Primer sets used in the present study.

<p>Primer sets used in the present study.</p

Schematic diagram of UCA1 regulation by Ets-2.

<p>Dashed line from UCA1 to Akt indicates that the association between UCA1 and Akt may be indirect.</p

Ets-2 Regulates Cell Apoptosis via the Akt Pathway, through the Regulation of Urothelial Cancer Associated 1, a Long Non-Coding RNA, in Bladder Cancer Cells

<div><p>The majority of the human genome is transcribed and generates non-coding RNAs (ncRNAs) that fail to encode protein information. Long non-coding RNAs (lncRNAs) are emerging as a novel class of ncRNAs, but our knowledge about these ncRNAs is limited. Previously, our laboratory has identified that a lncRNA, Urothelial cancer associated 1 (UCA1), played an important role in bladder cancer. Despite the recent interest in UCA1 as a diagnostic marker for bladder cancer, little is known about its transcriptional regulation. To elucidate the regulation of UCA1 gene expression, we have characterized the human UCA1 gene promoter. A 2.0-kb fragment of its 5′ flanking region was cloned into a luciferase reporter vector. Deletion and mutation analysis suggested that an Ets-2 binding site was critical for UCA1 gene promoter activity. Further analysis of this site by gel shifting, chromatin immune precipitation (ChIP), and co-transfection experiments showed that transcription factor Ets-2 directly bound to the UCA1 promoter region and stimulated UCA1 promoter activity in bladder cancer cells. Taking into account the anti-apoptosis function of Ets-2, our data suggested that Ets-2 regulates apoptosis process by regulating the expression of UCA1, moreover UCA1 may be involved in the activation of Akt signaling pathway by Ets-2 in bladder cancer cells.</p></div

UCA1 may participate in the inactivation of Akt apoptotic pathway induced by Ets-2 down-regulation.

<p>Ets-2 and apoptosis related proteins were detected by Western blot. GAPDH was used as an internal control. NSC: non-specific control siRNA.</p

Ets-2-binding sites contribute to promoter activation of the UCA1.

<p>(A) Luciferase assay. The Ets-2 binding sites (GGGAAG) were replaced with <i>Hind</i>III sites (AAGCTT) by site-directed mutagenesis (upper panel). Relative luciferase activities of the wild-type (wt) and the mutant vectors (mutant) were measured by luciferase assay. Values represent means ± SD of at least three independent experiments. *<i>P</i><0.05. (B) <i>In vitro</i> detection of Ets-2 transcription factor binding to the promoter region of the UCA1 gene by EMSA assay. One predominant band was observed when biotin-labeled Ets-2 probe was incubated with the nuclear extract (lane 2). Ets-2 binding could not be inhibited by the mutant probe (lane 3) while it was competed with the unlabeled cold probe (lane 4). (C) <i>In vivo</i> detection of Ets-2 transcription factor binding to the promoter region of the UCA1 gene promoter by ChIP assay. Input chromatin (Input), which represented portions of sonicated chromatin before immunoprecipitation, was used as a positive control. IgG antibody was used as a negtive control of ChIP assay.</p

Ets-2 is essential to the transcription regulation of the UCA1.

<p>(A) Expression of Ets-2 and UCA1 was measured by RT-PCR following Ets-2 specific siRNA or scrambled siRNA treatment in BLZ-211 cells. 18 S rRNA was used as an internal control. (B) Luciferase assay. Ets-2 specific siRNA or scrambled control siRNA was co-transfected with pGL3-UCA1-1200 into BLZ-211 cells. Values represent means ± SD of at least three independent experiments. *<i>P</i><0.05. NSC: non-specific control siRNA.</p

UCA1 may be involved in apoptosis induced by Ets-2 knockdown in BLZ-211 cells.

<p>(A) The induction of apoptosis following a 48-hour treatment of Ets-2 siRNA or scrambled control siRNAs in BLZ-211 cells was examined by using a fluorescence microscope. Cells were stained with Annexin V (Green) and PI (Red). Scale bar 25 µm. The images were representative of three independent experiments. (B) Apoptosis was quantified by flow cytometry (FCM). The FCM results were representative of three independent experiments. Cellular pro-apoptosis processes (upper right quadrant) were increased after Ets-2 knockdown in BLZ-211 cells (12.49±1.21% vs. 6.40±1.47%, *<i>P</i><0.05) while no significant change in BLS-211 cells (4.12±0.28% vs. 3.20±2.50%, <i>P</i>>0.05). NSC: non-specific control siRNA.</p