AC16 cells (American Type Culture Collection) were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator with 5% CO₂ at 37°C. For establishment of the H/R model, the cell culture atmosphere was N₂ (94%), O₂ (1%) and CO₂ (5%) to simulate hypoxia for 6 h, followed by 12-h reoxygenation under normal atmospheric culture conditions as previously described (17).

ENCORI database (http://starbase.sysu.edu.cn/index.php) was used to predict the interactions among lncRNA SNHG1, miR-450b-5p and IGF1 (Figs. S1 and S2).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA was reverse transcribed into cDNA using a TIANScript II RT Kit (Tiangen Biotech Co., Ltd.) according to the manufacturer's protocol. Subsequently, qPCR was performed using a RealMastcrMix (SYBR-Green) kit (Tiangen Biotech Co., Ltd.). The following thermocycling conditions were used for qPCR: 95°C for 2 min; 40 cycles at 95°C for 20 sec, 60°C for 15 sec and 72°C for 30 sec. The sequences of the primers (obtained from Sangon Biotech) used for qPCR were as follows: lncRNA SNHG1 forward, 5′-AGGCTGAAGTTACAGGT-3′ and reverse, 5′-TTGGCTCCCAGTGTCTT-3′; miR-450b-5p forward, 5′-GCTTTTGCAATATGTTCCG-3′ and reverse, 5′-CAGTGCGTGTCGTGGAGT-3′; IGF1 forward, 5′-CCCAGAAGGAAGTACATTTG-3′ and reverse, 5′-GTTTAACAGGTAACTCGTGC-3′; U6 forward, 5′-AGAGAAGATTAGCATGGCCCCTG-3′ and reverse, 5′-ATCCAGTGCAGGGTCCGAGG-3′; and GAPDH forward, 5′-ACCACAGTCCATGCCATCAC-3′ and reverse, 5′-GTGAGGGAGATGCTCAGTGT-3′. miRNA and mRNA expression levels were quantified using the 2^−∆∆Cq method (18) and normalized to the internal reference genes U6 and GAPDH, respectively.

A SNHG1 overexpression plasmid (Oe-SNHG1; pBluescript vector) and the empty vector [Oe-negative control (NC)], miR-450b-5p mimic (forward, 5′-UUUUGCAGUAUGUUCCUGAAUA-3′ and reverse 5′-UUCAGGAACAUACUGCAAAAUU-3′; lentiviral vector pLVTHM), mimic NC (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACGUUCGGAGAATT-3′; lentiviral vector pLVTHM), IGF1 short hairpin (sh)RNA lentiviral particles (shRNA-IGF1; shRNA-IGF1#1, 5′-GAAGAATTGTGAAAGTTTA-3′ and shRNA-IGF2#1, 5′-GCTAGAGTGTCATAATAAA-3′) and non-targeting NC shRNA lentiviral particles (shRNA-NC; 5′-TTCTCCGAACGTGTCACGT-3′) were all purchased from Genecopoeia, Inc. Cells (1×10⁵ cells/well) were transfected with 20 nM overexpression plasmid, miRNA mimic, shRNA or the corresponding NC using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C for 48 h according to the manufacturer's protocol. At 48 h post-transfection, RT-qPCR and western blotting were used to detect transfection efficiency.

AC16 cells were seeded (8×10³ cells/well) into 96-well plates. At 48 h post-transfection, the medium was removed and 10 µl MTT (Sigma-Aldrich; Merck KGaA) was added to each well for 4 h. Subsequently, DMSO was added. The optical density was measured at a wavelength of 570 nm using a microplate analyzer.

Apoptosis was measured using a TUNEL staining kit (Beyotime Institute of Biotechnology). Transfected cells were fixed using 4% paraformaldehyde for 1 h at 4°C and then 0.1% Triton X-100 was added at room temperature for 5 min. Subsequently, cells were washed with PBS and incubated with TUNEL reagent for 1 h at 37°C. Subsequently, 50 µl DAB color development was performed for 10 min at 15°C according to the manufacturer's instructions. The cells were stained with DAPI for 10 min at room temperature in the dark. Stained cells were observed under a glass coverslip with PBS using a fluorescent microscope (magnification, ×200; Carl Zeiss AG). ImageJ software (version 1.8.0; National Institutes of Health) was used to count the total cells and the TUNEL⁺ cells. The number of apoptotic cells was calculated to be the average number of positive cells out of the total number of cells in six fields of view per slide.

