Hydrogen inhibits the proliferation and migration of gastric cancer cells by modulating lncRNA MALAT1/miR-124-3p/EZH2 axis

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. Gastric cancer cell lines MGC-803 and BGC-823 were treated with or without H2 /O2 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. H2 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 H2. 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. These data demonstrated that H2 might be developed as a therapeutics of gastric cancer and lncRNA MALAT1/miR-124-3p/EZH2 axis could be a target for intervention.


Background
Gastric cancer is the sixth most-common cancer with over a million new cases worldwide in 2018 and causes second most mortalities among malignancies [1]. While Helicobacter pylori 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 [2]. Genetically, the main oncogene of gastric cancer is CDH1 (E-cadherin) as many pathogenic variants are associated with familial diffuse-type gastric cancer [3,4]. However, many other genes including MSH2, PMS2,, BRCA1, PALB2, CTNNA1, and ATM, have been identified to increase the risk of gastric cancer [5][6][7][8][9][10][11][12].
Long noncoding RNAs (lncRNAs) are transcripts larger than 200 base-pairs and similar to mRNA biochemically and structurally but do not code for protein [13]. A large number of lncRNA have been implicated in different aspects of cancer biology [14]. LncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1)

Open Access
Cancer Cell International *Correspondence: xuweiqiangqp@yeah.net † Baocheng Zhu and Hengguan Cui contributed equally to this work 2 Qingpu Branch of Zhongshan Hospital, Fudan University, 1158 Park East Road, Qingpu District, Shanghai 201700, China Full list of author information is available at the end of the article 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 [16]. LncRNA MALAT1 enhanced the stemness, proliferation, migration, invasion, and drug resistance of gastric cancer cells [17][18][19][20].
Although hydrogen gas was found effective in treating mouse squamous cell carcinoma and thought it might be used to treat other cancer [21], 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 [22]. Increasing evidence showed the potential of hydrogen gas in preventing and relieving different cancers [23]. 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 [24]. Daily inhalation of hydrogen for 3 month resulted in the shrinkage of metastatic gallbladder cancer and improve of quality of life [25]. This study aims to explore the effects of hydrogen gas on gastric cancer cells and the underlying molecular mechanism.

Cell culture
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 °C and 5% CO 2 in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin (All from ThermoFisher, (Shanghai, China).

MTT assay
Seeded 5000 MGC-803 or BGC-823 cells per well in 96 well plates and cultured at 37 °C overnight before treated with or without hydrogen gas for 24 h. Added 20 µl of 5 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to each well and incubated at 37 °C for 4 h. The medium was carefully removed and 100 µl of MTT solvent (4 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 min before read at 590 nm.

Would healing scratch assay
After MGC-803 and BGC-823 cells in 24 well plates reached confluency, the cells were treated with 0.2 mg/ ml mitomycin C for 3 min. The center of the wells were scratched with a blue tip and photographed. The plates then cultured at 37 °C for 12 h and photographed. The gaps were measured with ImageJ (NIH, Bethesda, MD). The migration rate was calculated as (gap distance at 0 h − gap distance at 24 h)/gap distance at 0 h *100.

Quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA was extracted from using MiniBEST Universal RNA Extraction Kit (TaKaRa, Beijing, China) following manufacturer's manual. First strand cDNA was synthesized from 0.5 µg total RNA using Invitrogen SuperScript III Reverse Transcriptase kit (ThermoFisher, Shanghai, China). Noncoding small RNAs were first poly 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 TGC TGT GTG CCA ATG TTT CG and CAG CTG CCT GCT GTT TTC TG for MALAT1, CTG CTT CCT ACA TCG TAA GTG CAA and TTG CTC CCT CCA AAT GCT GGT for enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), CTG AGG AGC AGC TTC AGT CC and GAG TAG CCA TTG TCC ACG CT for β-catenin (CTNNB1), and CCG AGA ATG GGA AGC TTG TC and AAG CAC CAA CGA GAG GAG AA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH). TAA GGC ACG CGG TGA ATG C for miR-124-3p and CGC AAG GAT GAC ACG CAA ATTC for U6 with universal reverse primer GTG CAG GGT CCG AGGT. The relative gene expression was calculated using 2 − ΔΔCt method with GAPDH as internal control for lncRNA MALAT1 and EZH2 while U6 for miR-124-3p.

Mouse xenograft gastric cancer models
Animal 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 × 10 6 ) stably transfected with lncRNA MALAT1 or miR-124-3p were subcutaneously injected into the flank of a nude mouse (n = 7). Three days later, mice were exposed to hydrogen/ oxygen mixture (2:1 vol:vol) for 2 h daily. All mice were sacrificed 5 weeks after inoculation.

Statistical analyses
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.

