LncRNA FEZF1-AS1 Promotes TGF-β2-Mediated Proliferation and Migration in Human Lens Epithelial Cells SRA01/04

Posterior capsule opacification (PCO) is a common complication after cataract surgery attributed to the proliferation and migration of postoperative residual lens epithelial cells (LECs). The long noncoding RNA (lncRNA) FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1) promotes the proliferation and migration of multiple types of cancer cells. Here, we discovered that FEZF1-AS1 is markedly upregulated in TGF-β2-treated SRA01/04 cells. In addition, the proliferation and migration of SRA01/04 cells were enhanced following TGF-β2 treatment. FEZF1-AS1 knockdown inhibited the TGF-β2-induced proliferation and migration of SRA01/04 cells. Accordingly, FEZF1-AS1 overexpression promoted the TGF-β2-induced proliferation and migration of SRA01/04 cells. Finally, FEZF1-AS1 upregulated TGF-β2-induced SRA01/04 cell proliferation and migration via boosting FEZF1 protein levels. Our findings indicate that the dysregulation of FEZF1-AS1 participates in the TGF-β2-induced proliferation and migration of human lens epithelial cells (HLECs), which might be achieved, at least in part, through the induction of FEZF1 expression.


Introduction
Cataracts are the most common cause of blindness, and cataract surgery is their only cure. Phacoemulsification and extracapsular cataract extraction (ECCE) are the most common types of surgery to treat cataracts. However, posterior capsule opacification (PCO) is a common complication after cataract surgery. Decreased visual acuity induced by PCO occurs in 20-40% of patients 2-5 years postoperatively [1]. PCO is generally associated with the pathological progression of postoperative residual lens epithelial cells (LECs), including proliferation, migration, and epithelial-mesenchymal transition (EMT) [2].
Long noncoding RNAs (lncRNAs), which consist of more than 200 nucleotides (nt) and exhibited limited or no protein-coding capacity [3], are dysregulated in multiple human diseases, including PCO [4,5]. e long noncoding RNA (lncRNA) FEZ family zinc finger 1 antisense RNA 1 (FEZF1-AS1), which is located on the opposite strand of the FEZF1 gene, upregulates the mRNA level and protein expression of FEZF1 [6]. In addition, FEZF1-AS1 promotes the proliferation and migration of colorectal carcinoma [6], pancreatic ductal adenocarcinoma [7], and osteosarcoma [8]. However, the expression and role of FEZF1-AS1 in the pathogenesis of PCO are unclear.
In this study, we questioned whether FEZF1-AS1 regulated the proliferation and migration of HLECs via modulating FEZF1 expression. erefore, transforming growth factor-β2 (TGF-β2)-stimulated HLECs were used to mimic the PCO microenvironment. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect FEZF1-AS1 expression in HLECs following TGF-β2 stimulation. Meanwhile, the proliferation and migration of HLECs were detected following TGF-β2 stimulation with FEZF1-AS1 knockdown or overexpression. Finally, FEZF1 expression was measured by western blot and inhibited by FEZF1 siRNAs to identify the effect of FEZF1 on the role of FEZF1-AS1. We found that FEZF1-AS1 upregulated TGF-β2-induced SRA01/ 04 cell proliferation and migration via boosting FEZF1 protein levels. Our study demonstrated the novel role and mechanism of FEZF1-AS1 in the pathogenesis of PCO.

Materials and Methods
2.1. Cell Culture and Treatment. SRA01/04 cells that were transformed via SV40 T-antigen [9] were obtained from Jennio Biotech Company (Guangzhou, China). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco/Brl, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in a humidified atmosphere containing 5% CO 2 . Cells treated with TGF-β2 (Sigma Aldrich, St Louis, USA) at a concentration of 10 ng/ml for 48 h were used as the TGF-β2 group.

