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MicroRNAs (miRNAs) are unique in that a single miRNA molecule regulates a vast number of RNA transcripts. Thus, aberrantly expressed miRNAs disrupt tightly controlled RNA networks in cancer cells. Our functional screening showed that expression of miR-124-3p was downregulated in pancreatic ductal adenocarcinoma (PDAC) tissues. Here, we aimed to investigate the anti-tumor roles of miR-124-3p in PDAC cells and to identify miR-124-3p-mediated oncogenic signaling in this disease. Ectopic expression of miR-124-3p inhibited cancer cell migration and invasion in PDAC cells. Moreover, restoration of miR-124-3p suppressed oncogenic signaling, as demonstrated by reduced phosphorylation of focal adhesion kinase, AKT, and extracellular signal-regulated kinase, in PDAC cells. Our in silico database analyses and luciferase reporter assays showed that two cell-surface matrix receptors, integrin α3 (ITGA3) and integrin β1 (ITGB1), were directly regulated by miR-124-3p in PDAC cells. Overexpression of ITGA3 and ITGB1 was confirmed in PDAC clinical specimens. Interestingly, a large number of cohort analyses from TCGA database showed that high expressions of ITGA3 and ITGB1 were significantly associated with poor prognosis of patients with PDAC. Knockdown of ITGA3 and ITGB1 by siRNAs markedly suppressed the migration and invasion abilities of PDAC cells. Moreover, downstream oncogenic signaling was inhibited by ectopic expression of miR-124-3p or knockdown of the two integrins. The discovery of anti-tumor miRNAs and miRNA-mediated oncogenic signaling may provide novel therapeutic targets for the treatment of PDAC.

// Keiichi Yonemori 1 , Naohiko Seki 2 , Tetsuya Idichi 1 , Hiroshi Kurahara 1 , Yusaku Osako 1 , Keiichi Koshizuka 2 , Takayuki Arai 2 , Atsushi Okato 2 , Yoshiaki Kita 1 , Takaaki Arigami 1 , Yuko Mataki 1 , Yuko Kijima 1 , Kosei Maemura 1 and Shoji Natsugoe 1 1 Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical Sciences, Kagoshima University, Sakuragaoka, Kagoshima 890-8520, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chuo-ku, Chiba 260-8670, Japan Correspondence to: Naohiko Seki, email: naoseki@faculty.chiba-u.jp Keywords: pancreatic ductal adenocarcinoma, microRNA, expression signature, miR-216b-3p , FOXQ1 Received: February 07, 2017 Accepted: June 26, 2017 Published: July 26, 2017 ABSTRACT We analysed the RNA sequence-based microRNA (miRNA) signature of pancreatic ductal adenocarcinoma (PDAC). Aberrantly expressed miRNAs were successfully identified in this signature. Using the PDAC signature, we focused on 4 clustered miRNAs, miR-216a-5p , miR-216a-3p , miR-216b-5p and miR-216b-3p on human chromosome 2p16.1. All members of the miR-216 cluster were significantly reduced in PDAC specimens. Ectopic expression of these miRNAs suppressed cancer cell aggressiveness, suggesting miR-216 cluster as anti-tumour miRNAs in PDAC cells. The impact of miR-216b-3p (passenger strand of pre- miR-216b ) on cancer cells is still ambiguous. Forkhead box Q1 ( FOXQ1 ) was directly regulated by miR-216b-3p and overexpression of FOXQ1 was confirmed in clinical specimens. High expression of FOXQ1 predicted a shorter survival of patients with PDAC by Kaplan–Meier analysis. Loss-of-function assays showed that cancer cell migration and invasion activities were significantly reduced by si FOXQ1 transfectants. We investigated pathways downstream from FOXQ1 by using genome-wide gene expression analysis. Identification of the miR-216-3p/FOXQ1 -mediated network in PDAC should enhance understanding of PDAC aggressiveness at the molecular level.

