Epstein-Barr Virus miR-BART17-5p Promotes Migration and Anchorage-Independent Growth by Targeting Kruppel-Like Factor 2 in Gastric Cancer

Epstein-Barr virus (EBV) infects more than 90% of the global population and is associated with a variety of tumors including nasopharyngeal carcinoma, Hodgkin lymphoma, natural killer/T lymphoma, and gastric carcinoma. In EBV-associated gastric cancer (EBVaGC), highly expressed EBV BamHI A rightward transcripts (BART) miRNAs may contribute to tumorigenesis with limited viral antigens. Despite previous studies on the targets of BART miRNAs, the functions of all 44 BART miRNAs have not been fully clarified. Here, we used RNA sequencing data from the Cancer Genome Atlas to find genes with decreased expression in EBVaGC. Furthermore, we used AGS cells infected with EBV to determine whether expression was reduced by BART miRNA. We showed that the expression of Kruppel-like factor 2 (KLF2) is lower in AGS-EBV cells than in the AGS control. Using bioinformatics analysis, four BART miRNAs were selected to check whether they suppress KLF2 expression. We found that only miR-BART17-5p directly down-regulated KLF2 and promoted gastric carcinoma cell migration and anchorage-independent growth. Our data suggest that KLF2 functions as a tumor suppressor in EBVaGC and that miR-BART17-5p may be a valuable target for effective EBVaGC treatment.


Introduction
Gastric cancer (GC) is the fifth most common cancer worldwide with a high incidence in East Asia. In South Korea, GC has a higher incidence rate than in other countries [1]. Despite efforts to treat advanced GC, therapies remain an active area of investigation because the causes and pathology of GC are diverse [2]. Therefore, GC needs to be studied at the molecular level.
The Cancer Genome Atlas (TCGA) has classified GC into four subtypes: Epstein-Barr virus (EBV), microsatellite instability (MSI), chromosomal instability (CIN), and genomically stable (GS). EBV GC accounts for 9% of all GC [3]. EBV infects epithelial cells through various mechanisms [4,5]. EBV GC is characterized by the hypermethylation of CpG islands in the promoter and the downregulation of tumor suppressor genes [6]. EBV has a modified latency 1 infection expressing EBV-encoded small RNAs (EBERs), Epstein-Barr nuclear antigen 1 (EBNA1), latent membrane protein 2A (LMP2A), and BamHI A rightward transcripts (BART) microRNAs (miRNAs) in EBV-associated GC (EBVaGC) [7]. As a limited number of viral proteins are expressed, it would be of interest to identify the role of BART miRNAs expressed at a high level in EBVaGC.
MicroRNA consists of 19-23 nucleotides single-stranded noncoding RNA that binds to the 3 UTR of mRNA and inhibits the translation of the target protein. EBV BART miRNAs have been reported to

Plasmid Constructs
The full-length 3 UTR of KLF2 mRNA was amplified from the gDNA of AGS cells. The amplified PCR product containing XhoI and NotI sites at each end was inserted between the Renilla luciferase coding sequence and the poly(A) site of the psiCHECK-2 plasmid (Promega, Madison, WI, USA) to produce psiC-KLF2. The primers for the 3 UTR of KLF2 containing XhoI and NotI sites are as follows: 5 -TCTAGGCGATCGCTCGAGCCGGGACGCCCCCGCCCA-3 and 5 -TTATTGCGGCCAG CGGCCGCCTCGGAAAATGAATCAGATTGTCA-3 . Mutations were introduced into the two seed match sequences of psiC_KLF2 to produce psiC_KLF2_M1, psiC_KLF2_M2, and psiC_KLF2_M1M2 using an EZ change site-directed mutagenesis kit (Enzynomics, Daejeon, South Korea).

Luciferase Reporter Assay
To investigate the direct effects of BART miRNAs on the expression of KLF2, HEK293T and AGS cells were seeded in a 96-well plate at 5.5 × 10 3 cells/well. After 24 h, cells were co-transfected with psiC-KLF2 and the BART miRNA mimic using Lipofectamine 2000 (Invitrogen). Luciferase activity was measured 48 h post-transfection using the Dual-Glo luciferase reporter assay system (Promega). For each sample, Renilla luciferase activity was normalized using the internal control firefly luciferase activity.

