Overexpression of long non-coding RNA SBF2-AS1 promotes cell progression in esophageal squamous cell carcinoma (ESCC) by repressing miR-494 to up-regulate PFN2 expression

Esophageal squamous cell carcinoma (ESCC) is an intractable esophageal cancer caused by smoking, alcohol consumption and nutritional deficiencies. Recently, long non-coding RNA SET-binding factor 2 antisense RNA 1 (SBF2-AS1) was validated as an oncogene in multiple cancers. However, the mechanism of SBF2-AS1 in ESCC progression is poorly understood. In the present research, we found that the expression of SBF2-AS1 and PFN2 was up-regulated, while miR-494 was down-regulated in ESCC tumors and cells using quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and transwell assay demonstrated that silencing of SBF2-AS1 suppressed proliferation, migration and invasion. Moreover, western blot showed that SBF2-AS1 deletion also inhibited epithelial to mesenchymal transition (EMT) by detecting MMP9, Vimentin and E-cadherin protein expression. We confirmed that miR-494 was a target of SBF2-AS1 by luciferase reporter system, RIP and RNA pull-down assay. In addition, miR-494 inhibitor reversed the repression induced by SBF2-AS1 silencing on ESCC cell proliferation, migration, invasion and EMT. Furthermore, PFN2 was negatively regulated by miR-494. Besides, restoration of PFN2 inversed the inhibition effects on cell proliferation, migration, invasion and EMT induced by SBF2-AS1 silencing in ESCC. In conclusion, SBF2-AS1 contributed to cell proliferation, migration, invasion and EMT in ESCC by enhancing PFN2 expression via sponging miR-494, providing promising biomarkers for ESCC diagnosis and treatment.


Quantitative real-time polymerase chain reaction (qRT-PCR)
QRT-PCR was performed following the method described in our earlier study (Y. J. Tian et al., 2019). Total RNA was obtained by incubating ESCC tissues and cells with TRIzol reagent (Invitrogen). Then, a NaniDrop ND-1000 Spectrophotometer (NaniDrop, Wilmington, MA, USA) was used to quantify RNA, and its purity was detected using the A260/280 ratio.
The amplification parameters were as follows: denaturation at 95˚C for 10 min, followed by 40 cycles of denaturation at 95˚C for 30s, annealing at 60˚C for 30s and extension at 72˚C for 1min. The primers for SBF2-AS1, miR-494, PFN2 GAPDH, and U6 were listed in Table S1.

Transwell assay
Cell migration and invasion were determined using Transwell chambers (24-well, Sigma-Aldrich). The upper chamber of transwell was treated with Matrigel (without Matrigel coating for migration assay). In briefly, transfected Eca-109 and KYSE-150 cells (7 × 10 4 cells per well) were resuspended in serum-free DMEM (Gibco). Then, the medium containing the cells were seeded into the upper chamber. Meanwhile, the lower chamber was supplemented with the medium containing 10% FBS (Gibco) as chemoattractant. After incubation for 48 h, the non-migrated and non-invasive cells were scraped, while the migrated and invaded cells at the lower chamber of transwell were fixed with methsnol and stained with 0.1% crystal violet (Sigma, St. Louis, MO, USA). The number of migrated and invaded cells was counted using a microscope (Lv et al., 2016).
Subsequently, the cytoplasmic RNA and nuclear RNA were incubated with Buffer SK and anhydrous ethanol, respectively. Then, the nuclear RNA and cytoplasmic RNA were eluted using the spin column. Finally, the expression of SBF2-AS1 in cytoplasmic and nucleus fractions was detected by qRT-PCR (Zhou et al., 2019).

Luciferase reporter assay
LncBase Predicted v.2 predicted that miR-494 was a target gene of SBF2-AS1 and starBase 2.0 predicted that PFN2 was a target gene of miR-494. Based on these prediction results,

RNA pull-down assay
A pull-down assay was performed as previously described (Gao et

Statistical analysis
Data were presented as means ± standard deviation (SD). Statistical analysis was performed by GraphPad Prism 7. The survival curve was plotted by Kaplan-Meier analysis. Paired Student's t-test or one-way analysis of variance (ANOVA) was exploited to compare two or more samples. The correlation between miR-494 and SBF2-AS1 or PFN2 was analyzed by Pearson's correlation coefficient. P value less than 0.05 (P<0.05) was considered statistically significant.

SBF2-AS1 was closely associated with poor prognosis of ESCC
Initially, the role of SBF2-AS was evaluated by measuring the expression of SBF2-AS using GEPIA2 (http://gepia2.cancer-pku.cn/#index) and qRT-PCR. As displayed in Fig. 1A 1D). Moreover, the survival rate was significantly reduced in ESCC patients with high SBF2-AS level. By contrast, low SBF2-AS level resulted in relatively high survival rate (Fig.   1C). Hence, overexpression of SBF2-AS contributed to poor prognosis of ESCC.

Depletion of SBF2-AS1 repressed cell proliferation, migration, invasion and EMT in ESCC
Functionally depletion of SBF2-AS1 was conducted to further explore the influence of SBF2-AS1 on proliferation, migration, invasion and EMT of ESCC cells. It was apparent that SBF2-AS1 expression was distinctly decreased in Eca-109 and KYSE-150 cells stably transfected with sh-SBF2-AS1 compared with sh-NC group, indicating that the transfection efficiency was extremely high (Fig. 2A). Cell proliferation was repressed by SBF2-AS1 silencing ( Fig. 2B-C). Meanwhile, SBF2-AS1 silencing evidently attenuated cell migration and invasion of ESCC cells (Fig. 2D-E). In addition, the expression of EMT associated protein MMP9, Vimentin and E-cadherin was analyzed by western blot. As illustrated in Fig.   2F, the expression of MMP9, Vimentin was reduced whereas E-cadherin expression was Biology Open • Accepted manuscript boosted, demonstrating that SBF2-AS1 knockdown restricted EMT process. Collectively, depletion of SBF2-AS1 inhibited cell proliferation, migration, invasion and EMT in ESCC.

