{"id":26842,"date":"2024-01-03T21:39:07","date_gmt":"2024-01-03T19:39:07","guid":{"rendered":"https:\/\/hho-bulgaria.com\/h2-suppresses-gastric-cancer-cells-via-malat1-mir-124-3p-ezh2-axis\/"},"modified":"2024-01-29T21:20:50","modified_gmt":"2024-01-29T19:20:50","slug":"h2-suppresses-gastric-cancer-cells-via-malat1-mir-124-3p-ezh2-axis","status":"publish","type":"post","link":"https:\/\/hho-bulgaria.com\/en\/h2-suppresses-gastric-cancer-cells-via-malat1-mir-124-3p-ezh2-axis\/","title":{"rendered":"H2 suppresses gastric cancer cells via MALAT1\/miR-124-3p\/EZH2 axis"},"content":{"rendered":"<div class=\"jig-ncbiinpagenav\" data-jigconfig=\"smoothScroll: false, allHeadingLevels: ['h2'], headingExclude: ':hidden,.nomenu'\" id=\"ui-ncbiinpagenav-1\">\n<div class=\"fm-sec half_rhythm no_top_margin\">\n<div class=\"fm-flexbox\">\n<div class=\"fm-citation\">\n<div class=\"citation-default\">\n<div class=\"part1\"><span role=\"menubar\"><a href=\"#\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Cancer Cell Int.<\/a><\/span> 2021; 21: 70. <\/div>\n<div class=\"part2\"><span class=\"fm-vol-iss-date\">Published online 2021 Jan 22. <\/span>  <span class=\"doi\"><span>doi:&nbsp;<\/span><a href=\"\/\/doi.org\/10.1186%2Fs12935-020-01743-5\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=7821405&amp;issue-id=372124&amp;journal-id=96&amp;FROM=Article%7CFront%20Matter&amp;TO=Content%20Provider%7CCrosslink%7CDOI\">10.1186\/s12935-020-01743-5<\/a><\/span><\/div>\n<\/div>\n<\/div>\n<div class=\"fm-ids\">\n<div class=\"fm-citation-pmcid\"><span class=\"fm-citation-ids-label\">PMCID: <\/span><span>PMC7821405<\/span><\/div>\n<div class=\"fm-citation-pmid\">PMID: <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/33482814\">33482814<\/a><\/div>\n<\/div>\n<\/div>\n<h1 class=\"content-title\">Hydrogen inhibits the proliferation and migration of gastric cancer cells by modulating lncRNA MALAT1\/miR-124-3p\/EZH2 axis<\/h1>\n<div class=\"half_rhythm\">\n<div class=\"contrib-group fm-author\"><a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhu%20B%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139672633418496\" co-class=\"co-affbox\">Baocheng Zhu<\/a>,<sup>#<\/sup><sup>1<\/sup> <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Cui%20H%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139672595785568\" co-class=\"co-affbox\">Hengguan Cui<\/a>,<sup>#<\/sup><sup>1<\/sup> and  <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Xu%20W%5BAuthor%5D\" class=\"affpopup\" co-rid=\"_co_idm139672592351360\" co-class=\"co-affbox\">Weiqiang Xu<\/a><sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup><sup>1,<\/sup><sup>2<\/sup><\/div>\n<div style=\"display:none\" class=\"contrib-group aff-tip\">\n<div id=\"_co_idm139672633418496\">\n<h3 class=\"no_margin\">Baocheng Zhu<\/h3>\n<p><sup>1<\/sup>Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, China <\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Zhu%20B%5BAuthor%5D\">Baocheng Zhu<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139672595785568\">\n<h3 class=\"no_margin\">Hengguan Cui<\/h3>\n<p><sup>1<\/sup>Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, China <\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Cui%20H%5BAuthor%5D\">Hengguan Cui<\/a><\/div>\n<\/div>\n<div id=\"_co_idm139672592351360\">\n<h3 class=\"no_margin\">Weiqiang Xu<\/h3>\n<p><sup>1<\/sup>Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, China <\/p>\n<p><sup>2<\/sup>Qingpu Branch of Zhongshan Hospital, Fudan University, 1158 Park East Road, Qingpu District, Shanghai, 201700 China <\/p>\n<div>Find articles by <a href=\"https:\/\/pubmed.ncbi.nlm.nih.gov\/?term=Xu%20W%5BAuthor%5D\">Weiqiang Xu<\/a><\/div>\n<\/div>\n<\/div>\n<\/div>\n<div class=\"half_rhythm\">\n<div class=\"togglers fm-copyright-license\"><a href=\"#\" class=\"pmctoggle\" rid=\"idm139672630393664_ai\">Author information<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139672630393664_an\">Article notes<\/a> <a href=\"#\" class=\"pmctoggle\" rid=\"idm139672630393664_cpl\">Copyright and License information<\/a> <a href=\"\/pmc\/about\/disclaimer\/\" style=\"margin-left: 1em\">PMC Disclaimer<\/a><\/div>\n<div class=\"fm-authors-info hide half_rhythm\" id=\"idm139672630393664_ai\" style=\"display:none\">\n<div class=\"fm-affl\" id=\"Aff1\"><sup>1<\/sup>Qingpu Branch of Zhongshan Hospital Affiliated to Fudan University, Shanghai, China <\/div>\n<div class=\"fm-affl\" id=\"Aff2\"><sup>2<\/sup>Qingpu Branch of Zhongshan Hospital, Fudan University, 1158 Park East Road, Qingpu District, Shanghai, 201700 China <\/div>\n<div><span class=\"fm-affl\">Weiqiang Xu, <\/span><span class=\"fm-affl\"><span class=\"email-label\">Email: <\/span><a href=\"mailto:dev@null\" data-email=\"ten.haey@pqgnaiqiewux\" class=\"oemail\">ten.haey@pqgnaiqiewux<\/a><\/span>.<\/div>\n<div><sup><img decoding=\"async\" src=\"\/corehtml\/pmc\/pmcgifs\/corrauth.gif\" alt=\"corresponding author\"><\/sup>Corresponding author.<\/div>\n<div><sup>#<\/sup>Contributed equally.<\/div>\n<\/div>\n<div class=\"fm-article-notes hide half_rhythm\" id=\"idm139672630393664_an\" style=\"display:none\">\n<div class=\"fm-pubdate half_rhythm\">Received 2020 May 18; Accepted 2020 Dec 31.<\/div>\n<\/div>\n<div class=\"permissions half_rhythm hide\" id=\"idm139672630393664_cpl\" style=\"display:none\">\n<div class=\"fm-copyright half_rhythm\"><a href=\"\/pmc\/about\/copyright\/\">Copyright<\/a> \u00a9 The Author(s) 2021<\/div>\n<div class=\"license half_rhythm\"><strong>Open Access<\/strong>This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article&#8217;s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article&#8217;s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit <a href=\"http:\/\/creativecommons.org\/licenses\/by\/4.0\/\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=7821405&amp;issue-id=372124&amp;journal-id=96&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">http:\/\/creativecommons.org\/licenses\/by\/4.0\/<\/a>. The Creative Commons Public Domain Dedication waiver (<a href=\"http:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=7821405&amp;issue-id=372124&amp;journal-id=96&amp;FROM=Article%7CFront%20Matter&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">http:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/<\/a>) applies to the data made available in this article, unless otherwise stated in a credit line to the data.<\/div>\n<\/div>\n<\/div>\n<div id=\"pmclinksbox\" class=\"links-box whole_rhythm hidden\" role=\"complementary\" aria-label=\"Related or updated information about this article.\"><\/div>\n<\/div>\n<div class=\"sec\"><\/div>\n<div id=\"ass-data\" class=\"tsec fm-sec whole_rhythm\" data-section=\"Featured_PMC_Datacitation\">\n<h2 class=\"nomenu\">Associated Data<\/h2>\n<dl data-length=\"80\" class=\"box-data-avail whole_rhythm no_bottom_margin\">\n<dt><a href=\"#\" rid=\"data-avl-stmnt\" data-ga-action=\"click_feat_toggler\" data-ga-label=\"Data Availability Statement\" class=\"pmctoggle\">Data Availability Statement<\/a><\/dt>\n<dd id=\"data-avl-stmnt\" style=\"display: none;\">\n<p>All data generated or analyzed during this study are included in the manuscript.