Wound Healing Assay
To evaluate the effect of miR-BART17-5p on cell migration, AGS (1 × 10 6 cells/well) and AGS-EBV (1.5 × 10 6 ) cells were each seeded into six-well plates and allowed to reach 90-95% confluence. A monolayer of cells covering the plate was scratched with a sterile 200 µL pipette tip and subsequently washed with phosphate-buffered saline to remove cell debris. The cells were transfected with miR-BART17-5p, miR-BART17-5p inhibitor, or siKLF2. The cells were then cultured in RPMI-1640 medium containing 0.1% FBS at 37 • C in an incubator with 5% CO 2 . The scratched wounds were observed by an Axiovert 200 (Carl Zeiss, Thornwood, NY, USA) microscope just after transfection (time 0) and 48 h after transfection. Photographs were taken to assess the level of migration in each group of transfected cells, and wound areas were measured by Image J 1.37v software (National Institutes of Health, Bethesda, MD, USA). Three independent experiments were performed.

Soft Agar Colony Formation Assay
To confirm if KLF2 was involved in anchorage-independent growth, a soft agar colony formation assay was performed. AGS and AGS-EBV were each transfected with 30 nM miR-BART17-5p mimic or inhibitor. After 24 h, the cells were harvested and seeded (3000 cells/well) in 0.6% Bacto Agar (214010; BD, Franklin Lakes, NJ, USA) mixed with culture medium (on top of 1% agar with medium) in six-well plates. Cells were cultured at 37 • C for 4-6 weeks. The images of the plates were captured under a microscope and camera. Then, the pictures were analyzed using Image J software. Three independent experiments were performed.

KLF2 Overexpression Vector
A human KLF2 expression vector (#60441, Addgene, Cambridge, MA, USA) was used for migration and soft agar colony formation assays.

Propidium Iodide (PI) Staining
AGS cells were trypsinized, washed twice with cold PBS and fixed in 70% ethanol at −20 • C overnight. The fixed cells were resuspended in PBS containing 20 µg/mL RNase A (Invitrogen) and 2.5 µg/mL PI (Sigma-Aldrich). The distribution of cells in each phase of the cell cycle was analyzed using FACSCalibur apparatus (BD Biosciences, San Diego, CA, USA) as described previously [42].

Statistical Analyses
The MTT assay data were analyzed using two-way analysis of variance (ANOVA) and the Student's t-test was used for other experiments. GraphPad Prism version 5.03 (GraphPad Software, San Diego, CA, USA) was used to analyze and draw graphs. p-values less than 0.05 were considered statistically significant. All results were expressed as the mean ± standard deviation (SD).

KLF2 Expression is Suppressed by EBV Infection in Gastric Carcinoma
To investigate the effect of EBV infection on Kruppel-like factor 2 (KLF2) expression in gastric cancer (GC) tissues, RNA sequencing data from TCGA were analyzed. The expression of five KLFs (KLF2, 4, 8, 9, and 15) was decreased more in GC tissues than in normal tissues (Figure 1a). We next examined whether the expression of these five KLFs was reduced in EBVaGC compared to other subtypes  (Figure 1b). In all four subtypes of GC, KLF8 and KLF15 had the least expression in GC tissues, while the expression of KLF4 showed no significant differences between EBVaGC and the other GC subtypes. The expression of KLF2 was the most reduced in EBVaGC compared to other subtypes of GC.
To identify whether the expression of KLF2 was affected by EBV infection in GC cell lines, qRT-PCR was performed for KLF2 using AGS and AGS-EBV cells, which differed only in EBV infection status. We found that the mRNA level of KLF2 was 67% lower (Figure 1c) and the protein level of KLF2 was 48.3% lower in AGS-EBV than in AGS (Figure 1d,e). In addition, qRT-PCR of KLF2 was performed for EBV-negative (MNK1, MKN28, NCI-N87, and SNU-484) and EBV-positive (NCC24, SNU-719, and YCCEL1) cell lines. The results showed that the expression of KLF2 was generally lower in the EBV-positive cell lines than in the EBV-negative cell lines (Figure 1f).

