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Circptpra Acts As a Tumor Suppressor in Bladder Cancer by Sponging Mir-636 and Upregulating KLF9

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Circptpra Acts As a Tumor Suppressor in Bladder Cancer by Sponging Mir-636 and Upregulating KLF9 www.aging-us.com AGING 2019, Vol. 11, No. 23 Research Paper CircPTPRA acts as a tumor suppressor in bladder cancer by sponging miR-636 and upregulating KLF9 Qingqing He1,2,*, Lifang Huang2,*, Dong Yan1,2,*, Junming Bi1,2, Meihua Yang1,2, Jian Huang1,2, Tianxin Lin1,2 1Department of Urology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China 2Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China *Equal contribution Correspondence to: Jian Huang, Tianxin Lin; email: [email protected], [email protected] Keywords: bladder cancer, circPTPRA, miR-636, KLF9, proliferation Received: September 4, 2019 Accepted: November 18, 2019 Published: December 10, 2019 Copyright: He et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT Growing evidence suggests that circular RNAs (circRNAs) play pivotal roles in cancer progression. In this study, bioinformatic analysis identified a dysregulated circRNA termed circPTPRA in bladder cancer (BC). By using qRT- PCR analysis, we verified that circPTPRA is down-regulated in clinical BC specimens compared with the matched non-tumor samples, while correlation analyses showed that low circPTPRA expression is associated with poor prognosis, advanced tumor stage and larger tumor size. Based on these findings, we conducted functional assays and revealed that circPTPRA inhibits BC cell proliferation in vitro and tumor growth in vivo. In addition, RNA pull-down, miRNA capture, FISH, and luciferase reporter assays demonstrated that circPTPRA can directly sponge miR-636. Cell transfection experiments showed that miR-636 promotes the proliferation of BC cells by decreasing the expression of Krüppel Like Factor 9 (KLF9) upon binding to the 3’UTR of its mRNA. Further analysis confirmed that circPTPRA competitively sponges miR-636 to upregulate the KLF9 expression, leading to decreased proliferation of BC cells. Our investigation indicates that circPTPRA acts as a tumor suppressor in BC, and suggests that this circRNA may be a novel prognostic biomarker and therapeutic target in BC. INTRODUCTION elucidating the mechanisms underlying BC cell proliferation is crucial to design new approaches to Bladder cancer is the ninth most common cancer prevent metastasis. worldwide with an estimated 549, 000 newly diagnosed cases and 200, 000 deaths in 2018 [1, 2]. Most BC cases Circular RNAs (circRNAs) constitute a large class of are diagnosed as non-muscle invasive BC (NMIBC), single-stranded, non-coding RNA molecules that which has favorable prognosis following transurethral attracted great research interest in recent years. resection and intravesical perfusion [3]. Unfortunately, CircRNAs are generated by back-splicing of pre-mRNA NMIBC always relapses and progresses to muscle and form covalently closed, continuous loop structures invasive BC (MIBC), which often metastasizes to without terminal 5’ caps and 3’ polyadenylated tails [6, lymph node and distant organs [4]. Despite standard 7]. Initially, circRNAs were misinterpreted as the treatment strategies such as surgery, radiotherapy and byproducts of splicing errors [8]. With the development chemotherapy, BC metastasis is seldom controlled and of high-throughput sequencing and bioinformatic leads to poor prognosis. The proliferation of BC and its analysis, many circRNAs have been discovered in infiltration depth promote metastasis [5]. Thus, multiple species; based on functional assays, regulatory www.aging-us.com 11314 AGING roles for circRNAs in biological processes, including verified to exclude the possibility that it resulted from a cancer, have been proposed [9–11]. These include gene rearrangement event. Thus, we designed divergent translation modulation by microRNA (miRNA) primers for circPTPRA and convergent primers for sponging, transcriptional regulation, protein binding, PTPRA. Agarose gel electrophoresis indicated that and encoding protein [12–15]. circPTPRA was only amplified by divergent primers using cDNA but not gDNA from BC cells (Figure 1B). The most studied circRNA role is arguably miRNA By browsing the human reference genome sponging. By inhibiting the association of miRNAs with (GRCh37/hg19), we identified that circPTPRA was the UTRs of mRNAs, circRNAs are considered derived from the exons 8 and 9 of the PTPRA gene competitive endogenous RNAs (ceRNAs) that modulate (Figure 1C). Sanger sequencing of PCR products of protein translation [12]. Emerging evidence revealed divergent primers validated the existence of the back- that several circRNAs may