miR-148-3p inhibits gastric cancer cell malignant phenotypes and chemotherapy resistance by targeting Bcl2

ABSTRACT

Gastric cancer (GC) is the fourth most common cancer in the world. This work was designed to explore the biological effects of miR-148-3p on GC. Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was utilized to analyze the mRNA expression of miR-148-3p in GC cell lines. The mimics and inhibitors of miR-148-3p were carefully transfected into GC cells to up-regulate or down-regulate miR-148-3p expression. Observe the effect on miR-148-3p expression change to GC cell proliferation, colony formation, tumorigenesis, chemotherapy sensitivity, transwell migration, and invasion. Use online database tool to predict the miR-148-3p promising targets, and can be verified via RT-qPCR, Western blot, and luciferase report. We found that miR-148-3p expression level in GC cells was markedly down-regulated (P < 0.05), as compared with human normal gastric mucosal cells GES-1. Otherwise, miR-148-3p overexpression could effectively inhibit the cell proliferation, cell cycle progress, colony formation, anti-apoptosis, anti-migration and anti-invasion in gastric cancer cells, whereas miR-148-3p inhibition exhibited the opposite phenomenon (P < 0.05). Further research revealed that Bcl2 set as a direct downstream target of miR-148-3p. Our study firstly confirmed that, miR-148-3p might play a crucial role in tumorigenesis, as well as development of gastric cancer by targeting Bcl2, and could become a promising target for gastric cancer treatment.

KEYWORDS:

• miR-148-3p
• Bcl2
• cell proliferation and invasion
• gastric cancer
• chemotherapy resistance

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Publisher’s Note

1. Introduction

The incidence of gastric cancer (GC) has been declining in most parts of the world, but it is still the fourth most common cancer in the world [1]. According to the latest statistics report on cancer in China in 2017, more than 80% gastric cancer patients are last-phase cases [2,3]. For patients with relapsed or metastatic gastric cancer, multi-chemotherapy might be the only selection. Nevertheless, the effectiveness of palliative and supportive treatment is about 25% to 50%, while the median overall survival is only 6–12 months [4]. Cisplatin (CDDP) is the first-line regimen used to treat patients with locally advanced or metastatic [5]. Studies have shown that the mechanism of cytotoxicity induced by CDDP is to bind with DNA and form DNA adducts. In addition, CDDP could active series important signaling pathways, and lead to apoptosis by binding to the DNA of nuclear and mitochondrial [6]. However, just as clinical studies shown, whether it is intrinsic or acquired cancer, the development of multidrug resistance will gradually reduce the chemotherapy efficacy.The main mechanisms for intrinsic and acquired drug resistance include the reduced intracellular drug accumulation, cell apoptosis, changed cell cycle, enhanced DNA repair mechanisms, as well as accelerated metabolism of pharmaceutical [7]. However, an exact mechanism for how cisplatin effectively kills tumor cells remains obscure.

MicroRNA (MiRNA) is a small endogenous non-coding RNA, which served as a critical factor in gene modulation and degradation of target proteins in post-transcription [8]. Several studies have reported that miRNA is not merely closely related to the biological functions of tumor, but also has an effect on drug resistance [9]. miR-124 suppresses cisplatin resistance in lung cancer cells via regulating STAT3. MiR-148-3p is regarded as an anti-oncogene in prostate adenocarcinoma and glioma [10,11], Moreover, a study using bioinformatics analyses combining TCGA with GEO datasets demonstrated that miR-148a-3p plays an important role in CDDP cytotoxicity of GC cells by mitochondrial fission induction and cyto-protective autophagy suppression [12].

B cell lymphoma/leukemia-2 (Bcl2) family proteins are a key regulatory component of the intracellular apoptosis pathway, and this pathway is essential for the development of cancer [13]. Bcl2 could be uniformly expressed in chronic lymphocytic leukemia and promote the survival of leukemia cells [14]. Furthermore, Bcl2 could promote cell migration as well as invasion in colorectal cancer, and gastric cancer [15,16]. The above studies have proven that Bcl2 can promote the tumorigenesis to a certain extent.

By consulting a large number of documents and querying the prediction of miRNAs targets in TargetScan, we discovered miR-148-3p and Bcl2 have a targeting relationship. However, the drug resistance mechanism and biological functions for miR-148-3p and Bcl2 on the gastric cancer cells have not been researched yet.

