA2-treated cells as compared (Figure 2b). GC cells that were stably transfected recombinant short with the Scrambled RNA-treated cells (Figure 2g). Furthermore, hairpin RNA (shRNA) and Scrambled shRNA, were used for colony- western blot analysis indicated that BCL-2 expression and cleaved formation assays. The shRNA1 and shRNA2 depleted the TINCR caspase-3 levels were altered in the siRNA-treated GC cells, cellular level to 3 or 5% of normal, respectively (Supplementary confirming that TINCR is involved in cell apoptosis (Figure 2h). Figure S1b, lower panel). The resulting data showed that shRNA1 These results indicated that TINCR may drive GC development.

Oncogene (2015) 5648 – 5661 © 2015 Macmillan Publishers Limited TINCR contributes to the oncogenic potential of GC T-p Xu et al 5653 TINCR regulates cell cycle progression and apoptosis by (Supplementary Table S3; Figure 3a). To further study the involved influencing KLF2, CDKN1A/P21 and CDKN2B/P15 expression pathways activated by TINCR, we analyzed these genes using In order to probe the TINCR-associated pathway on an unbiased data collected from the Kyoto Encyclopedia of Genes and Genomes basis, we assessed the gene expression profiles of GC cells in which (KEGG) and Gene Ontology (GO) databases (Fisher analysis TINCR expression was suppressed. We performed RNA transcrip- P-valueo0.01, false discovery rate o0.01). We found that cell tome sequencing from control or TINCR-depleted SGC7901 cells cycle and apoptosis were both involved in the affected biological using two independent siRNAs (siRNA1 and siRNA2) against TINCR. process in TINCR-depleted cells (Figures 3b and c). Using SGC7901 cells were treated with a control (Scrambled) or TINCR- quantitative reverse transcription PCR, we validated the changes specific siRNAs for 48 h. Analyses of the RNA transcriptome in the cellular levels of a large number of mRNAs involved in cell sequencing data from triplicate samples revealed that a common cycle progression and apoptosis (Figure 3d). set of 326 mRNAs showed ≥ 1.5-fold increased abundance in TINCR- On the basis of our RNA sequencing data, KLF2 mRNA was the most depleted cells (Supplementary Table S3; Figure 3a). Silencing upregulated transcript among the transcripts that were commonly TINCR also reduced the abundance (⩽1.5-fold) of 304 genes regulated upon TINCR downregulation (Supplementary Figure S1c).

Figure 3. RNA transcriptome sequencing analysis in SGC7901 cells revealed that TINCR depletion reduces the expression of genes involved in cell cycle progression and apoptosis, and altered KLF2, CDKN1A/P21 and CDKN2B/P15 expression were involved in TINCR-mediated cell growth and apoptosis. (a) Mean-centered, hierarchical clustering of 630 transcripts altered (⩾1.5-fold change) in scrambled siRNA-treated cells and siRNA-TINCR–treated cells, with three repeats (Scr denotes scrambled siRNA, si-T1 and si-T2 represent siRNA1-TINCR and siRNA2-TINCR, respectively). The (b) KEGG pathway and (c) Gene Ontology analysis for all genes with altered expressions between the scrambled siRNA- treated and siRNA-TINCR–treated cells in vitro. Cell cycle and apoptosis were both among the top signiﬁcant biological processes for genes whose transcript levels were changed in the TINCR-depleted GC cells. (d) Quantitative reverse transcription PCR analyses in control (scr) vs siTINCR (siRNA1 and siRNA2) -treated GC cells reveal altered mRNA levels of genes involved in G1/S transition and apoptosis upon TINCR depletion. Error bars represent s.d., n = 3. *Po0.05, **Po0.01. (e) Ectopic expression of TINCR decreased KLF2, CDKN1A/P21 and CDKN2B/P15 mRNA levels. Error bars represent s.d., n = 3. *Po0.05, **Po0.01. (f,g) Modulating TINCR expression signiﬁcantly affected KLF2, CDKN1A/P21 and CDKN2B/P15 protein expression. Gastric cancer cells were stably transfected with either TINCR siRNA/Scrambled (f), or pcDNA3.1-TINCR/ Empty vector (g), followed by the detection of KLF2, CDKN1A/P21 and CDKN2B/P15 proteins using western blot. The level of GAPDH is used as loading control.