influence tumor splicing junction site of circPTPRA (Figure 1C). proliferation, invasion and tumor immunity, and may Additionally, an actinomycin D assay revealed that aid prognosis assessment in several cancers [16–18]. circPTPRA was more stable than the linear PTPRA For example, circPVT1 promotes gastric cancer mRNA, and its half-life was more than 24h (Figure 1D, proliferation by sponging members of the miR-125 1E). Moreover, an RNase R assay showed that family and was proposed as an independent prognostic circPTPRA was resistant to RNase R, whereas PTPRA marker for overall survival [16]. CircABCB10 mRNA was not (Figure 1F). To identify the location of modulates proliferation and progression of breast cancer circPTPRA in BC cells, we conducted a nuclear and by sponging miR-1271 [18], and circTP63 promotes cytoplasmic extraction assay which indicated that lung squamous cell carcinoma progression by circPTPRA was mostly located in the cytoplasm of BC competitively binding to miR-873-3p and upregulating cells (Figure 1G). The same result was obtained through FOXM1 [19]. Although several studies assessed the FISH assay (Figure 1H). roles of circRNAs in BC progression, the mechanisms by which they affect proliferation of BC cells remain Expression of circPTPRA in human BC specimens largely unknown [12, 20]. and clinical significance Here, we analyzed the circRNA profile of three clinical To further verify the expression of circPTPRA in BC, 64 BC samples and paired adjacent normal bladder tissues matched BC and adjacent normal specimens were retrieved from the GEO database (GEO97239), and analyzed by qRT-PCR. Results confirmed that identified circPTPRA (circBase ID: hsa_circ_0006117) circPTPRA was downregulated in BC tissues compared as a dysregulated circRNA in BC. Subsequently, the with normal tissues (Figure 2A). Additionally, we correlation between circPTPRA expression and clinical analyzed the expression of circPTPRA in 104 BC parameters, and functional assays were conducted to specimens and found that both advanced tumor stage (T2- investigate potential functions of circPTPRA in BC and T4) and tumor size (≥3cm) correlated with low identify miRNA and gene targets. Our results indicate circPTPRA expression (Figure 2B, 2C). Then we divided circPTPRA acts as a tumor suppressor in BC, and patient samples into high and low circPTPRA groups, and suggest a potential role for this novel circRNA as a the Chi-square test indicated that circPTPRA expression prognostic biomarker and therapeutic target. was indeed associated with tumor stage and size, but not with other clinical parameters (Table 1). Moreover, RESULTS survival analyses indicated poor prognosis for BC patients with low circPTPRA expression (Figure 2D). Identification and characterization of circPTPRA circPTPRA inhibits the proliferation of BC cells in To identify dysregulated circRNAs in BC, we screened vitro three matched BC and adjacent normal bladder tissue sets retrieved from the GEO database (GEO97239). We To illustrate the function of circPTPRA in BC cells, found that circPTPRA (circBase ID: hsa_circ_0006117) gain- and loss-of-function assays were performed in was significantly downregulated in BC samples, and vitro. First, two siRNAs targeting the back-splicing selected this circRNA for further characterization. junction site of circPTPRA were designed (Figure 3A). Expression analysis using qRT-PCR indicated lower The expression of circPTPRA was significantly circPTPRA levels in two BC cell lines, T24 and UM- decreased after transfection of these siRNAs in BC UC-3, compared to normal bladder epithelial SV-HUC- cells, but without obvious changing in the level of 1 cells (Figure 1A). Because circPTPRA originates PTPRA mRNA (Figure 3B). After construction of BC from PTPRA (protein tyrosine phosphatase receptor cell lines with stable expression of circPTPRA, qRT- type A) mRNA, the existence of circPTPRA was www.aging-us.com 11315 AGING Figure 1. Characterization of circPTPRA in BC cell lines. (A) Expression of circPTPRA in normal SV-HUC-1 cells and two BC cell lines (T24 and UM-UC-3). (B) Gel electrophoresis of qRT-PCR products resulting from divergent and convergent primers. GAPDH was used as internal control. (C) Schematic diagram depicting the circPTPRA’s origin from exons 8 and 9 of the PTPRA gene. Sanger sequencing confirmed the back-splicing junction site (blue arrow). (D, E) Analysis of PTPRA mRNA and circPTPRA by qRT-PCR in BC cell lines after actinomycin D treatment. (F) PTPRA mRNA and circPTPRA levels measured by qRT-PCR after RNase R treatment in BC cell lines. (G) Cellular localization of circPTPRA in BC cell lines, as assessed by cytoplasmic and nuclear fractionation assay. (H) FISH assay of indicating
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