Sodium Selenite Regulates the Proliferation and Apoptosis of Gastric Cancer Cells by Suppressing the Expression of LncRNA HOXB-AS1

Gastric carcinoma has a high incidence, accounting for approximately 6% of all cancers worldwide. The in vivo antitumor effect of sodium selenite on gastric carcinoma has been demonstrated. This study therefore aimed to further explore its targets in gastric cancer in vitro and elucidate its mechanism of action. The effects of inorganic sodium selenite (Na2SeO3) on apoptosis, proliferation, and invasion of gastric cancer cells were investigated, and the interaction between Na2SeO3 and expression of long noncoding RNA homeobox B cluster antisense RNA 1 (HOXB-AS1) was investigated to elucidate the specific mechanism of action of selenium on gastric cancer cell proliferation through regulation of HOXB-AS1. Na2SeO3 downregulated the expression of HOXB-AS1 in the human gastric cancer (HGC) cell lines, HGC-27, NCI-N87, and KATO III cells, while inhibiting their proliferation and invasion and inducing apoptosis. The upregulation of HOXB-AS1 produced the opposite results. Na2SeO3 was used to stimulate HGC-27 cells, which caused HOXB-AS1 overexpression. The cell counting kit-8 (CCK-8) assay revealed a decrease in cell proliferation, while western blotting, flow cytometry, and transwell migration assays showed the expression of apoptosis-related (Bad, Bcl-2, and cleaved-caspase-3) and invasion-related (MMP2, E-cadherin, and N-cadherin) proteins, indicating increased apoptosis and decreased invasion. We therefore conclude that Na2SeO3 inhibits the malignant progression of gastric cancer by downregulating the expression of HOXB-AS1 and thus could be used as a potential drug for its treatment.


Introduction
Gastric carcinoma ranks ffth and third in terms of the incidence and mortality rates of malignant tumors worldwide [1]. Although radical surgery is the preferred treatment modality for gastric cancer, standard treatment also involves perioperative chemotherapy [2,3]. Most chemotherapeutic agents are toxic and exert adverse efects. In addition, 50% of patients with advanced gastric cancer show varying degrees of recurrence after receiving standard adjuvant therapy [4]. century have shown that sodium selenite, sodium selenate, selenomethionine, selenium methylselenocysteine, and methylselenic acid all exert obvious antitumor efects [10,11].
Long noncoding RNAs (lncRNAs) are RNA molecules that are longer than 200 nucleotides but have no protein coding ability [12]. In recent years, substantial evidence has suggested that lncRNAs play an important role in cancer by participating in proliferation, migration, apoptosis, and other tumor cell activities [13,14]. Yan et al. found that the overexpression of lncRNA-MUF could activate the Wnt/β-catenin signaling pathway and promote epithelial mesenchymal transition by forming a complex with membrane-binding protein A2, thus promoting the development of liver cancer [15]. lncRNA H19 promotes cell proliferation by competitively binding to miR-200a and derepressing β-catenin expression in colorectal cancer [16]. Selenium can further regulate the transcription of genes and afect lncRNA expression [17]. HOXB-AS1, a newly discovered lncRNA, and a similar encoded short peptide, HOXB-AS3, have been reported to inhibit the proliferation and metabolism of colon cancer cells [18,19]. However, the mechanism underlying the interaction between selenium and HOXB-AS1 in gastric cancer remains unclear.
In this study, the efects of Na 2 SeO 3 and HOXB-AS1 on apoptosis, proliferation, and invasion of gastric cancer cells as well as the interaction between selenium and HOXB-AS1 were investigated. Our results suggest that Na 2 SeO 3 inhibits gastric cancer pathogenesis by downregulating HOXB-AS1 expression.

Cell Culture.
Human gastric cancer (HGC) cells (HGC-27, NCI-N87, and KATO III) and a human gastric epithelial cell line (GES-1) were obtained from the Shanghai Cell Bank of the Chinese Academy of Science and cultured at 37°C in 5% CO 2 . Te cells were passaged at a ratio of 1:2-4 after reaching a confuency of >90%. HGC cells were grown in the RPMI-1640 medium (Hyclone) supplemented with 10% fetal bovine serum (FBS). GES-1 cells were cultured in Dulbecco's modifed Eagle's medium (DMEM) (Hyclone) supplemented with 10% FBS.