Total protein was isolated from cells using RIPA lysis buffer (Thermo Fisher Scientific, Inc.) and then quantified using a BCA kit (Bio-Rad Laboratories, Inc.). Proteins (30 µg) were separated via 12% SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk at room temperature for 1.5 h, the membranes were incubated at 4°C overnight with primary antibodies targeted against: Bcl-2 (1:1,000; cat. no. ab32124; Abcam), Bax (1:1,000; cat. no. ab182733; Abcam), cytochrome c (1:1,000; cat. no. ab133504; Abcam), cleaved caspase 3 (1:1,000; cat. no. ab32042; Abcam), IGF1 (1:1,000; cat. no. ab134140; Abcam), phosphorylated (p)-PI3K (1:1,000; cat. no. ab278545; Abcam), p-Akt (1:1,000; cat. no. ab8805; Abcam), PI3K (1:1,000; cat. no. ab191606; Abcam), Akt (1:1,000; cat. no. ab8805; Abcam) and GAPDH (1:1,000; cat. no. ab8245; Abcam). Subsequently, the membranes were probed with horseradish peroxidase-conjugated secondary antibodies (1:5,000; cat. no. ab150113 or ab9482; Abcam) for 2 h at room temperature. Signals were visualized using Super Signal ECL (Thermo Fisher Scientific, Inc.) and semi-quantified using ImageJ software (version 1.46; National Institutes of Health).

The level of SOD was detected using the SOD Assay Kit (cat. no. A001-3-2; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. MDA concentrations were determined using an ELISA kit (cat. no. A003-1-2; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. All above were used in accordance with the manufacturer's protocol. GSH-Px enzyme activity was measured using a GSH-Px Assay Kit (cat. no. A005-1-2, Nanjing Jiancheng Bioengineering Institute) according to a manufacturer's protocol.

The amplified PCR products of the 3′ untranslated regions of SNHG1 and IGF1 were ligated into the miRNA target expression vector pmirGLO-dual-luciferase (Promega Corporation). A QuickChange II XL site-directed mutagenesis kit (Agilent Technologies, Inc.) was used to generate the SNHG1 (SNHG1 MUT) and IGF (IGF1 MUT) mutants. Cells were co-transfected with miR-450b-5p mimic or miR-NC and SNHG1 MUT, SNHG1 wild-type (WT), IGF1 WT or IGF1 MUT using Lipofectamine 2000 reagent, all at a final concentration of 100 nmol/l. At 48 h post-transfection, the luciferase activities were assayed using a dual-luciferase reporter gene assay system (Promega Corporation) according to the manufacturer's protocol. Renilla luciferase activity was used for normalization and results were recorded using a GloMax 96 Microplate Luminometer (Promega Corporation).

Data are presented as the mean ± standard deviation. Comparisons among multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. All statistical analyses were performed using SPSS software (version 22.0; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference. Each experiment was repeated at least three times.

After AC16 cells were induced by H/R, the expression levels of lncRNA SNHG1 were detected using RT-qPCR. The results showed that lncRNA SNHG1 expression in the H/R group was significantly decreased compared with that in the control group (Fig. 1A). Subsequently, lncRNA SNHG1 was overexpressed in cells (Fig. 1B). The MTT assay showed that cell viability was significantly decreased following H/R induction compared with that in the control group. SNHG1 overexpression significantly increased cell viability compared with that in the H/R + Oe-NC group (Fig. 1C). Cell apoptosis was detected using a TUNEL assay and western blotting. Compared with the control group, apoptosis was significantly increased following H/R induction, which was accompanied by significantly increased expression levels of Bax, cytochrome c and cleaved caspase 3 and significantly decreased expression levels of Bcl-2. Compared with the H/R + Oe-NC group, apoptosis was significantly decreased in the H/R + Oe-SNHG1 group, which was accompanied by significantly decreased expression levels of Bax, cytochrome c and cleaved caspase 3 and significantly increased expression levels of Bcl-2 (Fig. 1D and E). The levels of MDA, SOD and GSH-Px were detected using ELISAs. The results demonstrated that the level of MDA was significantly increased, whereas the levels of SOD and GSH-Px were significantly decreased in the H/R group compared with those in the control group. However, SNHG1 overexpression significantly inhibited the effects of H/R induction on the levels of oxidative stress-related indicators (Fig. 2A-C). The aforementioned results indicated that lncRNA SNHG1 overexpression inhibited H/R-induced apoptosis and oxidative stress levels in AC16 cells.

