A total of 47 patients with Rb (age range, 4 months to 14 years; mean age, 5.54±3.09 years) were enrolled in the No. 215 Hospital of Shaanxi Nuclear Industry (Xianyang, China) between March 2017 and July 2019. The Rb tissues and adjacent normal tissues (1 cm from the edge of the tumour tissues) were collected following an enucleation procedure. Tumour tissues were histologically confirmed and the adjacent tissues without histopathological changes were considered as the normal control group. None of the patients received any preoperative radiotherapy and/or chemotherapy. Each patient or their parents provided written informed consent. The protocol was approved by the Ethics Committee of the No. 215 Hospital of Shaanxi Nuclear Industry (approval no. EC-20200924-1017).

The human retinal epithelial ARPE-19 cell line and the human Rb Y79, HXO-RB44, WERI-Rb-1 and SO-RB50 cell lines were obtained from the American Type Culture Collection. These cell lines were selected according to previous studies (9,31-34). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and placed at 37˚C in a humidified incubator containing 5% CO₂.

The plasmids used in the present study were synthesised by Hanbio Biotechnology Co., Ltd. and included the short hairpin (sh)RNA-MIAT (sh-MIAT; 5'-UCCUCCGAACCUGGCA CGU-3'), shRNA-negative control (sh-NC; 5'-UUCUCCGAAC GUGUCACGU-3'), miR-665 mimics (5'-ACCAGGAGGCU GAGGCCCCU-3'), mimics-NC (miR-NC; 5'-UUCUCCGAA CGUGUCACGUTT-3'), miR-665 inhibitor (5'-AGGGGCCU CAGCCUCCUGGU-3'), inhibitor NC (5'-CAGUACUUUUGU GUAGUACAA-3'), LASP1 overexpression vector (pcDNA-LASP1) and pcDNA-negative control (pcDNA-NC). Transfections (all the above molecules at 20 nM) were performed using Lipofectamine^® 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturers' instructions. After 48 h, cells were collected for subsequent experiments.

Total RNA was extracted from Rb tissues and the Y79, HXO-RB44, WERI-Rb-1 and SO-RB50 cell lines using TRIzol^® (Invitrogen; Thermo Fisher Scientific, Inc.). According to the manufacturer's instructions, RNA was reversed transcribed into cDNA using the First-Strand cDNA Synthesis kit (APeXBIO Technology LLC). RT-qPCR was performed using the SYBR-Green FAST Mastermix (Qiagen GmbH). The following thermocycling conditions were used for the qPCR: Initial denaturation at 95˚C for 3 min, followed by 40 cycles at 95˚C for 15 sec (denaturation), 60˚C for 30 sec (annealing), 72˚C for 1 min (elongation) and a final extension at 72˚C for 5 min. GADPH and U6 were used as internal references. The relative expression levels were normalized to endogenous control and were expressed as 2^-ΔΔCq (35). The respective sequences of primers were as follows: MIAT forward, 5'-TCTTCATGTCAGAACACGCTTTA-3' and reverse, 5'-AAGGTCACCCGAGGTCCAA-3'; miR-665 forward, 5'-GCCGAGACCAGGAGGCTGAG-3' and reverse, 5'-CTCAACTGGTGTCGTGGA-3'; LASP1 forward, 5'-GGT GCGGCAAGATCGTGTA-3' and reverse, 5'-TGCAGGTCT CGCAATGGAA-3'; GAPDH forward, 5'-CCAGGTGGTCTC CTCTGA-3' and reverse, 5'-GCTGTAGCCAAATCGTTGT-3'; and U6 forward, 5'-CTCGCTTCGGCAGCACA-3' and reverse, 5'-AACGCTTCACGAATTTGCGT-3'.

Cells were cultured in 96-well plates at a density of 5x10⁴ cells/ml for 96 h, followed by addition of 15 µl MTT (Procell Life Science & Technology, Co., Ltd.) and incubation for 2 h at 37˚C. Subsequently, 100 µl DMSO was added to dissolve the formazan. The optical density was measured at 570 nm using a microplate reader (Thermo Fisher Scientific, Inc.).

