ORIGINAL ARTICLE Year : 2023  |  Volume : 13  |  Issue : 6  |  Page : 268-276

G9a-targeted chaetocin induces pyroptosis of gastric cancer cells

Mian-Qing Huang1, Gui-Lan Tao2, Li-Fang Han3, Shu-Hong Tian3, Peng Zhou4
1 School of Life Science, Hainan University; Center for Drug Safety Evaluation, Hainan Medical University, Haikou, China
2 The First Affiliated Hospital of Hainan Medical University, Haikou, China
3 Center for Drug Safety Evaluation, Hainan Medical University, Haikou, China
4 Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs & Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China

Date of Submission07-Apr-2023Date of Decision28-Apr-2023Date of Acceptance15-Jun-2023Date of Web Publication26-Jun-2023

Correspondence Address:
Mian-Qing Huang
School of Life Science, Hainan University; Center for Drug Safety Evaluation, Hainan Medical University, Haikou
China
Peng Zhou
Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture and Rural Affairs & Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou
China

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/2221-1691.378601

Objective: To evaluate the effect of chaetocin on pyroptosis of gastric cancer cells and its underlying mechanisms.
Methods: The proliferation of gastric cancer cells was detected by trypan blue staining. Flow cytometry and Hoechst/propidium iodide double staining were used to detect apoptosis and pyroptosis. Cellular ultrastructure was observed by transmission electron microscopy. The levels of p-mixed lineage kinase domain-like (MLKL), gasdermin-D (GSDMD), gasdermin E (GSDME), N-GSDMD, and N-GSDME proteins were detected by Western blotting. In addition, lactate dehydrogenase (LDH) release assay was used to verify pyroptosis induced by chaetocin, and caspase 3 inhibition test and siRNA interference test were conducted to investigate pyroptosis mechanisms.
Results: Chaetocin at concentrations of 200 nmol/L to 600 nmol/L inhibited the proliferation of AGS, HGC27, MKN28, and SGC7901 gastric cancer cells in a dose-dependent and time-dependent manner by inducing apoptosis and pyroptosis. Significant ultrastructure changes, such as chromatin condensation, vacuolization, disrupted mitochondrial cristae, and increased nuclear occupancy, were observed after treatment with chaetocin in SGC7901 cells. Chaetocin at a concentration of 400 nmol/L significantly increased the number of pyroptotic cells, LDH release, and the ratio of N-GSDME/ GSDME (P<0.01), which were reversed by Z-DEVD-FMK. In addition, chaetocin did not affect the expression of GSDMD. G9a silencing abolished the effect of chaetocin on the expression levels of GSDME and N-GSDME and LDH release (P>0.05).
Conclusions: In addition to inducing apoptosis, chaetocin inhibits gastric cancer cells by inducing pyroptosis via the caspase 3/GSDME pathway. G9a was the target of chaetocin to induce pyroptosis of gastric cancer cells.

Keywords: Chaetocin; Gastric cancer; Pyroptosis; G9a; GSDME

How to cite this article:
Huang MQ, Tao GL, Han LF, Tian SH, Zhou P. G9a-targeted chaetocin induces pyroptosis of gastric cancer cells. Asian Pac J Trop Biomed 2023;13:268-76
How to cite this URL:
Huang MQ, Tao GL, Han LF, Tian SH, Zhou P. G9a-targeted chaetocin induces pyroptosis of gastric cancer cells. Asian Pac J Trop Biomed [serial online] 2023 [cited 2023 Jun 26];13:268-76. Available from: https://www.apjtb.org/text.asp?2023/13/6/268/378601

Significance:
Chaetocin induces apoptosis of many types of tumor cells. The current study demonstrated that chaetocin induced pyroptosis of gastric cancer cells via the caspase 3/GSDME pathway and G9a was the target of chaetocin to induce pyroptosis. Therefore, chaetocin can be further explored in combination with other chemotherapy drugs and developed as an anti-tumor agent.