5-Ethynyl-20-deoxyuridine (EdU)
Assay. An EdU kit (Cell Light EdU DNA imaging kit, RiboBio, Guangzhou, China) was used to evaluate cell proliferation according to the manufacturer's instructions. Images were detected and analyzed with a microscope (Olympus, Tokyo, Japan). e average ratio of EdU-stained cells (red) to DAPI-stained cells (blue) was used to evaluate cell proliferation activity.
2.6. Wound Healing Assay. SRA01/04 cells were seeded (2 × 10 6 /well) in a 6-well plate. A wound was made by scratching a confluent monolayer with a 200 μl pipette tip. Nonadherent cells were washed off with sterile PBS. e cells were placed in an incubator for 48 h. Pictures were taken by an inverted microscope (Nikon Ti, Japan) at a 50x magnification. e widths of the scratches were measured using Motic Image Plus 2.2S software (Shimadzu, Japan). e relative cell migration compared to the location at 0 h was calculated as a ratio (Figure 1(h)). e wound width (μm) was measured from at least three experiments ( Figure 2(b)).

Transwell Migration Assay.
Cell migration was determined by the Transwell assay. After the required treatment, 5 × 10 4 cells were transferred to the upper chambers of 8 μm hanging inserts in 24-well plates (Corning, USA) in serum-free DMEM. A volume of 800 μl of DMEM containing 10% FBS was then added to the lower chamber. After a 24 h incubation, the noninvaded cells were removed with cotton swabs. e invaded cells were fixed with 4% paraformaldehyde for 15 min and stained with 0.1% crystal violet (Beyotime) for 30 min and photographed. ree different microscopic fields were used to calculate the average number of migrated cells.
2.9. Statistical Analysis. All statistical analyses were performed using SPSS 19.0 software (SPSS, USA). Data are represented as the mean ± SEM; all experiments were performed at least in triplicate. Student's t-test or oneway analysis of variance (ANOVA) was used for statistical analysis. When a significant difference was apparent by ANOVA, the Dunnett test was used to compare multiple means. A value of P < 0.05 indicated statistical significance.

FEZF1-AS1 Is Upregulated and Accompanied by Increased Proliferation and Migration in TGF-β2-Treated
SRA01/04 Cells. Using real-time RT-PCR, we first detected FEZF1-AS1 levels and found that FEZF1-AS1 was upregulated in TGF-β2-treated SRA01/04 cells compared to its expression in the blank control group (Figure 1(a)). Meanwhile, the viability and proliferation of TGF-β2-treated SRA01/ 04 cells, which were detected by CCK-8 and EdU assays, respectively, increased compared to that observed in control cells (Figures 1(b)-1(d)). Previous studies found that FEZF1-AS1 knockdown repressed the proliferation of gastric cancer and lung adenocarcinoma cells, inhibited cell cycle progression by causing G1/S arrest, and decreased the levels of cyclindependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin-dependent kinase 6 (CDK6), and cyclin D1 [10,11]. Interestingly, western blotting demonstrated that CDK2, CDK4, CDK6, and cyclin D1 protein levels were upregulated in the TGF-β2-treated cells compared to their expression in the blank control cells (Figures 1(e) and 1(f)). Additionally, the wound healing assay showed that cell migration was increased in the TGF-β2-treated cells compared to that in the control cells (Figures 1(g) and 1(h)). ese data suggested that FEZF1-AS1 was upregulated and accompanied by the increased proliferation and migration of TGF-β2treated SRA01/04 cells, implying that FEZF1-AS1 was associated with the proliferation and migration of SRA01/04 cells after TGF-β2 stimulation.

