A total of 26 RB tissues were obtained from patients (17 males and 9 females; age range, 15–53 years) with RB who received surgical resection at Liaocheng People's Hospital between September 2014 and February 2017. Normal retinal tissues were collected from 10 patients with ruptured globes. All tissues were quickly snap-frozen in liquid nitrogen and stored at −80°C until RNA isolation. None of these subjects had been treated with radiotherapy or chemotherapy prior to recruitment in this study. This research was approved by the Ethics Committee of Liaocheng People's Hospital. Written informed consent was also provided by all subjects.

The three human RB cell lines Y79, WERI-RB1 and SO-RB50 and the normal retinal pigmented epithelial cell line ARPE-19 were acquired from the American Type Culture Collection (ATTC; Manassas, VA, USA) and cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin mixture (all from Gibco; Thermo Fisher Scientific, Inc., Grand Island, NY, USA). All cells were grown at 37°C under normoxic conditions of 95% air and 5% CO₂.

The oligonucleotides of the miR-758 mimics and their negative control (miR-NC) were chemically produced by Guangzhou RiboBio Co., Ltd. (Guangzhou, China). For PAX6 overexpression, pCMV-PAX6 plasmid and empty PAX6 plasmid were synthesised by Shanghai GenePharma Co., Ltd. (Shanghai, China). For the transfection experiments, cells were inoculated into 6-well plates at a density of 5×10⁵ cells/well one day prior to transfection and maintained in antibiotic-free DMEM containing 10% FBS. miRNA mimics or plasmid were transfected into cells using Lipofectamine^® 2000 (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the manufacturer's instructions.

TRIzol^® reagent (Invitrogen Life Technologies; Thermo Fisher Scientific, Inc.) was applied to isolate total RNA from tissue samples or cells. The quality and concentration of total RNA was determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.). For the quantification of miR-758 expression, total RNA was converted into cDNA using a TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Subsequently, miR-758 expression was detected by performing qPCR using a TaqMan MicroRNA Assay kit on an ABI 7300 Plus thermal cycler (both from Applied Biosystems; Thermo Fisher Scientific, Inc.). U6 snRNA served as an internal control for miR-758 expression. The standard SYBR-Green RT-PCR kit (Takara Biotechnology Co., Ltd., Dalian, China) was used to quantify the mRNA level of PAX6, with β-actin employed as an internal control. Relative expression was analysed using the 2^−∆∆Cq method (22).

After transfection for 24 h, cells were harvested and seeded in 96-well plates with a density of 3,000 cells/well. Following various incubation times (0–72 h), a CCK-8 assay was conducted to detect cell proliferative ability. Briefly, 10 µl of CCK-8 solution (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) was added into each well and further incubated at 37°C for 2 h. Finally, cell proliferation was determined by detecting the absorbance at a wavelength of 450 nm on a multifunction microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Transfected cells were harvested at 24 h post-transfection and prepared into a cell suspension. A total of 1,000 transfected cells were transferred into 6-well plates and incubated for 2 weeks at 37°C under 95% air and 5% CO₂. On day 15, the cells were fixed in 4% paraformaldehyde, stained with methyl violet (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) and washed with phosphate-buffered saline (PBS). The number of colonies (>50 cells/colony) was counted using an inverted microscope (Olympus IX53; Olympus Corp., Tokyo, Japan).

An Annexin V fluorescein isothiocyanate (FITC) Apoptosis Detection kit (BioLegend, San Diego, CA, USA) was employed to evaluate cell apoptosis. Briefly, transfected cells were collected at 48 h post-transfection, washed with cold PBS and suspended in 100 µl of binding buffer. Subsequently, the cells were further incubated with 500 µl of Annexin V-FITC and 5 µl of propidium iodide (PI) in the dark at room temperature for 15 min. Finally, cell apoptosis was detected by flow cytometry (FACScan; BD Biosciences, Franklin Lakes, NJ, USA) within 1 h and analysed with CellQuest software version 3.3 (BD Biosciences).

Transwell chambers with 8-µm pore size (Corning Inc., Corning, NY, USA) coated with or without Matrigel (BD Biosciences) were used for the detection of cellular migration and invasion, respectively.

At 48 h post-transfection, a total of 1×10⁵ transfected cells were suspended in FBS-free DMEM and placed into the top chambers. The bottom chambers were filled with 5 µl of DMEM containing 10% FBS. After 24 h of incubation at 37°C with 95% air and 5% CO₂, non-migrated and non-invaded cells were removed from the surface of the membrane with a cotton swab. The migrated and invaded cells were fixed with 100% methanol, stained with 0.5% crystal violet, washed with PBS and photographed using an inverted microscope. Values for migration and invasion were determined by counting five randomly selected fields per membrane.

TargetScan online software (www.targetscan.org) and microRNA.org (www.microrna.org/microrna/) were used to predict the potential targets of miR-758.

PAX6 was predicted as a major target gene of miR-758, and this association was further confirmed by luciferase reporter assay. To synthesise luciferase reporter plasmids, we amplified the 3′-UTR fragment of PAX6 containing wild-type (Wt) putative or mutant-type binding sites for miR-758 (GenePharma Co., Ltd.), inserted it into the pmirGLO luciferase reporter vector (Promega Corp., Madison, WI, USA) and labelled it as pmirGLO-Wt-PAX6-3′-UTR or pmirGLO-Mut-PAX6-3′-UTR, respectively. For the luciferase assay, the cells were seeded into 24-well plates and co-transfected with miR-758 mimics or miR-NC and pmirGLO-Wt-PAX6-3′-UTR or pmirGLO-Mut-PAX6-3′-UTR using Lipofectamine^® 2000, in accordance with the manufacturer's protocol. Luciferase activities were detected at 48 h after transfection using a Dual-Luciferase Reporter Assay System (Promega Corp.). Renilla luciferase activity was determined for normalisation.

The total protein of cells or tissues was extracted using ice-cold radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China). The concentration of total protein was quantified with a BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein were separated by 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes (PVDF; EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked with 5% skimmed dry milk in Tris-buffered saline-Tween (TBST) at room temperature for 2 h and incubated with primary antibodies at 4°C overnight. After washing with TBST thrice, the membranes were incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; cat. no. sc-2005; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) or goat anti-rabbit horseradish peroxidase-conjugated secondary antibodies (1:5,000 dilution; cat. no. sc-2004; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. The membranes were washed thrice with TBST and subjected to protein signal detection using an enhanced chemiluminescence reagent (Bio-Rad Laboratories). The primary antibodies used in the present study included mouse anti-human monoclonal PAX6 (1:500 dilution; cat. no. ab197768), rabbit anti-human monoclonal p-pi3k (1:500 dilution; cat. no. ab182651) and mouse anti-human monoclonal pi3k (1:500 dilution; cat. no. ab86714; all from Abcam, Cambridge, UK), mouse anti-human monoclonal p-Akt (1:1,000 dilution; cat. no. sc-81433) and mouse anti-human monoclonal Akt (1:1,000 dilution; cat. no. sc-56878; both from Santa Cruz Biotechnology, Inc.), and rabbit anti-human monoclonal GAPDH antibody (1:500 dilution; cat. no. ab181603; Abcam). GAPDH was employed as the internal reference.

The data were expressed as the median ± standard deviation (SD) and analysed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Differences between groups were compared with Student's t-test or one-way ANOVA, followed by the SNK multiple comparison test. The possible correlation between miR-758 and PAX6 mRNA in RB tissues was evaluated using Spearman's correlation analysis. P<0.05 indicated a statistically significant difference.

To illustrate the potential roles of miR-758 in RB, we first detected miR-758 expression in 26 RB tissues and 10 normal retinal tissues. The RT-qPCR analysis data revealed that miR-758 was significantly downregulated in RB tissues compared with that in normal retinal tissues (Fig. 1A, P<0.05). In addition, miR-758 expression level was determined in the three RB cell lines (Y79, WERI-RB1 and SO-RB50) and in the normal retinal pigmented epithelium cell line ARPE-19. Compared with that in ARPE-19, the expression levels of miR-758 were markedly reduced in all three RB cell lines (Fig. 1B, P<0.05). These results indicated that the dysregulation of miR-758 may be involved in the progression of RB.

To explore the role of miR-758 in the progression of RB, we transfected miR-758 mimics into Y79 and WERI-RB1 cells to upregulate the endogenous miR-758 expression level. Following transfection, miR-758 was markedly overexpressed in Y79 and WERI-RB1 cells transfected with miR-758 mimics compared with that in cells transfected with miR-NC (Fig. 2A, P<0.05). The effect of miR-758 overexpression on cellular proliferation was investigated using a CCK-8 assay. As displayed in Fig. 2B, the proliferation of Y79 and WERI-RB1 cells transfected with miR-758 mimics was delayed compared with that in cells transfected with miR-NC (P<0.05).

Colony formation assay was performed to further confirm the inhibition of RB cell proliferation caused by miR-758 overexpression. The results revealed that the colony numbers of Y79 and WERI-RB1 cells transfected with miR-758 mimics were significantly decreased relative to those in the miR-NC groups (Fig. 2C, P<0.05). Furthermore, flow cytometric assay revealed that the enforced expression of miR-758 increased the percentage of cell apoptosis in Y79 and WERI-RB1 cells (Fig. 2D, P<0.05). These findings revealed that miR-758 may be important in regulating the proliferation and apoptosis of RB cells.