Hydrogen inhibited the proliferation and migration of gastric cancer cells
As hydrogen gas has been shown to inhibit cancer progression [25] and lung cancer cell proliferation [26], we first examined whether it could impact the proliferation of gastric cancer cells. After 24 h exposure to hydrogen gas, the viable MGC-803 and BGC-823 cells were reduced more than 20% compared to normally cultured ones (Fig. 1a). Moreover, H2 gas drastically reduced the motility of MGC-803 cells by about 60% and BGC-823 cells by more than 50% (Fig. 1b, c).

Hydrogen gas inhibited the expression of MALAT1 and EZH2
Microarray analysis identified that H 2 gas significantly inhibited the expression of lncRNA MALAT1 and transcription factor EZH2 of MGC-823 cells (Fig. 2a). Real-time quantitative PCR confirmed that 24 h H 2 exposure reduced the RNA transcript levels of MALAT1 and EZH2 about 50% in both MGC-803 and BGC-823 cells (Fig. 2b). The protein levels of EZH2 of MGC-803 and BGC-823 cells treated with H 2 were reduced more than 50% compared to control cells (Fig. 2c).

LncRNA MALAT1 and miR-124-3p mediated the inhibition of tumor growth by H 2
Inhalation of H 2 drastically inhibited tumor growth in mouse xenografted with BGC-823 cells (p < 0.01, H 2 vs. Ctrl) (Fig. 7), which was consistent with clinical observation of different cancers [23,25]. Overexpression of lncRNA MALAT1 partially suppressed H 2 caused inhibition of tumor growth whereas miR-124-3p counteracted the effect of lncR MALAT1 (Fig. 7a). EZH2 protein level of gastric tumor tissue was reduced more than 50% by H 2 exposure, which was enhanced by miR-124-3p and suppressed by lncRNA MALAT1 (Fig. 7b).  [27]. 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 [28]. H 2 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 [29]. PI3K/AKT1 signaling was shown to mediate the protection of H 2 on I/R induced cardiac injuries [30]. 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 [24]. Daily H 2 inhalation stopped the growth of primary and metastatic tumors in a metastatic Fig. 2 The expression of lncRNA MALAT1 and its target genes in gastric cancer cells was inhibited by H 2 . a Heatmap of genes differently expressed in MGC-803 cells cultured with or without H 2 . b Transcript levels of lncRNA MALAT1, EZH2, were analyzed by quantitative real-time PCR. c Protein levels of EZH2 were assessed by immunoblot. CK & Ctrl, Normal culture conditions. *p < 0.01 compared to Ctrl gallbladder cancer patient with the levels of tumor biomarkers returning to normal [25]. H 2 treatment inhibited CD47 expression and induced apoptosis of A549 lung cancer cells [31]. Another study showed that SMC3 mediated the inhibition of lung cancer progression by H 2 in in vitro study and xenograft mouse model [26]. The current study showed that H 2 could inhibit the progression and migration of gastric cancer cells. Further studies about the effects of H 2 on gastric cancer progression in animal models are warranted. Long noncoding RNA MALAT1 was identified as a factor associated with metastasis of non-small cell lung cancer [32] and regulated gene expression through different mechanism [16]. 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 [19,33]. LncRNA MALAT1 regulated miR-30e/ATG5 expression to modulate autophagy and apoptosis of SGC7901 gastric Fig. 5 The upregulation of EZH2 by lncRNA MALAT1 was antagonized by miR-124-3p. a The miR-124-3p binding site in EZH2 3'UTR predicted using TargetScan. b 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. c The effects of lncRNA MALAT1 and miR-124-3p on EZH2 protein level were examined by western blot. Ctrl, Normal culture conditions. *p < 0.01 compared to control; # p < 0.01 compared to MALAT1; & p < 0.01 compared to 124-3p cancer cells and conferred resistance to cisplatin [34]. 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 [20]. The current study demonstrated that lncRNA MALAT1 promoted gastric cancer proliferation and migration through modulating miR-124-3p/EZH2 axis.

Discussion
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 [35]. ITGB3 [36] and ZEB1 [37] 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 [37]. LncRNA XIST and miR-124-3p competitively regulated EZH2 expression and modulated the proliferation, migration, and invasion of laryngeal squamous cancerous cells [38]. miR-124-3p was antagonized by lncRNA MALAT1 in inhibiting the expression of Slug and metastasis of hepatocellular carcinoma [39]. This study demonstrated that H 2 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. 8).

Conclusions
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 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 µm. Ctrl, Normal culture conditions. *p < 0.01 compared to control; # p < 0.01 compared to MALAT1; & p < 0.01 compared to 124-3p 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 2 should be further studied for treating gastric cancers and lncRNA MALAT1/ miR-124-3p/EZH2 axis would be a novel intervention target.  were grafted into nude mice subcutaneously. Mice were then exposed to H2 for 2 h daily for 5 weeks. a Picture of representative tumors (upper panel) was shown and tumor size (lower panel) was measured. b EZH2 protein level of tumor tissue was evaluated by western blot. *p < 0.01 compared to Ctrl; ^p < 0.01 compared to H 2 + 124-3p; # p < 0.01 compared to H 2 + MALAT1; & p < 0.01 compared to H 2