Role and mechanism of the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis in the regulation of myocardial ischemia reperfusion injury

Abstract

The increasing rates of morbidity and mortality caused by ischemic heart disease pose a serious threat to human health. Long non‑coding (lnc)RNA small nucleolar RNA host gene 1 (SNHG1) has a protective effect on the myocardium. In the present study, the role of lncRNA SNHG1 in myocardial ischemia reperfusion injury (MIRI) and the underlying mechanisms were investigated. After hypoxia/reoxygenation (H/R) induction, the expression levels of lncRNA SNHG1 were detected using reverse transcription‑quantitative PCR. After lncRNA SNHG1 overexpression via cell transfection, cell viability was detected using an MTT assay, apoptotic rates were detected using TUNEL staining, apoptosis‑related protein expression levels were detected using western blotting and respective kits were used to measure the oxidative stress levels. The Encyclopedia of RNA Interactomes database predicted the presence of binding sites between lncRNA SNHG1 and microRNA (miR)‑450b‑5p, and between miR‑450b‑5p and insulin‑like growth factor 1 (IGF1). These interactions were then verified using luciferase reporter assays. Subsequently, the regulatory mechanism underlying the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis in MIRI was investigated by overexpressing miR‑450b‑5p and knocking down IGF1 expression in H/R‑induced cells. Finally, the expression of PI3K/Akt signaling pathway‑related proteins was detected using western blotting. lncRNA SNHG1 expression was significantly downregulated in H/R‑induced AC16 cells. lncRNA SNHG1 overexpression significantly inhibited apoptosis and decreased oxidative stress levels in H/R‑induced AC16 cells, which was mediated via regulation of the miR‑450b‑5p/IGF1 axis and activation of the PI3K/Akt signaling pathway. Therefore, the present study suggested that activation of the PI3K/Akt signaling pathway via the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis inhibited the apoptosis and oxidative stress levels of H/R‑induced AC16 cells.

Keywords: apoptosis; long non‑coding RNA small nucleolar RNA host gene 1/microRNA‑450b‑5p/insulin‑like growth factor 1; myocardial ischemia reperfusion injury; oxidative stress.

Figure 1.

lncRNA SNHG1 overexpression inhibits H/R-induced apoptosis in AC16 cells. Reverse transcription-quantitative PCR was performed to detect the expression levels of lncRNA SNHG1 in (A) AC16 cells (**P<0.01 vs. control) and (B) transfected AC16 cells (***P<0.001 vs. Oe-NC). (C) MTT assays were performed to detect cell viability. (D) TUNEL assays were performed to detect cell apoptosis. (E) Western blotting was performed to measure the expression levels of apoptosis-related proteins. ***P<0.001 vs. control; ^##P<0.01, ^###P<0.001 vs. H/R + Oe-NC. lncRNA, long non-coding RNA; SNHG1, small nucleolar RNA host gene 1; H/R, hypoxia/reoxygenation; Oe, overexpression; NC, negative control; Cyto c, cytochrome c.

Figure 2.

Long non-coding RNA SNHG1 overexpression inhibits H/R-induced oxidative stress in AC16 cells. (A) MDA, (B) SOD and (C) GSH-Px were detected using commercial kits. ***P<0.001 vs. H/R; ^###P<0.001 vs. H/R + Oe-NC. SNHG1, small nucleolar RNA host gene 1; H/R, hypoxia/reoxygenation; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; Oe, overexpression; NC, negative control.

Figure 3.

lncRNA SNHG1 overexpression upregulates IGF1 expression by sponge adsorption of miR-450b-5p. Binding sides of (A) lncRNA SNHG1 and miR-450b-5p, and (B) miR-450b-5p and IGF1. (C) RT-qPCR was performed to detect the expression levels of (C) miR-450b-5p and (D) IGF1. ***P<0.001 vs. control. (E) RT-qPCR was performed to assess the transfection efficiency of miR-450b-5p mimic. ***P<0.001 vs. mimic NC. (F) Luciferase reporter assays confirmed the binding sides between lncRNA SNHG1 and miR-450b-5p, and between miR-450b-5p and IGF1. ***P<0.001 vs. mimic NC + IGF1 or mimic NC + SNHG1. (G) RT-qPCR was performed to assess the effect of lncRNA SNHG1 overexpression on miR-450b-5p expression. ***P<0.001 vs. control; ^###P<0.001 vs. H/R + Oe-NC. (H) RT-qPCR and (I) western blotting were performed to assess the effect of lncRNA SNHG1 and miR-450b-5p overexpression on IGF1 expression. ***P<0.001 vs. control; ^###P<0.001 vs. H/R + mimic NC; ^@P<0.05 vs. H/R + Oe-NC. lncRNA, long non-coding RNA; SNHG1, small nucleolar RNA host gene 1; IGF1, insulin-like growth factor 1; miR, microRNA; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control; H/R, hypoxia/reoxygenation; Oe, overexpression; WT, wild-type; MUT, mutant.

Figure 4.

Long non-coding RNA SNHG1/miR-450b-5p/IGF1 axis affects the apoptosis of H/R-induced AC16 cells. (A) Reverse transcription-quantitative PCR and (B) western blotting were performed to assess the transfection efficiency of shRNA-IGF1#1 and shRNA-IGF1#2. ***P<0.001 vs. shRNA-NC. (C) MTT assays were performed to detect cell viability. (D) Western blotting was performed to detect the expression of apoptosis-related proteins. (E) TUNEL assays were performed to detect apoptosis. ***P<0.001 vs. control; ^###P<0.001 vs. H/R; ^@P<0.05, ^@@P<0.01 and ^@@@P<0.001 vs. H/R + Oe-SNHG1 + mimic NC; ^$P<0.05 and ^$$$P<0.001 vs. H/R + Oe-SNHG1 + shRNA-NC. SNHG1, small nucleolar RNA host gene 1; miR, microRNA; IGF1, insulin-like growth factor 1; H/R, hypoxia/reoxygenation; shRNA, short hairpin RNA; NC, negative control; Oe, overexpression; Cyto c, cytochrome c.

Figure 5.

Long non-coding RNA SNHG1/miR-450b-5p/IGF1 axis affects oxidative stress levels of H/R-induced AC16 cells. (A) MDA, (B) SOD and (C) GSH-Px were detected using commercial kits. ***P<0.001 vs. control; ^###P<0.001 vs. H/R; ^@@@P<0.001 vs. H/R + Oe-SNHG1 + mimic NC; ^$$$P<0.001 vs. H/R + Oe-SNHG1 + shRNA-NC. SNHG1, small nucleolar RNA host gene 1; miR, microRNA; IGF1, insulin-like growth factor 1; H/R, hypoxia/reoxygenation; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; Oe, overexpression; NC, negative control; shRNA, short hairpin RNA.

Figure 6.

Long non-coding RNA SNHG1/miR-450b-5p/IGF1 axis regulates the PI3K/Akt signaling pathway, and affects apoptosis and oxidative stress levels in H/R-induced AC16 cells. Western blotting was performed to detect the expression of PI3K/Akt pathway-related proteins. ***P<0.001 vs. control; ^###P<0.001 vs. H/R; ^@@@P<0.001 vs. H/R + Oe-SNHG1 + mimic NC; ^$P<0.05 and ^$$P<0.01 vs. H/R + Oe-SNHG1 + shRNA-NC. SNHG1, small nucleolar RNA host gene 1; miR, microRNA; IGF1, insulin-like growth factor 1; H/R, hypoxia/reoxygenation; Oe, overexpression; NC, negative control; shRNA, short hairpin RNA; p, phosphorylated.