Increased B-type-natriuretic peptide promotes myocardial cell apoptosis via the B-type-natriuretic peptide/long non-coding RNA LSINCT5/caspase-1/interleukin 1β signaling pathway

Abstract

Chronic heart failure (CHF) is the final stage of various heart diseases, and is increasingly recognized as a major health problem in the elderly. Previous studies demonstrated that B‑type‑natriuretic peptide (BNP) is an established biomarker of CHF. Furthermore, BNP also regulates cell proliferation, differentiation and apoptosis. Recent evidence has revealed that BNP affects myocardial cell apoptosis during myocardial ischemia‑reperfusion injury. Long non‑coding RNAs (lncRNAs) are emerging as novel molecular compounds involved in gene regulation, and have important roles in numerous human diseases. However, the mechanism underlying the BNP and lncRNA-induced regulation of myocardial cell apoptosis remains to be elucidated. The present study reported that lncRNA LSINCT5, upregulated by BNP, is able to regulate myocardial cell apoptosis via the activation of the caspase‑1/interleukin (IL)‑1β signaling pathway. BNP-induced apoptosis of HCM cells was observed using flow cytometry, and involved caspase‑1. In addition, expression profiling using a human lncRNA polymerase chain reaction array revealed that LSINCT5 was highly expressed in BNP-treated myocardial cells, as compared with untreated cells. The role of lncRNA LSINCT5 in HCM cell apoptosis was also investigated. The results of the present study indicated that LSINCT5 silencing by small interfering RNA inhibits caspase‑1/IL‑1β signaling, and suppresses apoptosis in BNP-treated HCM cells. Therefore, high expression levels of BNP promote the apoptosis of myocardial cells through the lncRNA LSINCT5 mediator, which activates the caspase‑1/IL‑1β signaling pathway. These findings uncovered a novel pathogenic mechanism, and provided a potential therapeutic target for CHF.

Figure 1

BNP overexpression is followed by increased apoptotic activity in HCM cells. (A) Flow cytometric analysis of HCM cells treated with various concentrations of BNP, and stained with Annexin V-FITC and PI. (B) Western blots of caspase-1, -3, -7 and-8 in BNP-stimulated HCM cells. The protein expression levels were normalized to those of GAPDH. BNP, B-type-natriuretic peptide; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Figure 2

Expression levels of LSINCT5 are the most significantly altered in HCM cells stimulated by BNP. (A) Heatmap analysis of lncRNAs in HCM cells treated with various concentrations of BNP was conducted using a method of hierarchical clustering by GeneSpring GX 7.3. Rows, samples; columns, lncRNAs. Color key indicates lncRNA expression value: Red, highest; blue, lowest. (B) mRNA expression levels of lncRNA LSINCT5 as determined by reverse transcription- quantitative polymerase chain reaction. lncRNA, long non-coding RNA; BNP, B-type-natriuretic peptide. ^*P<0.05; ^**P<0.01, compared with the 0 pg/ml group.

Figure 3

lncRNA LSINCT5 knockdown by siRNA inhibits caspase-1/IL-1β signaling in BNP-treated HCM cells. (A) Silencing efficiency of LSINCT5 was determined by RT-qPCR. Representative images of (B) the relative mRNA and (C) the protein expression levels of caspase-1, -3, -7 and-8 in HCM cells treated with si-LSINCT5-2. GAPDH was used as an internal control. (D) Western blot analysis and (E) RT-qPCR analysis of the expression levels of caspase-1 and IL-1β in siRNA-LSINCT5-2 and siRNA-scramble-treated HCM cells. Results are presented as the mean ± standard deviation from three independent experiments performed in duplicate. Relative expression is expressed in arbitrary units. lncRNA, long non-coding RNAs; IL, interleukin; BNP, B-type-natriuretic peptide; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative polymerase chain reaction. ^*P<0.05; ^**P<0.01, compared with the control group.

Figure 4

lncRNA LSINCT5 knockdown by siRNA prevents BNP-induced apoptosis. (A) Cell proliferation was assessed by immunofluorescence under a confocal microscope at 24 and 72 h (magnification, x100). (B) The expression of caspase-1 and IL-1β was detected by western blot analysis, GAPDH was used as an internal control. (C) Effects of lncRNA LSINCT5 knockdown on BNP-induced HCM apoptosis determined by flow cytometry. The cells were stained with Annexin V-FITC and PI. lncRNA, long non-coding RNA; BNP, B-type-natriuretic peptide; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate; PI, propidium iodide.