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. 2018 Sep 24;9(10):1000.
doi: 10.1038/s41419-018-1029-4.

Silencing Long Non-Coding RNA Kcnq1ot1 Alleviates Pyroptosis and Fibrosis in Diabetic Cardiomyopathy

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Free PMC article

Silencing Long Non-Coding RNA Kcnq1ot1 Alleviates Pyroptosis and Fibrosis in Diabetic Cardiomyopathy

Fan Yang et al. Cell Death Dis. .
Free PMC article

Abstract

Diabetes cardiomyopathy (DCM) is a critical complication of long-term chronic diabetes mellitus and is characterized by myocardial fibrosis and myocardial hypertrophy. It has been suggested that DCM is related to pyroptosis, a programmed cell death associated with inflammation. The long non-coding RNA Kcnq1ot1 is involved in different pathophysiological mechanisms of multiple diseases, including acute myocardial damage and arrhythmia. Our previous study found that Kcnq1ot1 was elevated in left ventricular tissue of diabetic mice. However, whether Kcnq1ot1 is capable of regulating pyroptosis and fibrosis in high glucose-treated cardiac fibroblasts remains unknown. The aim of the study was to investigate the mechanisms of Kcnq1ot1 in DCM. Our study revealed that silencing Kcnq1ot1 by a lentivirus-shRNA improved cardiac function and fibrosis, ameliorated pyroptosis, and inhibited TGF-β1/smads pathway in C57BL/6 mice. In vitro, experiments revealed that Kcnq1ot1 and pyroptosis were activated in cardiac fibroblasts treated with 30 mmol/l glucose. Furthermore, Kcnq1ot1 knockdown by a small interfering RNA decreased caspase-1 expression. Bioinformatic prediction and luciferase assays showed that Kcnq1ot1 functioned as a competing endogenous RNA to regulate the expression of caspase-1 by sponging miR-214-3p. In addition, silencing Kcnq1ot1 promoted gasdermin D cleavage and the secretion of IL-1β, thus repressing the TGF-β1/smads pathway in high glucose-treated cardiac fibroblasts through miR-214-3p and caspase-1. Therefore, Kcnq1ot1/miR-214-3p/caspase-1/TGF-β1 signal pathway presents a new mechanism of DCM progression and could potentially be a novel therapeutic target.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. Silencing Kcnq1ot1 attenuates cardiac function and structure in diabetic mice
a The expression of Kcnq1ot1 in left ventricle was detected by qRT-PCR. M-mode echocardiograms of left ventricle b, ejection fraction (EF) c and shortening fraction (FS) d are shown. e Hematoxylin-eosin (HE) and Masson’s trichrome staining were performed. Scale bar, 50 μm. f The expression levels of collagen I and collagen III were detected by western blot. g Relative protein expression of TGF-β1, p-smad2, and p-smad3 were detected by western blot. *P < 0.05 compared with the control group, #P < 0.05 compared with the DM + Scr-shRNA group. n = 5 in each group
Fig. 2
Fig. 2. Kcnq1ot1 is involved in the regulation of pyroptosis in vivo
a Immunohistochemistry analysis was performed to detect the expression NLRP3, caspase-1, IL-1β, and GSDMD-N. Scale bar, 20 μm. bd qRT-PCR was conducted to analyze the mRNA expression of NLRP3, caspase-1 and IL-1β. e Western blot was conducted to determine the protein expression of NLRP3, caspase-1 and IL-1β. *P < 0.05 compared with the control group, #P < 0.05 compared with the DM + Scr-shRNA group. n = 5 in each group
Fig. 3
Fig. 3. Kcnq1ot1 and pytoptosis are activated in HG-treated cardiac fibroblasts
qRT-PCR was performed to measure the Kcnq1ot1 expression level in the serums of non-diabetic and diabetic patients a. *P < 0.05 compared with the non-diabetic group. n = 6 in each group. Cardiac fibroblasts of neonatal C57BL/6 mice were incubated with 5.5 mmol/L glucose (Control) or 30 mmol/L (high glucose, HG) for 24 h. The expression levels of Kcnq1ot1 were detected by qRT-PCR b. The expression levels of NLRP3, caspase-1 and IL-1β were determined by qRT-PCR c, immunofluorescence dg and western blot h, i. *P < 0.05 compared with the control group. n = 3 in each group
Fig. 4
Fig. 4. miR-214-3p has both Kcnq1ot1 and caspase-1-binding sites
HG-treated cardiac fibroblasts were transfected with siRNA against Kcnq1ot1 and the negative control. The Kcnq1ot1 expression was detected by qRT-PCR a. qRT-PCR b and western blot c were performed to determine the mRNA and protein expression levels of caspase-1. Bioinformatic prediction revealed that miR-214-3p contained potential binding sites for both Kcnq1ot1 d and caspase-1 e. The relative expression of miR-214-3p was analyzed in cardiac fibroblasts f, serums of patients g, and myocardium of mice h. i miR-214-3p expression after transfection with si-Kcnq1ot1. Fibroblasts were transfected with miR-214-3p mimics or miR-214-3p inhibitor, and the expressions of miR-214-3p and caspase-1 were determined jl. *P < 0.05 compared with the HG + NC group, #P < 0.05 compared with the HG + AMO-NC group. n = 3 in each group
Fig. 5
Fig. 5. Kcnq1ot1 silencing alleviates caspase-1 via miR-214-3p
a The HG-treated fibroblasts were transfected with si-Kcnq1ot1 with or without AMO-214-3p. The relative expression of Kcnq1ot1 was detected. b Relative miR-214-3p expression in each group. c The mRNA expression of caspase-1 in each group. d The caspase-1 expression was determined by immunofluorescence. e Western blot was conducted to detect the protein expression of cleaved caspase-1. *P < 0.05 compared with the si-NC + AMO-NC group, #P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-NC group, &P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-214-3p group. n = 3 in each group
Fig. 6
Fig. 6. The texts overlap in figure 6g. So I want to replace it with a new figure 6 in the attachment. Kcnq1ot1/miR-214-3p pathway regulates inflammation in fibroblasts
a, b qRT-PCR was conducted to detect expression of NLRP3 and IL-1β. c, d The expression levels of NLRP3 and IL-1β were determined by immunofluorescence. eg Western blot was performed to determine the expression of NLRP3, IL-1β, and GSDMD-N. *P < 0.05 compared with the si-NC + AMO-NC group, #P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-NC group, &P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-214-3p group. n = 3 in each group
Fig. 7
Fig. 7. Kcnq1ot1 regulates fibrosis in HG-induced cardiac fibroblasts
ac Immunofluorescence was conducted to detect the expression levels of collagen I, collagen III, and TGF-β1. d The protein expression levels of collagen I and III were detected by western blot. e, f The protein expression levels of TGF-β1, p-smad2, and p-smad3 were determined by western blot. *P < 0.05 compared with the si-NC + AMO-NC group, #P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-NC group, &P < 0.05 compared with the HG + si-Kcnq1ot1 + AMO-214-3p group. n = 3 in each group

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