Tumor suppressive microRNA-1 mediated novel apoptosis pathways through direct inhibition of splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) in bladder cancer Hirofumi Yoshino a , Hideki Enokida a,⇑ , Takeshi Chiyomaru a , Shuichi Tatarano a , Hideo Hidaka a , Takeshi Yamasaki a , Takenari Gotannda a , Tokushi Tachiwada a , Nijiro Nohata b , Takashi Yamane a , Naohiko Seki b , Masayuki Nakagawa a a Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan b Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan article info Article history: Received 25 November 2011 Available online 9 December 2011 Keywords: SRSF9/SRp30c MicroRNA miR-1 Apoptosis Caspase-3/7 Bladder cancer abstract We have previously found that restoration of tumor suppressive microRNA-1 (miR-1), induced cell apop- tosis in bladder cancer (BC) cell lines. However, the apoptosis mechanism induced by miR-1 was not fully elucidated. Alternative splicing of mRNA precursors provides cancer cells with opportunities to translate many oncogenic protein variants, which promote cell proliferation and survival under unpreferable con- dition for cancer development. Serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splicing, plays a critical role for regulating apoptosis by splicing apoptosis-related genes. How- ever, transcriptional regulation of SR proteins, themselves, has not been elucidated. In this study, we focused on splicing factor serine/arginine-rich 9 (SRSF9/SRp30c) on the basis of our previous genome-wide gene expression analysis using miR-1-transfected BC cell lines because putative target sites of miR-1 are existed in 3 0 -untranslated region (UTR) of SRSF9 mRNA. The expression levels of mRNA of SRSF9 were extremely reduced in the miR-1 transfectants. A luciferase activity significantly decreased in the transfec- tants suggesting that actual binding occurred between miR-1 and 3 0 UTR of SRSF9 mRNA. Loss-of-function assays demonstrated that significant inhibitions of cell proliferation, migration, and invasion were observed in the si-SRSF9 transfectants. Apoptosis assays demonstrated that cell apoptosis fraction increased and that caspase-3/7 was activated in the si-SRSF9 transfectants. Our data indicated that tumor suppressive miR-1 induces apoptosis through direct inhibition of SRSF9 in BC. The identification of molec- ular mechanisms between miRNAs and SR proteins could provide novel apoptosis pathways and their epigenetic regulations and offer new strategies for BC treatment. Ó 2011 Elsevier Inc. All rights reserved. 1. Introduction Bladder cancer (BC) is a common malignancy with high mortal- ity in the world [1]. The disease divides into non-muscle-invasive tumors and muscle-invasive cancer. Although 70–80% of BC are diagnosed as non-muscle-invasive tumors at the first treatment, recurrence occur about 70% of them. Among recurrent tumors, 10–15% proceed to muscle invasion and metastasis [2]. There have been significant advances in medical science and treatment, including surgical techniques and adjuvant chemotherapy; how- ever, the biology of BC is incompletely understood, and muscle- invasive BC is especially difficult to cure. Therefore, new treatment modalities based on novel molecular networks in BC are desired. Alternative splicing is indispensable for allowing individual genes to express multiple mRNAs, which provide proteins with di- verse and even antagonistic functions [3]. In cancer field, different splice variants caused by splice change are associated with prolif- eration, adhesion, differentiation, and invasion [4]. Serine/arginine- rich splicing factor 9 (SRSF9) is a member of the serine/arginine-rich (SR) protein family, which involved in alternative pre-mRNA splic- ing. Recent studies have revealed that changes in levels of various SR proteins might be associated with different mRNA products, and it can be linked to cancer development, and that depletion of SRSF9 reduced cancer viability in ovarian cancer and prostate cancer (PC) [5–7]. However, functional role of SRSF9 is still unknown as of now. MicroRNAs (miRNAs) are a class of small non-coding RNA mol- ecules of 20–22 nucleotides that post-transcriptionally modulate protein expression. Although their biological functions remain 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.12.011 ⇑ Corresponding author. Address: Department of Urology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshi- ma 890-8520, Japan. Fax: +81 99 265 9727. E-mail address: enokida@m.kufm.kagoshima-u.ac.jp (H. Enokida). Biochemical and Biophysical Research Communications 417 (2012) 588–593 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc