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Silencing lncRNA AFAP1-AS1 Inhibits the Progression of Esophageal Squamous Cell Carcinoma Cells via Regulating the miR-498/VEGFA Axis

Purpose In view of the continuous increase of the mortality rate, esophageal squamous cell carcinoma (ESCC) develops into a major health concern. In this study, we aimed to investigate the underlying mechanism of long noncoding RNA (lncRNA) actin filament-associated protein 1 antisense RNA (AFAP1-AS1)/microRNA-498 (miR-498)/vascular endothelial growth factor A (VEGFA) in ESCC cells. Methods The expression levels of AFAP1-AS1, miR-498 and VEGFA in ESCC tissues and cells were detected using quantitative real-time polymerase chain reaction (qRT-PCR). The effects of AFAP1-AS1 on ESCC cells proliferation and apoptosis were measured by methyl thiazolyl tetrazolium (MTT) and flow cytometry, respectively. Transwell assay was carried out to determine cell migration. In addition, VEGFA and cell behaviors-related proteins were determined by Western blot analysis. The targeted relationships of AFAP1-AS1 were verified by dual-luciferase reporter and RNA pull-down assays. Results The expression levels of lncRNA AFAP1-AS1 and VEGFA mRNA were upregulated, but miR-498 was downregulated in ESCC tissues and cells. Moreover, miR-498 was directly targeted by AFAP1-AS1 and there was a negative correlation between miR-498 and AFAP1-AS1. Functionally, AFAP1-AS1 silencing inhibited the proliferation and migration and induced apoptosis of ESCC cells. Interestingly, miR-498 inhibition rescued the effects of AFAP1-AS1 knockdown on cell proliferation, apoptosis and migration and restored the expression levels of tumor-developing marker proteins of AFAP1-AS1 silencing in Eca109 and KYSE-30 cells. Furthermore, VEGFA was verified as a direct target of miR-498 and reversed the effects of miR-498 overexpression on cell behaviors of ESCC in vitro. Conclusion Downregulation of AFAP1-AS1 impeded the proliferation and migration and induced apoptosis of ESCC cells by regulating miR-498/VEGFA axis, which might serve as a novel biomarker for the diagnosis and treatment of ESCC.


Introduction
Esophageal cancer is one of the most aggressive cancers, characterized as the highest incidence in Eastern Asia. 1 According to statistics, China alone accounted for 53.8% of cases and 51.9% of deaths from esophageal cancer in 2008. 2 Based on the histological types, esophageal cancer is classified into squamous cell carcinoma and adenocarcinoma. Particularly, esophageal squamous cell carcinoma (ESCC) is the most common histological subtype in less-developed regions of the world, including Eastern Asia. 3 Although the treatments of ESCC have been continuously developed, such as surgery, chemotherapy and radiotherapy, patients with ESCC still have a dismal prognosis. 4,5 Thus, it is important to uncover several helpful therapeutic strategies for the treatment of ESCC.
Nowadays, long non-coding RNAs (lncRNAs) have received widespread attention from researchers. By definition, lncRNAs are composed of 200 nucleotides in length and do not have functional protein-coding capacity. Numerous evidence has shown that lncRNAs participate in extensive biological processes and play oncogene or tumor suppressor roles in several diseases. 6,7 In ESCC, several lncRNAs with aberrant expression have been reported to function as potential oncogenes, such as taurine up-regulated 1 (TUG1), 8 SPRY4 intronic transcript (SPRY4-IT1), 9 colon cancer-associated transcript 2 (CCAT2) 10 and Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1). 11 Actin filamentassociated protein 1 antisense RNA (AFAP1-AS1) is a lncRNA located on chromosome 4p16.1 with 6810 base pair (bp) in length has been reported to play a vital role in the molecular mechanism of human cancers. 12 AFAP1-AS1 is found to be markedly upregulated in non-small-cell lung cancer (NSCLC) tissues, and the ectopic expression can facilitate the proliferation of NSCLC cells. 13 According to previous findings, disordered expression of AFAP1-AS1 is identified to be associated with prognosis of cholangio carcinoma, 14 gastric cancer, 15 hepatocellular carcinoma (HCC), 16 nasopharyngeal carcinoma (NPC), 17 pancreatic ductal adenocarcinoma (PDAC), 18 colorectal cancer 19 and gallbladder cancer. 20 Importantly, AFAP1-AS1 has also been demonstrated to be upregulated in esophageal adenocarcinoma (EAC) tissues and cells. 21 Furthermore, Luo et al revealed that AFAP1-AS1 overexpression promoted ESCC cell proliferation and inhibited cell apoptosis. 22 However, in progression of ESCC, the deeper molecular function of AFAP1-AS1 is still not well elucidated.
MicroRNA (miRNA) is a shorter noncoding RNA that regulating gene expression by binding to the 3ʹ-untranslated region (3ʹUTR) of target mRNAs. Recent studies have demonstrated the dysregulation of miRNAs is closely associated with cancer progression. 23 In both ESCC tissues and cell lines, miRNA-146a is downregulated and its unusual expression level affects cell growth and metabolism through regulating IRS2 mRNA expression. 24 Previous study showed that miR-31 is decreased in esophageal cancer cells, and overexpression of miR-31 can hinder esophageal cancer cell growth, migration and invasion. 25 In ESCC, miR-130b as an oncogenic gene regulates the expression of (phosphatase and tensin homolog deleted on chromosome ten) PTEN. 26 Downregulated miR-126 is connected with poor prognosis of ESCC. 27 Thus, miRNA-based treatments will be recommended as the emerging strategies for drug designs. Interestingly, miR-498 is reported to play important roles in mediating the pathogenesis of ESCC and restrain ESCC cell activities like cell proliferation and barrier penetration, 28 which is the opposite of the effects of AFAP1-AS1 in ESCC. However, whether AFAP1-AS1 can regulate miR-498 and its roles in ESCC progression remain unclear.
Vascular endothelial growth factor (VEGF) was an oncogene in multiple all known cancers, 29-31 including ESCC. 32 Previous studies showed that VEGFA could activate PI3K-Akt/PKB, 33 Akt/mTOR 34 signaling pathway and play a critical role in cell proliferation, migration and angiogenesis in ESCC. 35 All evidences revealed that VEGF might participate in ESCC progression, while the mechanism of VEGF in ESCC and the interaction among AFAP1-AS1, miR-498 and VEGFA need to be further elucidated.
Based on the previous researches, the present research aimed to investigate the potential functional mechanisms of AFAP1-AS1 and miR-498 on cell growth and metastasis in ESCC.

Patients and Tissue Samples
A total of 42 ESCC patients with no selection bias were selected to participate in this study at Taizhou People's Hospital. The patients did not receive radiotherapy or chemotherapy before surgery. The main inclusion criteria contain age, gender, differentiation, depth of invasion (pT), lymph node metastasis (pN), clinical stage and tumor location. The detailed information of these ESCC patients was shown in Tables S1-S3. While surgical resection of ESCC was undertaken, 42 ESCC tissues and 35 adjacent normal specimens were obtained, and samples were promptly frozen until use. All experiments were performed in accordance with the Guidelines "Declaration of Helsinki". Experiments were approved by the ethics committee at "Taizhou People's Hospital". Informed consents were obtained from human participants of this study.

RNA Pull-Down Assay
Biotinylated AFAP1-AS1 was transfected into ESCC cells using lipofectamine 3000 (Invitrogen). After transfection for 2 hrs, RNA-protein pull-down kit (Millipore, Billerica, MA, USA) was serviced for RNA pull-down assay, and then cells were lysed and incubated with streptavidincoupled beads. Next, RNA was eluted and the enrichment of miR-498 was identified by qRT-PCR assay.
Optical density (OD) values of cell lysates were detected with a microplate reader (Bio-Rad, Richmond, CA, USA).

Cell Apoptosis Assay
ESCC cells were collected after transfection with miR-498 mimics/inhibitor or pcDNA VEGFA. The FITC-AnnexinV Apoptosis Detection Kit (BD Pharmingen, San Diego, CA, USA) was used according to the manufacturer's instructions. After stained with the fluorescein isothiocyanate (FITC)-AnnexinV and propidium iodide (PI), the apoptotic cells were recognized with a flow cytometry system (Bio-Rad) and analyzed via Cell Quest software.