Screening EBV BART miRNAs that May Target KLF2
We confirmed that KLF2 expression was reduced in EBVaGC ( Figure 1). In addition, previous studies have reported that BART miRNAs are significantly expressed in EBV latency type 1 [43]. To check if KLF2 expression was suppressed by BART miRNAs in EBV-positive cells, an RNA hybrid program was used to predict BART miRNAs with the potential to target KLF2. Four BART miRNAs have seed match sequences for the 3'UTR of KLF2, and three BART miRNAs were expected to target more than two sites on the 3'UTR of KLF2 ( Figure 2a). To check whether these miRNAs directly target the KLF2 3'UTR, a luciferase reporter vector containing the 3 UTR of KLF2 (psiC_KLF2) was constructed. psiC_KLF2 and each BART miRNA mimic were co-transfected and a luciferase assay was conducted. The results showed that luciferase activities were reduced when miR-BART11-5p (33%) or miR-BART17-5p (22%) was transfected (Figure 2b) into HEK293T cells. When AGS cells were co-transfected with psiC_KLF2 vector and either miR-BART11-5p or miR-BART17-5p mimics, luciferase activity was suppressed by miR-BART17-5p but not by miR-BART11-5p (Figure 2c). There were two miR-BART17-5p seed match sites in the 3 UTR of KLF2 (Figure 2a). To determine whether they play important roles in reducing KLF2 expression, one or both of the two seed match sites were mutated (Figure 2d). The psiC_KLF2 luciferase reporter vector with mutated site 1 (1894-1923) seed sequence was named psiC_KLF2_M1 and the vector with mutated site 2 (1995-2002) was named psiC_KLF2_M2. The vector containing both mutated 1 and 2 sites was named psiC_KLF2_M1M2. AGS cells were co-transfected with miR-BART17-5p and each of the vectors for a luciferase assay. While luciferase activity was reduced by 26.2% when the wild-type psiC_KLF2 was co-transfected with miR-BART17-5p; luciferase activity was less suppressed when psiC_KLF2_M1 (22.1%) or psiC_KLF2_M2 (12%) was co-transfected with miR-BART17-5p ( Figure 2e). When psiC_KLF2_M1M2 was co-transfected, luciferase activity was not affected by miR-BART17-5p (Figure 2e). These results indicate that miR-BART17-5p binds to both of the sites and M2 is the major site to which miR-BART17-5p binds.
Target site 2 wt miR-BART17-5p Three independent experiments were conducted to confirm reproducibility. Luciferase activity was normalized using internal firefly luciferase activity. The luciferase activity of the cells transfected with each BART miRNA is expressed as a ratio to the luciferase activity obtained from the scrambled control transfected cells. Error bars indicate SDs. (d) Schematic drawing shows the location of the predicted target sites 1 and 2 for miR-BART17-5p on the 3 UTR of KLF2 mRNA (upper panel). Two sites in the 3 UTR of KLF2 that seed match with miR-BART17-5p were mutated individually or together, and the mutated sequences are shown in red (lower panel). (e) Luciferase assay was carried out using AGS cells co-transfected with psiC_KLF2 (wildtype or mutants) and 30 nM miR-BART17-5p. Each value represents the mean ± SD in four independent experiments. Abbreviation: n.s, not significant.

miR-BART17-5p Promotes Migration and Anchorage-Independent Growth in AGS Cells
In previous results, miR-BART17-5p negatively regulated the expression of KLF2, therefore we tried to confirm the role of miR-BART17-5p in EBVaGC. To analyze the effect of miR-BART17-5p on cell proliferation, migration, and anchorage-independent growth, miR-BART17-5p was artificially delivered to EBV-negative AGS cells. Cell proliferation was not affected by miR-BART17-5p (Figure 4a). A soft agar colony formation assay showed that miR-BART17-5p promoted anchorage-independent growth by 2.1-fold compared to the scrambled control (Figure 4b,c). A wound-healing assay also showed that migration increased 1.3-fold when miR-BART17-5p was delivered to AGS cells compared with the scrambled control (Figure 4d,e).