Therefore, this study aims to explore the bioregulation of miR-148-3p in GC, the molecular mechanism of miR-148-3p/Bcl-2 in GC and drug resistance, to lay a theoretical foundation for drug development and possible clinical application in the future.

2. Materials and Methods

2.1 Cell culture and transfection

Human GC cell lines (BGC-823, SUN-1, MKN-7 and SGC-7901), as well as human normal gastric mucosal cell lines (GES-1) were culturedin DMEM medium consist of 10% FBS and 100 U/mL penicillin mixed with streptomycin at 37°C in a humidified atmosphere of 5% CO₂. When cells grew to about 85%, cells were collected with 25% trypsinization. After digestion, the resultant cell suspensions were used to be cultured continuously. Subsequently, SCG-7901, as well as BGC-823 cells, were utilized for transfection. Liposome ™ 2000 kit was used for transfecting into miR-148-3p inhibitors, miR-148-3p mimics, miR-negative control, targeted Bcl2 RNA inhibition, overexpression of targeted Bcl2 RNA, as well as negative control, respectively.

2.2 Quantitative RT-PCR

Total RNA was extracted from cultured cells with TRIzol reagent. Collect 5 μg of reverse transcribed synthetic cDNA based on the instructions of the manufacturer, and take 1 μL of synthetic cDNA to amplify after transcription. The quantity and procedure were listed as follow: 1 μL cDNA, 0.4 μL up-stream and down-stream primers (2 μmol/L), 10 μL Transscript® Tip Green qPCR SuperMix (2X), 20 μL of nucleic acid-free water, 0.4 μL passive reference dye (50X) and U6 as miR-148-3p internal reference. The relative expression levels of mRNA were calculated by using the 2 − ΔΔCt method [17]. β‐actin was served as internal control.2.

2.3 In vitro cytotoxicity study

The proliferation potential in BGC-823 as well as SGC-7901 cells were performed with CCK-8 kit. After 48 h transfection, the cells were seeded into 96-well plates (3 × 10⁴cells/mL). 100 μL cells/well were incubated at 37°C with 5% CO₂. After co-culturing for 24 h, 48 h and 72 h, adding 10 μL CCK8 solution and further incubating for another 2 h, respectively. Microplate reader was utilized for absorbance testing under 450 nm [18]. Cell proliferation was measured and draw a growth curve. All samples were tested by three times (n = 3).

2.4 Cell cycle and apoptosis

The cell cycle was measured with FLOW cytometer. In short, wash of cells twice using PBS and fixed with 75% alcohol, refrigerated overnight at −20°C. Before the test, RNase was used for treating and propidium iodide (PI) was utilized for staining. The cell cycle was measured via FLOW cytometry, and the fluorescence value of PI was analyzed, accordingly.

Annexin V-FITC (fluorescein isothiocyanate)/PI analysis was utilized for cell apoptosis. In brief, cells were cultured with CDDP and collected with trypsin without ethylenediaminetetraacetic acid after 48 h. After that, 1 mL of 1× Annexin V-FITC/PI solution was added, and co-incubated for 20 min at 4°C in dark. Cell staining was detected using FACS Calibur FLOW cytometer [19].

2.5 Transwell tumor cell migration and invasion assay

Cell migration assay. A total of 5 × 10⁴ cells (SGC-7901 cell and BGC-823 cell), 100 μl cell suspension were placed in the upper chamber, respectively. Meanwhile, fresh medium with 20% FBS, was added to the lower chamber, which was aimed to induce the migration of SGC-7901 as well as BGC-823 cells. After 24 h of incubation, cells without migration were removed with a cotton swab. The migranted cells were immobilized with methanol, then staining with crystal violet. Finally, inverted microscope (Olympus Corporation, Tokyo, Japan) was utilized to count cell quantity.

Cell invasion assay: A total of 5 × 10⁴ cells (SGC-7901, as well as BGC-823 cells) under different treatments were seeded into 24-well insert transwell chambers (Corning, NY, USA), and the lower chamber contained medium with 20% FBS (0.5 mg/mL). After incubation for 24 h, 4% paraformaldehyde was added and incubated for 20 min. Next, 0.1% crystal violet was used tostain cells and lasted for 5 min. Count and photograph the number of cells, which was penetrating the back of the membrane under a light microscope [20].