  1. Introduction 

Gastric cancer is a major threat to human health with a 5-year survival rate of less than 20%. Global cancer statistics released by the International Agency for Research on Cancer under the GLOBOCAN 2020 program reported gastric cancer as the fifth most common with 1089103 new cases and a morbidity rate of 5.6%. The mortality was 7.7% in 2020, accounting for 768793 deaths and making this the fourth most deadly cancer[1]. Early diagnosis of gastric cancer is difficult and most patients are diagnosed at an advanced stage. Furthermore, the efficacy of anti-gastric cancer drugs is unsatisfactory and surgical treatment is the last resort for most patients[2]. Therefore, there is an urgent need to develop superior anti-gastric cancer medicines.

G9a, a histone methyl transferase (HMT) generates H3K9me1 and H3K9me2 by transferring a methyl group from S-adenosylmethionine to histone H3 at lysine K9 (H3K9). The resulting modification has gene regulatory effects[3],[4]. G9a is encoded by the EHMT2 gene and belongs to the Su (var)3-9, Enhancer-of-zeste, Trithorax (SET) domain-containing Su(var)3-9 family. Its N-terminus contains Pre-SET, I-SET, and Post-SET domains with characteristic ankyrin repeats[5],[6]. In order to explore the relationship between G9a expression and the prognosis of patients, a search was performed on the Kaplan Meier Plotter website. High G9a expression in tumor tissues was associated with a significantly reduced survival rate and mean survival time, suggesting an impact of G9a on gastric cancer progression.

Chaetocin is biosynthesized by the fungus, Chaetomium, and is a naturally-occurring HMT inhibitor[7],[8]. Dithiodiketopiperazine rings contribute to its pharmacological effects, such as anti-tumor activity and reversal of HIV latency[9]. Chaetocin is thought to induce apoptosis, contributing to its anti-tumor cytotoxicity, but no reports on tumor cell pyroptosis have been found[10],[11],[12]. Therefore, the current study aimed at investigating the effect of chaetocin on pyroptosis of gastric cancer cells and its underlying mechanisms.

  2. Materials and methods 

2.1. Reagents and antibodies

The reagents and antibodies were used as follows: Chaetocin and Z-DEVD-FMK (Selleck Chemicals, Shanghai, China), anti-mixed lineage kinase domain-like (MLKL) (phospho S358), anti-cleaved N-terminal gasdermin-D (GSDMD), anti-cleaved N-terminal gasdermin E (GSDME), anti-G9a (Abcam, Cambridge, UK), anti-beta-actin primary antibodies and HRP-conjugated secondary antibody (Servicebio Technology, Wuhan, China).

2.2. Cell culture

Gastric cancer cell lines AGS (poorly differentiated), HGC27 (undifferentiated), MKN28, (highly differentiated) and SGC7901 (moderately differentiated) were purchased from Otwo Biotech (Otwo Biotech, Shenzhen, China) and cultured in RPMI 1640 medium (Hyclone, Utah, USA) with 10% fetal bovine serum (Tianhang, Huzhou, China) at 37 °C in a humidified environment with 5% CO2.

2.3. Cell viability

Aliquots of 1×105 cells were seeded into 24-well plates and cultured for 24 h. Cells from 3 wells were harvested at 24 h, 48 h, 72 h, 96 h, and 120 h for viability measurement by staining with 0.08% trypan blue (Solarbio, Beijing, China) for 2 min at room temperature and counting in a hemocytometer under an inverted microscope.

2.4. Flow cytometry

Cells were digested, centrifuged, and washed with phosphate-buffered saline (PBS). Cell death was detected by flow cytometry based on Annexin V-FITC/propidium iodide (PI) double labeling following the instructions of the FITC Annexin-V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA)[13]. Caspase 3 activity was suppressed in gastric cancer cells with Z-DEVD-FMK (ZDF) to distinguish GSDMD or GSDME pyroptotic pathway. Cells were treated with chaetocin at different concentrations for 24 h immediately after incubating with 50 μmol/L ZDF for 30 min. Stained cells were detected by BD Accuri C6 flow cytometer (BD Biosciences) and results were analyzed by BD Accuri C6 software.

2.5. Hoechst/PI double staining

Cells were cultured in 24-well plates. Then the medium was removed and washed with PBS before double-staining with Hoechst 33342/PI for 20 min according to the instructions of the Hoechst 33342/PI Double Staining Kit (Solarbio, Beijing, China). Fluorescence was assessed by ZEISS Axio Vert. A1 fluorescence microscope.