miR-BART17-5p Inhibitor Suppresses Migration and Anchorage-Independent Growth in AGS-EBV Cells
We then investigated the effect of endogenously expressed miR-BART17-5p on the tumorigenesis of AGS-EBV by delivering a miR-BART17-5p inhibitor into cells. A soft agar colony formation assay revealed that anchorage-independent growth was reduced by 50% following miR-BART17-5p inhibition (Figure 5a,b). In addition, wound-healing of AGS-EBV cells was hindered by 44.6% following transfection with the miR-BART17-5p inhibitor in comparison with the control inhibitor (Figure 5c,d).

Knocking Down KLF2 Using siRNA Induces Migration and Anchorage-Independent Growth in AGS Cells
KLF2 siRNA was transfected to confirm that the phenotype changes following miR-BART17-5p transfection were manifested by the downregulation of KLF2. KLF2 expression in AGS was sharply decreased by siKLF2 transfection (Figure 6a) compared with control siRNA (siNC) transfection. After delivering the siKLF2, anchorage-independent growth of AGS on soft agar was increased 1.8-fold compared to cells transfected with the siNC (Figure 6d,e). Cell migration was also increased 1.3-fold by the siKLF2 compared with the siNC (Figure 6f,g). In contrast, cell proliferation and cell cycle were not affected following KLF2 knockdown using the siKLF2 (Figure 6b,c).

KLF2 Overexpression Inhibits Migration and Anchorage-Independent Growth in AGS-EBV Cells
To confirm KLF2 function, a KLF2 overexpression vector was delivered to AGS-EBV, in which KLF2 expression was lower than in AGS. Following KLF2 overexpression (Figure 7a), anchorage-independent growth was reduced by 71% in AGS-EBV cells (Figure 7b,c). In addition, migration of AGS-EBV cells was suppressed by 36% compared to the control vector transfection (Figure 7d,e).