2.6 Western blot

The cells were lysed, and total proteins were extracted and examined for concentration using BCA Protein Assay Kit. The concentration was adjusted to 4 μg/μL, and 12% SDS-PAGE was utilized for electrophoretic separation of proteins. After that, sample was transferred onto PVDF membranes. Subsequently, PVDF membrane was sealed via 5% skimmed milk for 2 hours, incubated with diluted Bcl2 (1:1000, Abcam, Cambridge, UK), and diluted primary antibody of β-Actin (1:2000, Santa Cruz, Biotech, CA, USA) passthenight at 4°C. free Bcl2 and primary antibody were removed by washing 3 times with PBST. After incubation with a secondary antibody goat anti-mouse labeled with horseradish peroxidasey (1: 500). Finally, the membranes were measured using ECL reagent [18].

2.7 Dual luciferase reporter assay

Cells were co-transfectd with pGL3-BCL2-WT or pGL3-BCL2-MUT carrier (Promega Corporation, Madison, WI, USA) with miR-148-3p mimic carrier (Promega Corporation, Madison, WI, USA), and co-transfect SPC-A1 cells with pGL3-BCL2-WT or pGL3-BCL2-MUT carrier (Promega Corporation, Madison, WI, USA). After transfection for 48 h, cells were lysed and detectedd the activity of luciferase. The firelly luciferase activity was calculated through Luciferase Reporting Analysis (Promega, Madison, WI, USA), and Renilla luciferase activity was utilized as the standard [21].

2.8 Animal tumor xenograft models

The sterile BALB/c nude mice (4–6 weeks) were provided by Changsha Silaike Jingda Experimental Animal Co., Ltd (Hunan, China). All animal experiments were authorized through the Animal Ethics Committee of Zhengzhou University, as well as executed according to the Guidelines for Evaluation and Accreditation Association of Laboratory Animal Care. The cells of SGC-7901/miR-148-3p/NC, BGC-823/miR-148-3p/NC, SGC-7901/miR-148-3p/Mimics, BGC-823/miR-148-3p/Mimics, SGC-7901/miR-148-3p/Inhibitors as well as BGC-823/miR-148-3p/Inhibitors were constructed using lentivirus (Gene Pharma) as a carrier. For tumor-bearing nude mice, suspend the 2 × 10⁶ cells of SGC-7901 and BGC-823 in 0.2 mL of non-serum DMEM medium, which was mixed with 50% phenol red Matrigel, respectively. Subsequently, under the sterile conditions, 2 × 10⁶ cells of SGC-7901 and BGC-823 in a volume of 0.2 mL were carefully injected on the dorsal flank of mice (4 ~ 6 weeks, n = 6) without anesthesia, respectively. Check the health of the mice every day. In tumor-bearing nude mice, CDDP (5 mg/kg) was administered once a week for four periods. Tumor volume in tumor-bearing nude mice was tested 2 or 3 days. The tumor volumes were counted as: V (mm³) = (length×width×width×3.14)/6. The tumor-bearing nude mice weight was measured per 3 days. Since the experiment ended, the nude mice have been humanely killed [22].

2.9 Human samples

Human gastric cancer and their corresponding non-tumorous gastric tissues were collected at the time of surgical resection from 42 patients with gastric adenocarcinoma from 2020 to 2021 at our hospital. Human tissues were immediately frozen in liquid nitrogen and stored at −80°C refrigerator. Signed informed consent was obtained from all patients and the study was approved by the Seventh Medical Center of PLA General Hospital Clinical Research Ethics Committee.

2.10 Statistical analysis

The data are detailed as mean ± standard deviation. However, statistical analysis for two different groups was followed via t test. The variance analysis (ANOVA) was utilized for statistical comparisons among groups. All analyses data were conducted through SPSS 21.0 software (IBM, Armonk, NY, USA), and the two-tailed value was regarded as statistically significant (P < 0.05).

3. Results

GC is one of the most popular malignancy globally. Understanding the pathogenesis and progression mechanism of GC, may offer us new strategies for GC treatment. miR-148a-3p has been reported play an important role in many kinds of tumors and CDDP cytotoxicity of GC. However, its mechanisms need further elucidation. In the present study, we conducted a series of in vitro and in vivo assays, aimed to explore the molecular mechanism of miR-148a-3p/Bcl-2 in GC, to lay a theoretical foundation for drug development and possible clinical application in the future.

