Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Aug 14, 2015; 21(30): 9093-9102
Published online Aug 14, 2015. doi: 10.3748/wjg.v21.i30.9093
Mechanisms of pyruvate kinase M2 isoform inhibits cell motility in hepatocellular carcinoma cells
Yan-Ling Chen, Jun-Jiao Song, Xiao-Chun Chen, Wei Xu, Qiang Zhi, Yun-Peng Liu, Hong-Zhi Xu, Jin-Shui Pan, Jian-Lin Ren, Bayasi Guleng
Yan-Ling Chen, Jun-Jiao Song, Xiao-Chun Chen, Wei Xu, Qiang Zhi, Yun-Peng Liu, Hong-Zhi Xu, Jin-Shui Pan, Jian-Lin Ren, Bayasi Guleng, Department of Gastroenterology, Zhongshan Hospital affiliated to Xiamen University, Xiamen 361004, Fujian Province, China
Author contributions: Chen YL and Song JJ contributed equally to this work; Chen YL, Song JJ, Ren JL and Guleng B designed the study; Chen YL, Song JJ, Chen XC, Xu W, Zhi Q and Liu YP carried out the study; Xu HZ and Pan JS contributed new reagents and annalytic tools; Chen YL, Song JJ and Liu YP analyzed the data; Liu YP and Guleng B wrote the paper.
Supported by National Natural Science Foundation of China, No. 81370505, No. 81370591, No. 81225025, No. 81100285 and No. 91229201; grants from Ministry of Health Foundation for State Key Clinical Department, 863 and 973 programs in China, No. 2012AA02A201 and No. 2013CB944903.
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. Bayasi Guleng, Department of Gastroenterology, Zhongshan Hospital affiliated with Xiamen University, No. 201 Hubin South Road, Xiamen 361004, Fujian Province, China. bayasi8@gmail.com
Telephone: +86-592-2292371 Fax: +86-592-2212328
Received: September 26, 2014
Peer-review started: September 27, 2014
First decision: December 2, 2014
Revised: January 8, 2015
Accepted: June 9, 2015
Article in press: June 10, 2015
Published online: August 14, 2015
Processing time: 324 Days and 19 Hours
Abstract

AIM: To investigate biological mechanisms underlying pyruvate kinase M2 isoform (PKM2) regulation of cell migration and invasion in hepatocellular carcinoma cells.

METHODS: HepG2 and Huh-7 hepatocellular carcinoma cell lines were stably transfected and cultured in DMEM (HyClone, Logan, UT, United States). To investigate the effects of PKM2 on cellular proliferation, hepatocellular carcinoma cells were subjected to the Cell Counting Kit-8 (Dojindo, Kamimashiki-gun, Kumamoto, Japan). And investigate the effects of PKM2 on cell signal pathway related with migration and invasion, Western immunoblotting were used to find out the differential proteins. All the antibody used was purchaseed from Cell Signal Technology. In order to explore cell motility used Transwell invasion and wound healing assays. The transwell plate with 0.5 mg/mL collagen type I (BD Bioscience, San Jose, CA)-coated filters. The wound-healing assay was performed in 6-well plates. Total RNA was extracted using TRIzol reagent (Invitrogen, CA, United States) and then reverse transcription was conducted. Quantitative reverse transcription-polymerase chain reaction (PCR) analysis was performed with the ABI 7500 real-time PCR system (Applied Biosystems). We further use digital gene expression tag profiling and identification of differentially expressed genes.

RESULTS: The cells seeded in four 96-well plates were measured OD450 by conducted Cell Counting Kit-8. From this conduction we observed that both HepG2 and Huh-7 hepatocellular carcinoma cells with silenced PKM2 turn on a proliferate inhibition; however, cell migration and invasion were enhanced compared with the control upon stimulation with epidermal growth factor (EGF). Our results indicate that the knockdown of PKM2 decreased the expression of E-cadherin and enhanced the activity of the EGF/EGFR signaling pathway, furthermore up-regulate the subsequent signal molecular the PLCγ1 and extracellular signal-regulated kinase 1/2 expression in the hepatocellular carcinoma cell lines HepG2 and Huh-7, which regulates cell motility. These variations we observed were due to the activation of the transforming growth factor beta (TGFβ) signaling pathway after PKM2 knockdown. We also found that the expression of TGFBRI was increased and the phosphorylation of Smad2 was enhanced. Taken together, our findings demonstrate that PKM2 can regulate cell motility through the EGF/EGFR and TGFβ/TGFR signaling pathways in hepatocellular carcinoma cells.

CONCLUSION: PKM2 play different roles in modulating the proliferation and metastasis of hepatocellular carcinoma cells, and this finding could help to guide the future targeted therapies.

Keywords: Pyruvate kinase; Migration; Epidermal growth factor/EGFR signaling pathway; Transforming growth factor beta signaling pathway; Hepatocellular carcinoma

Core tip: The present study is to explore the effects of pyruvate kinase M2 (PKM2) on motility of human hepatocellular carcinoma cells. Here, our results revealed that silenced PKM2 activated the epidermal growth factor (EGF)/EGFR and transforming growth factor beta (TGFβ)/TGFR signaling pathways, in addition, up-regulate the expression of downstream targets, and this is the first research to connect PKM2 with the vital EGF and TGFβ pathways in hepatocellular carcinoma cell motility.