CCK-8 Assay.
Te CCK-8 (Solarbio) assay was performed to evaluate cell viability. Cells were seeded in 96-well plates (3 × 10 3 cells/well) and cultured overnight at 37°C in 5% CO 2 . After treatment, 10 μL of the CCK-8 solution was added to each well and incubated for 4 h. Te absorbance of each well was measured at 450 nm with a microplate reader.

Flow
Cytometry. Apoptosis was investigated using fow cytometry. A cell suspension containing 1 × 10 6 cells was washed and centrifuged with ice-coldphosphate-bufered saline at 800 × g for 5 min. Te cells were then stained with 10 μL annexin V-FITC and 10 μL propidium iodide for 20 min in the dark before being subjected to fow cytometry.

Transwell Migration
Assay. HGC-27 cells were suspended in a serum-free medium with 1% FBS and seeded (1 × 10 5 cells/well) in the upper chamber of the transwell; 0.75 mL of the serum-free medium with 10% FBS was then added to the lower chamber. After 48 h of coincubation at 37°C, the HGC-27 cells on the bottom surface of the lower chamber were fxed with 4% formaldehyde for 20 min at room temperature and stained with 0.5% crystal violet for 30 min. Cells that did not migrate to the lower chamber were counted under an optical microscope (200× magnifcation).

Statistical
Analysis. Data are presented as the mean-± standard deviation. One-way ANOVA was used to compare the diferences between the groups using SPSS 21.0. Statistical signifcance was set at P ≤ 0.05. and 1(c)) compared to the controls. In addition, apoptosisrelated and migration-related protein expression (Figures 1(f ) and 1(g)) was detected in HGC-27 cells with or without drug treatment. Te protein expression of Bad, cleaved-caspase-3, and E-cadherin was signifcantly upregulated in groups treated with Na 2 SeO 3 compared to that in the control group (P < 0.01). In contrast, the expression of Bcl-2, MMP2, and N-cadherin proteins was signifcantly downregulated compared to the control group (P < 0.01, Figures 1(d) and 1(e)). In all cases, the efects of Na 2 SeO 3 were dose-dependent.