3.1 miR-148-3p inhibits gastric cancer cell proliferation and promotes cell apoptosis

To explore the promising function of miR-148-3p in GC, we detected miR-148-3p expression level in GC cell lines. In comparison with normal gastric epithelial cell lines GES-1, miR-148-3p expressed level in GC cell lines was lower. The expression of miR-148-3p in gastric cancer tissue and pan-cancerous tissue in 42 GC patients was also analyzed. As shown in Figure 1(a), miR-148-3p was significantly down-regulated in GC tissues. Then, miR-148-3p mimics and inhibitors were used to transfected into SGC7901 and BGC823 cells, respectively (Figures 1(b,c)). Figures 1(d,e) shows, miR-148a-3p mimics inhibited the SGC7901 cells proliferation, whereas miR-148-3p inhibitors could promote BGC823 cells proliferation. We further analyzed the influence of miR-148-3p to gastric cancer cell cycle. As Figures 1(f,g) shown, miR-148-3p mimics significantly reduced the G2/M phase cell ratio, but dramatically increased the G0/G1 phase cell ratio. In contrast, miR-148-3p inhibitors represented opposite phenomenon.

Figure 1. miR-148-3p inhibits in vitro proliferation for gastric cancer. A. In comparison with normal gastric epithelial cells, miR-148-3p expressed on gastric cancer cells is significantly down-regulated. miR-148-3p was also notably downregulated in GC tissues. B and C. Effect of transfected miR-148-3p mimics or inhibitors on miR-148-3p expressed in gastric cancer cells. D and E. Effects on transfection of miR-148-3p mimics or inhibitors to the gastric cancer cells proliferation. F and G. Effect on transfection of miR-148-3p mimics or inhibitors to the gastric cancer cell cycle. ** P < 0.01, *** P < 0.001.

3.2 The miR-148-3p potential for drug resistance, migration, and invasion of GC cells

The purpose of experiment is to study the of miR-148-3p potential effects on drug resistance, cell migration and cell invasion. Meanwhile, we also evaluated the effect of miR-148-3p on cell sensitivity to CDDP. miR-148-3p overexpression promoted SGC7901 cell sensitivity to CDDP, while miR-148-3p inhibitors inhibited BGC823 cells sensitivity to CDDP (Figures 2(a,b). We also explored whether miR-148-3p regulate GC cell migration, as well as cell invasion. The results of Figures 2(c,d) showed that, miR-148-3p mimics significantly inhibited cell migration, as well as cell invasion of GC, whereas miR-148-3p inhibitors exhibited the opposite phenomenon.

Figure 2. miR-148-3p inhibits gastric cancer cell migration, invasion and chemotherapy resistance. A and B. Effects on miR-148-3p mimics as well as inhibitors in cisplatin-induced gastric cancer cells apoptosis. C. Functions of miR-148-3p mimics to cell invasion as well as cell migration. D. Effect on miR-148-3p inhibitors to cell invasion and cell migration. ** P < 0.01, *** P < 0.001.

3.3 Bcl2 is s a direct target for miR-148-3p

To investigate the mechanisms underlying the regulation by miR-148a-3pin GC cells, we analyzed the potential targets of miR-148-3p using the online database tool TargetScan. The results indicated that Bcl2 is only a common miR-148-3p predicted target gene, and the binding site of miR-148-3p and 3ʹ-untranslated regions (3ʹ-UTR) of Bcl2 showed in Figure 3(c). In order to verify the prediction, we analyzed Bcl2 expression in GC cells after transfection of miR-148-3p mimics or inhibitors. As shown in Figures 4(a,b), miR-148-3p mimics significantly inhibited the mRNA and Bcl2 protein expression, while miR-148-3p inhibitors up-regulated the expression of Bcl2. In addition, we also constructed a luciferase reporter plasmid recombined with wild-type (Wt) or mutant (Mut) 3ʹ-UTR of Bcl2, which is demonstrated in Figure 3(c). Luciferase activity report has shown that co-transfection of miR-148-3p mimics significantly reduce the luciferase activity of Bcl2 3ʹ-UTR cells in wild type (Wt), but has no significant effect on mutant (Mut) cells. However, the miR-148-3p inhibitors significantly increased the luciferase activity of Wt-type cells, rather than the luciferase activity of Bcl2 3ʹ-UTR cells in non-Mut-type (Figure 3(d,e)). The above results have proved that miR-148-3p may negatively regulate Bcl2 by combining with its 3ʹ-UTR.