<\/p>\n<\/dd>\n<\/dl>\n<\/div>\n<div id=\"Abs1\" lang=\"en\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><span role=\"menubar\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"menuitem\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/span><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Abs1title\">Abstract<\/h2>\n<p><!--article-meta--><\/p>\n<div>\n<div id=\"sec-a.j.b.m.a\" class=\"sec sec-first\">\n<h3 id=\"sec-a.j.b.m.atitle\">Background<\/h3>\n<p id=\"Par4\" class=\"p p-first-last\">Gastric cancer is one of the most prevalent and deadly malignancies without efficient treatment option. This study aimed to investigate the effect of hydrogen gas on the behavior of gastric cancer cells.<\/p>\n<\/div>\n<div id=\"sec-a.j.b.m.b\" class=\"sec\">\n<h3 id=\"sec-a.j.b.m.btitle\">Methods<\/h3>\n<p id=\"Par5\" class=\"p p-first-last\">Gastric cancer cell lines MGC-803 and BGC-823 were treated with or without H<sub>2<\/sub> \/O<sub>2<\/sub> gas mixture (66.7%:33.3% v\/v). Proliferation and migration were assessed by MTT and scratch wound healing assays respectively. The expression of lncRNA MALAT1, miR-124-3p, and EZH2 was analyzed by real-time quantitative PCR and\/or western blot. Tumor growth was estimated using xenograft mouse model.<\/p>\n<\/div>\n<div id=\"sec-a.j.b.m.c\" class=\"sec\">\n<h3 id=\"sec-a.j.b.m.ctitle\">Results<\/h3>\n<p id=\"Par6\" class=\"p p-first-last\">H<sub>2<\/sub> gas significantly inhibited gastric tumor growth in vivo and the proliferation, migration, and lncRNA MALAT1 and EZH2 expression of gastric cancer cells while upregulated miR-124-3p expression. LncRNA MALAT1 overexpression abolished all the aforementioned effects of H<sub>2<\/sub>. LncRNA MALAT1 and miR-124-3p reciprocally inhibited the expression of each other. MiR-124-3p mimics abrogated lncRNA MALAT1 promoted EZH2 expression and gastric cancer cell proliferation and migration.<\/p>\n<\/div>\n<div id=\"sec-a.j.b.m.d\" class=\"sec sec-last\">\n<h3 id=\"sec-a.j.b.m.dtitle\">Conclusions<\/h3>\n<p id=\"Par7\" class=\"p p-first-last\">These data demonstrated that H<sub>2<\/sub> might be developed as a therapeutics of gastric cancer and lncRNA MALAT1\/miR-124-3p\/EZH2 axis could be a target for intervention.<\/p>\n<\/div>\n<\/div>\n<div class=\"sec\"><strong class=\"kwd-title\">Keywords: <\/strong><span class=\"kwd-text\">Hydrogen, Gastric cancer, LncRNA MALAT1, miR-124-3p, EZH2, Proliferation, Migration<\/span><\/div>\n<\/div>\n<div id=\"Sec1\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Sec1title\">Background<\/h2>\n<p id=\"Par8\" class=\"p p-first\">Gastric cancer is the sixth most-common cancer with over a million new cases worldwide in 2018 and causes second most mortalities among malignancies [<a href=\"#CR1\" rid=\"CR1\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">1<\/a>]. While <em>Helicobacter pylori<\/em> infection is the most prominent cause of gastric cancer, many nutritional and life style factors, such as drinking, smoking, physical activity, overweight, intake of fruit and vegetable, are also significantly associated with the development of gastric cancer [<a href=\"#CR2\" rid=\"CR2\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">2<\/a>]. Genetically, the main oncogene of gastric cancer is CDH1 (E-cadherin) as many pathogenic variants are associated with familial diffuse-type gastric cancer [<a href=\"#CR3\" rid=\"CR3\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">3<\/a>, <a href=\"#CR4\" rid=\"CR4\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">4<\/a>]. However, many other genes including <em>MSH2<\/em>, <em>PMS2<\/em>,, <em>BRCA1, PALB2, CTNNA1, and ATM<\/em>, have been identified to increase the risk of gastric cancer [<a href=\"#CR5\" rid=\"CR5\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">5<\/a>\u2013<a href=\"#CR12\" rid=\"CR12\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">12<\/a>].<\/p>\n<p id=\"Par9\">Long noncoding RNAs (lncRNAs) are transcripts larger than 200 base-pairs and similar to mRNA biochemically and structurally but do not code for protein [<a href=\"#CR13\" rid=\"CR13\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">13<\/a>]. A large number of lncRNA have been implicated in different aspects of cancer biology [<a href=\"#CR14\" rid=\"CR14\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">14<\/a>]. LncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) has been shown to promote the proliferation and migration of cancer cells, epithelial-mesenchymal transition, and metastasis of many cancer types but recently found to function as a tumor suppressor in breast and colorectal cancers [<a href=\"#CR16\" rid=\"CR16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">16<\/a>]. LncRNA MALAT1 enhanced the stemness, proliferation, migration, invasion, and drug resistance of gastric cancer cells [<a href=\"#CR17\" rid=\"CR17\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">17<\/a>\u2013<a href=\"#CR20\" rid=\"CR20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a>].<\/p>\n<p id=\"Par10\" class=\"p p-last\">Although hydrogen gas was found effective in treating mouse squamous cell carcinoma and thought it might be used to treat other cancer [<a href=\"#CR21\" rid=\"CR21\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">21<\/a>], its clinical application was not widely explored until 2007 Ohsawa et al. demonstrated that hydrogen gas selectively eliminated the hydroxyl radical and attenuated focal ischemia and reperfusion caused oxidative stress and brain injury [<a href=\"#CR22\" rid=\"CR22\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">22<\/a>]. Increasing evidence showed the potential of hydrogen gas in preventing and relieving different cancers [<a href=\"#CR23\" rid=\"CR23\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">23<\/a>]. Drinking hydrogen-rich water for 6 weeks reduced reactive oxygen metabolites in the blood, maintained blood oxidation potential, improved the quality of life of malignant liver cancer patients after radiotherapy [<a href=\"#CR24\" rid=\"CR24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a>]. Daily inhalation of hydrogen for 3 month resulted in the shrinkage of metastatic gallbladder cancer and improve of quality of life [<a href=\"#CR25\" rid=\"CR25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>]. This study aims to explore the effects of hydrogen gas on gastric cancer cells and the underlying molecular mechanism.<\/p>\n<\/div>\n<div id=\"Sec2\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Sec2title\">Materials and methods<\/h2>\n<div id=\"Sec3\" class=\"sec sec-first\">\n<h3 id=\"Sec3title\">Cell culture<\/h3>\n<p id=\"Par11\" class=\"p p-first\">Human gastric cancer cell lines MGC-803 and BGC-823 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37 \u00b0C and 5% CO<sub>2<\/sub> in Dulbecco\u2019s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100&nbsp;U\/ml of penicillin and 100 \u00b5g\/ml of streptomycin (All from ThermoFisher, (Shanghai, China).