Regulation of actin-binding protein ANLN by antitumormiR-217inhibits cancer cell aggressiveness in pancreatic ductal adenocarcinoma

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.

Molecular pathogenesis of pancreatic ductal adenocarcinoma: Impact of passenger strand of pre‐miR‐148a on gene regulation

“…Quantitative real‐time PCR (qRT‐PCR) of clinical samples was carried out by extracted total RNA from frozen specimens, immunohistochemistry was performed by PDAC tissues from formalin‐fixed paraffin‐embedded. Two human PDAC cell lines (PANC‐1 and SW1990) were used in this study as described previously …”

“…Protocols for total RNA extraction from clinical specimens and cell lines were described in previous studies. The procedure for quantification of miRNAs and mRNAs was described earlier . TaqMan qRT‐PCR probes and primers were obtained from Thermo Fisher Scientific (Waltham, MA, USA) as follows: miR‐148a‐5p (product ID: 002134); miR‐148a‐3p (product ID: 000470); PHLDA2 (product ID: Hs00169368_m1); LPCAT2 (product ID: Hs01044164_m1); AP1S3 (product ID: Hs00950999_m1); SMS (product ID: Hs01924834_u1); ENDOD1 (product ID: Hs00826684_m1); UHMK1 (product ID: Hs00332674_m1).…”

“…Protocols for functional assays (cell proliferation, migration and invasion) were described previously …”

“…Antibodies against PHLDA2 (product ID: ab99209; Abcam, Tokyo, Japan), poly (ADP‐ribose) polymerase (PARP) antibody (product ID: 9542; Cell Signaling Technology, Danvers, MA, USA) and GAPDH (product ID: SAF6698; Wako) were used in this study. The procedures for western blotting and tissue immunohistochemistry were as described previously …”

“…To identify genes targeted by miR‐148a‐5p and miR‐148a‐3p , we used a combination of in silico analyses and genome‐wide gene expression analyses as described previously . Expression data by microarray analyses were deposited into the Gene Expression Omnibus (GEO) database (accession numbers , ).…”