Discussion
We found that KLF2 expression was lower in EBVaGC than in EBV-negative GC. In the process of testing the hypothesis that EBV miRNAs inhibit KLF2 expression, we found that miR-BART17-5p directly targeted the KLF2 3 UTR to reduce mRNA and protein expression. We also found that miR-BART17-5p promoted cell migration and anchorage-independent growth by reducing the expression of KLF2.
The expression of miR-BART17-5p increases in plasma with the progression of NPC tumors [44]. miR-BART17-5p is also a biomarker that is associated with poor prognosis, since it is only detected in the serum of patients with recurrent NPCs [17]. Although there is a considerable expression of miR-BART17-5p in EBVaGC [20,21], little is known about the role of miR-BART17-5p in EBVaGC. In NPC, LMP1 has been reported to be a target of miR-BART17-5p [9]. As LMP1 is rarely detected in EBVaGC [45][46][47], miR-BART17-5p may target LMP1 and almost abrogate LMP1 expression in EBVaGC. Even when miR-BART17-5p is bound to the 3 UTR of cellular N-myc downstream-regulated gene 1 (NDRG1) [11], miR-BART17-5p did not affect NDRG1 expression, curiously [48]. Our study shows that highly expressed miR-BART17-5p targets KLF2 in EBVaGC, revealing the role of this BART miRNA.
Although KLF2 acts as a tumor suppressor in several cancers, including GC [49,50], reports show that KLF2 acts as an oncogene in liver cancer [51]. Our results support that KLF2 is a target of miR-BART17-5p and acts as a tumor suppressor in EBVaGC. Even though KLF2 expression in GC tissues including EBVaGC were not tested in this study, we analyzed EBVaGC patient data from TCGA [3]. We found that KLF2 expression was lower in GC than in normal tissues, and that KLF2 expression was lower in EBVaGC than in other GCs (Figure 1a,b). In addition, all three naturally EBV infected GC cell lines as well as AGS-EBV cells showed low-level KLF2 expression (Figure 1f). This is consistent with microarray data showing a greater reduction in the expression of KLF2 in EBV-infected cell lines than in EBV-uninfected cell lines [10]. Thus, KLF2 may be expressed at low levels in EBVaGC.
In previous studies, EBV infection in gastric epithelial and gastric carcinoma cell lines increased anchorage-independent growth despite the restricted viral protein expression [10,52]. It was speculated that this phenotype is due to BART miRNAs. Wang et al. [53]. showed that miR-BART7 promoted anchorage-independent growth. Additionally, several studies showed that BART miRNAs promoted cell migration [54,55]. Based on our KLF2 knockdown and overexpression data, the phenotype induced by miR-BART17-5p may be due to the decreased expression of KLF2. Previous reports showed that KLF2 suppressed anchorage-independent growth by inhibiting the expression of Gli1 in liver cancer [56] as well as migration by inhibiting MMP2, N-cadherin, and vimentin in several cancers [48,49]. In our study, reducing KLF2 expression may have caused anchorage-independent growth and cell motility through the mechanisms described above.
In the present study, the expression level of KLF2 in AGS-EBV was lower than the level of KLF2 following miR-BART17-5p transfection to AGS. This suggests that not only miR-BART17-5p but also other EBV genes may down-regulate KLF2 expression. The expression of EZH2, a histone methyltransferase, is increased in NPC [57]. EBV infection in primary B cells induced EZH2 expression [58]. In addition, RNA sequencing data from TCGA showed that the expression of EZH2 in EBVaGC was higher than in normal tissues and other subtypes of GC [3]. Since KLF2 is silenced by EZH2 in GC [27], increased expression of EZH2 in EBVaGC may have silenced KLF2. Li et al. [59] reported that Helicobacter pylori (H. pylori) induced miR-25 to reduce the expression of KLF2. As GC can be co-infected with EBV and H. pylori, both miR-25 and miR-BART17-5p may exert a simultaneous effect on the expression of KLF2. It is not clear which pathway would be more important for the development of GC based on our data, as we did not test the effect of H. pylori infection. Further studies would be required to clarify this point.
Although in a previous study KLF2 was reported to be a tumor suppressor that affected cell proliferation in GC [34], cell proliferation was not affected by either siKLF2 or miR-BART17-5p in the present study. KLF2 was shown to inhibit cell proliferation by increasing the expression of p21, a cell cycle blocker [60]. However, siKLF2 transfection did not affect the cell cycle and the KLF2 overexpression vector transfection did not affect the expression of p21 in our experiments. Recently, a study reported that FOXO4 binds to the activation domain of KLF2 and co-operates to induce p21 expression [61]. RNA sequencing data from TCGA showed that FOXO4 is decreased in EBVaGC [3]. This is consistent with a previous study where the expression of FOXO4 was reduced by LMP1 and LMP2A in AGS-EBV [62]. Therefore, even when KLF2 was overexpressed in AGS-EBV cells, p21 may not be induced due to low FOXO4 expression in the cells. In addition, we previously reported that miR-BART17-5p did not affect cell proliferation, which is consistent with this study [63]. Many studies suggest that the growth rate of cancer cells does not necessarily correlate with cell migration and invasion abilities [64,65]. Thus, we propose that miR-BART17-5p may play a role in the metastatic process rather than in tumor growth of EBVaGC.

Conclusions
Our data suggest that miR-BART17-5p, which is highly expressed in EBVaGC, plays an oncogenic role by inhibiting a tumor suppressor KLF2 expression. miR-BART17-5p increased cell motility and anchorage-independent growth, features associated tumor metastasis. Therefore, miR-BART17-5p may serve as a potential therapeutic target of EBVaGC. Inhibitors of miR-BART17-5p may be useful alone or in combination with other therapeutic agents to treat EBVaGC.