<\/p>\n<p id=\"Par12\" class=\"p p-last\">Hydrogen\/oxygen gas mixture (66.7%:33.3% v\/v) was produced with a Hydrogen\/Oxygen Generator (Asclepius Meditec, Shanghai, China). Hydrogen treatment was executed in an adjustable three gas cell culture incubator (Puhe Bio, Wuxi, China). MALAT1 overexpression vector (GenScript, Nanjing, China) and miR-124-3p mimics (GeneCopoeia, Guangzhou, China) were transfected using Lipofectamine 3000 (ThermoFisher, Shanghai, China).<\/p>\n<\/div>\n<div id=\"Sec4\" class=\"sec\">\n<h3 id=\"Sec4title\">MTT assay<\/h3>\n<p id=\"Par13\" class=\"p p-first-last\">Seeded 5000 MGC-803 or BGC-823 cells per well in 96 well plates and cultured at 37 \u00b0C overnight before treated with or without hydrogen gas for 24&nbsp;h. Added 20 \u00b5l of 5&nbsp;mg\/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to each well and incubated at 37 \u00b0C for 4&nbsp;h. The medium was carefully removed and 100 \u00b5l of MTT solvent (4&nbsp;mM HCl and 0.1% Nondet P-40 (NP40) in isopropanol) was added to every well. The plates were incubated at room temperature with orbital shaking for 15&nbsp;min before read at 590&nbsp;nm.<\/p>\n<\/div>\n<div id=\"Sec5\" class=\"sec\">\n<h3 id=\"Sec5title\">Would healing scratch assay<\/h3>\n<p id=\"Par14\" class=\"p p-first-last\">After MGC-803 and BGC-823 cells in 24 well plates reached confluency, the cells were treated with 0.2&nbsp;mg\/ml mitomycin C for 3&nbsp;min. The center of the wells were scratched with a blue tip and photographed. The plates then cultured at 37 \u00b0C for 12&nbsp;h and photographed. The gaps were measured with ImageJ (NIH, Bethesda, MD). The migration rate was calculated as (gap distance at 0&nbsp;h \u2212&nbsp;gap distance at 24&nbsp;h)\/gap distance at 0&nbsp;h *100.<\/p>\n<\/div>\n<div id=\"Sec6\" class=\"sec\">\n<h3 id=\"Sec6title\">Quantitative real\u2010time polymerase chain reaction (RT-qPCR)<\/h3>\n<p id=\"Par15\" class=\"p p-first-last\">\nTotal RNA was extracted from using MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China) following manufacturer\u2019s manual. First strand cDNA was synthesized from 0.5&nbsp;\u00b5g total RNA using Invitrogen SuperScript III Reverse Transcriptase kit (ThermoFisher, Shanghai, China). Noncoding small RNAs were first poly (A) tailed with <em>E. coli<\/em> Poly(A) Polymerase (M0276, NEB, Ipswich, MA) and then reverse transcribed using Moloney Murine Leukemia Virus (M-MuLV, MMLV) Reverse Transcriptase (M0253, NEB) with GTCGCAGTGCAGGGTCCGAGGTGGCGATTTTTTTTTTTTTTTTTTTTTTT(A\/G\/C)(A\/G\/T\/C) as primer. Real-time RT-PCR amplication was carried on an ABI StepOne Plus (Applied Biosystems, Foster City, CA) using SYBR Premix Ex TaqTM kit (Takara, Beijing, China). The PCR primers were TGCTGTGTGCCAATGTTTCG and CAGCTGCCTGCTGTTTTCTG for MALAT1, CTGCTTCCTACATCGTAAGTGCAA and TTGCTCCCTCCAAATGCTGGT for enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), CTGAGGAGCAGCTTCAGTCC and GAGTAGCCATTGTCCACGCT for \u03b2-catenin (CTNNB1), and CCGAGAATGGGAAGCTTGTC and AAGCACCAACGAGAGGAGAA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). TAAGGCACGCGGTGAATGC for miR-124-3p and CGCAAGGATGACACGCAAATTC for U6 with universal reverse primer GTGCAGGGTCCGAGGT. The relative gene expression was calculated using 2<sup>\u2212\u2009\u0394\u0394Ct<\/sup> method with GAPDH as internal control for lncRNA MALAT1 and EZH2 while U6 for miR-124-3p.<\/p>\n<\/div>\n<div id=\"Sec7\" class=\"sec\">\n<h3 id=\"Sec7title\">\nWestern blot<\/h3>\n<p id=\"Par16\" class=\"p p-first-last\">MGC-803 and BGC-823 cells were lyzed with RIPA lysis buffer (1% v\/v NP-40, 20&nbsp;mM Tris-HCL pH 7.4, 5&nbsp;mM sodium pyrophosphate, 5&nbsp;mM EDTA) supplemented with protease and phosphatase inhibitor cocktail (Millipore Sigma, Burlington, MA). Total protein samples (40 \u00b5g) were resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% nonfat milk in TBST (50&nbsp;mM Tris, pH 7.5; 150&nbsp;mM NaCl; 0.1% Tween 20) for 30&nbsp;min, incubated with primary antibodies at 4 \u00b0C overnight, washed and incubated with proper horseradish peroxidase conjugated goat against mouse or rabbit IgG antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 60&nbsp;min before visualized with enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL). The primary antibodies used were anti-EZH2 (05-1319, Millipore Sigma) and anti-\u03b2-actin (A5441, Millipore Sigma).<\/p>\n<\/div>\n<div id=\"Sec8\" class=\"sec\">\n<h3 id=\"Sec8title\">Mouse xenograft gastric cancer models<\/h3>\n<p id=\"Par17\" class=\"p p-first-last\">\nAnimal protocol was approved by Institutional Animal Care and Usage Committee of Zhongshan Hospital Affiliated to Fudan University (ZHFU20200374). Female BALB\/c nude mice (5 weeks old) were purchased from Cavens Laboratory Animal Inc (Changzhou, China) and acclimatized for a week. BGC-823 cells (1\u2009\u00d7\u200910<sup>6<\/sup>) stably transfected with lncRNA MALAT1 or miR-124-3p were subcutaneously injected into the flank of a nude mouse (n\u2009=\u20097). Three days later, mice were exposed to hydrogen\/oxygen mixture (2:1 vol:vol) for 2&nbsp;h daily. All mice were sacrificed 5 weeks after inoculation.<\/p>\n<\/div>\n<div id=\"Sec9\" class=\"sec sec-last\">\n<h3 id=\"Sec9title\">Statistical analyses<\/h3>\n<p id=\"Par18\" class=\"p p-first-last\">All experiments were performed 3 times with triplicates. Statistical analyses were performed using GraphPad Prism 6 (San Diego, CA). The differences among treatments were assessed with analysis of variance followed by Bonferroni tests. A p value less than 0.05 was considered statistical significant.<\/p>\n<\/div>\n<\/div>\n<div id=\"Sec10\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Sec10title\">Results<\/h2>\n<div id=\"Sec11\" class=\"sec sec-first\">\n<h3 id=\"Sec11title\">Hydrogen inhibited the proliferation and migration of gastric cancer cells<\/h3>\n<p id=\"Par19\" class=\"p p-first\">As hydrogen gas has been shown to inhibit cancer progression [<a href=\"#CR25\" rid=\"CR25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>] and lung cancer cell proliferation [<a href=\"#CR26\" rid=\"CR26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">26<\/a>], we first examined whether it could impact the proliferation of gastric cancer cells. After 24&nbsp;h exposure to hydrogen gas, the viable MGC-803 and BGC-823 cells were reduced more than 20% compared to normally cultured ones (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig1\" rid-ob=\"ob-Fig1\" co-legend-rid=\"lgnd_Fig1\" rel=\"noopener\"><span>1<\/span><\/a>a). Moreover, H2 gas drastically reduced the motility of MGC-803 cells by about 60% and BGC-823 cells by more than 50% (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig1\" rid-ob=\"ob-Fig1\" co-legend-rid=\"lgnd_Fig1\" rel=\"noopener\"><span>1<\/span><\/a>b, c).