© 2015 Macmillan Publishers Limited Oncogene (2015) 5648 – 5661 TINCR contributes to the oncogenic potential of GC T-p Xu et al 5654 Interestingly, CDKN1A/P21 and CDKN2B/P15, the two important TINCR targets KLF2 via SMD growth inhibitors of cell cycle checkpoints, were also upregulated KLF2 serves a tumor suppressor function in many malignancies25 after TINCR depletion (Supplementary Figure S1c). Next we and is a bona fide SMD target.18 Previous work has identified that manipulated TINCR expression in GC cells and monitored its STAU1 recruits UPF1 via protein–protein interaction to induce KLF2 effect on KLF2, CDKN1A/P21 and CDKN2B/P15 expression. Knock- mRNA degradation in adipogenesis.18 Therefore, we sought to down of TINCR by specific siRNAs substantially increased their determine whether KLF2 is a bona fide SMD target in GC cells. First, expression, whereas ectopic expression of TINCR reduced their we used chemically synthesized siRNAs to knock down endogenous mRNA levels (Figures 3d and e). Consistently, western blot STAU1 and UPF1 in GC cells, and both of them were considered to analysis revealed that the knockdown/overexpression of TINCR be appropriate for STAU1 and UPF1 knockdown, respectively increased /decreased, respectively, their protein level in GC cells (Supplementary Figure S1g, Figure S1h). The mRNA level of KLF2 (Figures 3f and g). increaseduponSTAU1-depletedandUPF1-depletedGCcells (Figure 4c). Second, we performed RNA immunoprecipitation (RIP) fi TINCR binds to cytoplasmic protein STAU1 assays with STAU1 antibody and showed a signi cant enrichment of KLF2 by STAU1 antibody compared with the IgG control, indicating To detect the distribution of TINCR in GC cells, we fractionated GC that STAU1 is associated with KLF2 mRNA (Figure 4a). Third, to cell lines into nuclear and cytoplasmic fractions and thoroughly detect whether STAU1 binds to the 3’UTR of KLF2 mRNA, cells were separated the nucleus from the cytoplasm. The results showed transfected with the following test plasmids: pLUC-KLF2 3’UTR, the that TINCR was present in the cytoplasmic fraction (mean ± s.d., STAU1-FLAG expression vector, pLUC-ARF1 STAU1-binding site, and 74.96 ± 4.67%) and nuclear fraction (25.04 ± 5.83%) of SGC7901 cells, phCMV-MUP reference plasmid, which encodes major urinary in the cytoplasmic fraction (58.41 ± 4.32%) and nuclear fraction protein (MUP) mRNA. The two latter of these served as a positive (41.59 ± 3.83%) of BGC823 cells, and in the cytoplasmic fraction and a negative control, respectively, for STAU1-FLAG binding.15 (53.82 ± 3.11%) and nuclear fraction (46.18 ± 2.63%) of MGC803 cells Anti-FLAG antibody could immunopurify Rluc-KLF2 3’UTR and (Supplementary Figure S1d). These data revealed that most TINCR Rluc-ARF1 STAU1-binding site , but not MUP mRNA (Figure 4d). molecules are located within the cytoplasm in GC cells, indicating These results indicate that KLF2 is a bona fide SMD target in GC cells. that TINCR is likely to bind STAU1 protein, which is mainly expressed To further determine whether TINCR is required for the co-IP of in cytoplasm, and has a posttranscriptional regulation function. RNA STAU1 with KLF2 mRNA, SGC7901 cells that transiently transfected IP revealed significant TINCR enrichment by STAU1 antibody with control siRNA or siRNA against TINCR were immunoprecipitated compared with the IgG control (Figure 4a). In addition, the RNA using anti-STAU1 antibody. Compared with the control siRNA, siRNA- pull-down assay revealed that TINCR is bound to STAU1 protein as TINCR reduced by ~ 1.5-fold the co-IP of STAU1 with KLF2 mRNA well as the UPF1 SMD factor (Figure 4b). These data indicate that (Figure 4e). Furthermore, the RNA pull-down assay revealed that TINCR can act together with STAU1 protein. A recent study has TINCR interacted with KLF2 mRNA (Figure 4f) and that the depletion revealed that STAU1 recognizes an RNA duplex formed by an of STAU1 significantly reduced the interaction of TINCR with KLF2 imperfect base pairing between an Alu element of an mRNA target mRNA (Figure 4g), corroborating that STAU1 is required for the of SMD and another Alu sequence in an lncRNA with a half-STAU1- association between TINCR and KLF2 mRNA. More importantly, the binding site. STAU1-binding to this