Migration and invasion assays were employed to determine the role of miR-758 on cell metastasis in RB cells. As shown in Fig. 3A, overexpression of miR-758 suppressed the migratory capacities of Y79 and WERI-RB1 cells compared with those in the miR-NC groups (P<0.05). Similarly, the number of invaded cells was lower in the miR-758 mimics-transfected Y79 and WERI-RB1 cells than those in cells transfected with miR-NC (Fig. 3B, P<0.05). These results indicated that miR-758 may have suppressive roles in RB metastasis.

To gain insights into the mechanisms underlying the action of miR-758 in RB, we conducted bioinformatics analysis to predict the potential targets of miR-758. PAX6, a major highly conserved putative target of miR-758, plays oncogenic roles in RB (23–25) and was therefore selected for further confirmation (Fig. 4A). To clarify whether miR-758 can directly interact with the 3′-UTR of PAX6, we performed luciferase reporter assays in Y79 and WERI-RB1 cells transfected with miR-758 mimics or miR-NC in the presence of pmirGLO-Wt-PAX6-3′-UTR or pmirGLO-Mut-PAX6-3′-UTR. Luciferase activities were considerably reduced in Y79 and WERI-RB1 cells co-transfected with miR-758 mimics and the luciferase reporter plasmid carrying the wild-type miR-758 binding sequences (P<0.05). However, miR-758 upregulation did not affect the luciferase activities of the plasmid carrying the mutant PAX6 3′-UTR (Fig. 4B). To further determine the regulatory roles of miR-758 on endogenous PAX6 expression, we utilised RT-qPCR and western blot analysis to assess the expression levels of PAX6 in Y79 and WERI-RB1 cells after transfection with miR-758 mimics or miR-NC. The ectopic expression of miR-758 decreased the mRNA (Fig. 4C, P<0.05) and protein (Fig. 4D, P<0.05) expression of PAX6 in both Y79 and WERI-RB1 cells. In summary, these data demonstrated that PAX6 is a direct target of miR-758 in RB.

To examine the possible clinical relevance of PAX6 in RB, we detected PAX6 mRNA and protein expression in RB and normal retinal tissues. As indicated in Fig. 5A and B, the mRNA and protein levels of PAX6 were significantly higher in the RB tissues than those in the normal retinal tissues (P<0.05). We analysed the correlation between miR-758 and PAX6 mRNA in RB tissues using Spearman's correlation analysis. An inverse correlation was validated between miR-758 and PAX6 mRNA level in RB tissues (Fig. 5C; r=−0.5946, P=0.0014). These data further indicated that PAX6 is a direct target of miR-758 in RB.

After determining that PAX6 is a direct target of miR-758, we further performed a series of rescue experiments to investigate whether miR-758 mediates its tumour-suppressive roles in RB cells by inhibiting PAX6. PAX6 overexpression plasmid pCMV-PAX6 or empty pCMV plasmid, together with miR-758 mimics, were transfected into Y79 and WERI-RB1 cells. After transfection, western blot analysis confirmed that the decreased PAX6 protein induced by miR-758 overexpression was restored in Y79 and WERI-RB1 cells after co-transfection with pCMV-PAX6 (Fig. 6A, P<0.05). Functional assays revealed that restored PAX6 expression significantly abrogated the effects of miR-758 overexpression on cell proliferation (Fig. 6B, P<0.05), colony formation (Fig. 6C, P<0.05), apoptosis (Fig. 6D, P<0.05), migration (Fig. 6E, P<0.05) and invasion (Fig. 6F, P<0.05). These results revealed that miR-758 may exert tumour-suppressive effects in RB cells, at least partly, by downregulating PAX6 expression.

PAX6 participates in the regulation of the PI3K/Akt pathway (26,27). Therefore, we investigated whether miR-758 could regulate the PI3K/Akt pathway in RB cells via PAX6 modulation. Y79 and WERI-RB1 cells were transfected with miR-758 mimics in the presence of pCMV-PAX6 or pCMV. Following transfection for 72 h, the ectopic expression of miR-758 significantly reduced p-PI3K and p-Akt protein levels in Y79 and WERI-RB1 cells relative to those in the miR-NC groups. Furthermore, the downregulation of p-PI3K and p-Akt protein levels caused by miR-758 overexpression was restored in Y79 and WERI-RB1 cells after co-transfection with pCMV-PAX6 (Fig. 7). These results confirmed that miR-758 may act as a tumour suppressor in RB by negatively regulating the PAX6/PI3K/Akt pathway.