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig1\" co-legend-rid=\"lgnd_Fig1\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/\" target=\"figure\" rid-figpopup=\"Fig1\" rid-ob=\"ob-Fig1\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592327664\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/\" target=\"figure\" rid-figpopup=\"Fig1\" rid-ob=\"ob-Fig1\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig1_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig1_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig1_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592327664\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig1\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig1\/\" target=\"figure\" rid-figpopup=\"Fig1\" rid-ob=\"ob-Fig1\" rel=\"noopener\">Fig. 1<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>Gastric cancer cell proliferation and migration were inhibited by H<sub>2<\/sub> gas. <strong>a<\/strong>&nbsp;MGC-803 and BGC-823 cells were cultured with or without H<sub>2<\/sub> for 24&nbsp;h and assessed by MTT assay. <strong>b<\/strong>&nbsp;MGC-803 and BGC-823 cell migration was assayed by scratch wound healing assay. <strong>c<\/strong>&nbsp;Quantitative analysis of migration data. Scale bar: 25 \u00b5m. Ctrl, Normal culture conditions. *p\u2009&lt;\u20090.01 compared to Ctrl<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par20\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec12\" class=\"sec\">\n<h3 id=\"Sec12title\">Hydrogen gas inhibited the expression of MALAT1 and EZH2<\/h3>\n<p id=\"Par21\" class=\"p p-first\">Microarray analysis identified that H<sub>2<\/sub> gas significantly inhibited the expression of lncRNA MALAT1 and transcription factor EZH2 of MGC-823 cells (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" co-legend-rid=\"lgnd_Fig2\" rel=\"noopener\"><span>2<\/span><\/a>a). Real-time quantitative PCR confirmed that 24&nbsp;h H<sub>2<\/sub> exposure reduced the RNA transcript levels of MALAT1 and EZH2 about 50% in both MGC-803 and BGC-823 cells (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" co-legend-rid=\"lgnd_Fig2\" rel=\"noopener\"><span>2<\/span><\/a>b). The protein levels of EZH2 of MGC-803 and BGC-823 cells treated with H<sub>2<\/sub> were reduced more than 50% compared to control cells (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" co-legend-rid=\"lgnd_Fig2\" rel=\"noopener\"><span>2<\/span><\/a>c).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig2\" co-legend-rid=\"lgnd_Fig2\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592316768\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig2_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig2_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig2_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592316768\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig2\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig2\/\" target=\"figure\" rid-figpopup=\"Fig2\" rid-ob=\"ob-Fig2\" rel=\"noopener\">Fig. 2<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>The expression of lncRNA MALAT1 and its target genes in gastric cancer cells was inhibited by H<sub>2<\/sub>. <strong>a<\/strong>&nbsp;Heatmap of genes differently expressed in MGC-803 cells cultured with or without H<sub>2<\/sub>. <strong>b<\/strong>&nbsp;Transcript levels of lncRNA MALAT1, EZH2, were analyzed by quantitative real-time PCR. <strong>c<\/strong>&nbsp;Protein levels of EZH2 were assessed by immunoblot. CK &amp; Ctrl, Normal culture conditions. *p\u2009&lt;\u20090.01 compared to Ctrl<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par22\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec13\" class=\"sec\">\n<h3 id=\"Sec13title\">H<sub>2<\/sub> inhibited EZH2 expression through downregulating LncRNA MALAT1<\/h3>\n<p id=\"Par23\" class=\"p p-first\">Overexpressing MALAT1 upregulated the mRNA (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>a) and protein (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>b) levels of EZH2 in MGC-803 and BGC-823 cells. Furthermore, LncRNA MALAT1 abrogated the inhibition of EZH2 expression by H<sub>2<\/sub> (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>a, b).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig3\" co-legend-rid=\"lgnd_Fig3\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592304544\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig3_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig3_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig3_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592304544\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig3\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" rel=\"noopener\">Fig. 3<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>H<sub>2<\/sub> inhibited gastric cancer cell proliferation and migration through regulating lncRNA MALAT1. <strong>a<\/strong>&nbsp;Transcript levels of lncMALAT1 and EZH2 of MGC-803 cells were measured by quantitative real-time PCR. <strong>b<\/strong>&nbsp;Protein level of EZH2 was assessed by immunoblot. <strong>c<\/strong>&nbsp;MGC-803 and BGC-823 cells transfected with or without lncRNA MALAT1 expression vector were cultured with or without H<sub>2<\/sub> for 24&nbsp;h and assessed by MTT assay. <strong>d<\/strong>&nbsp;Scratch wound healing assay was used to examine the effects of H<sub>2<\/sub> and lncRNA MALAT1 on MGC-803 and BGC-823 cell migration. Scale bar: 25 \u00b5m. Ctrl, Normal culture conditions. *p\u2009&lt;\u20090.01 compared to Ctrl; <sup>#<\/sup>p\u2009&lt;\u20090.01 compared to MALAT1; <sup>&amp;<\/sup>p\u2009&lt;\u20090.01 compared to H<sub>2<\/sub><\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par24\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec14\" class=\"sec\">\n<h3 id=\"Sec14title\">LncRNA MALAT1 relieved the inhibitory effect of H<sub>2<\/sub> on gastric cancer cell proliferation and migration<\/h3>\n<p id=\"Par25\" class=\"p p-first-last\">As previously showed that H<sub>2<\/sub> gas inhibited the proliferation and migration of MGC-803 and BGC-823 cells, we next examined the roles of LncRNA MALAT1 on the effects of H<sub>2<\/sub>. LncRNA MALAT1 overexpression resulted in about 25% increase of MGC-803 and BGC-823 cell proliferation (p\u2009&lt;\u20090.01 MALAT1 vs. Ctrl) and abolished the inhibition of proliferation imposed by H<sub>2<\/sub> (p\u2009&lt;\u20090.01 for MALAT1\u2009+\u2009H<sub>2<\/sub> vs. MALAT1 and MALAT1\u2009+\u2009H<sub>2<\/sub> vs. H<sub>2<\/sub>) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>c). Overexpression of LncRNA MALAT1 increased MGC-803 and BGC-823 cell migration by about 40% (p\u2009&lt;\u20090.01 MALAT1 vs. Ctrl) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>D). Moreover, MALAT1 abrogated the inhibitory effect of H<sub>2<\/sub> gas on the migration of MGC-803 gastric cancer cells (p\u2009&lt;\u20090.01 for MALAT1\u2009+\u2009H<sub>2<\/sub> vs. MALAT1 and MALAT1\u2009+\u2009H<sub>2<\/sub> vs. H<sub>2<\/sub>) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig3\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig3\" rid-ob=\"ob-Fig3\" co-legend-rid=\"lgnd_Fig3\" rel=\"noopener\"><span>3<\/span><\/a>d).