ZNF561-AS1 Regulates Cell Proliferation and Apoptosis in Myocardial Infarction Through miR-223-3p/NLRP3 Axis

Figures

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  Figure 1.. NLRP3 is overexpressed in mice MI models and hypoxia-induced myocardial cells. (A) RT-qPCR and WB detected the NLRP3 level in infarct zone, border zone, and remote zone after MI for 4, 24, and 72 h, respectively. (B, C) RT-qPCR and WB measured IL-1β and IL-18 expression in infarct zone, border zone, and remote zone after MI for 4, 24, and 72 h, respectively. (D) The expression of NLRP3, IL-1β, and IL-18 was assessed in hypoxia HCMs compared with the normal group through RT-qPCR and WB. (E) The expression of NLRP3 was detected in hypoxia HCMs transfected with shRNAs targeting NLRP3 via RT-qPCR and WB. (F) RT-qPCR examined the expression of IL-1β and IL-18 after NLRP3 silencing in hypoxia HCMs. (G) RT-qPCR and WB evaluated NLRP3 level in normal HCMs transfected with pcDNA3.1/NLRP3. (H) The expression of IL-1β and IL-18 was examined in normal HCMs transfected with pcDNA3.1/NLRP3 using RT-qPCR. HCM: human cardiomyocytes; MI: myocardial infarction; NLRP3: NLR family pyrin domain containing 3; NLRP3: NLR family pyrin domain containing 3; RT-qPCR: reverse transcription quantitative polymerase chain rection; WB: western blot. **P < 0.01.

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  Figure 2.. Silencing of NLRP3 promotes proliferation and suppresses apoptosis of hypoxia-induced myocardial cells. (A) EdU assays testified the proliferation ability of hypoxia HCMs transfected with shRNAs targeting NLRP3 or of normal HCMs transfected with pcDNA3.1/NLRP3. (B, C) Flow cytometry and TUNEL assays examined the apoptosis rate of hypoxia HCMs transfected with shRNAs targeting NLRP3 or of normal HCMs transfected with pcDNA3.1/NLRP3. HCM: human cardiomyocytes; NLRP3: NLR family pyrin domain containing 3; shRNA: short hairpin RNA; DAPI: 4’,6-diamidino-2-phenylindole. **P < 0.01.

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  Figure 3.. NLRP3 is directly targeted by miR-223-3p. (A) Bioinformatics programs including RegRNA, miRTarBase, and TargetScan predicted miR-223-3p was the potential miRNA combining with NLRP3. (B) RT-qPCR examined miR-223-3p expression in normal and hypoxia HCMs. (C) MiR-223-3p expression was detected in hypoxia HCMs transfected with miR-223-3p mimics. (D, E) RT-qPCR and WB examined NLRP3 expression after miR-223-3p upregulation in hypoxia HCMs or after miR-223-3p inhibition in normal HCMs. (F) Ago2-RIP assay indicated the enrichment of NLRP3 and miR-223-3p in Ago2 groups. (G) RNA pull-down experiments showed the enrichment of NLRP3 in Biotin-miR-223-3p WT groups. (H) StarBase predicted the binding site between NLRP3 and miR-223-3p. (I) The affinity between NLRP3 and miR-223-3p was further investigated via luciferase reporter experiments. HCM: human cardiomyocytes; miRNA: microRNA; NLRP3: NLR family pyrin domain containing 3; RT-qPCR: reverse transcription polymerase chain reaction; WT: wild type. **P < 0.01.

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  Figure 4.. MiR-223-3p is the downstream gene of ZNF561-AS1. (A) StarBase and DIANA screened out two lncRNAs (KCNQ1OT1 and ZNF561-AS1) that combined with miR-223-3p. (B) RT-qPCR analysis indicated ZNF561-AS1 expression was overtly higher in hypoxia HCMs. (C) RT-qPCR detected the expression of ZNF561-AS1 in hypoxia HCMs transfected with shRNAs targeting ZNF561-AS1 and NLRP3 expression was tested via RT-qPCR along with WB in hypoxia HCMs after ZNF561-AS1 was inhibited. (D, E) FISH and nuclear separation assays showed the location of ZNF561-AS1. (F) Ago2-RIP assays manifested the enrichment of ZNF561-AS1, miR-223-3p, and NLRP3 in Ago2 groups. (G) RNA pull-down experiments displayed the enrichment of ZNF561-AS1 in Bio-miR-223-3p WT groups. (H) The binding site between ZNF561-AS1 and miR-223-3p was observed using starBase. (I) Luciferase reporter assays were implemented for further validation of the affinity between miR-223-3p and ZNF561-AS1. FISH: fluorescence in situ hybridization; HCM: human cardiomyocytes; lncRNA: long non-coding RNAs; NLRP3: NLR family pyrin domain containing 3; RNA: ribonucleic acid; WB: Western blot; WT: wild type. **P < 0.01.

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  Figure 5.. ZNF561-AS1 regulates hypoxia HCM proliferation and apoptosis through miR-223-3p/NLRP3 axis. (A) EdU assay examined cell proliferation of hypoxia HCMs transfected with sh-ZNF561-AS1#1 or sh-ZNF561-AS1#1+pcDNA3.1/NLRP3. (B, C) Flow cytometry and TUNEL assays detected the apoptosis rate of hypoxia HCMs transfected with sh-ZNF561-AS1#1 or sh-ZNF561-AS1#1+pcDNA3.1/NLRP3. HCM: human cardiomyocytes; NLRP3: NLR family pyrin domain containing 3; DAPI: 4’,6-diamidino-2-phenylindole. **P < 0.01.