Long Noncoding RNA WDFY3-AS2 Represses the Progression of Esophageal Cancer through miR-18a/PTEN Axis

Background Understanding the role of lncRNAs in the development of human malignancies is necessary for the targeted therapy of malignant tumors, including esophageal cancer (EC). Nevertheless, the specific role and regulatory mechanism of lncRNA WDFY3-AS2 in EC are still unclear. Here, we examined the functional role and regulatory mechanism of WDFY3-AS2 in EC. Materials and Methods RT-qPCR assay was applied to measure the expression of WDFY3-AS2 and miR-18a in EC samples and cells. The luciferase reporter and RIP assays were used to check the relationship between WDFY3-AS2, miR-18a, and PTEN. Counting Clock Kit-8 (CCK-8) assay was carried out to detect cell viability, and transwell assays were used for measuring cell migration and invasion. Results Underexpression of WDFY3-AS2 was found in EC specimens and cells, which predicted a poor prognosis of EC patients. Reexpression of WDFY3-AS2 repressed the progression of EC via inhibiting cell proliferation, migration, and invasion. Additionally, WDFY3-AS2 was negatively correlated with miR-18a and positively with PTEN. Furthermore, we discovered that the expression of PTEN decreased by miR-18a mimic was rescued by WDFY3-AS2 overexpression. Conclusions WDFY3-AS2 modulates the expressional level of PTEN as a competitive endogenous RNA via sponging miR-18a in EC, which suggests that the WDFY3-AS2/miR-18a/PTEN pathway might be involved in the progression of EC.


Introduction
Esophageal cancer (EC) is a malignant tumor with rapid progression and poor prognosis and is one of the most fatal diseases worldwide [1]. EC-related risk factors include consumption of foods containing amines nitrite and fungal infection, as well as irritation from hot foods and drinks [2]. Although significant progress has been made in the treatment of EC, such as chemotherapy and surgical resection, the prognosis of EC patients is still poor, and the 5-year survival rate is low, only about 10% [3,4]. us, it is essential to understand the pathogenesis of EC and discover the possible therapeutic targets for EC.
Long noncoding RNA (lncRNA) is a noncoding RNA that cannot encode proteins, and its length exceeds 200 base pairs [5]. Previous reports have discovered that LncRNAs play essential roles in the occurrence and development of EC [6]. In addition, there are enough pieces of evidence that dysregulation of lncRNAs that modulate cancer-related pathways could affect the progression of tumors, including EC [7]. For instance, ZNF750 was involved in ESCC progression and emphasized its significance as a biomarker for metastasis and prognosis [8]. Liang et al. discovered that lnc01980 acted as an oncogenic lncRNA in modulating ESCC proliferation, migration, and invasion [9]. Furthermore, Shen et al. verified that knockdown of AGAP2-AS1 showed suppressive effects on the migration and invasion of EC through miR-195-5p/FOSL1 pathway [10]. LncRNA WDFY3-AS2 was first found to be increased in hepatocellular carcinoma [11]. Moreover, WDFY3-AS2 was decreased in triple-negative breast tumors and served as a potential prognostic factor in TNBC development [12]. Li et al. showed that overexpression of WDFY3-AS2 inhibited tumor growth, cell migration, and invasion [13]. To our knowledge, there are no reports on the role of WDFY3-AS2 in ESCC progression.
Previous literature demonstrated that miR-18a is significantly increased in hepatocellular cancer [14] and gliomas [15]. Also, miR-18a was reported to involve in nasopharyngeal carcinoma by suppressing SMG1 and activating the mTOR pathway [16]. Nair et al. displayed that miR-18a promoted the proliferative and migratory ability of ER-positive breast cancer cells via activating Wnt signaling [17]. In EC, miR-18a was highly expressed in plasma of EC patients [18]. However, there are few reports about the role of miR-18a in EC.
In the present study, we hypothesized that WDFY3-AS2 plays a critical role in regulating EC development with the implication of miR-18a and PTEN. us, we investigated the regulatory mechanism of WDFY3-AS2 in the regulation of EC cell proliferation, invasion, migration, and the relationships between WDFY3-AS2, miR-18a, and PTEN. Our findings may provide new insights into the role of lncRNA-miRNA-mRNA in EC.

Patients.
irty-six EC tissue samples and the corresponding normal tissue samples were collected from EC patients at Rizhao People's Hospital, Rizhao, Shandong, China. None of EC patients received preoperative radiotherapy or chemotherapy before surgery. e tissue samples were stored at −80°C for further use. All patients provided written informed consent and the study was approved by the ethics committee of the Rizhao People's Hospital, Rizhao, Shandong, China (Approval no. 2018020078).