2.6. Transmission electron telescope

SGC7901 cells were treated with chaetocin for 24 h and gently scraped into suspension in clean 1.5 mL EP tubes and centrifuged at 1500 rpm for 10 min. The supernatant was discarded and the cells were fixed with 2.5% fresh glutaraldehyde. Ultrathin sections were made for staining, as described previously[14], and photographed with a Hitachi JEM-1400 Flash transmission electron microscope.

2.7. Western blotting

Cells were harvested, centrifuged, and washed with PBS, and cell lysates containing phenylmethylsulfonyl fluoride and phosphoprotease inhibitors were prepared. Protein concentrations were determined in a loading buffer with BCA protein quantitative assay kit (Servicebio). The proteins were separated by electrophoresis on 10% polyacrylamide gel (Servicebio) and electroporated onto PVDF membranes (Servicebio), blocked with 5% skimmed dry milk for 2 h, and incubated with primary antibodies against p-MLKL (ab187091; 1:1000;Abcam), N-GSDMD (ab215203; 1:1000; Abcam), N-GSDME (ab196436; 1:1000; Abcam), G9a (ab185050; 1:1000;Abcam) and β-actin (GB15001; 1:1500; Servicebio) overnight and secondary antibody conjugated with horseradish peroxidase (GB23301; 1:4000; Servicebio) for 2 h. Protein bands were visualized for luminescence with an ECL Kit (Servicebio), scanned, and analyzed with a Tanon-5200Multi gel imaging system (Tanon, Shanghai, China). The optical density values of the target bands were analyzed using ImageJ software (National Institutes of Health)[15].

2.8. Lactate dehydrogenase (LDH) release

After cells were treated with chaetocin for 24 h, LDH activity in the medium was measured according to the instructions of the LDH Cytotoxicity Assay Kit (Yeasen, Shanghai, China). The percentage of LDH activity was calculated as the ratio of LDH OD490 in the cell medium to total cellular LDH with optical densities determined by a microplate reader (Thermo Fisher Scientific).

2.9. siRNA knockdown

Two siRNAs (Invitrogen) for silencing of the EHMT2 gene encoding G9a were synthesized with the sequences: siEHMT2-1: 5ʹ-GCACAAGCACATCGAGGTGAT-3ʹ, siEHMT2-2: 5ʹ- GCAACATCAGCCGCTTCATCA-3ʹ. Aliquots of 1×105 SGC7901 cells were seeded into 6-well plates and grown for 24 h before transfection with 50 pmol siRNA for 72 h, as directed by the instructions of Lipofectamine RNAiMAX (Invitrogen). Cells were treated with 400 nmol/L chaetocin and Western blotting and LDH release assays were performed 24 h later.

2.10. Bioinformatics analysis

The GEPIA website (http://gepia.cancer-pku.cn) was used to analyze the differential expression of EHMT2 gene between normal gastric tissue and gastric cancer.

2.11. Statistical analysis

Statistical analyses were performed using GraphPad Prism version 7.0 for Windows and data are expressed as mean ± standard deviation (SD). One-way ANOVA was used to assess statistical differences between the two groups. A value of P<0.05 was considered statistically significant.

  3. Results 

3.1. Chaetocin inhibits the proliferation of gastric cancer cells

Treatment with 50, 100, 200, 400, and 600 nmol/L chaetocin produced a time-dependent decline in survival rates of AGS and SGC7901 cells [Figure 1]A and [Figure 1]D. The survival rate of AGS and SGC7901 cells was zero after 120 h of treatment with 400 nmol/L chaetocin. The viability of SGC7901 cells was declined with increasing concentration. Cell morphology changed from normal to aberrant, such as rounding, vacuolization, detached cells [Figure 1]E. AGS cells showed the greatest sensitivity to all doses of chaetocin cytotoxicity with fewer than 40% survival rate after 24 h of treatment [Figure 1]A. Surprisingly, the viability of HGC27 and MKN28 cells was increased after treatment with 50 nmol/L chaetocin for 24-72 h. Whereas at 100-600 nmol/L, chaetocin inhibited the viability of HGC27 and MKN28 cells in a time- and concentration-dependent manner [Figure 1]B and [Figure 1]C.