<\/p>\n<\/div>\n<div id=\"Sec15\" class=\"sec\">\n<h3 id=\"Sec15title\">miR-124-3p expression in gastric cancer cells was regulated by H<sub>2<\/sub> and lncRNA MALAT1<\/h3>\n<p id=\"Par26\" class=\"p p-first\">Using LncBase Predicted v.2 (<a href=\"http:\/\/carolina.imis.athena-innovation.gr\/diana_tools\" data-ga-action=\"click_feat_suppl\" ref=\"reftype=extlink&amp;article-id=7821405&amp;issue-id=372124&amp;journal-id=96&amp;FROM=Article%7CBody&amp;TO=External%7CLink%7CURI\" target=\"_blank\" rel=\"noopener\">http:\/\/carolina.imis.athena-innovation.gr\/diana_tools<\/a>), has-miR-124-3p was identified as a regulator and target of lncRNA MALAT1. Four strong miR-124-3p binding sites (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" co-legend-rid=\"lgnd_Fig4\" rel=\"noopener\"><span>4<\/span><\/a>a) and many other imperfect binding sites of miR-124-3p were identified in MALAT1. The miR-124-3p level of MGC-803 and BGC-823 cells was increased more than 100% by H<sub>2<\/sub> treatment (p\u2009&lt;\u20090.01, H<sub>2<\/sub> vs. Ctrl) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" co-legend-rid=\"lgnd_Fig4\" rel=\"noopener\"><span>4<\/span><\/a>b). On the other hand, lncRNA MALAT1 overexpression reduced miR-124-3p level more than 40% (p\u2009&lt;\u20090.01 MALAT1 vs. Ctrl). Moreover, lncRNA MALAT1 and H<sub>2<\/sub> gas antagonized the effect of each other on the expression of miR-124-3p (p\u2009&lt;\u20090.01 for MALAT1\u2009+\u2009H<sub>2<\/sub> vs. MALAT1 and MALAT1\u2009+\u2009H<sub>2<\/sub> vs. H<sub>2<\/sub>) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" co-legend-rid=\"lgnd_Fig4\" rel=\"noopener\"><span>4<\/span><\/a>b).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig4\" co-legend-rid=\"lgnd_Fig4\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592281264\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig4_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig4_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig4_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592281264\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig4\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig4\/\" target=\"figure\" rid-figpopup=\"Fig4\" rid-ob=\"ob-Fig4\" rel=\"noopener\">Fig. 4<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>LncRNA MALAT1 mediated the effects of H<sub>2<\/sub> on miR-124-3p expression in gastric cancer cells. <strong>a<\/strong>&nbsp;The binding sites of miR-124-3p within lncRNA MALAT1 transcript sequence predicted using LncBase Predicted v.2 of Diana Tools. <strong>b<\/strong>&nbsp;The expression level of miR-124-3p in MGC-803 and BGC-823 cells transfected with or without lncRNA MALAT1 expression vector and cultured with or without H<sub>2<\/sub> for 24&nbsp;h was assayed by quantitative real-time PCR. *p\u2009&lt;\u20090.01 compared to control; <sup>#<\/sup>p\u2009&lt;\u20090.01 compared to MALAT1; <sup>&amp;<\/sup>p\u2009&lt;\u20090.01 compared to H<sub>2<\/sub><\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par27\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec16\" class=\"sec\">\n<h3 id=\"Sec16title\">LncRNA MALAT1 upregulated EZH2 expression through inhibiting miR-124-3p<\/h3>\n<p id=\"Par28\" class=\"p p-first\">As miR-124-3p was regulated by lncRNA MALAT1 and EZH2 was identified as a direct target of miR-124-3p (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" co-legend-rid=\"lgnd_Fig5\" rel=\"noopener\"><span>5<\/span><\/a>a), the effect of miR-124-3p on the regulation of EZH2 expression by lncRNA MALAT1 was assessed. Transfection of miR-124-3p mimics reduced EZH2 mRNA level about 50% (p\u2009&lt;\u20090.01, miR-124-3p vs. Ctrl) and suppressed the upregulation of EZH2 by lncRNA MALAT1 (p\u2009&lt;\u20090.01 for miR-124-3p\u2009+\u2009MALAT1 vs. MALAT1 and miR-124-3p\u2009+\u2009MALAT1 vs. miR-124-3p) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" co-legend-rid=\"lgnd_Fig5\" rel=\"noopener\"><span>5<\/span><\/a>b). Moreover, lncRNA MALAT1 and miR-124-3p reciprocally inhibited the expression of one another (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" co-legend-rid=\"lgnd_Fig5\" rel=\"noopener\"><span>5<\/span><\/a>b). The protein level of EZH2 was reduced about 60% by miR-124-3p (p\u2009&lt;\u20090.01, miR-124-3p vs. Ctrl) and miR-124-3p abrogated the lncRNA MALAT1 induced EZH2 upregulation (p\u2009&lt;\u20090.01 for miR-124-3p\u2009+\u2009MALAT1 vs. MALAT1 and miR-124-3p\u2009+\u2009MALAT1 vs. miR-124-3p) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" co-legend-rid=\"lgnd_Fig5\" rel=\"noopener\"><span>5<\/span><\/a>c).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig5\" co-legend-rid=\"lgnd_Fig5\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592269760\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig5_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig5_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig5_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592269760\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig5\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig5\/\" target=\"figure\" rid-figpopup=\"Fig5\" rid-ob=\"ob-Fig5\" rel=\"noopener\">Fig. 5<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>The upregulation of EZH2 by lncRNA MALAT1 was antagonized by miR-124-3p. <strong>a<\/strong>&nbsp;The miR-124-3p binding site in EZH2 3\u2019UTR predicted using TargetScan. <strong>b<\/strong>&nbsp;The expression levels of lncRNA MALAT1, miR-124-3p, and EZH2 after lncRNA MALAT1 and\/or miR-124-3p overexpression were analyzed by quantitative real-time PCR. <strong>c<\/strong>&nbsp;The effects of lncRNA MALAT1 and miR-124-3p on EZH2 protein level were examined by western blot. Ctrl, Normal culture conditions. *p\u2009&lt;\u20090.01 compared to control; <sup>#<\/sup>p\u2009&lt;\u20090.01 compared to MALAT1; <sup>&amp;<\/sup>p\u2009&lt;\u20090.01 compared to 124-3p<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par29\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec17\" class=\"sec\">\n<h3 id=\"Sec17title\">miR-124-3p abolished lncRNA MALAT1 induced proliferation and migration of gastric cancer cells<\/h3>\n<p id=\"Par30\" class=\"p p-first\">Viable MGC-803 and BGC-823 cells were reduced more than 20% by miR-124-3p mimics (p\u2009&lt;\u20090.01, miR-124-3p vs. Ctrl) and the promotion of MGC-803 and BGC-823 cell proliferation by lncRNA MALAT1 was inhibited by miR-124-3p (p\u2009&lt;\u20090.01 for miR-124-3p\u2009+\u2009MALAT1 vs. MALAT1, and miR-124-3p\u2009+\u2009MALAT1 vs. miR-124-3p) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig6\" rid-ob=\"ob-Fig6\" co-legend-rid=\"lgnd_Fig6\" rel=\"noopener\"><span>6<\/span><\/a>a). miR-124-3p also inhibited the migration rate of MGC-803 and BGC-823 cells by about 50% (p\u2009&lt;\u20090.01, miR-124-3p vs. Ctrl) and abolished lncRNA MALAT1 induced increase of cell motility (p\u2009&lt;\u20090.01 for miR-124-3p\u2009+\u2009MALAT1 vs. MALAT1 and miR-124-3p\u2009+\u2009MALAT1 vs. miR-124-3p) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig6\" rid-ob=\"ob-Fig6\" co-legend-rid=\"lgnd_Fig6\" rel=\"noopener\"><span>6<\/span><\/a>b).