Silenced SOX2‐OT alleviates ventricular arrhythmia associated with heart failure by inhibiting NLRP3 expression via regulating miR‐2355‐3p

Figures

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  Figure 1. Expression of SOX2‐OT and NLRP3 is increased in rats with VA‐HF. (A) SOX2‐OT expression measured by qRT‐PCR. (B) NLRP3, ASC, caspase‐1, IL‐1β, and TGF‐β1 expression measured by Western blot analysis. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HF, heart failure; IL‐1β, interleukin‐1β; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; qRT‐PCR, quantitative reverse transcriptase‐polymerase chain reaction; SOX2‐OT, SOX2‐overlapping transcripts; TGF‐β1, transforming growth factor‐β1; VA, ventricular arrhythmia. *p < .05 (n = 6 per group)

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  Figure 2. Silenced SOX2‐OT expression inhibits NLRP3 inflammasomes, reduces ROS levels and myocardial histopathological changes in rats V with VA‐HF. (A) Expression of SOX2‐OT measured by qRT‐PCR 6 weeks after injection of si‐SOX2‐OT. (B–D) Ventricular chambers stained with H&E, Masson trichrome, and Picro Sirius Red 6 weeks after injection of si‐SOX2‐OT (200×). (E) Caspase‐1, NLRP3, ASC, IL‐ 1β, and TGF‐β1 expression measured by western blot analysis 6 weeks after injection of si‐SOX2‐OT. (F) Levels of ROS measured by flow cytometry 6 weeks after injection of si‐SOX2‐OT. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; H&E, hematoxylin‐eosin; IL‐1β, interleukin‐1β; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; qRT‐PCR, quantitative reverse transcriptase‐polymerase chain reaction; ROS, reactive oxygen species; si‐NC, small interfering negative control; SOX2‐OT, SOX2‐overlapping transcripts; TGF‐β1, transforming growth factor‐β1; VA‐HF, ventricular arrhythmia associated with heart failure. *p < .05 (n = 6 per group)

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  Figure 3. MicroR‐2355‐3p is a potential target miRNA (miR) of SOX2‐OT. (A) MicroRNA binding with SOX2‐OT and the 3′‐UTR of NLRP3 in miR‐2355‐3p was analyzed using starBase 3.0 and target 7.2. (B) Expression of miR‐2355‐3p measured by qRT‐PCR in sham, HF, VA‐HF‐control, and VA‐HF groups. (C) Expression miR‐2355‐3p measured by qRT‐PCR in si‐NC and si‐SOX2‐OT at 6 weeks after injection in rats with VA‐HF. (D) Binding between miR‐2355‐3p and SOX2‐OT. (E) Binding between miR‐2355‐3p and 3′‐UTR NLRP3 analyzed by dual‐luciferase reporter gene assays. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HF, heart failure; IL‐1β, interleukin‐1β; Mut, mutant; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; qRT‐PCR, quantitative reverse transcriptase‐polymerase chain reaction; ROS, reactive oxygen species; si‐NC, small interfering negative control; SOX2‐OT, SOX2‐overlapping transcripts; TGF‐β1, transforming growth factor‐β1; UTR, untranslated region; VA, ventricular arrhythmia; WT, wild‐type. *p < .05

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  Figure 4. MicroR‐2355‐3p reverses SOX2‐OT effects in rats with VA‐HF. (A) SOX2‐OT expression measured by qRT‐PCR 6 weeks after coinjection of si‐SOX2‐OT and anti‐miR‐2355‐3p. (B) Ventricular chambers stained with H&E, Masson trichrome, and Picro Sirius Red 6 weeks after coinjection of si‐SOX2‐OT and anti‐miR‐2355‐3p (200×). (C) Caspase‐1, NLRP3, ASC, IL‐1β, and TGF‐β1 expression measured by Western blot analysis 6 weeks after coinjection of si‐SOX2‐OT and anti‐miR‐2355‐3p. (D) Levels of ROS measured by flow cytometry 6 weeks after coinjection of si‐SOX2‐OT and anti‐miR‐2355‐3p. Anti‐miR‐NC group: coinjected with si‐SOX2‐OT and anti‐miR‐2355‐3p negative control; anti‐miR‐2355‐3p group: coinjected with si‐SOX2‐OT and anti‐miR‐2355‐3p. GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; HF, heart failure; H&E, hematoxylin‐eosin; IL‐1β, interleukin‐1β; NLRP3, nucleotide‐binding oligomerization domain‐like receptor family pyrin domain containing 3; miR, microRNA; qRT‐PCR, quantitative reverse transcriptase‐polymerase chain reaction; ROS, reactive oxygen species; si, small interfering; SOX2‐OT, SOX2‐overlapping transcripts. *p < .05 (n = 6 per group)