Cell
Culture. Immortalized esophageal epithelial cell SHEE and human esophageal cancer cell lines (ECA109, YES2) were purchased from BeNa Culture Collection (Beijing, China). e cells were cultured in RPMI 1640 (Gibco, Invitrogen, Germany) medium containing 10% FBS at 37°C 5% CO 2 . e complete medium was updated regularly every 2-3 days. When the degree of fusion reached 80%, the cells were separated and passaged as usual.

Transwell
Assays. 3 × 10 4 cells/well were added to the upper chamber with a serum-free medium. e lower chamber was fixed with a fresh medium supplement with 10% FBS. For migration assay, ECA109 cells were incubated at 37°C and 5% CO 2 for 24 h. e invasion test procedure was the same except that the upper chamber was coated with a matrix. After incubation for 48 h, the migration and invasion cells were stained with 0.1% crystal violet. Finally, a phase-contrast microscope was applied to observe EC cells. e expressional levels of WDFY3-AS2 and miR-18a were measured by qRT-PCR assay. IgG was used as a negative control.

2
Journal of Oncology

Statistical Analysis.
e data were shown as mean ± SD and analyzed using SPSS 19.0 or GraphPad Prism 8.0. e statistical differences were assessed by Student's t-test or one-way ANOVA. P < 0.05 was considered as statistically significant.

Low Expression of WDFY3-AS2 in EC and Its Clinical
Significance. We first investigated WDFY3-AS2 expression in EC tissue samples and cells by RT-qPCR. Downregulation of WDFY3-AS2 was found in EC tissue samples compared to corresponding normal tissue samples (Figure 1(a)). Moreover, the expression level of WDFY3-AS2 in EC cells was also higher than that in normal EC cells SHEE (Figure 1(b)). 36 EC patients were divided into a high expression group and a low expression group according to the average of WDFY3-AS2 expression. Results discovered that the higher expression of WDFY3-AS2, the higher the survival time of EC patients (Figure 1(c)). In addition, WDFY3-AS2 is closely associated with tumor grading and TNM stage ( Table 1). All results indicate that WDFY3-AS2 is involved in the progress of EC.

Upregulation of WDFY3-AS2 Represses Proliferation,
Migration, and Invasion of EC Cells. Next, we investigated the functional role of WDFY3-AS2 in EC cells. pcDNA3.1-WDFY3-AS2 and sh-WDFY3-AS2 vectors were adopted to overexpress and knockdown WDFY3-AS2 in ECA109 cells. As shown in Figure 2(a), WDFY3-AS2 expression was remarkably increased or decreased in ECA109 cells after overexpression or knockdown of WDFY3-AS2. en, we detected the viability of EC cells by CCK-8 assay.

WDFY3-AS2 Acted as a Sponge for miR-18a in EC.
To explore the molecular mechanism of WDFY3-AS2 in EC, bioinformatic analysis was first carried out to predict the possible binding site of WDFY3-AS2. miR-18a was obtained as the candidate miRNA, and the predicted binding site of miR-18a in WDFY3-AS2 is shown in Figure 3(a). en, miR-18a was chosen for the subsequent investigations. Upregulation of WDFY3-AS2 decreased miR-18a expression and downregulation of WDFY3-AS2 increased the expression of miR-18a (Figure 3(b)). Afterwards, luciferase reporter assay and RIP assay were carried to verify the direct binding ability of miR-18a and WDFY3-AS2. e luciferase assay results displayed that the luciferase activity was inhibited or fortified by transfected with WDFY3-AS2-WT and miR-18a mimic or inhibitor (Figure 3(c)). However, there has no response to the alterations of miR-18a in WDFY3-AS2-Mut (Figure 3(d)). RIP assay showed that the expressions of WDFY3-AS2 and miR-18a were abundant in Ago2 groups versus IgG groups (Figure 3(e)). e results demonstrated that WDFY3-AS2 could directly bind to miR-18a in EC.

Overexpression of miR-18a
Enhanced Proliferation, Migration, and Invasion of EC Cells. Next, we detected the expressional level of miR-18a in EC specimens and results found that miR-18a increased significantly in EC samples compared to the normal samples (Figure 4(a)). Considering the negative relationship between WDFY3-AS2 and miR-18a in EC tissues (Figure 4(b)), we investigated the functional role of miR-18a in EC proliferation, migration, and invasion. As shown in Figure 4(c), ECA109 cells transfected with miR-18a mimic displayed significantly increased cell viability compared to ECA109 cells transfected with NCmimic, while decreased cell viability in ECA109 cells transfected with miR-18a inhibitor. Transwell migration and invasion assays displayed that overexpression of WDFY3-AS2 inhibited the migration and invasion of ECA109 cells, whereas knockdown of WDFY3-AS2 exerted the opposite effects (Figures 4(d) and 4(e)).