sequence leads to mRNA KLF2 mRNA half-life was significantly increased upon the down- degradation in a UPF1/2-dependent manner.17 The lack of any regulation of STAU1 or TINCR, whereas it was decreased after TINCR fi change in STAU1 expression in the TINCR knockdown cells overexpression (Figure 4h). Our ndings suggest that TINCR affects (Supplementary Figure S1e) and the lack of any change in TINCR KLF2 mRNA stability and expression through SMD. transcript levels in the STAU1-depleted GC cells (Supplementary Figure 1f, upper panel) indicate that TINCR is not a direct KLF2 inhibits GC cell proliferation and induces apoptosis by degradation target of STAU1 and does not affect STAU1 transcrip- activating CDKN1A/P21 and CDKN2B/P15 tion and translation. In addition, STAU1 does not affect the location KLF2 expression was induced after transfection with a FLAG-tagged of TINCR in GC cells (Supplementary Figure 1f, lower panel). KLF2 expression vector and was knocked down by a siRNA targeting

Figure 4. TINCR interacts with cytoplasmic protein STAU1 and affects KLF2 mRNA stability and expression by forming the TINCR–STAU1 complex. (a) Interaction of TINCR or KLF2 mRNA with STAU1. RIP experiments were performed using the STAU1 antibody to immunoprecipitate (IP) in total-cell extracts of SGC7901 cells, and relative enrichment was determined as RNA associated with STAU1 IP relative to an input control (relative ARF1 enrichment serve as a positive control and U1 snRNA as a negative control that do not interact with STAU1). Error bars represent s.d., n = 3. **Po0.01. (b) Biotinylated TINCR or antisense RNA was incubated with total-cell extracts (SGC7901 cells), targeted with streptavidin beads and washed; the associated proteins were resolved in a gel. Western blotting analysis of the specific association of STAU1 as well as UPF1 with TINCR (n = 3). A nonspecific protein (GAPDH) is shown as a control. (c) KLF2 mRNA expression upon TINCR, STAU1 and UPF1 depletion, as detected by quantitative reverse transcription (qRT–PCR). Error bars represent s.d., n = 3. **Po0.01. (d) IP of STAU1-FLAG. SGC7901 cells were transiently co-transfected with (1) STAU1-FLAG expression plasmid; (2) RLuc-KLF2 3’UTR; (3) phCMV-MUP, which encodes MUP mRNA that lacks an STAU1-binding site (SBS) and serves as a negative control for STAU1-FLAG binding; and (4) Rluc-ARF1 SBS, which contains an ARF1 SBS downstream of the translation termination codon of C-terminally deleted Renilla luciferase and serves as a positive control for STAU1-FLAG binding. After cell lysis, total RNA and protein were purified from the lysate before and after IP using FLAG antibody or nonspecific rabbit (r) IgG. The three leftmost lanes represent twofold serial dilutions of RNA and demonstrate that the RT–PCR is semiquantitative. Schematic representations of the pLUC-KLF2 3’UTR and pLUC-ARF1 SBS test plasmids (above). RT–PCR analysis demonstrates that KLF2 3’UTRs and ARF1 SBS bind STAU1-FLAG, whereas MUP mRNA does not (below). Results are representative of three independently performed experiments. (e) Suppression of the interaction between KLF2 mRNA and STAU1 upon TINCR depletion, as detected by RIP experiments. SGC7901 cells were transfected with control (Scrambled) or siTINCR, and cellular extract was prepared for RIP assay using STAU1 antibody 24 h after transfection. Levels of STAU1 protein of cellular extract both in the control (Scrambled) or the siTINCR group were detected by western blot. GAPDH is used as loading control. Error bars represent s.d., n = 3. *Po0.05. (f) Biotinylated TINCR RNA pulls down the full- length KLF2 mRNA detected by RT–PCR analysis. A nonspecific RNA (GAPDH) is shown as a control. (g) STAU1 depletion reduced the interaction between TINCR and KLF2 mRNA. SGC7901 cells were transfected with control (Scrambled) or si-STAU1, and the cell lysates were incubated with biotin-labeled TINCR; after pulldown, mRNAs were extracted, and KLF2 mRNA was detected by qRT–PCR. Error bars represent s.d., n = 3.*Po0.05; **Po0.01. (h) TINCR and STAU1 control KLF2 mRNA stability. RNA stability assays were performed in SGC7901 cells using Actinomycin D to disrupt RNA synthesis, and the degradation rates of the KLF2 mRNA were measured over 12 h. *Po0.05; **Po0.01.