// Tetsuya Idichi 1 , Naohiko Seki 2 , Hiroshi Kurahara 1 , Keiichi Yonemori 1 , Yusaku Osako 1 , Takayuki Arai 2 , Atsushi Okato 2 , Yoshiaki Kita 1 , Takaaki Arigami 1 , Yuko Mataki 1 , Yuko Kijima 1 , Kosei Maemura 1 and Shoji Natsugoe 1 1 Department of Digestive Surgery, Breast and Thyroid Surgery, Graduate School of Medical Sciences, Kagoshima University, Kagoshima, Japan 2 Department of Functional Genomics, Chiba University Graduate School of Medicine, Chiba, Japan Correspondence to: Naohiko Seki, email: naoseki@faculty.chiba-u.jp Keywords: microRNA, miR-217 , pancreatic ductal adenocarcinoma, ANLN , tumor-suppressor Received: January 11, 2017 Accepted: May 08, 2017 Published: May 29, 2017 ABSTRACT Analysis of our microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC) revealed that microRNA-217 ( miR-217 ) was significantly reduced in cancer tissues. The aim of this study was to investigate the antitumor roles of miR-217 in PDAC cells and to identify miR-217 -mediated molecular pathways involved in PDAC aggressiveness. The expression levels of miR-217 were significantly reduced in PDAC clinical specimens. Ectopic expression of miR-217 significantly suppressed cancer cell migration and invasion. Transcription of actin-binding protein Anillin (coded by ANLN ) was detected by our in silico and gene expression analyses. Moreover, luciferase reporter assays showed that ANLN was a direct target of miR-217 in PDAC cells. Overexpression of ANLN was detected in PDAC clinical specimens by real-time PCR methods and immunohistochemistry. Interestingly, Kaplan–Meier survival curves showed that high expression of ANLN predicted shorter survival in patients with PDAC by TCGA database analysis. Silencing ANLN expression markedly inhibited cancer cell migration and invasion capabilities of PDAC cell lines. We further investigated ANLN -mediated downstream pathways in PDAC cells. “Focal adhesion” and “Regulation of actin binding protein” were identified as ANLN -modulated downstream pathways in PDAC cells. Identification of antitumor miR-217 / ANLN -mediated PDAC pathways will provide new insights into the potential mechanisms underlying the aggressive course of PDAC.

We analysed the RNA sequence-based microRNA (miRNA) signature of pancreatic ductal adenocarcinoma (PDAC). Aberrantly expressed miRNAs were successfully identified in this signature. Using the PDAC signature, we focused on 4 clustered miRNAs, miR-216a-5p, miR-216a-3p, miR-216b-5p and miR-216b-3p on human chromosome 2p16.1. All members of the miR-216 cluster were significantly reduced in PDAC specimens. Ectopic expression of these miRNAs suppressed cancer cell aggressiveness, suggesting miR-216 cluster as anti-tumour miRNAs in PDAC cells. The impact of miR-216b-3p (passenger strand of pre-miR-216b) on cancer cells is still ambiguous. Forkhead box Q1 (FOXQ1) was directly regulated by miR-216b-3p and overexpression of FOXQ1 was confirmed in clinical specimens. High expression of FOXQ1 predicted a shorter survival of patients with PDAC by Kaplan–Meier analysis. Loss-of-function assays showed that cancer cell migration and invasion activities were significantly reduced by siFOXQ1 transfectants. We investigated pathways downstream from FOXQ1 by using genome-wide gene expression analysis. Identification of the miR-216-3p/FOXQ1-mediated network in PDAC should enhance understanding of PDAC aggressiveness at the molecular level.

Analysis of our microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC) revealed that microRNA-217 (miR-217) was significantly reduced in cancer tissues. The aim of this study was to investigate the antitumor roles of miR-217 in PDAC cells and to identify miR-217-mediated molecular pathways involved in PDAC aggressiveness. The expression levels of miR-217 were significantly reduced in PDAC clinical specimens. Ectopic expression of miR-217 significantly suppressed cancer cell migration and invasion. Transcription of actin-binding protein Anillin (coded by ANLN) was detected by our in silico and gene expression analyses. Moreover, luciferase reporter assays showed that ANLN was a direct target of miR-217 in PDAC cells. Overexpression of ANLN was detected in PDAC clinical specimens by real-time PCR methods and immunohistochemistry. Interestingly, Kaplan–Meier survival curves showed that high expression of ANLN predicted shorter survival in patients with PDAC by TCGA database analysis. Silencing ANLN expression markedly inhibited cancer cell migration and invasion capabilities of PDAC cell lines. We further investigated ANLN-mediated downstream pathways in PDAC cells. “Focal adhesion” and “Regulation of actin binding protein” were identified as ANLN-modulated downstream pathways in PDAC cells. Identification of antitumor miR-217/ANLN-mediated PDAC pathways will provide new insights into the potential mechanisms underlying the aggressive course of PDAC.