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Growth Arrest-Specific Gene 2 Suppresses Hepatocarcinogenesis by Intervention of Cell Cycle and P53-Dependent Apoptosis

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Growth Arrest-Specific Gene 2 Suppresses Hepatocarcinogenesis by Intervention of Cell Cycle and P53-Dependent Apoptosis World Journal of W J G Gastroenterology Submit a Manuscript: https://www.f6publishing.com World J Gastroenterol 2019 August 28; 25(32): 4715-4726 DOI: 10.3748/wjg.v25.i32.4715 ISSN 1007-9327 (print) ISSN 2219-2840 (online) ORIGINAL ARTICLE Basic Study Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis Ran-Xu Zhu, Alfred Sze Lok Cheng, Henry Lik Yuen Chan, Dong-Ye Yang, Wai-Kay Seto ORCID number: Ran-Xu Zhu Ran-Xu Zhu, Dong-Ye Yang, Wai-Kay Seto, Department of Gastroenterology and Hepatology, (0000-0001-8366-925X); Alfred Sze The University of Hong Kong–Shenzhen Hospital, Shenzhen 518053, Guangdong Province, Lok Cheng (0000-0001-8692-3807); China Henry Lik Yuen Chan (0000-0003-3173-733); Dong-Ye Alfred Sze Lok Cheng, School of Biomedical Sciences, The Chinese University of Hong Kong, Yang (0000-0001-9724-6784); Wai- Hong Kong, China Kay Seto (0000-0003-2474-3055). Henry Lik Yuen Chan, Department of Medicine and Therapeutics, The Chinese University of Author contributions: Zhu RX and Hong Kong, Hong Kong, China Cheng ASL conceived and designed the study; All authors Corresponding author: Ran-Xu Zhu, MD, PhD, Doctor, Department of Gastroenterology and provided material support; Zhu RX Hepatology, The University of Hong Kong–Shenzhen Hospital, No. 1 Haiyuan Road, Futian performed the experiments and District, Shenzhen 518053, Guangdong Province, China. [email protected] collected the data; Zhu RX and Telephone: +86-755-86913333 Cheng ASL analyzed the data; Zhu RX wrote the manuscript; All Fax: +86-755-86913333 authors reviewed the manuscript; Zhu RX and Cheng ASL revised the manuscript; Zhu RX and Chan HLY provided financial support; Abstract Cheng ASL and Chan HLY BACKGROUND provided study supervision; All authors gave final approval of the Growth arrest-specific gene 2 (GAS2) plays a role in modulating in reversible version of the article to published. growth arrest cell cycle, apoptosis, and cell survival. GAS2 protein is universally expressed in most normal tissues, particularly in the liver, but is depleted in some Supported by the National Natural tumor tissues. However, the functional mechanisms of GAS2 in hepatocellular Science Foundation of China, No. carcinoma (HCC) are not fully defined. 81702777; and Natural Science Foundation of Guangdong AIM Province, No. 2015A030310053. To investigate the function and mechanism of GAS2 in HCC. Institutional review board statement: The study was METHODS reviewed and approved by the GAS2 expression in clinic liver and HCC specimens was analyzed by real-time University of Hong Kong- PCR and western blotting. Cell proliferation was analyzed by counting, MTS, and Shenzhen Hospital Ethics colony formation assays. Cell cycle analysis was performed by flow cytometry. Committee. Cell apoptosis was investigated by Annexin V apoptosis assay and western Conflict-of-interest statement: No blotting. potential conflicts of interest ware disclosed. RESULTS GAS2 protein expression was lower in HCC than in normal tissues. Open-Access: This article is an Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type open-access article which was p53, while knockdown of GAS2 promoted the proliferation of hepatocytes (P < selected by an in-house editor and 0.05). Furthermore, GAS2 overexpression impeded the G1-to-S cell cycle fully peer-reviewed by external reviewers. It is distributed in transition and arrested more G1 cells, particularly the elevation of sub G1 (P < accordance with the Creative 0.01). Apoptosis induced by GAS2 was dependent on p53, which was increased WJG https://www.wjgnet.com 4715 August 28, 2019 Volume 25 Issue 32 Zhu RX et al. Mechanism of GAS2 inhibiting hepatocarcinogenesis Commons Attribution Non by etoposide addition. The expression of p53 and apoptosis markers was further Commercial (CC BY-NC 4.0) enhanced when GAS2 was upregulated, but became diminished upon license, which permits others to distribute, remix, adapt, build downregulation of GAS2. In the clinic specimen, GAS2 was downregulated in upon this work non-commercially, more than 60% of HCCs. The average fold changes of GAS2 expression in tumor and license their derivative works tissues were significantly lower than those in paired non-tumor tissues (P < 0.05). on different terms, provided the original work is properly cited and CONCLUSION the use is non-commercial. See: GAS2 plays a vital role in HCC cell proliferation and apoptosis, possibly by http://creativecommons.org/licen regulating the cell cycle and p53-dependent apoptosis pathway. ses/by-nc/4.0/ Manuscript source: Unsolicited Key words: Growth arrest-specific gene 2; Cell cycle; Apoptosis; Hepatocellular manuscript carcinoma; p53-dependent signaling pathway Received: March 28, 2019 ©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved. Peer-review started: March 28, 2019 First decision: April 16, 2019 Revised: July 