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PYCR1 Interference Inhibits Cell Growth and Survival Via C-Jun N-Terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathway

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PYCR1 Interference Inhibits Cell Growth and Survival Via C-Jun N-Terminal Kinase/Insulin Receptor Substrate 1 (JNK/IRS1) Pathway Zhuang et al. J Transl Med (2019) 17:343 https://doi.org/10.1186/s12967-019-2091-0 Journal of Translational Medicine RESEARCH Open Access PYCR1 interference inhibits cell growth and survival via c-Jun N-terminal kinase/ insulin receptor substrate 1 (JNK/IRS1) pathway in hepatocellular cancer Juhua Zhuang1†, Yanan Song2†, Ying Ye2, Saifei He2, Xing Ma1, Miao Zhang2, Jing Ni1, Jiening Wang2* and Wei Xia1* Abstract Background: Liver cancer is the second leading causes of cancer-related death globally. Pyrroline-5-carboxylate reductase 1 (PYCR1) plays a critical role in metabolic profles of tumors. Therefore, it is necessary to explore the mecha- nisms of PYCR1 on cell growth and survival in hepatocellular carcinoma (HCC). Methods: Protein and mRNA expression levels of PYCR1 in 140 pairs of tumor and adjacent normal liver tissues of HCC patients were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction (qRT- PCR). Expressions of PYCR1 were inhibited in BEL-7404 cells and SMMC-7721 cells using gene interference technology. The cell proliferation was detected by Celigo and MTT assay. The colony formation assay was also performed. The cell apoptosis was measured by fow cytometric assay. The efect of PYCR1 interference on tumor growth was observed by xenograft nude mice assay in vivo. The downstream pathway of PYCR1 interference was searched by microarray and bioinformatics analysis, and validated by qRT-PCR and western blot. Results: PYCR1 levels were signifcantly up-regulated in HCC tumor tissues than adjacent normal liver tissues in both protein and mRNA levels (P < 0.01). In vitro, the cell proliferation was signifcantly slower in shPYCR1 group than shCtrl group in BEL-7404 and SMMC-7721 cells (P < 0.001). The colony number was signifcantly smaller after PYCR1 interfer- ence (P < 0.01). The percentage of apoptosis cells signifcantly increased in shPYCR1 group (P < 0.01). In vivo, PYCR1 interference could obviously suppress tumor growth in xenograft nude mice. The volume and weight of tumors were signifcantly smaller via PYCR1 interference. The c-Jun N-terminal kinase (JNK) signaling pathway signifcantly altered, and insulin receptor substrate 1 (IRS1) were signifcantly down-regulated by PYCR1 interference in both mRNA and protein levels (P < 0.001). Conclusion: PYCR1 interference could inhibit cell proliferation and promote cell apoptosis in HCC through regluting JNK/IRS1 pathway. Our study will provide a drug target for HCC therapy and a potential biomarker for its diagnosis or prognosis. Keywords: PYCR1, Hepatocellular carcinoma, JNK, IRS1, Insulin resistance *Correspondence: [email protected]; [email protected] †Juhua Zhuang and Yanan Song contributed equally to this work and should be considered as co-frst authors 1 Department of Nuclear Medicine, The Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine, 358 Datong Road, Pudong, Shanghai 200137, People’s Republic of China 2 Central Laboratory, The Seventh People’s Hospital of Shanghai University of Traditional Chinese Medicine, 358 Datong Road, Pudong, Shanghai 200137, People’s Republic of China © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhuang et al. J Transl Med (2019) 17:343 Page 2 of 10 Background Helsinki, and the study protocol was approved by the Liver cancer is the second leading causes of cancer- Institutional Research Ethics Committee in 2007 (No. related death globally [1]. 90% of primary liver cancer 2007-019). Te mRNA profles used in the study were patients belong to hepatocellular carcinoma (HCC) [2]. obtained from the Cancer Genome Atlas (TCGA) data- Te incidence and mortality rates for HCC have greatly base (http://cance rgeno me.nih.gov). increased during the past two decades, and the major- ity of HCC patients occur in eastern/south-eastern Asia Immunohistochemistry (IHC) and Africa [3]. Although surgical hepatic resection and Tissues were fxed in 10% bufered formalin, embedded liver transplantation techniques have improved in recent in parafn and cut into 4 μm sections. Deparafnized years, the overall 5-year survival rate is still only around sections were treated with methanol containing 3% 5% and the long-term prognosis remains discouraging [4, hydrogen peroxide for 10 min before conducting antigen 5]. Terefore, a better understanding of the key molecules retrieval using a microwave oven at 95 °C for 5 min and and regulative pathways involved in the etiology and pro- cooling at 25 °C for 2 h. After washing with PBS, block- gression of HCC may lead to improved treatments. ing serum was applied for 20 min. Te