For the migration assay, transfected Y79 and HXO-RB44 cells (5x10⁴ cells/ml) were re-suspended in serum-free DMEM and seeded in the upper chamber of a Transwell insert (8 µm pore size; BD Biosciences). Simultaneously, DMEM containing 10% FBS was added in the lower chamber. For the invasion assay, Matrigel matrix (Becton-Dickinson and Company) was used to coat the membranes before cell seeding. Following overnight incubation at 37˚C, cells in the lower chamber were fixed with 4% paraformaldehyde at 37˚C for 1 h and stained with 0.1% crystal violet for 15 min at 37˚C. The stained cells were imaged using an inverted light microscope (Olympus Corporation) and analysed with ImageJ software (version 1.46; National Institutes of Health).

The targeting association between MIAT and miR-665 was analyzed using the StarBase software (version 2.0; http://starbase.sysu.edu.cn). Additionally, the targeting interaction between miR-665 and LASP1 was predicted using TargetScan software (v7.2; http://www.targetscan.org/vert_72/). The predicted binding region sequences (MIAT, 5'-GGGUUCCAGGCUCCUGG-3'; LASP1, 5'-CUCCUGGU-3') were inserted into pGL3 vector (Promega Corporation) to construct the wild-type (wt) phenotype. For construction of the mutant (mut) phenotype, the mutation sequences (MIAT mut, 5'-CCCUUCGUCGGAGGACC-3'; LASP1 mut, 5'-GAGGACCA-3') were inserted into pGL3. Y79 and HXO-RB44 cells were then co-transfected with MIAT/LASP1-wt or MIAT/LASP1-mut (80 ng) and miR-665 mimics/miR-NC (50 nM) using Lipofectamine 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37˚C for 48 h, followed by detection of the luciferase activity using a dual-luciferase reporter assay system (Promega Corporation). The activity of firefly luciferase was normalized to that of Renilla luciferase.

Transfected cells were lysed on ice with RIPA (Beyotime Institute of Biotechnology) containing 10 mmol/l PMSF (Beyotime Institute of Biotechnology). The protein concentration was detected using a BCA Protein Assay Kit (Abcam). A total of 50 µg of protein/lane was separated via 10% SDS-PAGE and transferred onto PVDF membranes. Following blocking with 5% skimmed milk for 2 h at 25˚C, membranes were incubated with the primary antibodies against LASP1 (1:1,000; cat. no. SAB1402251; Sigma-Aldrich; Merck KGaA) and α-tubulin (1:1,000; cat. no. T6199; Sigma-Aldrich; Merck KGaA) at 4˚C overnight. Membranes were then incubated with the HRP-conjugated anti-mouse IgG secondary antibody (1:5,000; cat. no. sc-2005; Santa Cruz Biotechnology, Inc.) for 1 h at 37˚C. Bands were detected using enhanced chemiluminescence substrate (Amersham; Cytiva). Relative expression level of LASP1 was normalized to endogenous control α-tubulin. The immunoblots were visualized using an ECL detection kit (Thermo Fisher Scientific, Inc.) using Gel-Pro analyzer (version 4.0; Media Cybernetics, Inc.).

The SPSS 20.0 software (IBM Corp.) was used to perform statistical analysis. All cell experiments were performed three times. Data are presented as the mean ± standard deviation. Student's t-test was used to assess the differences between two groups. One-way ANOVA followed by Tukey's post hoc test was used to evaluate the differences among multiple groups. χ² test was used to analyse data from Table I. The linear correlation was assessed by Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

The expression of MIAT was initially detected in Rb tissues using RT-qPCR. The results demonstrated that MIAT expression was upregulated in Rb tissues compared with that in adjacent tissues (Fig. 1A; P<0.01). Furthermore, a significantly increased expression level of MIAT was observed in the tissues from patients with Tumour-Node-Metastasis (TNM) stage III/IV compared with that in the tissues from patients with TNM stage I/II (Fig. 1B; P<0.01). As shown in Table I, MIAT expression was significantly associated with intraocular international retinoblastoma classification (IIRC) stage (P=0.0035), TNM stage (P=0.012) and optic nerve invasion (P=0.044). In addition, MIAT mRNA expression was detected in the Rb WERI-Rb-1, SO-RB50, HXO-RB44 and Y79 cell lines, and the normal ARPE-19 cell line. The results from RT-qPCR demonstrated that MIAT expression level was significantly increased in all Rb cell lines compared with that in the ARPE-19 cell line (Fig. 1C; P<0.01). Since the Y79 and HXO-RB44 cell lines exhibited the highest MIAT expression levels amongst all Rb cell lines, they were selected for subsequent experiments.