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig6\" co-legend-rid=\"lgnd_Fig6\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/\" target=\"figure\" rid-figpopup=\"Fig6\" rid-ob=\"ob-Fig6\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672592260672\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/\" target=\"figure\" rid-figpopup=\"Fig6\" rid-ob=\"ob-Fig6\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig6_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig6_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig6_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672592260672\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig6\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig6\/\" target=\"figure\" rid-figpopup=\"Fig6\" rid-ob=\"ob-Fig6\" rel=\"noopener\">Fig. 6<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>miR-124-3p blocked lncRNA MALAT1 induced proliferation and migration of gastric cancer cells. <strong>a<\/strong>&nbsp;MGC-803 cells transfected with lncRNA MALAT1 and\/or miR-124-3p were cultured for 24&nbsp;h and assessed by MTT assay. <strong>b<\/strong>&nbsp;The migration of MGC-803 and BGC-823 cells with lncRNA MALAT1 and\/or miR-124-3p overexpression was assayed by scratch wound healing assay. Scale bar: 25 \u00b5m. Ctrl, Normal culture conditions. *p\u2009&lt;\u20090.01 compared to control; <sup>#<\/sup>p\u2009&lt;\u20090.01 compared to MALAT1; <sup>&amp;<\/sup>p\u2009&lt;\u20090.01 compared to 124-3p<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par31\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec18\" class=\"sec sec-last\">\n<h3 id=\"Sec18title\">LncRNA MALAT1 and miR-124-3p mediated the inhibition of tumor growth by H<sub>2<\/sub><\/h3>\n<p id=\"Par32\" class=\"p p-first\">Inhalation of H<sub>2<\/sub> drastically inhibited tumor growth in mouse xenografted with BGC-823 cells (p\u2009&lt;\u20090.01, H<sub>2<\/sub> vs. Ctrl) (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" co-legend-rid=\"lgnd_Fig7\" rel=\"noopener\"><span>7<\/span><\/a>), which was consistent with clinical observation of different cancers [<a href=\"#CR23\" rid=\"CR23\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">23<\/a>, <a href=\"#CR25\" rid=\"CR25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>]. Overexpression of lncRNA MALAT1 partially suppressed H<sub>2<\/sub> caused inhibition of tumor growth whereas miR-124-3p counteracted the effect of lncR MALAT1 (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" co-legend-rid=\"lgnd_Fig7\" rel=\"noopener\"><span>7<\/span><\/a>a). EZH2 protein level of gastric tumor tissue was reduced more than 50% by H<sub>2<\/sub> exposure, which was enhanced by miR-124-3p and suppressed by lncRNA MALAT1 (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" co-legend-rid=\"lgnd_Fig7\" rel=\"noopener\"><span>7<\/span><\/a>b).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig7\" co-legend-rid=\"lgnd_Fig7\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/a><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672595637440\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" rel=\"noopener\"><\/a><a class=\"inline_block ts_canvas\" href=\"\/core\/lw\/2.0\/html\/tileshop_pmc\/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&amp;p=PMC3&amp;id=7821405_12935_2020_1743_Fig7_HTML.jpg\" target=\"tileshopwindow\" rel=\"noopener\"><\/p>\n<div class=\"ts_bar small\" title=\"Click on image to zoom\"><\/div>\n<p><img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig7_HTML.jpg\" title=\"Click on image to zoom\" class=\"tileshop\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig7_HTML.jpg\"><\/a><\/div>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672595637440\" style=\"display: none;\"><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig7\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig7\/\" target=\"figure\" rid-figpopup=\"Fig7\" rid-ob=\"ob-Fig7\" rel=\"noopener\">Fig. 7<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>H2 inhibited gastric tumor growth via lncRNA MALAT1\/miR-124-3p axis. BGC-823 cells stably expressing lncRNA MALAT1 and\/or miR-124-3p were grafted into nude mice subcutaneously. Mice were then exposed to H2 for 2&nbsp;h daily for 5 weeks. <strong>a<\/strong>&nbsp;Picture of representative tumors (upper panel) was shown and tumor size (lower panel) was measured. <strong>b<\/strong>&nbsp;EZH2 protein level of tumor tissue was evaluated by western blot. *p\u2009&lt;\u20090.01 compared to Ctrl; <sup>^<\/sup>p\u2009&lt;\u20090.01 compared to H<sub>2<\/sub>\u2009+\u2009124-3p; <sup>#<\/sup>p\u2009&lt;\u20090.01 compared to H<sub>2<\/sub>\u2009+\u2009MALAT1; <sup>&amp;<\/sup>p\u2009&lt;\u20090.01 compared to H<sub>2<\/sub><\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par33\" class=\"p p-last\">\n<\/div>\n<\/div>\n<div id=\"Sec19\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Sec19title\">Discussion<\/h2>\n<p id=\"Par34\" class=\"p p-first\">H<sub>2<\/sub> gas inhibited the proliferation and migration of MGC-803 and BGC-823 gastric cancer cells and downregulated the expression of lncRNA MALAT1and EZH2. The inhibition of EZH2 by H<sub>2<\/sub> was relieved by lncRNA MALAT1 overexpression. LncRNA MALAT1 also alleviated H<sub>2<\/sub> caused inhibition of the proliferation and migration of MGC-803 and BGC-823 gastric cancer cells. MicroRNA hsa-miR-124-3p was identified as a target and regulator of lncRNA MALAT1 and suppressed the expression of EZH2. H<sub>2<\/sub> upregulated miR-124-3p expression, which was abrogated by lncRNA MALAT1 overexpression. LncRNA MALAT1 promoted proliferation and migration of MGC-803 and BGC-823 cells were abolished by miR-124-3p mimics. H<sub>2<\/sub> exposure drastically reduced gastric cancer growth in xenograft mouse model and inhibited EZH2 expression in cancer tissue, which was enhanced by miR-124-3p and suppressed by lncRNA MALAT1.