Figure 3. Bcl2 is a straight target for miR-148-3p. A and B. Effects on miR-148-3p mimics and inhibitors to Bcl2 expression as well as its mRNA on gastric cancer cells, respectively. C. TargetScan was applied to predict the Wt and Mut miR-148-3p target sequences of Bcl2 3ʹUTR. D and E. Gastric cancer cells relative luciferase activity was examined by co-transfecting with Wt or Mut Bcl2 3ʹUTR reporter carrier as well as miR-148-3p mimics or inhibitors. *** P < 0.001.

Figure 4. miR-148-3p inhibits the action of Bcl2. A to J. Bcl2 (a) inhibits miR-148-3p overexpression to simulate SGC7901 cells proliferation (b), apoptosis resistance (c), invasion (d) and migration (e); whereas Bcl2 low expression (f) reverses miR-148-3p inhibitors on BGC823 cell proliferation (g), apoptosis resistance (h), invasion (i) and migration (j). ** P < 0.01, *** P < 0.001.

3.4 MiR-148-3p inhibits GC by reducing the expression of Bcl-2

To clarify whether Bcl2 is an effector of miR-148-3p in GC, we conducted a rescue assay by co-transfecting miR-148-3p mimics and Bcl2 constructs into SGC7901 cells (Figure 4(a)). Just as shown in Figure 4(b), Bcl2 overexpression significantly increased the survival rate of SGC7901 cells. These data demonstrated that Bcl2 significantly reversed miR-148-3p mimic-mediated cells apoptosis in CDDP-treated cells (Figure 4(c)). Transwell assay indicated that Bcl2 over-expressed can inhibit the gastric cancer cell migration, as well as cell invasion induced by miR-148-3p mimics (Figures 4(d,e)). In addition, we also evaluated the impact on co-transfected miR-148-3p inhibitors, as well as Bcl2 siRNA on BGC823 cells (figure 4(f)). Just as shown in Figure 4(g), Bcl2 siRNA significantly reversed the promoting function on cell proliferation through miR-148-3p inhibitors. Furthermore, in cells transfected with miR-148-3p inhibitors, Bcl2 siRNA significantly enhanced the sensitivity of CDDP (Figure 4(h)). Moreover, Bcl2 siRNA also inhibited miR-148-3p inhibitor-mediated BGC823 cell migration and invasion (Figures 4(I,J)). The above results further confirmed that miR-148-3p could play an anticancer effect by targeting the expression of Bcl2.

3.5 MiR-148-3p enhances gastric cancer cells growth and chemotherapy resistance in vivo

In order to verify our findings, we further studied miR-148-3p function for gastric tumor-bearing nude mice in vivo. miR-148-3p mimics observably inhibited SGC7901 cells tumorigenicity in nude mice, whereas miR-148-3p inhibitors promoted BGC823 cells growth in vivo. In addition, miR-148-3p mimics can enhance the anti-cancer effect of CDDP, while miR-148-3p inhibitors could antagonize the effect of CDDP in vivo (Figure 5(a-h)).

Figure 5. In vivo, miR-148-3p inhibits cell growth and chemotherapy resistance of GC. A and B. miR-148-3p mimics can suppress GC cell growth in tumor-bearing mice. C and D. miR-148-3p inhibitors promoted GC growth in vivo. E and F: miR-148-3p mimics strengthened cisplatin GC cells sensitivity in vivo. G and H: miR-148-3p inhibitors reduced cisplatin GC cells sensitivity in vivo. ** P < 0.01, *** P < 0.001.

4. Discussion

Gastric cancer is the fourth most common cancer in the world. Currently, surgical treatment and systemic chemotherapy are the main treatments for gastric cancer [23,24]. However, since the majority of patients were found at late stage, who didn’t have the chance of surgery. Therefore, the most effective method was chemotherapy. Due to the existence of chemotherapy resistance, many patients suffered from failed treatment, and caused death [25,26].