Oncogene (2015) 5648 – 5661 © 2015 Macmillan Publishers Limited TINCR contributes to the oncogenic potential of GC T-p Xu et al 5655 KLF2 in GC cells. KLF2-overexpressing cells were selected by adding SGC7901 cells (Figure 5d). The proliferation suppression and pro- G418. After KLF2 induction, MTT assays (Figure 5a) and colony- apoptotic effect mediated by KLF2 transfection were also associated formation assays (Figure 5b) revealed a marked reduction in with an increase in the expression levels of the cell cycle-inhibiting proliferation relative to the empty vector-transfected GC cells. genes CDKN2B/P15 and CDKN1A/P21, as determined by western blot Enhanced annexin V expression was also detected on flow assays (Figure 5e). Conversely, KLF2 knockdown by siRNA in the GC cytometry (Figure 5c). Conversely, transient KLF2 depletion of the cells reduced CDKN2B/ P15 and CDKN1A/P21 expression GC cells by siRNA increased cell viability, as revealed by the MTT (Supplementary Figure S2b, Figure S2c). To confirm whether these assay (Supplementary Figure S2a). We extended the study of the two genes could be direct targets of the transcription factor KLF2, KLF2 growth-inhibitory role to in vivo athymic(nu/nu) mouse models, we performed a computational screen. We found two tandem the results demonstrated that KLF2-transfected cells developed putative KLF2 binding sites at the regions − 220 to − 210 bp (E1) and significantly smaller tumors than empty vector-transfected − 66 to − 56 bp (E2) in the CDKN1A/P21 promoter, and three putative

Figure 5. KLF2 inhibits proliferation, induces apoptosis and directly activates the cell cycle-inhibiting genes CDKN2B/ P15 and CDKN1A /P21 in GC cells. (a) MTT and (b) colony-formation assays reveal that stable KLF2-expressing GC cells grow more slowly than cells transfected with the control vector. (c) Enhanced apoptosis relative to empty vector-transfected cells upon KLF2-expressing vector transfection. The apoptotic rates of cells were detected by ﬂow cytometry. (d) Effects of KLF2 overexpression on tumor growth in a xenograft mouse model. SGC7901 cells were transfected with empty vector or KLF2 expression vector and then injected into mouse ﬂanks as described in the text. Tumor growth was measured every 2 days after injection, and the tumors were harvested at day 16 and weighed. (e) GC cells transfected with a FLAG-tagged KLF2 (pCMV-Tag2B-KLF2) expression vector show increased CDKN2B/P15 and CDKN1A /P21 expressions, as determined by western blot. (f, g) qChIP assay demonstrated endogenous KLF2 binding to the CDKN1A/P21 and CDKN2B/P15 gene promoters. qChIP assay showed KLF2 enrichment on (f) CDKN1A/P21 promoter around the E2 region and (g) the CDKN2B/P15 promoter around the E2 and E3 region transfected with FLAG-tagged KLF2 overexpression vector (upper panel) or siRNAs (lower panel) in SGC7901 cells. Error bars represent s.d., n = 3 except for indicated. *Po0.05, **Po0.01.

Figure 6. The KLF2 expression level in GC tissues and cell lines. (a) Real-time PCR analysis of KLF2 expression in the normal gastric epithelium cell line (GES1) and GC cells. Bars: s.d.; n = 3. *Po0.05. (b) KLF2 expression is examined by quantitative reverse transcription PCR in 80 paired human GC tissues and adjacent noncancerous tissues (Wilcoxon's signed-rank test, Po0.01). Data are represented as log2 fold change (tumor/ normal), deﬁned as ‘o − 1’ for underexpression and ‘41’ for overexpression. (c) Analysis of the relationship between TINCR expression (ΔCt value) and KLF2 mRNA level (ΔCt value) in 80 GC tissues. (d) Kaplan–Meier analysis of disease-free survival based on KLF2 expression in all 80 patients. The patients were divided into a low KLF2 expression group (n = 59) and a high KLF2 expression group (n = 21) according to whether their samples showed up or downregulation of TINCR compared with the expression levels in the corresponding adjacent noncancerous tissue samples. (e) Representative KLF2 protein levels and scores in GC tissues (T) and adjacent noncancerous tissues (ANT) was analyzed by immunohistochemistry. (f) Correlation analysis performed between KLF2 expression levels and clinical TNM stages.

