Fig 2: Ltb4r1 expression elevates in myocardial tissues of MI mice following CHD and silencing Ltb4r1 ameliorated CHD-related myocardial injury. (A) Screening of common differentially expressed genes in microarray data GSE18703 and GSE46395 retrieved from the GEO database (https://www.ncbi.nlm.nih.gov/geo/). (B) Ltb4r1 expression in MI mice injected with sh-Ltb4r1 analyzed by microarray data GSE18703. (C) Ltb4r1 expression in MI mice injected with sh-Ltb4r1 analyzed by microarray data GSE46395. (D) Ltb4r1 expression in cardiomyocytes, CFs, endothelial cells, and PBMSCs determined by RT-qPCR. (E) Ltb4r1 expression in myocardial cells of mice determined by RT-qPCR. (F) Ltb4r1 protein level in myocardial cells of MI mice determined using Western blot analysis, normalized to GAPDH. (G) Echocardiography of LVIDD, LVIDs, LVEF, LVFS, in myocardial tissues of MI mice. (H) Hemodynamic analysis of LV and dP/dt in myocardial tissues of MI mice. (I) The infarct size in myocardial tissues of MI mice detected using TTC staining. Hypoxia-induced MI cardiomyocytes treated with sh-Ltb4r1. (J), Ltb4r1 mRNA level in hypoxia-induced MI cardiomyocytes determined using RT-qPCR, normalized to GAPDH; (K) Ltb4r1 protein level in hypoxia-induced MI cardiomyocytes determined using Western blot analysis, normalized to GAPDH. (L) Levels of IL-1β, IL-6, and IL-18 in hypoxia-induced MI cardiomyocytes measured using ELISA. (M) Apoptosis of hypoxia-induced MI cardiomyocytes detected using TUNEL staining (× 400). (N) Protein levels of Cleaved caspase-3, Bax, and Bcl-2 in hypoxia-induced MI cardiomyocytes determined using Western blot analysis, normalized to GAPDH. * p < 0.05 vs. sham-operated mice injected with sh-NC or normal mice and # p < 0.05 vs. MI cardiomyocytes treated with sh-NC or hypoxia-induced MI cardiomyocytes treated with sh-NC. Data among groups were analyzed by one-way ANOVA/Tukey’s test.

Fig 3: miR-22-3p could target Ltb4r1. (A) Prediction of miRNAs that bind to Ltb4r1 and RT-qPCR analysis of four candidate miRNA expression in MI model by miRWalk (http://mirwalk.umm.uni-heidelberg.de/) and RAID v2.0 (http://www.rna-society.org/raid/index.html). (B) The putative binding sites of miR-22-3p and Ltb4r1 3'UTR by the online website (http://bioinformatics.psb.ugent.be/webtools/venn/). (C) Luciferase activity of PGLO-Ltb4r1 WT and PGLO-Ltb4r1 MUT detected using dual-luciferase reporter gene assay upon treatment with NC and miR-22-3p mimic. (D) miR-22-3p expression and Ltb4r1 mRNA level in myocardial tissues of MI mice or sham-operated mice determined using RT-qPCR, upon treatment with AgomiR-22-3p, AntagomiR-22-3p, sh-Ltb4r1 or sh-NC. (E) Ltb4r1 protein level in myocardial tissues of MI mice determined using Western blot analysis upon treatment with AgomiR-22-3p, AntagomiR-22-3p, sh-Ltb4r1 or sh-NC. (F) miR-22-3p expression and Ltb4r1 mRNA level in hypoxia-induced MI cardiomyocytes determined using RT-qPCR. (G) Ltb4r1 protein level in hypoxia-induced MI cardiomyocytes determined using Western blot analysis, normalized to GAPDH. * p < 0.05 vs. hypoxia-induced cardiomyocytes treated with NC mimic or sham-operated mice or cardiomyocytes treated with empty vector, # p < 0.05 vs. MI mice injected with AgomiR NC or hypoxia-induced MI cardiomyocytes treated with NC mimic, and & p < 0.05 vs. MI mice injected with AntagomiR-22-3p + sh-NC or hypoxia-induced MI cardiomyocytes treated with miR-22-3p inhibitor + sh-NC. Unpaired t-test was used to analyze data between two groups and one-way ANOVA/Tukey’s test to analyzed data among multiple groups.

Fig 4: NEAT1 sponging miR-22-3p reduces Ltb4r1 expression. (A) Prediction of binding sites between miR-22-3p and NEAT1 by the Lncbase v.2 website (http://carolina.imis.athena-innovation.gr/diana_tools/web/index.php?r=lncbasev2/index). (B) The expression of NEAT1 in myocardial tissues and cells of MI mice determined by RT-qPCR. (C) The verification of the binding between miR-22-3p and NEAT1 3'UTR using dual-luciferase reporter gene assay. (D) Co-localization of NEAT1 detected by FISH (× 400). (E) miR-22-3p enrichment detected by RNA-pull down. (F) Binding of NEAT1 and miR-22-3p with AGO2 tested by RIP assay. MI mice were injected with sh-NEAT1. (G) miR-22-3p expression and mRNA levels of NEAT1 and Ltb4r1 in myocardial tissues of MI mice determined using RT-qPCR. Hypoxia-induced MI cardiomyocytes were treated with sh-NEAT1. (H) miR-22-3p expression and mRNA levels of NEAT1 and Ltb4r1 in hypoxia-induced MI cardiomyocytes determined using RT-qPCR. * p < 0.05 vs. NC, Bio-probe NC, IgG, MI mice injected with sh-NC, or hypoxia-induced MI cardiomyocytes treated with sh-NC. Unpaired t-test was used to analyze data between two groups and one-way ANOVA/Tukey’s test to analyzed data among multiple groups.

Fig 5: miR-22-3p contributes to alleviation of CHD-related myocardial injury by downregulating Ltb4r1. MI mice injected with AgomiR-22-3p or AntagomiR-22-3p and sh-Ltb4r1. (A) Quantification of LVEF, LVIDD, LVIDs, LVEF, LVFS, in myocardial tissues of MI mice. (B) Hemodynamic analysis of LV and dP/dt in myocardial tissues of MI mice. (C) The infarct size in myocardial tissues of MI mice detected using TTC staining upon treatment with MI cardiomyocytes treated with exogenous miR-22-3p mimic or miR-22-3p inhibitor and sh-Ltb4r1. (D), Levels of IL-1β, IL-6, and IL-18 in hypoxia-induced MI cardiomyocytes measured using ELISA. (E), Representative images of apoptosis of hypoxia-induced MI cardiomyocytes detected by TUNEL staining (× 400). (F) Protein levels of Cleaved caspase-3, Bax, and Bcl-2 in hypoxia-induced MI cardiomyocytes determined using Western blot analysis, normalized to GAPDH. * p < 0.05 vs. MI + NC mimic and # p < 0.05 vs. AntagomiR-22-3p + sh-NC or MI + miR-22-3p inhibitor + sh-NC. Data among group were analyzed by one-way ANOVA/Tukey’s test.