Figure 1: Chaetocin inhibits the proliferation of gastric cancer cells (A: AGS, B: HGC27, C: MKN28, and D: SGC7901). Four gastric cancer cell lines were treated with chaetocin at 50-600 nmol/L and observed for 24-120 h by trypan blue staining under a microscope. Cell survival ratios at different times were calculated to plot the dose-response-time curves. (E) Cellular morphology was observed after chaetocin treatment at different concentrations for 24 h and the representative images of SGC7901 cells are shown at a magnification of 200×.

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3.2. Chaetocin induces apoptosis and pyroptosis of gastric cancer cells

The quadrant of Annexin V-FITC single staining (blue) indicated apoptotic cells and double-stained (green) cells had incomplete cell membranes due to the occurrence of necrosis or pyroptosis. [Figure 2]A shows that chaetocin induced apoptosis in four gastric cancer cells, with the most significant proapoptotic effects on AGS cells at 200 nmol/L and SGC7901 cells at 400 nmol/L. Chaetocin also induced other cell death with incomplete cell membranes, with the best effect in all four cell lines at a concentration of 400 nmol/L. The data of flow cytometry demonstrated that SGC7901 cells had the lowest spontaneous mortality rate among the four cell lines, thus the following mechanism study was conducted mainly on this cell line. The level of p-MLKL, a necrosis marker protein was not altered in AGS, HGC27, MKN28, and SGC7901 cells following 24 h of 400 nmol/L chaetocin treatment [Figure 2]C. Thus, cells showing Annexin V-FITC/PI double staining can only be pyroptotic. This result was further confirmed by the subsequent detection of the pro-pyroptotic protein, GSDME. The combination of p-MLKL and flow cytometry results indicated that chaetocin induced both apoptosis and pyroptosis simultaneously in gastric cancer cells. The merged pink fluorescence images by Hoechst 33342/PI double staining also identified SGC7901 cells with incomplete cell membranes [Figure 2]B which were also pyroptotic and the number of pyroptotic SGC7901 cells was increased with increasing chaetocin concentration. Ultrastructural examination of SGC7901 cells by transmission electron microscope revealed that chaetocin induced noticeable changes in SGC7901 cells. Control untreated SGC7901 cells had well-ordered organelles with typical structures and a relatively small nuclear volume. Treatment with 100 nmol/L chaetocin increased the nucleus-to-cytoplasm ratio accompanied by apoptotic characteristics, such as chromatin condensation, intermediate vacuolization, and a decreased number of abnormally shaped mitochondria. SGC7901 cells exposed to 200 nmol/L chaetocin had more increased chromatin condensation, massive cytoplasm vacuolization, few organelles, disrupted mitochondrial cristae, and increased nuclear occupancy. Large-scale nuclear membrane disruption, intranuclear vacuolization, visible chromatin in the cytoplasm, severe cytoplasmic vacuolization, and almost no organelles with intact structures could be seen after treatment with 400 nmol/L chaetocin [Figure 2]D. Although the cell volume did not alter greatly compared with untreated SGC7901 cells, the chaetocin-treated cells had noticeably increased nuclear occupancy. In summary, chaetocin promoted pyroptosis of gastric cancer cells in addition to inducing apoptosis.

Figure 2: Chaetocin induces pyroptosis of gastric cancer cells in addition to apoptosis. (A) AGS, HGC27, MKN28, and SGC7901 were treated with 100, 200, and 400 nmol/L chaetocin for 24 h. Flow cytometry was performed with Annexin V-FITC and propidium iodide (PI) staining. (B) SGC7901 cells were stained with Hoechst 33342 and PI after 24 h treatment with chaetocin. Fluorescent images were observed under ZEISS Axio Vert.A1 inverted fluorescence microscope (magnification: 200×). (C) AGS, HGC27, MKN28, and SGC7901 were treated with 400 nmol/L chaetocin for 24 h and p-MLKL protein level was evaluated by Western blotting. (D) Ultramicrostructure of SGC7901 cells under a transmission electron microscope after treatment with 100, 200, and 400 nmol/L chaetocin for 24 h (magnification: 1200×). CHA: chaetocin.