© 2015 Macmillan Publishers Limited Oncogene (2015) 5648 – 5661 TINCR contributes to the oncogenic potential of GC T-p Xu et al 5658 binding sites at the regions − 289 to − 279 bp (E1), − 130 to − 120 bp inoculated into flanks of mice, where those infected with (E2) and − 73 to − 48 bp (E3) in the CDKN2B/P15 promoter. We then adenoviral vector carrying sh-Scrambled as control were inocu- conducted ChIP assays. The qChIP analyses demonstrated that lated into the opposite flank of the same mouse. Ad-shRNA endogenous KLF2 bound to the promoter region around E2 of significantly inhibited the tumorigenicity in vivo, as tumor weight CDKN1A/P21 (Figure 5f) and around E2 and E3 of CDKN2B/P15 and size were obviously decreased compared with the controls (Figure 5g) in the transfected SGC7901 cells. To further assess the (Figure 7a). We considered shRNA to be appropriate for TINCR biological roles of KLF2 in CDKN1A/P21 and CDKN2B/P15 expression, knockdown until the subcutaneous tumors were harvested we applied loss- and gain-of-function approaches. We found that (Figure 7b). Moreover, we detected stronger Ki-67 expression in the ectopic expression of KLF2-FLAG-tagged expression vector tumors derived from Scrambled RNA than those derived from (Figures 5f and g, upper panel) and siRNA knockdown (Figures 5f TINCR-shRNA (Figures 7c and d and Supplementary Table S4). and g, lower panel) increased and reduced, respectively, KLF2 Intriguingly, knockdown of TINCR markedly increased the levels of enrichment on the CDKN1A/P21 and CDKN2B/P15 promoters. KLF2, CDKN1A/P21 and CDKN2B/P15 in the xenograft tumors To investigate whether KLF2 was involved in the TINCR- (Figures 7c and d, and Supplementary Table S4). These data depleted induced cell proliferation inhibition and apoptosis support our hypothesis that TINCR contributes to the malignant increase in GC, we carried out rescue experiments with phenotype by inhibiting KLF2, CDKN1A/P21 and CDKN2B/P15. SGC7901 cells. MTT assay indicated that the co-transfection could partially rescue the TINCR silencing-mediated suppression of SGC7901 cell proliferation (Supplementary Figure S2d). Flow DISCUSSION cytometric analysis indicated that co-transfection could partially Recent findings have suggested that many lncRNAs have rescue the increased cell apoptosis induced by TINCR depletion important roles in GC. Two well-known lncRNAs, MALAT1 and (Supplementary Figure S2e). Moreover, we found that the co- HOTAIR, were recently reported to drive GC development and transfection of siTINCR and siKLF2 could rescue the increased promote peritoneal metastasis.27 Our previous work also identified expression of CDKN2B/P15 and CDKN1A/P21 proteins induced by an lncRNA, named FENDRR, that regulates GC metastasis by TINCR depletion (Supplementary Figure S2c). Thus, KLF2 transcrip- affecting fibronectin1 expression.28 Moreover, some lncRNAs, such tional induction is an important step in the proliferation inhibition as GAS5, MEG3 and H19, were reported to regulate proliferation – mediated by TINCR depletion. and/or apoptosis in GC cells.29 31 Therefore, the identification of gastric cancer-associated lncRNAs may provide a missing piece of KLF2 expression is downregulated in cancer tissues and cell lines the well-known oncogenic and tumor suppressor network puzzle. fi and is inversely correlated with TINCR expression In this study, we found that TINCR expression was signi cantly upregulated in GC tissues compared with corresponding non- To assess the relationship between KLF2 and TINCR expression in cancerous tissues. We also explored the mechanism of tissue- GC, we detected KLF2 mRNA by quantitative PCR in 80 pairs of specific TINCR expression and found that SP1 is a key transcription GC tissues and in 4 GC cell lines. We also used immunohisto- factor in controlling lncRNA TINCR expression. Accumulating data chemistry to measure KLF2 protein levels in 42 pairs of GC