The ENCORI database predicted the presence of binding sites between lncRNA SNHG1 and miR-450b-5p, as well as between miR-450b-5p and IGF1 (Fig. 3A and B). In addition, compared with that in the control group, the expression of miR-450b-5p was significantly increased in H/R-induced cells, whereas the expression of IGF1 was significantly decreased (Fig. 3C and D). Cell transfection was used to overexpress miR-450b-5p, which was confirmed via RT-qPCR (Fig. 3E). Subsequently, the luciferase reporter gene assay demonstrated that in SNHG1 WT, the luciferase activity significantly decreased when transfected with the miR-450b-5p mimic compared with the mimic NC group. However, no significant change in luciferase activity was exhibited in the SNHG1 MUT group. In the IGF1 WT group, luciferase activity was significantly decreased following transfection with the miR-450b-5p mimic compared with the mimic NC group. However, no significant change in luciferase activity was exhibited following miR-450b-5p mimic transfection in the IGF1 MUT group. These results verified the targeted binding between lncRNA SNHG1 and miR-450b-5p and between miR-450b-5p and IGF1 (Fig. 3F). In addition, following lncRNA SNHG1 overexpression, the expression of miR-450b-5p in H/R-induced cells was significantly decreased, whereas the expression of IGF1 was significantly increased. The expression of IGF1 in the H/R + miR-450b-5p mimic group was significantly increased compared with that in the H/R + mimic NC group (Fig. 3G-I). These results indicated that lncRNA SNHG1 overexpression upregulated IGF1 expression by sponging miR-450b-5p.

IGF1 expression was knocked down using shRNAs and successful knockdown was confirmed via RT-qPCR (Fig. 4A). shRNA-IGF1#2 was selected for subsequent experiments as it displayed the optimum knockdown efficiency. Cells were divided into the following groups: i) Control; ii) H/R; iii) H/R + Oe-SNHG1; iv) H/R + Oe-SNHG1 + mimic NC; v) H/R + Oe-SNHG1 + miR-450b-5p mimic; vi) H/R + Oe-SNHG1 + shRNA-NC; and vii) H/R + Oe-SNHG1 + shRNA-IGF1. The MTT and TUNEL assay results demonstrated that cell viability was significantly decreased and apoptosis was significantly increased in the H/R + Oe-SNHG1 + shRNA-IGF1 group compared with that in the H/R + Oe-SNHG1 + mimic NC group. This was accompanied by a significant increase in the expression levels of the apoptosis-related proteins Bax, cytochrome c and cleaved caspase 3, whereas Bcl-2 expression was significantly decreased. Compared with that in the H/R + Oe-SNHG1 + shRNA-NC group, cell viability was significantly decreased and apoptosis was significantly increased in the H/R + Oe-SNHG1 + shRNA-IGF1 group, which was accompanied by increased Bax, cytochrome c and cleaved caspase 3 expression levels and decreased Bcl-2 expression levels (Fig. 4A-D). These results indicated that miR-450b-5p overexpression and IGF1 knockdown could reverse the effects of SNHG1 overexpression on cell viability and apoptosis. Subsequently, the levels of oxidative stress-related indicators were detected. Compared with those in the H/R + Oe-SNHG1 + mimic NC group, SOD and GSH-Px levels were significantly decreased and MDA levels were significantly increased in the H/R + Oe-SNHG1 + miR-450b-5p mimic group. Compared with those in the H/R + Oe-SNHG1 + shRNA-NC group, the levels of SOD and GSH-Px were significantly decreased and the levels of MDA were significantly increased in the H/R + Oe-SNHG1 + shRNA-IGF1 group (Fig. 5A-C).

Additionally, the expression levels of PI3K/Akt signaling pathway-related proteins were dysregulated. Compared with that in the control group, the expression of p-PI3K and p-AKT was significantly decreased following H/R induction, whereas SNHG1 overexpression resulted in significant upregulation of p-PI3K and p-Akt expression levels. miR-450b-5p overexpression or IGF1 knockdown significantly inhibited the effects of SNHG1 overexpression on the PI3K/Akt signaling pathway (Fig. 6).