<\/p>\n<p id=\"Par35\">H<sub>2<\/sub> gas has been shown to improve many conditions by clearing detrimental ROS [<a href=\"#CR27\" rid=\"CR27\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">27<\/a>]. Inhalation of high concentration of hydrogen gas protected rat retinal ganglion cells from retinal ischemia\/reperfusion (I\/R) injury caused oxidative stress, inflammation, and apoptosis [<a href=\"#CR28\" rid=\"CR28\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">28<\/a>]. H<sub>2<\/sub> inhalation ameliorated middle cerebral artery occlusion induced cerebral I\/R injury evidenced by reduced 3-nitrotyrosine and 8-hydroxy-2-deoxyguanosine production, tampered inflammation, and reduced neural apoptosis [<a href=\"#CR29\" rid=\"CR29\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">29<\/a>]. PI3K\/AKT1 signaling was shown to mediate the protection of H<sub>2<\/sub> on I\/R induced cardiac injuries [<a href=\"#CR30\" rid=\"CR30\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">30<\/a>]. Malignant liver cancer patients drinking hydrogen-rich water during radiotherapy significantly reduced blood hydroperoxide level and increased biological antioxidant activities, leading to improved quality of life [<a href=\"#CR24\" rid=\"CR24\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">24<\/a>]. Daily H<sub>2<\/sub> inhalation stopped the growth of primary and metastatic tumors in a metastatic gallbladder cancer patient with the levels of tumor biomarkers returning to normal [<a href=\"#CR25\" rid=\"CR25\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">25<\/a>]. H<sub>2<\/sub> treatment inhibited CD47 expression and induced apoptosis of A549 lung cancer cells [<a href=\"#CR31\" rid=\"CR31\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">31<\/a>]. Another study showed that SMC3 mediated the inhibition of lung cancer progression by H<sub>2<\/sub> in in vitro study and xenograft mouse model [<a href=\"#CR26\" rid=\"CR26\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">26<\/a>]. The current study showed that H<sub>2<\/sub> could inhibit the progression and migration of gastric cancer cells. Further studies about the effects of H<sub>2<\/sub> on gastric cancer progression in animal models are warranted.<\/p>\n<p id=\"Par36\">Long noncoding RNA MALAT1 was identified as a factor associated with metastasis of non-small cell lung cancer [<a href=\"#CR32\" rid=\"CR32\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">32<\/a>] and regulated gene expression through different mechanism [<a href=\"#CR16\" rid=\"CR16\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">16<\/a>]. LncRNA MALAT1 was overexpressed in gastric cancers compared to adjacent normal tissue and promoted gastric cancer cells proliferation, migration, and resistance to cisplatin through activating PI3K\/AKT pathway [<a href=\"#CR19\" rid=\"CR19\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">19<\/a>, <a href=\"#CR33\" rid=\"CR33\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">33<\/a>]. LncRNA MALAT1 regulated miR-30e\/ATG5 expression to modulate autophagy and apoptosis of SGC7901 gastric cancer cells and conferred resistance to cisplatin [<a href=\"#CR34\" rid=\"CR34\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">34<\/a>]. MALAT1 was shown to directly bind to and stabilize Sox2 mRNA, which led to the increase of stemness and chemo-and radioresistance of gastric cancer cells [<a href=\"#CR20\" rid=\"CR20\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">20<\/a>]. The current study demonstrated that lncRNA MALAT1 promoted gastric cancer proliferation and migration through modulating miR-124-3p\/EZH2 axis.<\/p>\n<p id=\"Par37\">MicroRNA miR-124-3p was significantly downregulated in gastric cancer tissues and its level was inversely associated with histological grade, TNM stage and lymph node metastasis. Moreover, low miR-124-3p expression was correlated with lower overall and disease-free survival rates [<a href=\"#CR35\" rid=\"CR35\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">35<\/a>]. ITGB3 [<a href=\"#CR36\" rid=\"CR36\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">36<\/a>] and ZEB1 [<a href=\"#CR37\" rid=\"CR37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">37<\/a>] were found to be targeted by miR-124-3p in promoting gastric cancer proliferation and invasion. Moreover, Circular RNA circ-PVT1 upregulated ZEB1 expression by sponging miR-124-3p and enhanced paclitaxel resistance of gastric tumor and gastric cancer cells [<a href=\"#CR37\" rid=\"CR37\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">37<\/a>]. LncRNA XIST and miR-124-3p competitively regulated EZH2 expression and modulated the proliferation, migration, and invasion of laryngeal squamous cancerous cells [<a href=\"#CR38\" rid=\"CR38\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">38<\/a>]. miR-124-3p was antagonized by lncRNA MALAT1 in inhibiting the expression of Slug and metastasis of hepatocellular carcinoma [<a href=\"#CR39\" rid=\"CR39\" class=\" bibr popnode\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">39<\/a>]. This study demonstrated that H<sub>2<\/sub> gas curbed gastric cancer growth in vivo and gastric cancer cell proliferation and migration in vitro by reducing lncRNA MALAT1 level, which in turn upregulated miR-124-3p and downregulated EZH2 expression (Fig.&nbsp;<a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig8\/\" target=\"figure\" class=\"fig-table-link figpopup\" rid-figpopup=\"Fig8\" rid-ob=\"ob-Fig8\" co-legend-rid=\"lgnd_Fig8\" rel=\"noopener\"><span>8<\/span><\/a>).<\/p>\n<p><!--fig ft0--><!--fig mode=article f1--><\/p>\n<div class=\"fig iconblock whole_rhythm\" id=\"Fig8\" co-legend-rid=\"lgnd_Fig8\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig8\/\" target=\"figure\" rid-figpopup=\"Fig8\" rid-ob=\"ob-Fig8\" rel=\"noopener\"><!--fig\/graphic|fig\/alternatives\/graphic mode=\"anchored\" m1--><\/p>\n<div class=\"figure\" data-largeobj=\"\" data-largeobj-link-rid=\"largeobj_idm139672595599584\"><img decoding=\"async\" loading=\"lazy\" class=\"fig-image\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig8_HTML.jpg\" title=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Fig8_HTML.jpg\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Fig8_HTML.jpg\"><\/div>\n<p><\/a><\/p>\n<div class=\"largeobj-link align_right\" id=\"largeobj_idm139672595599584\" style=\"display: none;\"><a href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig8\/\" target=\"figure\" rid-figpopup=\"Fig8\" rid-ob=\"ob-Fig8\" rel=\"noopener\"><\/a><a target=\"object\" rel=\"noopener\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig8\/?report=objectonly\">Open in a separate window<\/a><\/div>\n<div class=\"icnblk_cntnt\" id=\"lgnd_Fig8\">\n<div><a class=\"figpopup\" href=\"https:\/\/www.ncbi.nlm.nih.gov\/pmc\/articles\/PMC7821405\/figure\/Fig8\/\" target=\"figure\" rid-figpopup=\"Fig8\" rid-ob=\"ob-Fig8\" rel=\"noopener\">Fig. 8<\/a><\/div>\n<p><!--caption a7--><\/p>\n<div class=\"caption\">\n<p>A proposed model for how H<sub>2<\/sub> inhibits gastric cancer growth and invasion. Please refer to the text for details. <img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Figa_HTML.gif\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Figa_HTML.gif\"><br \/>\nactivation; <img decoding=\"async\" loading=\"lazy\" alt=\"An external file that holds a picture, illustration, etc.