SBF2-AS1 acted as sponge of miR-494
The interaction between SBF2-AS1 and miR-494 was further determined by qRT-PCR and luciferase reporter system. Obviously, SBF2-AS1 was highly expressed in the cytoplasm of Eca-109 and KYSE-150 cells in comparison with nuclear, clarifying that SBF2-AS1 mainly exerted its function in the cytoplasm of cells (Fig. 3A-B). Bioinformatics analysis by LncBase Predicted v.2 showed the existence of putative binding sites between SBF2-AS1 and miR-494 (Fig. 3C). In addition, down-regulation of miR-494 expression was observed in ESCC tumors and cells compared with the matched normal ones (Fig. 3D-E). There was also a negative linear relation between expression of SBF2-AS1 and miR-494 (r=-0.67, P<0.001) (Fig. 3F). What's more, miR-494 expression in ESCC cells was enhanced by miR-494 transfection (Fig. 3G). Reduction of luciferase activity in Eca-109 and KYSE-150 cells co-transfected with WT-SBF2-AS1 and miR-494 validated the interaction between SBF2-AS1 and miR-494 ( Fig. 3H-I). As expected, RIP assay showed that the enrichment of
These findings implicated that SBF2-AS1 could regulate ESCC cell progression by targeting miR-494.
To disclose the functional role of PFN2, the expression of PFN2 mRNA and protein in ESCC tumors and cells was detected. As illustrated in Fig. 5B-D, PFN2 mRNA and protein levels were up-regulated in ESCC tumors compared with normal tissues. Up-regulation of PFN2 mRNA and protein expression was also discovered in ESCC cells in comparison with normal cells (Fig. 5E-F). Meanwhile, we noticed that miR-494 was negatively correlated with PFN2 (r=-0.605, P<0.001) (Fig. 5G). Importantly, luciferase activity was reduced in ESCC cells co-transfected with PFN2 3'UTR-WT and miR-494. However, there was no evident change Biology Open • Accepted manuscript of luciferase activity in PFN2 3'UTR-MUT transfection group (Fig. 5H-I). In addition, miR-494 transfection repressed PFN2 protein expression in ESCC cells (Fig. 5J). Also, miR-494 inhibitor rescued SBF2-AS1 silencing-induced repression on PFN2 protein expression (Fig. 5K-L). All the data revealed that PFN2 was negatively regulated by miR-494 in ESCC.
Moreover, the production of protein MMP9 and Vimentin was blocked by SBF2-AS1 silencing and boosted by restoration of PFN2. By contrast, the expression of E-cadherin displayed the opposite trend (Fig. 6F-G). Altogether, SBF2-AS1 accelerated cell proliferation, migration, invasion and EMT in ESCC cells by enhancing PFN2.

Illustration of the association between SBF2-AS1/miR-494/PFN2 axis and ESCC cell progression
We illustrated the interrelation between SBF2-AS1/miR-494/PFN2 axis and ESCC cell Biology Open • Accepted manuscript proliferation, migration, invasion and EMT. SBF2-AS1 could repress miR-494 to enhance PFN2 expression and further facilitating EMT by boosting MMP9, Vimentin protein expression and blocking E-cadherin protein expression. Hence, SBF2-AS1 was able to accelerate ESCC cell proliferation, migration and invasion by promoting EMT through regulating miR-494/PFN2 axis (Figure 7). We firstly investigated the role of SBF2-AS1 by online tool GEPIA2 and qRT-PCR.

DISCUSSION
Up-regulation of SBF2-AS1 expression was observed in ESCC tumors and cells. As expected, knockdown of SBF2-AS1 attenuated ESCC cell proliferation, migration, invasion and EMT, implicating the oncogenic role of SBF2-AS1. Luciferase reporter system confirmed the interaction between miR-494 and SBF2-AS1 or PFN2. At the same time, we discovered that the expression of miR-494 was down-regulated, whereas PFN2 was up-regulated in ESCC tumors and cells compared with normal ones. Next, the molecular mechanism of ESCC cell progression was further investigated by rescue experiment. MiR-494 inhibitor alleviated SBF2-AS1 silencing-induced suppression on ESCC cell proliferation, migration, invasion and EMT. Consistently, restoration of PFN2 inversed the inhibition on cell proliferation, migration, invasion and EMT in ESCC induced by SBF2-AS1 silencing. In short, SBF2-AS1 Biology Open • Accepted manuscript accelerated cell proliferation, migration and invasion in ESCC by facilitating EMT through miR-494/PFN2 axis.

CONCLUSION
In conclusion, we clarified the specific molecular mechanism of SBF2-AS1 in ESCC tumorigenesis and progression. We discovered that SBF2-AS1 positively accelerated cell proliferation, migration, invasion and EMT in ESCC cells by sponging miR-494 and up-regulating PFN2 expression. Our study was capable of representing novel targeted methods for ESCC therapy.

Funding
None.

Declaration of Interests
The authors have no interests to disclose.