has revealed that SP1 is overexpressed in GC, and activated SP1 tissues. The results revealed that the mRNA levels of KLF2 were expression represents a potential risk for poor prognosis and generally reduced in GC tissues and cells compared with contributes to GC development and progression.32,33 Specifically, matched normal tissues and cells, respectively (Figure 6a and TINCR expression levels could be used to discriminate the cancer b). Further analysis revealed that TINCR expression was inversely tissues from non-tumorous tissues. Moreover, patients with higher correlated with KLF2 level in GC (Pearson’s correlation coefficient TINCR levels appeared to have a greater depth of tumor invasion, r2 = − 0.562, Po0.001; Figure 6c and Supplementary Table S2). higher tumor stage and shorter DFS than the lower group. Our We divided the samples according to whether they showed up or results indicate that TINCR expression provided a significantly downregulation of KLF2 compared with the expression level in predictive value and prognostic marker for patients with GC. the corresponding adjacent noncancerous tissue samples, and Our data revealed that silencing TINCR expression led to found that low KLF2 expression indicated disease progression significant inhibition of cell proliferation and tumorigenicity and (Figure 6d). We also found that 80% normal gastric tissues promotion of GC cell apoptosis, whereas TINCR overexpression showed a KLF2-positive signal, whereas most tumor-derived contributed to cell growth. Knockdown of TINCR expression tissues exhibited low KLF2 protein levels compared with paired contributed to G1 phase arrest and an S phase reduction, whereas normal tissues (Figure 6e) and displayed lower with disease ectopic overexpression of TINCR promoted cell cycle progression. progression (Figure 6f and Supplementary Table S2). These data To explore the underlying biological mechanisms, we performed indicated that the KLF2 level was mostly opposite to the TINCR RNA transcriptome sequencing to find the altered gene expres- expression level in GC and that low KLF2 expression was sion in control or TINCR-depleted cells. KEGG database and GO associated with GC progression. analysis revealed that cell cycle and apoptosis were involved in the most leading affected biological process in TINCR-depleted TINCR knockdown inhibits tumorigenesis of GC cells in vivo cells, corroborating the cell cycle arrest and apoptosis phenotype. To confirm the impact of TINCR on GC cells growth in vivo,we The G1–S transition in the cell cycle in mammalian cells is generated xenograft models. SGC7901 cells, which were infected controlled by cyclins, cyclin-dependent kinases (CDKs) and their by recombinant adenoviral vector producing-shRNA, were inhibitors, and the deregulation of CDK inhibitors is a common

Figure 7. Targeting TINCR decreased tumor growth and increased KLF2, CDKN1A/P21 and CDKN2B/P15 expression in vivo.(a) Effects of TINCR depletion on tumor growth in a xenograft mouse model. SGC7901 cells were transfected with control shRNA or TINCR-shRNA and then injected into mouse ﬂanks as described in the text. Tumor growth was measured every 4 days after 7 days of injection (right), and tumors were harvested at day 28 and weighed (left). (b) TINCR transcript expression was quantiﬁed by quantitative reverse transcription PCR after treatment of SGC7901 cells with sh-TINCR (shRNAs) and Scramble-control shRNA in subcutaneous tumors harvested at day 28. Data shown are the mean ± s.d. of three independent experiments. **Po0.01. (c) Representative Ki-67, KLF2, CDKN1A/P21 and CDKN2B/P15 protein levels and scores in xenograft tumors evaluated by immunohistochemistry. (d) Correlation analysis performed between Ki-67, KLF2, CDKN1A/P21 and CDKN2B/P15 expression levels and the different xenograft tumors groups, respectively. (e) Summary diagram describes that TINCR regulates GC cell proliferation.