Cell Migration Assay
Approximate 2×10 4 cells were harvested and added into the upper chamber of a well without Matrigel with 100 μL serum-free medium. Normal medium containing 10% fetal bovine serum (FBS; Hyclone) was added in the bottom wells as a stimulation of migration. The cells were then incubated for 24 hrs at 37°C, and the cells on the bottom were fixed with 20% methanol for 30 mins and stained with 0.1% crystal violet for 15 mins. Lastly, the stained cells, which had migrated through the transwell plate, were photographed and counted using a light microscope (Nikon, Tokyo, Japan).

Localization of Nucleus and Cytoplasm
To further understand the function of lncRNA AFAP1-AS1 in ESCC cells. The NE-PER™ Nuclear and Cytoplasmic Extraction Reagents Kit (Thermo Scientific, Waltham, MA, USA) was utilized to detect the distribution of AFAP1-AS1. According to the manufacturers' instructions, the cytoplasm and nuclear components of ESCC cells were separated and collected. Then, the qRT-PCR was conducted to examine the expression of AFAP1-AS1 in cell cytoplasm and nucleus, respectively. GAPDH is cytoplasm positioning control, U6 is the nucleus positioning control.

Statistical Analysis
The SPSS software 20.0 was used for statistical analysis, and then results were shown as mean ± standard deviation (SD). GraphPad5.0 software was employed to display the data in graphs. Significance was tested using Student's t-test between groups. P < 0.05 was considered to be statistically significant.

AFAP1-AS1 and VEGFA Were Upregulated, Whereas miR-498 Was Downregulated in ESCC Tissues and Cell Lines
To expose the biological functional roles of AFAP1-AS1, miR-498, and VEGFA in ESCC, qRT-PCR was performed to detect the expression levels of them. The results showed that the relative levels of AFAP1-AS1 and VEGFA were significantly upregulated, while miR-498 was markedly downregulated in ESCC tissues compared with normal tissues ( Figure 1A-C). According to the median of AFAP1-AS1, miR-498 and VEGFA expression in ESCC tissues, patients were divided into two groups: Low expression (n=21) and high expression (n=21). The statistical analysis presented that AFAP1-AS1, miR-498 and VEGFA were correlated with the malignancy of ESCC (Tables S1-S3). Moreover, similar alterations were observed of the expression levels of AFAP1-AS1 and VEGFA in ESCC cells (Eca109 and KYSE-30) compared with that in HET-1A cell ( Figure 1D-F). From these data, we speculated that AFAP1-AS1, miR-498 and VEGFA might be involved in the development of ESCC.

AFAP1-AS1 Downregulated miR-498 Expression by Competitively Binding to miR-498
It is reported that lncRNA functions as a molecular sponge of miRNA to liberate mRNA transcript targeted by miRNA, thereby affecting cancer progression. 36 Based on the contrary expression of miR-498 and AFAP1-AS1 in ESCC tissues and cell lines, the binding sites between them were predicted by LncBase Predicted v.2.0 and shown in Figure 2A. Next, we detected the location of AFAP1-AS1 in ESCC cells, the results showed that AFAP1-AS1 was mostly distributed in the cytoplasm in Eca109 and KYSE-30 cells ( Figure 2B and C). Dualluciferase reporter analysis showed that luciferase activity in Eca109 and KYSE-30 cells co-transfected with AFAP1-AS1 WT and miR-498 mimics was obviously declined compared with that in cells co-transfected with AFAP1-AS1 WT and NC mimics, and there was no distinct change in the cells transfected with AFAP1-AS1 MUT ( Figure 2D and E). At the same time, RNA pull-down further confirmed the interaction between miR-498 and AFAP1-AS1 in Eca109 and KYSE-30 cell ( Figure 2F and G). Subsequently, we analyzed the efficiency of AFAP1-AS1 overexpression and interference. The data showed that AFAP1-AS1 level was significantly increased after transfection with AFAP1-AS1 overexpression vector in Eca109 and KYSE-30 cells ( Figure 2H). Moreover, after transfection with si-AFAP1-AS1#1, si-AFAP1-AS1#2 and si-AFAP1-AS1#3, the expression level of AFAP1-AS1 was significantly downregulated and si-AFAP1-AS1#1 played the most obvious role in Eca109 and KYSE-30 cells ( Figure 2I). So si-AFAP1-AS1#1 was chosen for further experiments. Interestingly, miR-498 level was negatively regulated by AFAP1-AS1, when AFAP1-AS1 overexpression vectors were transfected, miR-498 expression level was significantly downregulated in Eca109 and KYSE-30 cells ( Figure 2J). Simultaneously, AFAP1-AS1 knockdown could steeply augment the expression of miR-498 in the two ESCC cells ( Figure 2K). In addition, we found miR-498 expression was negatively correlated with AFAP1-AS1 expression in ESCC tissues (R 2 = 0.548, P < 0.0001) ( Figure 2L). In sum, these data suggested that miR-498 was directly targeted by AFAP1-AS1 in ESCC cells.