\nObject name is 12935_2020_1743_Figb_HTML.gif\" src=\"https:\/\/hho-bulgaria.com\/wp-content\/uploads\/2024\/01\/12935_2020_1743_Figb_HTML.gif\"><br \/>\ninhibition<\/p>\n<\/div>\n<\/div>\n<\/div>\n<p id=\"Par38\" class=\"p p-last\">\n<\/div>\n<div id=\"Sec20\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Sec20title\">Conclusions<\/h2>\n<p id=\"Par39\" class=\"p p-first-last\">This study demonstrated the efficacy of molecular hydrogen in inhibiting the growth of gastric cancer by reducing the proliferation and migration of gastric cancer cells, which was through downregulating lncRNA MALAT1 and polycomb-group family member EZH2 and upregulating miR-124-3p. Moreover, miR-124-3p inhibited EZH2 expression and it reciprocally repressed the expression of each other with lncRNA MALAT1. These data indicated that H<sub>2<\/sub> should be further studied for treating gastric cancers and lncRNA MALAT1\/miR-124-3p\/EZH2 axis would be a novel intervention target.<\/p>\n<\/div>\n<div id=\"ack-a.l.c\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"ack-a.l.ctitle\">Acknowledgements<\/h2>\n<div class=\"sec\">\n<p>Not applicable.<\/p>\n<\/div>\n<\/div>\n<div id=\"glossary-a.l.a\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"glossary-a.l.atitle\">Abbreviations<\/h2>\n<div class=\"bk-sec\">\n<div>\n<table class=\"default-table glossary\">\n<tbody>\n<tr>\n<td>MTT<\/td>\n<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide<\/td>\n<\/tr>\n<tr>\n<td>lncRNA<\/td>\n<td>Long noncoding RNA<\/td>\n<\/tr>\n<tr>\n<td>miRNA<\/td>\n<td>MicroRNA<\/td>\n<\/tr>\n<tr>\n<td>MALAT1<\/td>\n<td>Metastasis associated lung adenocarcinoma transcript 1<\/td>\n<\/tr>\n<tr>\n<td>EZH2<\/td>\n<td>Enhancer of zeste 2 polycomb repressive complex 2 subunit<\/td>\n<\/tr>\n<tr>\n<td>GAPDH<\/td>\n<td>Glyceraldehyde 3-phosphate dehydrogenase<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n<\/div>\n<\/div>\n<\/div>\n<div id=\"notes-a.l.d\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"notes-a.l.dtitle\">Authors\u2019 contributions<\/h2>\n<p>WX conceived, designed the study and wrote the manuscript. BZ and HC carried out the study and data analysis. All authors read and approved the final manuscript.<\/p>\n<\/div>\n<div id=\"notes-a.l.e\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"notes-a.l.etitle\">Funding<\/h2>\n<p>Not applicable.<\/p>\n<\/div>\n<div id=\"notes-a.l.f\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"notes-a.l.ftitle\">Availability of data and materials<\/h2>\n<p>All data generated or analyzed during this study are included in the manuscript.<\/p>\n<\/div>\n<div id=\"FPar1\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"FPar1title\">Ethics approval and consent to participate<\/h2>\n<p id=\"Par42\">Not applicable.<\/p>\n<\/div>\n<div id=\"FPar2\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"FPar2title\">Consent for publication<\/h2>\n<p id=\"Par43\">Not applicable.<\/p>\n<\/div>\n<div id=\"FPar3\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"FPar3title\">Competing interests<\/h2>\n<p id=\"Par44\">The authors declare no potential conflict of interest.<\/p>\n<\/div>\n<div id=\"fn-group-a.l.b\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"fn-group-a.l.btitle\">Footnotes<\/h2>\n<p><!--back\/fn-group--><\/p>\n<div class=\"fm-sec half_rhythm small\">\n<p class=\"fn sec\" id=\"fn-a.l.b.a\">\n<p class=\"p p-first\"><strong>Publisher\u2019s note<\/strong><\/p>\n<p class=\"p p-last\">Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.<\/p>\n<\/p>\n<p class=\"fn sec\" id=\"fn-a.l.b.b\">\n<p class=\"p p-first-last\">Baocheng Zhu and Hengguan Cui contributed equally to this work<\/p>\n<\/p>\n<\/div>\n<\/div>\n<div id=\"Bib1\" class=\"tsec sec\">\n<div class=\"goto jig-ncbiinpagenav-goto-container\"><a class=\"tgt_dark page-toc-label jig-ncbiinpagenav-goto-heading\" href=\"#\" title=\"Go to other sections in this page\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Go to:<\/a><\/div>\n<h2 class=\"head no_bottom_margin ui-helper-clearfix\" id=\"Bib1title\">References<\/h2>\n<div class=\"ref-list-sec sec\" id=\"reference-list\">\n<div class=\"ref-cit-blk half_rhythm\" id=\"CR1\">1. <span class=\"element-citation\">Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. 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href=\"https:\/\/scholar.google.com\/scholar_lookup?journal=Cancer+Med&amp;title=miR-124-3p+availability+is+antagonized+by+LncRNA-MALAT1+for+Slug-induced+tumor+metastasis+in+hepatocellular+carcinoma&amp;author=RJ+Cui&amp;author=JL+Fan&amp;author=YC+Lin&amp;author=YJ+Pan&amp;author=C+Liu&amp;volume=8&amp;issue=14&amp;publication_year=2019&amp;pages=6358-69&amp;pmid=31466138&amp;doi=10.1002\/cam4.2482&amp;\" target=\"_blank\" rel=\"noopener noreferrer\" ref=\"reftype=other&amp;article-id=7821405&amp;issue-id=372124&amp;journal-id=96&amp;FROM=Article%7CCitationRef&amp;TO=Content%20Provider%7CLink%7CGoogle%20Scholar\" role=\"button\" aria-expanded=\"false\" aria-haspopup=\"true\">Google Scholar<\/a>]<\/span><\/span><\/div>\n<\/div>\n<\/div>\n<div style=\"display: none; width: 200px; top: -100px; left: -100px;\" aria-live=\"assertive\" aria-hidden=\"true\" class=\"ui-helper-reset ui-ncbipopper-wrapper ui-ncbilinksmenu\">\n<ul id=\"ui-ncbiinpagenav-2\">\n<li><a href=\"#Abs1title\">Abstract<\/a><\/li>\n<li><a href=\"#Sec1title\">Background<\/a><\/li>\n<li><a href=\"#Sec2title\">Materials and methods<\/a><\/li>\n<li><a href=\"#Sec10title\">Results<\/a><\/li>\n<li><a href=\"#Sec19title\">Discussion<\/a><\/li>\n<li><a href=\"#Sec20title\">Conclusions<\/a><\/li>\n<li><a href=\"#ack-a.l.ctitle\">Acknowledgements<\/a><\/li>\n<li><a href=\"#glossary-a.l.atitle\">Abbreviations<\/a><\/li>\n<li><a href=\"#notes-a.l.dtitle\">Authors\u2019 contributions<\/a><\/li>\n<li><a href=\"#notes-a.l.etitle\">Funding<\/a><\/li>\n<li><a href=\"#notes-a.l.ftitle\">Availability of data and materials<\/a><\/li>\n<li><a href=\"#FPar1title\">Ethics approval and consent to participate<\/a><\/li>\n<li><a href=\"#FPar2title\">Consent for publication<\/a><\/li>\n<li><a href=\"#FPar3title\">Competing interests<\/a><\/li>\n<li><a href=\"#fn-group-a.l.btitle\">Footnotes<\/a><\/li>\n<li><a href=\"#Bib1title\">References<\/a><\/li>\n<\/ul>\n<\/div>\n<\/div>\n","protected":false},"excerpt":{"rendered":"<p>Hydrogen inhibits the proliferation and migration of gastric cancer cells by modulating lncRNA MALAT1\/miR-124-3p\/EZH2 axis<\/p>\n","protected":false},"author":1,"featured_media":17899,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"footnotes":""},"categories":[130],"tags":[],"disease":[846],"body-organ":[1041],"applications":[679],"test_subjects":[1518],"report-topic":[1279],"class_list":["post-26842","post","type-post","status-publish","format-standard","has-post-thumbnail","hentry","category-hydrogen-health","disease-cancer","body-organ-stomach-2","applications-inhalation-2","test_subjects-mouse-2","report-topic-novel-therapy-2"],"acf":[],"yoast_head":"<!-- This site is optimized with the Yoast SEO plugin v28.1 - https:\/\/yoast.com\/product\/yoast-seo-wordpress\/ -->\n<title>H2 suppresses 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