Discussion
Te incidence of gastric cancer has been increasing in recent years, and mortality remains high due to recurrence and metastasis [20]. Te incidence of gastric cancer is related to diet, sex, age, region, Helicobacter pylori infection status, genetic polymorphisms, and other causes [21]. Te rate of early diagnosis is only 10%, meaning that most patients are diagnosed at an advanced stage [22]. Although surgical and perioperative techniques are continuously developing and the safety of gastric cancer resection has greatly improved, various postsurgical complications that seriously afect a patient's quality of life remain issues [23]. At present, most anticancer treatments are nonspecifc, killing both tumor and normal cells. Terefore, it is of great clinical signifcance to explore the pathogenesis of gastric cancer and to develop targeted treatments for its prevention and treatment.
Recent studies have shown that noncoding RNAs (ncRNAs), including ribosomal RNA, transfer RNA, microRNAs, and lncRNAs, are involved in many biological and pathological processes [24,25]. Cancer is an inherited disease that involves multiple changes in the genome, and diferential expression of lncRNAs has been closely related to the occurrence and development of tumors [20]. Seventyfve percent of the human genome is transcribed into RNA, but only a small portion of these transcripts encode proteins. Te number of lncRNA genes is very large, and their potential mechanisms of action in cancer largely remain to be explored [26]. For example, increased expression of lncRNA plasmacytoma-variant translocation 1 (PVT1) is associated with an advanced stage and a poor prognosis in patients with ovarian cancer. PVT1 inhibits the expression of miR-214 in ovarian cancer cells by regulating the epithelialmesenchymal transition process and its interaction with EZH2, which promotes the progression of ovarian cancer [27]. In addition, lncRNAs have been found to play important roles in the development and progression of various cancers [28]. Te expression of lncRNA-141 is decreased in gastric cancer cells, and it plays a tumor suppressor role by targeting signal transduction and transcriptional activator 4 [29]; LncRNA-141 is strongly expressed in nonsmall cell lung cancer (NSCLC) cells, where it promotes the proliferation, diferentiation, and migration of NSCLC cells by inhibiting the expression of PHLPP1 and PHLPP2 and modulating the PI3K/Akt signaling pathway [30]. HOX-BAS1 is located on chromosome 17: 48,543,551-48,551,250. Studies have shown that inhibition of miR-885-3p expression can antagonize HOXB-AS1 knockdown and further afect the expression of HOXB2 in human glioblastoma tissues and cells. Te proliferation, migration, and invasion of glioblastoma cells are regulated by HOXB-AS1, which modulates the miR-885-3p/HOXB2 axis [31]. Chen et al. previously showed that the inhibition of HOXB-AS1 expression in multiple myeloma (MM) cells blocks the binding between ELAVL1 and FUT4, thereby regulating the FUT4mediated Wnt/β-catenin pathway and resulting in decreased proliferation and increased apoptosis of MM cells [32]. Furthermore, HOXB-AS1 plays a key role in several cancers, including glioma and colon cancer, and is presumed to play an oncogenic role in gastric cancer. Moreover, lncRNA HOXB11-AS is overexpressed in gastric cancer and associated with a poor prognosis [33]. Currently, there are no studies on HOXB-AS1 and gastric cancer. Terefore, we hypothesized that HOXB-AS1 is a target for the treatment of gastric cancer. We found that HOXB-AS1 is highly expressed in HGC-27 cells. After HOXB-AS1 silencing, cell proliferation and migration abilities were reduced and the degree of apoptosis increased.
Since the discovery of an inverse relationship between the selenium content of local crops in the United States and the occurrence of cancer in 1969, the anticancer potential of selenium has gradually been discovered [34]. El Bayoumy et al. previously reported several potential actions of selenium in cancer prevention and treatment, including Evidence-Based Complementary and Alternative Medicine delaying the oxidative damage of DNA, lipids, and proteins; inhibiting the growth of tumor cells; changing the synthesis of DNA, RNA, and proteins; regulating the cell cycle; inducing apoptosis; regulating the expression of p53, COX-2, modifed transcription factor-activating protein P, and nuclear factor B [35,36]. Na 2 SeO 3 can induce apoptosis in tumor cells undergoing oxidative stress [37], and Gazi et al. found that Na 2 SeO 3 can inhibit the activation of androgen receptors mediated by interleukin-6 and inhibit the progression of prostate cancer by upregulating the expression of c-Jun [38]. Na 2 SeO 3 was found to exert antitumor efects by inhibiting tumor angiogenesis in a transplanted canine breast tumor cell model in mice [39]. Na 2 SeO 3 regulates the IDO1/kynurenine, TLR4, NF-κB, and Bcl2/Bax pathways and attenuates acetic acid-induced colitis in rats [40]. Liu et al. further verifed that Na 2 SeO 3 can increase ROS levels and inhibit the NF-κB signaling pathway, efectively inhibiting the growth, metastasis, and inducing apoptosis of renal cell carcinoma both in vitro and in vivo [41]. Tis strongly supports the in vivo antitumor efect of Na 2 SeO 3 and indicates that Na 2 SeO 3 is a promising therapeutic drug. Although it is often used as an anticancer agent for cancer treatment, the exact mechanism underlying the role of Na 2 SeO 3 in the development of a therapeutic response remains unclear [42,43]. In this study, we found that diferent concentrations of Na 2 SeO 3 promoted apoptosis in HGC-27 cells and inhibited cell proliferation in a dose-dependent manner. Na 2 SeO 3 plays an important role in cancer treatment through its important metabolite selenocysteine, a low-molecular-weightseleniumcontaining amino acid located at the active site of selenocysteine proteins and encoded by the UGA stop codon [44]. It has also been reported that Na 2 SeO 3 can regulate apoptosis and oxidative damage by reducing the generation of free radicals and inhibiting lipid peroxidation [45,46]. Currently, there are few reports on the role of lncRNAs in the anticancer potential of Na 2 SeO 3 . We found that Na 2 SeO 3 inhibited the expression of HOXB-AS1 in gastric cancer cells and regulated the expression of related proteins to inhibit cell proliferation and migration and promote apoptosis after the overexpression of HOXB-AS1 in HGC-27 cells.
In summary, we found that HOXB-AS1 is upregulated in HGC-27, NCI-N87, Kato III, and GES-1 cells. Mechanistic analysis showed that Na 2 SeO 3 inhibited the proliferation and invasion of gastric cancer cells and enhanced cell apoptosis by downregulating HOXB-AS1 expression. Terefore, the fndings of this study highlight the potential therapeutic role of Na 2 SeO 3 in gastric cancer and suggest that HOXB-AS1 may be a potential therapeutic target. Further in-depth studies are required to confrm these results.

Data Availability
All data used to support the fndings of this study have been included in this article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.