sections were Growing tumors alter their metabolic profles to meet incubated with primary antibodies overnight at 4 °C the biosynthetic and bioenergetics demands of increased and biotin-marked secondary antibodies for 20 min fol- cell growth and proliferation [6]. Many studies have lowed by a peroxidase-marked streptavidin for an addi- focused on the metabolic regulatory roles of amino tional 20 min. Te reaction was visualized by using 3, acids in cancers, especially the nonessential amino acids 3′-diaminobenzidine tetrahydrochloride. Te nuclei (NEAA) [7, 8]. Among these, the regulatory functions of were counterstained with hematoxylin. Reproducibility proline metabolism proposed 3 decades ago have been of staining was confrmed by reimmunostaining via the emphasized [9]. On one hand, the pyrrolidine ring makes same method in multiple, randomly selected specimens. proline a unique proteogenic amino acid with a distinc- tive role in protein folding and secondary structures [10]. Cell culture On the other hand, proline also plays key roles in many Te HCC cell lines used in this study included BEL-7404 aspects, such as cellular signaling processes [11], cellu- cells and SMMC-7721 cells. Tey were obtained from the lar bioenergetics [12], and cancer cell metabolism [13]. Type Culture Collection of the Chinese Academy of Sci- Recent discoveries paid attention to the broad efects of ences, Shanghai, China. BEL-7404 and SMMC-7721 cells proline metabolism on cancer cell growth and survival, were both maintained in 1640 medium (Gibco, Gaith- which implicated proline metabolic enzymes as potential ersburg, MD, USA), supplemented with 10% fetal bovine targets for therapeutic intervention [14–17]. Pyrroline- serum (Gibco, Gaithersburg, MD, USA), 100 U/ml peni- 5-carboxylate reductase 1 (PYCR1) plays a critical role in cillin and 0.1 mg/ml streptomycin (Gibco, Gaithersburg, proline biosynthesis and catalyzes the NADH-dependent MD, USA) at 37 °C in 100% air. conversion of pyrroline-5-carboxylate (P5C) to proline [18]. However, the role of PYCR1 in HCC cell growth and RNA interference survival is still unclear. Te sequences for RNA interference were 5′-TTC TCC In this study, we aim to analyze the relationship GAA CGT GTC ACG T-3′ for control, and 5′- CAG TTT between PYCR1 and HCC, explore the role of PYCR1 CTG CTC TCA GGA A-3′ for PYCR1 (GenePharma, regulating c-Jun N-terminal kinase/insulin receptor Shanghai, China). Te sequences were inserted into Age substrate 1 (JNK/IRS1) pathway, and clarify the mecha- I and EcoR I points of GV115 vector. Te shRNAs of nisms of PYCR1 afecting HCC cell growth and survival. GV-NC-GFP-shRNA and GV-PYCR1-GFP-shRNA were Te achievements will probably provide us a potential constructed as control lentivirus and shPYCR1 lentivirus, drug target for therapy or a biomarker for diagnosis and and positive clones were screened and identifed. Sub- prognosis. sequently, HCC cells were seeded in a 6-well plate at a density of 2 * 105/well for RNA interference. According Materials and methods to the results of preliminary experiment, HCC cells were Tissue specimens infected with a certain amount of control lentivirus and Te tumor and adjacent normal tissues used for immu- shPYCR1 lentivirus. At 72 h post-infection, the expres- nohistochemistry were collected from 90 HCC patients sion of reporter gene GFP was observed by the fuores- who underwent surgery at Shanghai Seventh People’s cent microscope. Te fuorescence rate was the positive Hospital between 2007 and 2009. Te informed consent infective rate. If the infective rate was larger than 70% was obtained from every study participant. Te study and the state of cells was normal, the further study could was conducted in accordance with the Declaration of be continued. Zhuang et al. J Transl Med (2019) 17:343 Page 3 of 10 Quantitative real‑time polymerase chain reaction the formazan crystals formed. Te absorbance at 490 nm (qRT‑PCR) was measured Glomax-Multi + Detection System (Pro- Total RNA from HCC cells was extracted using TRIzol mega, Madison, WI, USA). At least three independent Reagent (Invitrogen, Carlsbad, CA, USA). RNA concen- experiments were performed. tration was determined by NanoDrop 2000 spectropho- tometer (Termo Fisher Scientifc, Rockford, IL, USA). Colony formation assay A total of 20–100 ng RNA was reverse-transcribed using Cells were seeded in 6-well plates at a concentration of First-Strand cDNA Synthesis kits (Invitrogen, Carls- 400–1000 cells per well and changes culture with media bad, CA, USA) according to the manufacture’s protocol. every 72 h. Colonies were fx with 4% paraformaldehyde qRT-PCR was performed on ABI 7500 System (Applied and stained with GIEMSA solution (Dingguo, Shanghai, Biosystems, Foster City, CA, USA) and used to analyze China) after 14 days, then colonies were visualized by the expression levels of mRNA.
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