Long Non-coding RNA FEZF1-AS1 Promotes Growth and Reduces Apoptosis Through Regulation of miR-363-3p/PAX6 Axis in Retinoblastoma

Retinoblastoma is the most common malignancy in children's eyes with high incidence. Long non-coding RNAs (lncRNAs) play important roles in the progression of retinoblastoma. LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1) has been found to stimulate retinoblastoma. However, the mechanism of FEZF1-AS1 underlying progression of retinoblastoma is still unclear. In current study, FEZF1-AS1 was up-regulated in retinoblastoma tissues and cells. FEZF1-AS1 overexpression enhanced retinoblastoma cell viability, promoted cell cycle, and inhibited apoptosis. Conversely, FEZF1-AS1 knockdown reduced cell viability, cycle, and elevated apoptosis. The interaction between FEZF1-AS1 and microRNA-363-3p (miR-363-3p) was confirmed. FEZF1-AS1 down-regulated miR-363-3p and up-regulated PAX6. PAX6 was a target gene of miR-363-3p. EZF1-AS1 promoted retinoblastoma cell viability and suppressed apoptosis via PAX6. Further, we demonstrated that FEZF1-AS1 contribute to tumor formation in vivo. In conclusion, FEZF1-AS1 elevated growth and inhibited apoptosis by regulating miR-363-3p/PAX6 in retinoblastoma, which provide a new target for retinoblastoma treatment.


Introduction
Retinoblastoma is the most common malignancy in children's eyes (Stark 2016). Retinoblastoma is accompanied by multiple lesions and easily exhibits intracranial and systemic metastasis when the tumor grows and breaks through the eyeball (Garsed et al. 2018). About 8000 cases being diagnosed yearly worldwide (Pascual-Pasto et al. 2019). There are 3540 new estimated cases occurring in the United States and approximately 1000 new cases happening each year in China (Chen et al. 2007;Siegel et al. 2018). Some remedies have been applied into treatment of patients with retinoblastoma, including chemotherapy combined with focal therapy (Ortiz and Dunkel 2016). However, the survival rate from patients with retinoblastoma is still unsatisfactory in the developing nations (Li et al. 2018b). Thus, it is interesting to search the useful biomarker for retinoblastoma treatment.
Long non-coding RNAs (lncRNAs) are more than 200 nt in length and function in limit of protein-coding potential (Zhu et al. 2018). It has reported that lncRNAs show important roles in the progression of human tumors (Tsai et al. 2011;Tang et al. 2013), including retinoblastoma Yang and Peng 2018). LncRNA FEZF1 antisense RNA 1 (FEZF1-AS1), as one of lncR-NAs, produces a 2564 bp transcript and is localized chromosome 7q31.32. Accumulating evidences have revealed that FEZF1-AS1 was abnormal expressed in tumor tissues and cells, and showed oncogenic effects on liver cancer (Gong et al. 2018), ovarian cancer , and osteosarcoma . Interestingly, FEZF1-AS1 was demonstrated to promote proliferation, migration, and invasion in retinoblastoma (Quan and Wang 2019). However, the mechanism of FEZF1-AS1 underlying development of retinoblastoma is still unclear.
MicroRNAs (miRNAs), 20-24 nucleotide, are an abundant class of small and highly-conserved endogenous non-coding RNA molecules (Bartel 2004). MiR-NAs could bind to 3′-untranslated regions (UTR) of target mRNA and regulate different physiological processes (Sethi et al. 2013;Gu and Kay 2010). Accumulating evidences have shown that mRNAs played an important role in development of retinoblastoma (Reis et al. 2012). Notably, miR-363-3p has been found to take part in regulation of retinoblastoma (Ma et al. 2020). Thus, it is interesting to investigate the role of miR-363-3p in development of retinoblastoma.
In this study, the expression of FEZF1-AS1 was measured in retinoblastoma tissues and cells. The role of FEZF1-AS1 in progression of retinoblastoma was explored. Then, the interaction between FEZF1-AS1 and miR-363-3p was identified. We further investigated whether FEZF1-AS1 functions in progression of retinoblastoma through miR-363-3p. The study suggests that FEZF1-AS1 may provide a candidate target for retinoblastoma treatment.

Clinical Samples
The retinoblastoma specimens from 45 patients and 36 normal retinas were collected from The Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University. The research was carried out according to the World Medical Association Declaration of Helsinki and the Ethics Committee of The Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University. No subjects received preoperative radiotherapy or chemotherapy. Written informed consent was harvested from all subjects.