AFAP1-AS1 Silencing Inhibited ESCC Cells Proliferation and Migration and Led to Apoptosis via Regulating miR-498 Expression
Given the molecular mechanism between miR-498 and AFAP1-AS1, we researched the functional regulation in ESCC cells between them. Firstly, si-AFAP1-AS1#1 alone or along with miR-498 inhibitor was introduced into ESCC cells. As described in Figure 3A, the downregulated expression level of miR-498 in Eca109 and  Figure 3F). In addition, Western blot analysis showed that AFAP1-AS1 silencing generated a significant reduction in the protein levels of PCNA and MMP-9, and dramatically promoted protein level of c-caspase 3 in Eca109 and KYSE-30 cell. And the changes of AFAP1-AS1 knockdown on related protein expression level were rescued by silencing miR-498 in

VEGFA Was Directly Targeted by miR-498 in ESCC Cells
In order to illuminate the mechanism of VEGFA involved in regulating ESCC progression, bioinformatics software starBase v2.0 revealed putative binding sites between VEGFA 3ʹUTR and miR-498 ( Figure 4A). The dualluciferase reporter assay further proved that VEGFA was the target mRNA of miR-498, which miR-498 mimics significantly decreased the luciferase activity of VEGFA WT but not VEGFA MUT in Eca109 and KYSE-30 cells ( Figure 4B and C). In addition, the mRNA and protein levels of VEGFA in Eca109 and KYSE-30 cells were significantly reduced by miR-498 mimics, while miR-498 inhibitor observably elevated the mRNA and protein levels of VEGFA ( Figure 4D-G). Moreover, there was a negative interlink between the expression of miR-498 and VEGFA ( Figure 4H). These results suggested that miR-498 bound to 3ʹUTR of VEGFA mRNA and regulated VEGFA expression in ESCC cell lines.

VEGFA Reversed the Inhibitory Effect of miR-498 on ESCC Cell Progression
Considering the negative regulatory effect of VEGFA on the expression of miR-498 in ESCC, we further explored the functional effects of VEGFA in ESCC cell lines in vitro, As presented in Figure 5A, the declined expression level of VEGFA mRNA in Eca109 and KYSE-30 cells caused by miR-498 mimics could be improved by transfecting with VEGFA relative to that cells transfected with vector. Subsequently, MTT assay reflected a phenomenon that weakened cells vitality triggered by miR-498 overexpression was restored via VEGFA overexpression ( Figure 5B and C). Apoptotic assay indicated that reinforced expression of miR-498 significantly induced ESCC cell apoptosis, whereas ectopic expression of VEGFA abolished this effect ( Figure 5D). Transwell assay demonstrated that miR-498 overexpression significantly suppressed ESCC cell migration, and this effect was changed after transfection with VEGFA in vitro ( Figure 5E). Furthermore, miR-498 overexpression significantly decreased the protein levels of PCNA and MMP-9 and up-regulated c-caspase 3 protein expression in Eca109 and KYSE-30 cells, whereas overexpression of VEGFA reversed the effects of miR-498 overexpression on these proteins ( Figure 5F-I). All results  Silencing AFAP1-AS1 Downregulated VEGFA Expression by Sponging miR-498 in ESCC Cells Next, we aimed to explore whether AFAP1-AS1 could regulate VEGFA expression through miR-498 in ESCC. The positive correlation between AFAP1-AS1 and VEGFA was analyzed and exhibited in Figure 6A. Besides, qRT-PCR analysis indicated that VEGFA mRNA expression was inhibited in Eca109 and KYSE-30 cells transfected with si-AFAP1-AS1#1, whereas miR-498 inhibitor evidently improved expression of VEGFA ( Figure 6B). As well as VEGFA protein expression level, reduced by AFAP1-AS1, was markedly promoted following inhibition of miR-498 in Eca109 and KYSE-30 ( Figure 6C and D) cell. From the above, these data suggested that AFAP1-AS1 silencing downregulated VEGFA expression by sponging miR-498 in ESCC cells.