To explore the effects of MIAT on Rb progression in vitro, Y79 and HXO-RB44 cells were transfected with sh-MIAT or sh-NC and the transfection efficiency was detected by RT-qPCR. The results demonstrated that the expression level of MIAT was significantly decreased following transfection with sh-MIAT compared with sh-NC, which confirmed the successful transfection into Y79 and HXO-RB44 cells (Fig. 2A; P<0.01). Subsequently, transfected Y79 and HXO-RB44 cells were used to determine the proliferation and migratory and invasive abilities of Rb cells using MTT assay and Transwell assay. As presented in Fig. 2B, Y79 and HXO-RB44 cell proliferation was significantly decreased following transfection with sh-MIAT compared with that in the sh-NC group (P<0.01). Furthermore, both migratory and invasive abilities of Rb cell lines were decreased in the sh-MIAT group compared with those in the sh-NC group (Fig. 2C and D; P<0.01).

lncRNAs can act as competing endogenous RNAs or sponges of miRNAs to modulate cancer progression (36). Through the StarBase software, 58 target miRNAs of MIAT were predicted (data not shown). In the present study, miR-665 was selected due to its important role in Rb (23,24) (Fig. 3A). A DLR assay was used to confirm the target relationship between MIAT and miR-665 in Y79 and HXO-RB44 cells. The results demonstrated that the luciferase activity in MIAT wt/miR-665 mimics was decreased, which was not the case in the MIAT mut (Fig. 3B; P<0.01). The results from RT-qPCR indicated that the expression levels of MIAT and miR-665 in Rb cell lines (Fig. 3C; P<0.01) and Rb tissues (Fig. 3E; P=0.001, r=-0.4614) were negatively correlated. In addition, a decreased expression level of miR-665 was detected in Rb tissues compared with that in the adjacent tissues (Fig. 3D; P<0.01).

Both miR-665 mimics and miR-665 inhibitor were transfected into Y79 and HXO-RB44 cells, and the effects of miR-665 overexpression on Rb progression in vitro were subsequently evaluated. The results demonstrated that miR-665 was significantly upregulated following transfection with miR-665 mimics, whereas it was significantly downregulated following transfection with miR-665 inhibitor (Fig. 4A; P<0.01). Similar to the effects of MIAT knockdown on the progression of Rb in vitro, the results from the MTT assay and Transwell assay revealed that the proliferation and migratory and invasive abilities of Rb cells were inhibited following miR-665 overexpression (Fig. 4B-D; P<0.01).

As illustrated in Fig. 5A, the TargetScan software predicted a binding site between miR-665 and LASP1. A DLR assay was subsequently used to confirm the binding relationship, and a decrease in luciferase activity in the LASP1 wt/miR-665 mimics groups was observed in both Y79 and HXO-RB44 cells (Fig. 5B; P<0.01). Furthermore, LASP1 was overexpressed in Rb tissues compared with that in adjacent tissues (Fig. 5C; P<0.01). In addition, the results from Pearson's correlation analysis demonstrated a negative correlation between LASP1 and miR-665 expression (Fig. 5D; P<0.01, r=-0.4738); however, a positive correlation was observed between LASP1 and MIAT expression (Fig. 5E; P<0.01, r=0.4116) in Rb tissues. To further verify the interaction between the expression of miR-665 and LASP1, western blotting was performed to determine the protein expression of LASP1 following transfection of Y79 and HXO-RB44 cells with miR-665 mimics. The results revealed that LASP1 protein expression was decreased following miR-665 upregulation (Fig. 5F; P<0.01).

The results from western blotting revealed that LASP1 expression was increased following transfection with pcDNA-LASP1 (Fig. 6A; P<0.01). The results from MTT assay and Transwell assay demonstrated that the suppressive effects of sh-MIAT on the proliferation and migratory and invasive abilities of Y79 and HXO-RB44 cells (Fig. 2) were reversed following transfection with miR-665 inhibitor or pcDNA-LASP1 (Fig. 6B-D; P<0.01).