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3.3. The caspase 3/GSDME signaling pathway is involved in chaetocin-induced pyroptosis in gastric cancer cells

Caspase 3 activity was suppressed in AGS, HGC27, MKN28, and SGC7901 cells with ZDF to distinguish between GSDMD and GSDME pyroptotic pathways. Treatment with 400 nmol/L chaetocin increased the number of pyroptotic cells (P<0.01), which was reversed by ZDF in all gastric cancer cell lines except HGC27 (P<0.01) [Figure 3]A. LDH release was evaluated to confirm pyroptosis. Chaetocin treatment caused an increase in LDH release (P<0.01), however, ZDF substantially inhibited this effect (P<0.01) [Figure 3]B. Thus, the results suggest the involvement of caspase 3 in chaetocin-induced pyroptosis. GSDME, N-GSDME, GSDMD, and N-GSDMD levels in SGC7901 cells were assessed by Western blotting. Chaetocin treatment raised GSDME and N-GSDME levels but did not affect GSDMD and N-GSDMD levels. ZDF inhibited the upregulation of GSDME and N-GSDME levels caused by chaetocin while decreasing the N-GSDME/GSDME ratio (P<0.05) [Figure 3]C [Figure 3]D. Therefore, the above results suggest that chaetocin-induced pyroptosis in gastric cancer cells was caused by the caspase 3/GSDME signaling pathway rather than the GSDMD pathway.

Figure 3: Caspase 3/GSDME pathway contributes to chaetocin-induced pyroptosis in SGC7901 cells. (A) AGS, HGC27, MKN28, and SGC7901 were treated with 400 nmol/L chaetocin for 24 h immediately after incubating with 50 μmol/L Z-DEVD-FMK (ZDF) for 30 min. Pyroptotic cell percentage was measured by flow cytometry with Annexin V-FITC/PI double staining. Representative results for the SGC7901 cell line are shown in (B-D) after the same treatment as in (A). (B) LDH released was detected by LDH cytotoxicity assay kit. (C) The expressions of GSDME, N-GSDME, GSDMD, and N-GSDMD were evaluated by Western blot. (D) N-GSDME/GSDME ratios were calculated and analyzed by ImageJ V1.51 software. Bars represent mean ± SD (n=3). *P<0.05; **P<0.01.

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3.4. Silencing of EHMT2 gene abolishes chaetocin-induced pyroptosis in gastric cancer cells

G9a is encoded by the gene, EHMT2. Differential expression of EHMT2 in gastric cancer and healthy gastric tissues was analyzed by searching the GEPIA website. EHMT2 transcript levels were found to be higher in gastric cancer tissues than in normal tissues (P<0.05; [Figure 4]A), indicating a potential role for G9a as a biomarker in gastric cancer. EHMT2 was knocked down by siRNA in SGC7901 cells and treatment with 400 nmol/L chaetocin for 24 h produced no abnormal morphological changes compared to the control group [Figure 4]B [Figure 4]C. Similarly, chaetocin had no impact on GSDME and N-GSDME expression [Figure 4]C or LDH release in EHMT2-silenced SGC7901 cells (P>0.05) [Figure 4]D. These data suggest that chaetocin-induced pyroptosis was inhibited in the absence of G9a in SGC7901 cells.

Figure 4: Silencing of the EHMT2 gene abolishes chaetocin-induced pyroptosis. (A) Differential expression of EHMT2 in human stomach adenocarcinoma (red box) and normal gastric tissue (white box) (*P<0.05). EHMT2 transcripts per million (TPM) were analyzed using the GEPIA website (http://gepia.cancer-pku.cn) database. (B) G9a protein was detected in SGC7901 cells by Western blot after EHMT2-silencing with siRNA. (C) EHMT2-silenced cells were treated with 400 nmol/L chaetocin for 24 h. The cellular morphology was observed (magnification: 200×). GSDME and N-GSDME protein levels were measured by Western blotting. (D) LDH release in EHMT2-silenced SGC7901 cells after treatment with 100, 200, and 400 nmol/L chaetocin. Bars represent mean ± SD (n=3).