PTEN Served as the Target of miR-18a.
TargetScan database was used to discover the target gene of miR-18a, and PTEN was screened as the candidate gene ( Figure 5(a)). PTEN was reported to be highly expressed in diverse cancers as an oncogene [19,20]. We then tested PTEN expression in EC tissue specimens. As shown in Figure 5(b), PTEN was significantly increased in EC tissues. Moreover, we found that miR-18a was negatively associated with PTEN in EC tissues ( Figure 5(c)). Overexpression of miR-18a decreased the levels of PTEN in ECA109 cells, while knockdown of miR-18a increased PTEN expression (Figures 5(d) and 5(e)). e luciferase assay results showed that the luciferase activity was significantly inhibited or fortified by being transfected with miR-18a mimic or inhibitor in the PTEN-WT group ( Figure 5(f )). ese data suggested that PTEN was a direct target of miR-18a.

WDFY3-AS2 Attenuated the Development of EC via
Modulating miR-18a/PTEN Axis. Next, rescue assays were conducted to verify the role of WDFY3-AS2/miR-18a/PTEN in EC development. CCK-8 assay discovered that the decreased cell viability caused by overexpression of WDFY3-AS2 was attenuated by upregulation of miR-18a or downregulation of PTEN (Figure 6(b)). Transwell migration and invasion assays demonstrated that EC invasion and migration were repressed by increasing WDFY3-AS2 and restored by upregulation of miR-18a or depletion of PTEN (Figures 6(c) and 6(d)). e results stated that WDFY3-AS2 might inhibit EC progression by regulating PTEN via miR-18a.

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WDFY3-AS2-WT hsa-miR-18a WDFY3-AS2-MuT

Discussion
Esophageal cancer is a malignant tumor occurring in the esophageal mucosa and is a common gastrointestinal tumor in China [21]. Lack of potential biomarkers and unnoticed early symptoms of potential EC patients often lead to delayed diagnosis [22]. LncRNA has been reported to be involved in the progression and prognosis of various human tumors. In recent years, although lncRNAs have been studied to assist in the clinical diagnosis of cancer as a potential molecular biomarker, the effect is not evident in EC. Previous studies have found that WDFY3-AS2 was underexpressed in EC patients [23], which was consistent with our findings that WDFY3-AS2 decreased in EC tissues and cells. Moreover, we revealed that overexpression of WDFY3-AS2 inhibited cell proliferation, invasion, and migration, whereas inhibited WDFY3-AS2 showed the opposite effect, suggesting the inhibitory role of WDFY3-AS2 in EC progression. Previous literature has highlighted the critical role of lncRNAs and miRNAs in various biological processes of cancers, including EC [24,25]. In the current study, we demonstrated that miR-18a acted as a target of WDFY3-AS2. Moreover, WDFY3-AS2 increased PTEN expression by inhibiting miR-18a. ese findings were consistent with the previous study that WDFY3-AS2 binds to miR-18a and WDFY3-AS2 negatively regulated miR-18a expression in ovarian cancer cells. MiR-18a was upregulated in various cancers, including EC [26]. However, its functional role in EC has not been clarified. In this study, we discovered that miR-18a was highly expressed in EC tissues and EC cells. MiR-18a promoted EC cells proliferation, invasion, and migration and WDFY3-AS2 attenuated the development of EC via miR-18a/PTEN axis. e obtained results showed that PTEN was involved in EC progression. PTEN has been reported to have an oncogenic role in a variety of tumors. For instance, Xiang et al. discovered that PTEN repressed cell proliferation and inhibited apoptosis in lung cancer [27]. Besides, PTEN served as a promoter in gliomagenesis by facilitating cell proliferation, tumor growth, and inhibiting apoptosis [28]. Our study confirmed that the reexpression of WDFY3-AS2 suppressed EC cell proliferation, invasion, and migration by increasing PTEN expression via miR-18a. However, our research is in the preclinical stage; the mechanism of action involved is unclear. erefore, further evaluation of other relevant biomarkers is recommended.
In conclusion, our study suggests that WDFY3-AS2 was underexpressed in EC samples and cells. Low expression of WDFY3-AS2 was associated with poor prognosis of EC patients. Additionally, the reexpression of WDFY3-AS2 suppressed EC progression by miR-18a/PTEN axis. WDFY3-AS2 may be a novel therapeutic and prognostic target for EC.

Data Availability
e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.