14, 2019 Core tip: This study elucidated the function and mechanism of growth arrest-specific Accepted: July 19, 2019 gene 2 (GAS2) in hepatocellular carcinoma (HCC) progression. By overexpression and Article in press: July 19, 2019 knockdown approaches, we found that GAS2 inhibited the proliferation of HCC with Published online: August 28, 2019 wild-type p53. The effects of GAS2 on the cell cycle and apoptosis were investigated by flow cytometry, Annexin V apoptosis, and western blot assay, respectively. GAS2 P-Reviewer: Ozaki I, Ratnasari N suppressed HCC proliferation by deregulating the cell cycle and p53-dependent S-Editor: Yan JP apoptosis pathway. Thus, GAS2 is expected to be a promising anti-oncogene and L-Editor: Filipodia potential therapeutic target in HCC with wild-type p53. E-Editor: Qi LL Citation: Zhu RX, Cheng ASL, Chan HLY, Yang DY, Seto WK. Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis. World J Gastroenterol 2019; 25(32): 4715-4726 URL: https://www.wjgnet.com/1007-9327/full/v25/i32/4715.htm DOI: https://dx.doi.org/10.3748/wjg.v25.i32.4715 INTRODUCTION Liver cancer is currently one of the most common causes of cancer-related deaths globally. Liver cancer has become a major health problem in developing countries, where the highest incidence and mortality rates are in Eastern and Southeastern Asia and parts of Africa[1,2]. Particularly, approximately 50% of liver cancer cases and deaths worldwide are in China. Among these, hepatocellular carcinoma (HCC) accounts for about 70% to 90% of liver cancers worldwide[3,4]. The prognosis of HCC is poor with high mortality because of limited effective treatment options[5]. Growth arrest-specific (GAS) genes encode proteins implicated directly in reversible growth arrest. This family contains seven genes (GAS1–GAS7) that encode proteins exhibiting distinct biochemical properties[6-8]. Among these genes, the protein encoded by GAS2 is a cell death substrate of caspase-3 that exerts some effects on modulating microfilament and cellular morphological variation during apoptosis[9,10]. Through microfilament alterations regulated by GAS2, the actin cytoskeleton and cell shape are quickly reset to respond to growth arrest induced by environmental stimuli such as apoptosis, different proliferative stimuli with various growth factors. Therefore, GAS2 as a component of the microfilament system functions in mitogenesis, cell cycle, cell growth, apoptosis, and cell survival. GAS2 protein is broadly expressed in many normal tissues, and is particularly highly expressed in the liver; however, it is not expressed in some tumor tissues such as prostate and breast[11,12]. Although it has a potentially important role in cell survival, the role of GAS2 in HCC is largely unexplored. We hypothesized that GAS2 possesses anti-oncogenic properties in HCC cells. MATERIALS AND METHODS Patients and clinical specimens Clinical liver specimens were derived from 54 HCC patients with surgical treatment at The University of Hong Kong–Shenzhen Hospital (Guangdong Sheng, China). WJG https://www.wjgnet.com 4716 August 28, 2019 Volume 25 Issue 32 Zhu RX et al. Mechanism of GAS2 inhibiting hepatocarcinogenesis HCC tissue samples were obtained from curative hepatic tumor resection except for necrotic and hemorrhagic areas. The paired adjacent non-tumor tissues were more than 5 cm away from the tumor edge, where was estimated to have no tumor invasion. All tissue samples were snap-frozen immediately after resection and stored in a -80°C nitrogen canister until use. This study was executed according to the ethical guidelines of the 1975 Declaration of Helsinki and was authorized by the Institutional Review Boards at The University of Hong Kong–Shenzhen Hospital [(2014)84]. All HCC patients provided signed informed consent giving approval for the use of clinical specimens for research purposes. Human liver and HCC cell culture Human normal liver cell lines (LO2 and MIHA) and HCC cell lines (SK-Hep1, PLC5, Huh 7, and Hep3B) were obtained from the American Type Culture Collection (Manassas, VA, United States). All cells were routinely maintained in high-glucose Dulbecco’s Modified Eagle’s medium (Gibco, Gaithersburg, MD, United States) with 100 mL/L fetal bovine serum (Thermo Scientific HyClone, Buffalo, NY, United States) and 10 mL/L MEM Non-Essential Amino Acids (Gibco) at 37°C in a humidified incubator containing 50 mL/L CO2. Quantitative real-time polymerase chain reaction A total of 1 μg RNA sample extracted from samples by TriZol reagent (Invitrogen, Carlsbad, CA, United States) was mixed with DNase I (Invitrogen) and then synthesized to cDNA by SuperScript II reverse transcriptase (Invitrogen). Quantitative real-time PCR (qPCR) was performed on the 7500 Real-Time PCR System apparatus (Applied Biosystems, Framingham, MA, United States) to detect gene transcripts in the cDNA template mixed with SYBR Green (Applied Biosystems). The relative mRNA level of GAS2 (F5’-TG CAAATGCCCAAACAAGTTC-3’; GAS2- R5’-TTCTCCCACTCGGTATCTTCC TT-3’) was evaluated by relative quantification of an internal control gene expression based on a similar amplification efficiency.
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