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  4. Discussion 

Chaetocin can suppress growth of many tumor cell types, including myeloma, leukemia, non-small cell lung cancer, hepatocellular carcinoma, intrahepatic cholangiocarcinoma, melanoma, glioma, colon cancer, renal cancer, prostate cancer, breast cancer and ovarian cancer[16],[17],[18],[19],[20],[21]. In this study, chaetocin inhibited the proliferation of four gastric cancer cell lines with different degrees of differentiation in a time-dependent manner. Chaetocin inhibition tends to be more pronounced for cells from solid tumors. Mechanisms underlying the anti-tumor activity involve the induction of apoptosis, cell cycle arrest, autophagy, and suppression of tumor cell invasion, metastasis, and angiogenesis[10],[12],[22],[23],[24]. The current study reported that chaetocin induced pyroptosis of gastric cancer cells in addition to apoptosis.

Cookson et al. initially identified pyroptosis as a novel programmed cell death mode distinct from apoptosis[25]. In addition, caspase 1/4/5/11 had been found to cleave GSDMD into N-GSDMD increasing membrane permeability during pyroptosis. N-GSDMD creates oligomerized pores in the cell membrane to release cellular contents, triggering pyroptosis and inflammatory reactions[26],[27],[28]. It was later discovered that the gasdermin family, GSDMA, GSDMB, GSDMC, and GSDME/DFNA5, were also cleaved to yield N-terminal proteins with similar roles to N-GSDMD[29],[30],[31]. As a result, the Nomenclature Committee on Cell Death redefined pyroptosis as "a type of regulated cell death that critically depends on the formation of plasma membrane pores by members of the gasdermin protein, often (but not always) as a consequence of inflammatory activation"[32]. Chaetocin raised GSDME and N-GSDME levels in the current work accompanied by an increase in the number of pyroptotic cells, but did not affect GSDMD and N-GSDMD. Caspase 3 is thought to be involved in both apoptosis and pyroptosis and to either inactivate GSDMD or activate GSDME through the cleavage of GSDME to produce N-GSDME which increases membrane permeability and induce pyroptosis[33],[34]. Pyroptosis may be induced by the caspase 3/GSDME or the traditional GSDMD pathway in the treatment of cancers with most chemotherapeutic drugs. Caspase 3 inhibition abolished the upregulation of GSDME and N-GSDME caused by chaetocin, decreasing the N-GSDME/GSDME ratio and pyroptotic cell number. Thus, the caspase 3/GSDME pathway is responsible for chaetocin-induced pyroptosis in gastric cancer cells rather than the conventional GSDMD pathway. Further research is required to determine whether other gasdermins and associated pathways contribute to chaetocin-induced cell pyroptosis.

The reports of other researchers and our bioinformatics analysis indicate that G9a plays an important role in the formation and evolution of gastric cancer. In the present study, a lack of G9a expression could result in the abolition of pyroptosis induced by chaetocin. Thus, we infer that G9a may be one of the targets of chaetocin to induce pyroptosis of gastric cancer cells. However, the pharmacological action of chaetocin is not specific. In addition to HMTs, thioredoxin reductase 1, heat shock protein 90, and hypoxia-inducible factor-1 alpha/p300 complex have also been identified as target molecules of chaetocin[35],[36],[37]. Whether these other molecules are also involved in the chaetocin-induced regulation of pyroptosis remains to be addressed.

In conclusion, the present study shows that chaetocin suppresses proliferation of gastric cancer cells by simultaneously inducing apoptosis and pyroptosis. It activated the caspase 3/GSDME pathway by targeting G9a, resulting in increasing membrane permeability and inducing pyroptosis of gastric cancer cells. These findings give new insights into the cytotoxic mechanism of chaetocin, and lay a foundation for its further development as an anti-tumor agent and for possible combination therapy.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Acknowledgments

We would like to express our gratitude to EditSprings (https://www.editsprings.cn) for the expert linguistic services provided.

Funding

This work was supported by Natural Science Foundation of Hainan Province, China (No. 820MS048).

Authors’ contributions

MQH designed and performed experiments, analyzed data, prepared manuscript; GLT analyzed the data and revised the manuscript. LFH and SHT performed the experiments and analyzed the data. PZ designed experiments and revised the manuscript. All authors approved the final manuscript.

Publisher’s note

The Publisher of the Journal remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

 

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4]