Cell Proliferation Assay
Cells (2 × 10 3 ) were seeded in 96-well plates. Then, the cells were incubated for 24, 48, and 72 h. At different time points, 10 μl Cell Counting Kit-8 (CCK-8) reagents were added into plates. After incubation for 4 h, absorbance was examined at 450 nm and cell viability was assessed.

Cell Cycle Measurement
Cells (1 × 10 6 ) were collected and fixed with ice-cold 70% ethanol overnight at 4 °C. The cells were washed thrice with PBS. Then, propidium iodide (PI) (Takara, Dalian, China) was used to incubate cells for 30 min at 37 °C. Finally, the cells were detected using flow cytometry.

Cell Apoptosis Detection
Cells (3 × 10 5 ) were harvested and resuspended with Annexin V-FITC/PI binding buffer. Subsequently, cells were stained with 5 μl Annexin V-FITC and 5 μl PI at 37 °C. Following 20 min of incubation in dark, cells were analyzed via flow cytometry. Data were calculated through FACS Diva software.

RNA Immunoprecipitation (RIP) Assay
FEZF1-AS1overexpression was carried out in WERI-RB1 and Y79 cells. RIP lysis buffer was used to lysed cells and RIP assay was conducted via RIP RNA-Binding Protein Immunoprecipitation Kit (Abcam, Shanghai, China). Reaction system included Ago2 and IgG antibodies for RIP assays. The enrichments of FEZF1-AS1and miR-363-3p were identified using qPCR.

Immunohistochemistry
Tissues were fixed with 4% paraformaldehyde and 5-µm sections were harvested. The sections were deparaffinized and rehydrated and then blocked with goat serum for 10 min. Primary antibodies, including Ki67 and PAX6 (all from Abcam, Shanghai, China), were used to incubate the sections for 3 h at 37 °C. The secondary antibody covered the sections for 60 min at 37 °C. Nuclei were stained using DAPI. The photographs were acquired under a microscope.

Tumor Xenograft Experiment
The 4-week-old male BALB/c nude mice were obtained and housed in a specific pathogen-free (SPF) room. Y79 cells (1 × 10 6 ) expressing shFEZF1-AS1 were injected subcutaneously into the right fore-flank of nude mice. Size of the tumor was measured every week, and the weight of tumor was recorded. At 28 days post-injection, mice were euthanized and the tumors were excised. The volume was calculated through the following formula: V (mm 3 ) = (length × width 2 )/2. All studies were approved by the Guide for the Care and Use of Laboratory Animals and the Ethics Committee of The Affiliated Huaian No. 1 People's Hospital of Nanjing Medical University.

Statistical Analysis
Data were presented as mean ± SD. Statistical analysis was conducted via Graph-Pad Prism software 5.0. Student's t test and one-way analysis of variance (ANOVA) were used to analyze the data. The FEZF1-AS1 expression in relation to survival was assessed via the Kaplan-Meier analysis and the log-rank test. A p < 0.05 was considered significant.

FEZF1-AS1 Silencing Induced Retinoblastoma Cell Cycle Attest and Apoptosis
To explore the effect of FEZF1-AS1 on retinoblastoma cell cycle and apoptosis, the flow cytometry analysis was conducted. The results proved that FEZF1-AS1 overexpression reduced the cell population in the G1 phase and elevated cell population in Cell viability was examined using CCK-8 assay. n = 3. *p < 0.05. **p < 0.01 the S phase. Conversely, FEZF1-AS1 silencing showed an opposite effect on retinoblastoma cell cycle (Fig. 2a). Further, flow cytometry analysis was performed and the results demonstrated that FEZF1-AS1 overexpression decreased cell apoptosis and FEZF1-AS1 silencing increased cell apoptosis (Fig. 2b). These findings implied that FEZF1-AS1 silencing induced retinoblastoma cell cycle attest and apoptosis.