Oncogene (2015) 5648 – 5661 © 2015 Macmillan Publishers Limited TINCR contributes to the oncogenic potential of GC T-p Xu et al 5659 feature in tumor cells.34 CDKN1A/P21 and CDKN2B/P15 serve as upregulated upon TINCR knockdown. However, inconsistent with potent growth inhibitors of cell cycle checkpoints.34 Notably, a recent report,14 both are not signiﬁcantly changed upon TINCR CDKN1A/P21 and CDKN2B/P15 were found to be remarkably depletion in keratinocytes, perhaps due to the different cell types

© 2015 Macmillan Publishers Limited Oncogene (2015) 5648 – 5661 TINCR contributes to the oncogenic potential of GC T-p Xu et al 5660 and context. Interestingly, KLF2, which has been identified as an RNA preparation and quantitative real-time PCR upstream transcriptional factor that regulates CDKN2B/P15 and Total RNAs were extracted with TRIzol reagent (Invitrogen, Grand Island, 35 CDKN1A/P21 expression, was one of the most upregulated NY, USA), and quantitative real-time PCR analyses were conducted transcripts upon TINCR depletion. KLF2 is reported to exert tumor according to the manufacturer’s instructions (Takara, Dalian, China). The suppressor functions in many malignancies, but its role in GC is primers sequences have been listed in Supplementary Table S1. unclear. For the first time, we found that KLF2 could also inhibit cell proliferation and promote apoptosis by upregulating CDKN1A/P21 and CDKN2B/P15 expression in GC. Taken together, Isolation of cytoplasmic and nuclear RNA KLF2, CDKN1A/P21 and CDKN2B/P15 could be crucial TINCR Cytoplasmic and nuclear RNA were isolated and purified using the fi targets. Cytoplasmic & Nuclear RNA Puri cation Kit (Norgen, Belmont, CA, USA), ’ LncRNAs can act together with specific proteins to perform according to the manufacturer s instructions. various functions depending on their subcellular location.36,37 We found that TINCR is a predominantly cytoplasmic lncRNA in GC Construction of recombinant plasmids cells, indicating that its action in posttranscriptional gene Details of the construction of recombinant plasmids have been provided in regulation. The results of RNA IP and RNA pull-down assays show the Supplementary Materials and Methods. that TINCR could bind STAU1, which is consistent with previous data.14 STAU1 is a cytoplasmic protein and exerts multiple effects as a posttranscriptional regulator. On the basis of the interaction Luciferase reporter assay between TINCR and STAU1, we propose that TINCR targets Luciferase reporter assays were performed using a luciferase assay kit downstream transcripts through TINCR–STAU1 complex forma- (Promega, Madison, WI, USA), according to the manufacturer’s protocol. tion. Consistent with previous reports on adipogenesis,18 our The details have been given the in Supplementary Materials and Methods. study found that KLF2 is also a target of STAU1. In addition, KLF2 mRNA stability and the effects of binding to STAU1 are influenced by TINCR depletion. As evidenced above, TINCR may affect KLF2 RNA interference expression through SMD by TINCR–STAU1 complex formation. The details of RNA interference have been included in the Supplementary Furthermore, KLF2 functions as a transactivator of CDKN2B/P15 Materials and Methods, and the sequences of the oligonucleotides and CDKN1A/P21 in GC cells. The pathway via which TINCR synthesized for RNA interference have been listed in Supplementary regulates cell cycle and cells apoptosis has been depicted in Table S1. Figure 7e. The nuclear transcription factor SP1 induces TINCR overexpression. TINCR recruits STAU1 to the 3’UTR of KLF2 mRNA, Cell-proliferation assays degrading KLF2 through the UPF1-dependent mRNA decay Cell-proliferation assays and colony-formation assays were performed as mechanism. Subsequently, the reduced KLF2 abundance con- previously reported in Xu et al.28 tributes to decreased CDKN2B/P15 and CDKN1A/P21 transcripts, promoting cell cycle progression and tumorigenicity. In addition, TINCR or STAU1 may also affect mRNA stability of CDKN2B/P15 and Flow cytometry CDKN1A/P21 based on our previous work (these data were not Cell cycle and cell apoptosis were analyzed by flow cytometry and shown). Therefore, CDKN2B/P15 and CDKN1A/P21 expression detected as previously reported in Xu et al.28 could be regulated both at the transcriptional level by KLF2 and posttranscriptional level via TINCR or STAU1. Here we explored a novel mechanism via which TINCR induces the degradation of its RNA transcriptome sequencing target mRNA, this is the opposite of what was described regarding The details of RNA transcriptome sequencing have been provided in the keratinocyte differentiation by Kretz et al.14 Kretz et al. reported Supplementary Materials and Methods. that the binding of KRT80 and PGLYRP3 (two keratinocyte differentiation genes) to TINCR is independent of STAU1, whereas Bioinformatic analysis we found that the binding of KLF2 to TINCR is dependent on The details of this analysis have been included in the Supplementary STAU1 and that inducing target mRNA degradation is an Materials and Methods. important function of STAU1.15 Therefore, the promotion of mRNA degradation may serve as an important mechanism underlying TINCR functions. Western blot assay and antibodies We describe here a novel mechanism underlying GC cell Western blot assays were performed as previously reported in Xu et al.28 proliferation through a molecular cross-talk between TINCR, STAU1, KLF2, CDKN2B/P15 and CDKN1A/P21. Further insights into the functional and clinical implications of TINCR and its targets, ChIP which are identified as KLF2, CDKN2B/P15 and CDKN1A/P21 may ChIP assays were performed using the EZ ChIP Kit (Millipore, Bedford, MA, contribute to early GC diagnosis and help with GC treatment. USA), according to the manual. The primer sequences were listed in Supplementary Table S1.