Recently, the role of miRNA in tumors has also been investigated. During the process of tumor formation and progression, miRNA is capable of modulating the biological function of tumor cells by modulating regulating the expression of tumor suppressor genes or oncogenes [27,28]. MiR-148-3p had an effect of tumor suppressive in gastric cancer, which is confirmed by our research. Researchers have attached increasing importance to the relationship between microRNA and chemotherapy resistance. MicroRNA-217 could increase cisplatin sensitivity in non-small cell lung carcinoma by targeting KRAS19 [29]. Some researchers have also studied the role of miR-148-3p in tumors, but the effect of miR-148-3p on gastric cancer cisplatin sensitivity have not been investigated yet. Our work discovered the expression of miR-148-3p in gastric cancer cells was significantly decreased. However, the up-regulated miR-148-3p could effectively inhibit the gastric cancer cells proliferation, invasion and migration, as well as accelerate gastric cancer cells apoptosis. To explore the effect of miR-148-3p on cisplatin resistance, miR-148-3p expression level was regulated under the condition of cisplatin intervention. It was indicated that up-regulated miR-148-3p could increase cisplatin sensitivity on gastric cancer cells, whereas down-regulated miR-148-3p could decreased the cisplatin sensitivity on gastric cancer cells. Therefore, the experimental results indicated that miR-148-3p could improve chemotherapy efficacy and might become a potential target, which would bring hope for gastric cancer patients.

miRNA plays a biological effect through an imperfect pairing with the target mRNA 3ʹ-UTR [30]. Previous studies have shown that down-regulation of miR-148-3p can alleviate cerebral ischemia or reperfusion injury by targeting Sestrn 2 [31]. Feng et al. have shown that miR-148-3p inhibited prostate cancer formation by inhibiting KLF4 expression [10]. In gliomas, Li et al. have verified that miR-148-3p inhibit glioblastoma growth via targeting DNA methyltransferase-1 (DNMT1) [11]. In this study, we used an online database tool, TargetScan website, to predict target of miR-148-3p. The data indicated that Bcl2 might become a promising target for miR-148-3p, as well as the dual luciferase report and Western blot also confirmed that. Further studies have shown that Bcl2 over-expressed attenuate the action of miR-148-3p, and the silence of Bcl2 could augment the function of miR-148-3p in GC inhibition. The above experiment findings demonstrated that down-regulation of Bcl2 might mediate antitumor effect of miR-148-3p in gastric cancer cells.

Bcl2 apoptosis regulator (Bcl2) could encode a complete mitochondrial membrane, which can block the apoptosis of various cells such as lymphocytes [32]. The expression profile of the Bcl2 family has been reported that have some relationship with the tumor aggressiveness of B-cell malignancies. Several studies have reported that the abnormal expression of Bcl2 promoted the occurrence of various types of mature non-Hodgkin B-cell lymphoma in mice [33]. Further studies have shown that Bcl2 promotes cancer cell proliferation in a p53-dependent way through regulating the cyclin-related molecules expression, which includes cyclin A, cyclin D1, cyclin-dependent kinase 4, p21 as well as β-catenin [34,35]. Our work revealed that miR-148-3p could inhibit GC cell proliferation, cell cycle progress, migration, invasion and chemotherapy resistance by targeting Bcl2, and reduce the ontogenesis. However, the detailed mechanism of how Bcl2 to promote gastric cancer progression still needs further investigated.

5. Conclusion

In summary, we confirmed miR-148-3p could play a vital role in gastric cancer progression as well as chemotherapy resistance. All these experimental data manifested miR-148-3p is a key factor in suppressing gastric cancer cells proliferation, cell cycle progress, invasiveness, migration and cisplatin resistance. In addition, we also confirmed that Bcl2 acts as a directly functional target for miR-148-3p, which mediated antitumor effect of miR-148-3p in gastric cancer. All studies reveal that miR-148-3p could become a promising target for gastric cancer therapy. It is worth to further study and provide more theoretical evidences for gastric cancer clinical treatment.

Highlights

1. MiR-148-3p inhibits GC cell proliferation, migration and invasion and promotes cell apoptosis and cell sensitivity to CDDP.

2. Bcl2 is a direct target for miR-148-3p.

3. miR-148-3p might play a crucial role in GC by targeting Bcl2, and could become a promising target for gastric cancer treatment.

Informed consent

Informed consent was obtained from all individual participants included in the study.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.