FEZF1-AS1 Knockdown Impaired Tumor Formation In Vivo
The effect of FEZF1-AS1 on tumor formation was evaluated via tumor xenograft experiment in vivo. At 28 days post-injection, mice were euthanized, and the tumors were excised and photographed (Fig. 6a). As shown in Fig. 6b, FEZF1-AS1 knockdown inhibited tumor volume and weight. The results from qRT-PCR revealed that FEZF1-AS1 knockdown down-regulated FEZF1-AS1 expression and up-regulated miR-363-3p expression (Fig. 6c). Immunohistochemistry demonstrated that FEZF1-AS1 knockdown suppressed Ki67, which was the marker of proliferation, and PAX6 expression (Fig. 6d). The data indicated that FEZF1-AS1 knockdown impaired tumor formation in vivo.
Previous studies have shown that abnormal lncRNA expression was associated with progression of retinoblastoma. For example, lncRNA THOR was increased in the retinoblastoma patients and cells, enhanced cell growth, and suppressed cell apoptosis (Shang 2018). lncRNAUCA1 was up-regulated in retinoblastoma tissues and cells and elevated retinoblastoma cell proliferation and multidrug resistance (Yang et al. 2020). Interestingly, in our research, we demonstrated that FEZF1-AS1 was enhanced in retinoblastoma patients and cells. FEZF1-AS1 silencing inhibited cell viability in vivo and in vitro. Consistently, Quan et al. discovered that FEZF1-AS1 promoted proliferation in retinoblastoma (Quan and Wang 2019). Additionally, we found that FEZF1-AS1 could function in cell cycle and apoptosis. FEZF1-AS1 decreased the cell population in the G1 phase and increased cell population in the S phase. A decrease of cell apoptosis was caused by FEZF1-AS1 overexpression in retinoblastoma. We further proved that FEZF1-AS1 contributed to growth and inhibited apoptosis in retinoblastoma. Increasing evidences have revealed that FEZF1-AS1 could compete endogenous RNA (ceRNA) to bind to miRNAs and regulate tumorigenesis and progression. For instance, Ye et al. proved that FEZF1-AS1 promoted progression of pancreatic ductal adenocarcinoma through miR-107 (Ye et al. 2018). Li et al. discovered that FEZF1-AS1stimulated cell growth by miR-610 in multiple myeloma (Li et al. 2018a). FEZF1-AS1 was found to exert oncogenic effects in retinoblastoma (Quan and Wang 2019). Thus, We hypothesize that FEZF1-AS1 may participate in regulation of cell growth in retinoblastoma via sponging miRNA. Interestingly, we confirmed that miR-363-3p could targetFEZF1-AS1. In addition, FEZF1-AS1 was verified to reduce miR-363-3p level in vivo and in vitro. The data indicated that FEZF1-AS1 may regulate cell progression in retinoblastoma through sponging miR-363-3p. Reportedly, miR-363-3p was involved in the cell development in diseases via targeting mRNA. For example, miR-363-3p was proved to repress cell proliferation and invasion in osteosarcoma through targeting SOX4 ). Additionally, miR-363-3p took part in the inhibition of tumor growth and metastasis in colorectal cancer by SphK2 (Dong et al. 2018). As exhibited in our results, we identified the relationship between miR-363-3p and PAX6. Further, our study showed that FEZF1-AS1 silencing suppressed cell viability and cycle and promoted cell apoptosis. However, PAX6 overexpression exhibited opposite effects on FEZF1-AS1 silencing-caused cell viability, cycle and apoptosis. Notably, PAX6 was found to regulate the cell growth in retinoblastoma. For instance, Liu et al. revealed that miR-129-5p was demonstrated to inhibit cell progression in retinoblastoma through targeting PAX6 (Liu et al. 2019). Li et al. verified that miR-433 suppressed retinoblastoma cell proliferation and metastasis through direct targeting of PAX6 (Li et al. 2016). These findings indicated that FEZF1-AS1 participated in regulation of cell progression in retinoblastoma through miR-363-3p/PAX6. However, accumulating evidences have shown that more lncRNAs regulated tumor progression by sponging miR-363-3p (Xie et al. 2019;Wang et al. 2020). Moreover, the miR-363-3p inhibitor/mimic and FEZF1-AS1 overexpression/ knockdown should be reconfirmed in more retinoblastoma cell lines. In addition, the application of FEZF1-AS1 is not evaluated in clinical sample. Therefore, more experiments are still needed to be performed in the near future.
Author Contributions XL designed the study, supervised the data collection, and analyzed the data; XL interpreted the data and prepared the manuscript for publication; JL supervised the data collection, analyzed the data, and reviewed the draft of the manuscript. All authors have read and approved the manuscript.
Data Availability All data generated or analyzed during this study are included in this published article.