Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 14, 2015; 21(46): 13004-13019
Published online Dec 14, 2015. doi: 10.3748/wjg.v21.i46.13004
HuR mediated post-transcriptional regulation as a new potential adjuvant therapeutic target in chemotherapy for pancreatic cancer
Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas
Aldona Jakstaite, Aurelija Maziukiene, Giedre Silkuniene, Kristina Kmieliute, Antanas Gulbinas, Zilvinas Dambrauskas, Institute for Digestive System Research, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania
Antanas Gulbinas, Zilvinas Dambrauskas, Department of Surgery, Lithuanian University of Health Sciences, 50161 Kaunas, Lithuania
Author contributions: Jakstaite A carried out the molecular RT-PCR studies and drafted the manuscript; Silkuniene G performed the western blot analysis; Maziukiene A maintained cell cultures and did the transfection experiments; Kmieliute K carried out the microscopic analysis; Dambrauskas Z designed and planned the study and performed the statistical data analysis; Gulbinas A conceived the study, participated in its design and coordination, and helped to draft the manuscript; All authors read and approved the final manuscript.
Supported by The European Social Fund under the Global Grant measure.
Institutional review board statement: Ethical approval was issued by the Ethics Committee of the Lithuanian University of Health Sciences (Nr. BE-2-10 and BE-2-17).
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Dr. Aldona Jakstaite, Institute for Digestive System Research, Lithuanian University of Health Sciences, Eiveniu str. 4, 50161 Kaunas, Lithuania. aldona.jakstaite@gmail.com
Telephone: +370-37-60880820
Received: March 4, 2015
Peer-review started: March 5, 2015
First decision: April 24, 2015
Revised: May 9, 2015
Accepted: August 28, 2015
Article in press: August 31, 2015
Published online: December 14, 2015
Processing time: 279 Days and 22.6 Hours
Abstract

AIM: To investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA) and to assess the effects of HuR silencing on the expression of cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1) and the in vitro response to gemcitabine (GEM) treatment in pancreatic cell lines.

METHODS: We compared the expression of HuR, COX-2, and HO-1 in PDA and normal pancreatic tissue using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot. In addition, the HuR, COX-2 and HO-1 were analyzed in four types of cancer cell lines (MiaPaca2, Su.86.86, Capan-1, and Capan-2) with and without GEM treatment. Immunocytofluorescence analysis was used to investigate HuR localization in cells. Cell viability and response to GEM after HuR silencing were determined with the 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide test and the crystal violet clonogenic assay, respectively. To measure apoptosis, activation of caspases 3/7 was evaluated using immunofluorescence.

RESULTS: In PDA tissue obtained from patients not treated with GEM, HuR mRNA expression was 3.2 times lower (P < 0.05) and COX-2 and HO-1 mRNA expression was 2.3-fold and 7.2-fold higher (P < 0.05), respectively, than normal pancreatic tissue (from organ donor). qRT-PCR analysis showed that HuR, COX-2, and HO-1 mRNA were overexpressed in all cancer cell lines treated with the half maximal inhibitory concentration (IC50) dose of GEM compared with control cells (P < 0.05). Western blot analysis revealed that COX-2 and HO-1 levels were significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing increased the response to GEM treatment and decreased cell viability by 11.6%-53.7% compared to control cell lines. Caspases 3 and 7 were activated after HuR silencing and GEM treatment in all pancreatic cancer cell lines. In comparison, treatment with GEM alone did not activate caspases 3 and 7 in the same cell lines.

CONCLUSION: HuR mediated post-transcriptional upregulation of COX-2 and HO-1 expression after GEM treatment in pancreatic cancer cells. HuR silencing significantly increased the effectiveness of GEM treatment in vitro.

Keywords: HuR; Pancreatic cancer; Chemotherapy; Cyclooxygenase-2; Post-transcriptional regulation; Heme oxygenase-1

Core tip: The mRNA binding protein HuR is normally located in the nucleus and is activated by specific stressors. HuR has been reported to regulate the expression of many proteins by different post-transcriptional mechanisms, including mRNA trafficking, mRNA stabilization, and protein translation. HuR is also involved in the post-transcriptional modification of cytoprotective molecules, such as cyclooxygenase-2 and heme oxygenase-1, which may be related to the increased resistance of pancreatic cancer to chemotherapy. HuR silencing significantly increases the effectiveness of gemcitabine treatment in vitro.