Discussion
PCO is a common and significant complication following cataract surgery. At present, surgical intervention is the only cure for cataracts [1]; however, after cataract surgery, the growth of aberrant lens epithelial cells (LECs) across the lens capsule often leads to migration, fibrosis, and collagen deposition, leading to secondary visual loss known as PCO. Cell culture is the simplest method to study PCO and generally utilizes cell lines to analyze PCO characteristics. Experiments using these model systems permit the determination of factors that stimulate or inhibit proliferation, migration, differentiation, transdifferentiation, and matrix contraction [13]. SRA01/04 cells express mRNA for multiple growth factor receptors including TGF-β2, epidermal growth factor (EGF), and insulin-like growth factor 1 (IGF-1) [13]. SRA01/04 cells also express integrins α2 and α3.
is contradiction can be attributed to several things. First, the dose and stimulation time used were different. TGF-β2 at a concentration of 10 ng/ml was used for 48 h in our study, and 1 ng/ml TGF-β2 was used for 12 h in their study. Second, the cell lines were different. SRA01/04 cells were utilized in our study, while HLE-B3 cells and primary HLECs were utilized in their study. Four microRNAs (miRNAs), including miR-31, miR-124, miR-184, and miR-222, are differentially expressed between SRA01/04 and HLE-B3 cells [26]. ese differences in miRNA expression might promote different TGF-β2-induced and proliferation-associated patterns of gene expression. As a result, TGF-β2 induces a distinct proliferation response in the two HLEC cell lines. Finally, the cell proliferation assays used were different. We used CCK-8 and EdU incorporation assays, and the authors of the other studies performed a colorimetric WST-1 assay and proliferating cell nuclear antigen (PCNA) western blot. Which signaling pathways downstream of TGF-β2 in different HLECs are related to cell proliferation? e question requires future study. e Fez family zinc finger protein 1 (FEZF1) is a C 2 H 2 zinc finger transcription factor that plays critical roles in the development of the forebrain and olfactory system in vertebrates [27]. In mice, FEZF1 binds to and represses the expression of Hes family BHLH transcription factor 5 (Hes5), a transcription factor that inhibits neuronal differentiation and facilitates neurogenesis in the forebrain [28]. FEZF1 has also been implicated in the progression of human cancers. In gastric cancer cells, FEZF1 enhances proliferation and tumorigenic by binding to and activating Kirsten rat sarcoma viral oncogene homolog (KRAS) [29]. In addition, FEZF1 promotes cell migration and invasion in colorectal cancer cells [6]. We also found that FEZF1-AS1 promotes TGF-β2-induced SRA01/04 cell proliferation and migration via upregulating FEZF1 protein levels. In an in vitro experiment, FEZF1 promoted the proliferation, * *  migration, and invasion of glioma cells and inhibited cell apoptosis by activating the protein kinase B alpha (Akt)extracellular signal-regulated kinase (ERK) pathway [30]. e growth factor (such as EGF) induced proliferation of HLECs in the aqueous humor is dependent on the mitogenactivated protein kinase (MAPK)/ERK and Akt/PI3K signaling pathways [31]. Furthermore, EGF-induced cell migration is mediated by the ERK and phosphatidylinositol-3 kinase (PI3K)/AKT pathways in cultured HLECs [32].
us, we speculate that FEZF1 promotes the proliferation and migration of HLECs via upregulating the ERK and AKT signaling pathways, which requires intensive investigation.
We also found that FEZF1-AS1 upregulated FEZF1 protein levels (Figures 4(a) and 4(b)). FEZF1-AS1 increased the aggressive behavior of colorectal carcinoma cells by increasing the mRNA levels of its corresponding cognate gene, FEZF1, via modulating the transcription of FEZF1 or the stability of its mRNA [6], suggesting the mechanism by which FEZF1-AS1 upregulated FEZF1 protein levels. e detailed mechanism underlying the upregulation of FEZF1 expression FEZF1-AS1 requires future exploration.
In addition to upregulating FEZF1 expression, FEZF1-AS1 promoted cell proliferation and migration through other mechanisms. For example, the downregulation of FEZF1-AS1 inhibited the proliferation of gastric cancer (GC) cells, arrested the cell cycle at the G0/G1 stage, and suppressed the activation of the wingless (Wnt)/β-catenin signaling pathway [33].
However, there were several limitations of our study that should be noted; primary HLECs were not used, and FEZF1 mRNA levels were not detected. In summary, our study discovered that FEZF1-AS1 promotes the proliferation and migration of HLECs via upregulating FEZF1 expression, implying a novel therapeutic target for PCO.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
All authors declare that they have no conflicts of interest.