Discussion
During the past years, numerous dysregulated lncRNAs have been reported to be implicated in the carcinogenesis and progression of different malignancies. 37 Recently, study discovered that AFAP1-AS1 was aberrantly upregulated in esophageal adenocarcinoma (EAC) tissues and cells and could dysregulate biologic functions of EAC cells. 21 Moreover, Luo et al showed that AFAP1-AS1 was also upregulated in ESCC tissues and cell lines, and promoted ESCC cell growth and inhibited cell apoptosis, 22 which provide a clue that there may be a potential correlation between AFAP1-AS1 and the progression of ESCC. In this study, we found that AFAP1-AS1 was significantly upregulated in ESCC tissues compared with normal tissues, and this result was also confirmed in ESCC cell lines. Functionally, silencing of AFAP1-AS1 restrained ESCC cell proliferation and migration, and induced cell apoptosis in the two ESCC cell lines. More importantly, we found miR-498 was significantly downregulated and VEGFA was significantly upregulated in ESCC tissues and cells. Accordingly, the data suggested that AFAP1-AS1, miR-498 and VEGFA might play critical roles in ESCC. It has been the most common regulation patterns that lncRNAs acted as miRNA molecular sponges to regulate miRNA expression over the last few years. 38 For example, lncRNA AFAP1-AS1 promoted nasopharyngeal carcinoma cell migration by sponging miR-423-5p. 39 In addition, AFAP1-AS1 acted as a competing endogenous RNA (ceRNA) of miR-384 to regulate tumorigenicity of pancreatic cancer cells. 40 Furthermore, through targeting miR-103a-3p, AFAP1-AS1 facilitated cell proliferation and viability of Pituitary Adenoma. 41 Among this research, miR-498 was identified to be directly targeted by AFAP1-AS1 in ESCC cells. On the basis of previous researches, miR-498 was discovered to be located in 19q13.41 and downregulated in colorectal adenocarcinoma tissues. 42 miR-498 could affect telomerase expression 43 and was correlated with tumor progression in non-small cell lung cancer. 44 Moreover, miR-498 acted as a tumor suppressor and inhibited the proliferation of ovarian cancer cell 45 and ESCC cell. 46 In our study, dual-luciferase assay, RNA pull-down assay and gain or loss-of-function assay were performed, and our data supported that AFAP1-AS1 downregulated the expression of miR-498 by competitively binding to miR-498. Additionally, interference with miR-498 could restore the inhibition effect of si-AFAP1-AS1 transfection on cell proliferation and metastasis, and recover the promotion influence of si-AFAP1-AS1 in cell apoptosis in vitro. These evidences also revealed that AFAP1-AS1 functioned as an endogenous decoy for miR-498 to inhibit the growth and metastasis of ESCC cells.
Subsequently, we analyzed the target genes of miR-498 and found that VEGFA could be targeted by miR-498. VEGFA was initially discovered to be a regulator of angiogenesis. 47 Recently, increasing evidence has shown that VEGFA was involved in multiple biological processes, such as embryogenesis tumor survival, 48,49 proliferation and migration. 50 More than that, we found that interference with AFAP1-AS1 retarded the mRNA and protein levels of VEGFA, and the inhibitory impact of AFAP1-AS1 knockdown on the expression of VEGFA was attenuated via miR-498 inhibitor. These results revealed that there may be a directly competitive correlation between AFAP1-AS1 and VEGFA for binding miR-498.

Conclusion
To sum up, our study clarified that AFAP1-AS1 was upregulated in ESCC tissues and cell lines, and silencing of AFAP1-AS1 suppressed ESCC cell proliferation and migration, and promoted apoptosis. Furthermore, AFAP1-AS1 could act as a sponge of miR-498 to regulate VEGFA expression. Thus, our findings provided a theoretical basis for the diagnosis and treatment of ESCC.