MATERIALS AND METHODS RIP and RNA pulldown Cell lines We performed RIP experiments using the Magna RIP RNA-Binding protein The human gastric adenocarcinoma cancer cell lines MGC803, BGC823, immunoprecipitation kit (Millipore, Bedford, MA, USA) according to the MKN45 and SGC7901 and the normal gastric epithelium cell line (GES1) manufacturer’s instructions. The STAU1 and FLAG-tagged antibodies used were obtained from the Chinese Academy of Sciences Committee on Type for IP were from Millipore (Bedford, MA, USA; 03-116; RIPAb+ STAU1) and Culture Collection Cell Bank (Shanghai, China). Cell Signaling Technology (Boston, MA, USA; 8146S), respectively. The details of the primers for reverse transcription PCR and quantitative PCR Tissue samples and clinical data collection have been provided in Supplementary Table S1 and in the Supplementary The details of sample and data collection have been provided in the Materials and Methods. Supplementary Materials and Methods, and the clinicopathological Details of the RNA pull-down assay were available in the Supplementary characteristics of the GC patients have been summarized in Table 1. Materials and Methods.

Oncogene (2015) 5648 – 5661 © 2015 Macmillan Publishers Limited TINCR contributes to the oncogenic potential of GC T-p Xu et al 5661 Immunohistochemistry 13 Wan DF, Gong Y, Qin WX, Zhang PP, Li JJ, Wei L et al. Large-scale cDNA Immunohistochemical analysis was conducted as previously reported in Xu transfection screening for genes related to cancer development and progression. et al.28 The details of the scoring method to detect proteins expression Proc Natl Acad Sci USA 2004; 101: 17565–17565. were available in the Supplementary Materials and Methods. 14 Kretz M, Siprashvili Z, Chu C, Webster DE, Zehnder A, Qu K et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 2013; 493:231–U245. RNA stability assay 15 Kim YK, Furic L, DesGroseillers L, Maquat LE. Mammalian Staufen1 recruits To analyze RNA stability, GC cells were treated with actinomycin D (1 μg/ml). Upf1 to specificmRNA3'UTRssoastoelicitmRNAdecay.Cell 2005; 120: Cells were collected at different time points and the RNA was extracted 195–208. using Trizol reagent (Invitrogen, Grand Island, NY, USA). Reverse 16 Gong CG, Maquat LE. ALUstrious long ncRNAs and their roles in shortening mRNA transcription was performed using oligo (dT) primers and mRNA levels half-lives. Cell Cycle 2011; 10: 1882–1883. were measured using qRT–PCR. 17 Gong CG, Maquat LE. lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements. Nature 2011; 470:284. Tumor xenografts in animals 18 Cho H, Kim KM, Han S, Choe J, Park SG, Choi SS et al. Staufen1-mediated mRNA decay functions in adipogenesis. Mol Cell 2012; 46: 495–506. Athymic(nu/nu) mouse models were prepared as previously reported in 38 19 Kim YK, Furic L, Parisien M, Major F, DesGroseillers L, Maquat LE. Staufen1 Sun et al. regulates diverse classes of mammalian transcripts. EMBO J 2007; 26: 2670–2681. Statistical analysis 20 Black AR, Black JD, Azizkhan-Clifford J. Sp1 and kruppel-like factor family of 188 The details of the statistical analyses were available in the Supplementary transcription factors in cell growth regulation and cancer. J Cell Physiol 2001; : – Materials and Methods. 143 160. 21 Kaczynski J, Cook T, Urrutia R. Sp1-and Kruppel-like transcription factors. Genome Biol 2003; 4: 206. CONFLICT OF INTEREST 22 Wang F, Zhu Y, Huang Y, McAvoy S, Johnson WB, Cheung TH et al. Transcriptional repression of WEE1 by Kruppel-like factor 2 is involved in DNA damage-induced fl The authors declare no con ict of interest. apoptosis. Oncogene 2005; 24: 3875–3885. 23 Duhagon MA, Hurt EM, Sotelo-Silveira JR, Zhang XH, Farrar WL. Genomic profiling of tumor initiating prostatospheres. BMC Genomics 2010; 11: 324. 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