Korean Circ J.  2023 Jun;53(6):387-403. 10.4070/kcj.2022.0301.

LOXL1-AS1 Aggravates Myocardial Ischemia/Reperfusion Injury Through the miR-761/PTEN Axis

• He W
• Duan L
• Zhang L

• KMID: 2543051
• DOI: http://doi.org/10.4070/kcj.2022.0301

Abstract

Background and Objectives
Myocardial ischemia and reperfusion injury (MIRI) has high morbidity and mortality worldwide. We aimed to explore the role of long noncoding RNA lysyl oxidase like 1 antisense RNA 1 (LOXL1-AS1) in cardiomyocyte pyroptosis.
Methods
Hypoxia/reoxygenation (H/R) injury was constructed in human cardiomyocyte (HCM). The level of LOXL1-AS1, miR-761, phosphatase and tensin homolog (PTEN) and pyroptosis-related proteins was monitored by quantitative real-time polymerase chain reaction or western blot. Flow cytometry examined the pyroptosis level. Lactate dehydrogenase (LDH), creatine kinase-MB and cardiac troponin I levels were detected by test kits. Enzyme-linked immunosorbent assay measured the release of inflammatory cytokines. Dual-luciferase assay validated the binding relationship among LOXL1-AS1, miR-761, and PTEN. Finally, ischemia/reperfusion (I/R) animal model was constructed. Hematoxylin and eosin staining assessed morphological changes of myocardial tissue. NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and casepase-1 expression was determined by immunohistochemistry.
Results
After H/R treatment, LOXL1-AS1 and PTEN were highly expressed but miR-761 level was suppressed. LOXL1-AS1 inhibition or miR-761 overexpression increased cell viability, blocked the release of LDH and inflammatory cytokines (interleukin [IL]-1β, IL-18), inhibited pyroptosis level, and downregulated pyroptosis-related proteins (ASC, cleaved caspase-1, gasdermin D-N, NLRP3, IL-1β, and IL-18) levels in HCMs. LOXL1-AS1 sponged miR-761 to upregulate PTEN. Knockdown of miR-761 reversed the effect of LOXL1-AS1 down regulation on H/R induced HCM pyroptosis. LOXL1-AS1 aggravated the MIRI by regulating miR-761/PTEN axis in vivo.
Conclusions
LOXL1-AS1 targeted miR-761 to regulate PTEN expression, then enhance cardiomyocyte pyroptosis, providing a new alternative target for the treatment of MIRI.

Keyword

Pyroptosis; Myocardial ischemia; Reperfusion injury

Figure

• 

  Figure 1 Knocking down LOXL1-AS1 improved H/R-induced human cardiomyocyte pyroptosis. (A) Using qRT-PCR to detect the LOXL1-AS1 level. (B) Using qRT-PCR to examine the LOXL1-AS1 knockdown effect. (C) Using qRT-PCR to test the LOXL1-AS1 level. (D) Using MTT to determine cell viability. (E) Using the Release Assay Kit to monitor the LDH level. (F) Using ELISA to detect IL-1β and IL-18 secretion. (G) Using Western blot to measure ASC, cleaved caspase-1, GSDMD-N, NLRP3, IL-1β, and IL-18 levels. (H) Using flow cytometry to monitor the pyroptosis level. (I) Using immunofluorescence to detect NLRP3 expression. Data were presented as mean±standard deviation, and 3 separate experiments were performed in triplicate.ELISA = enzyme-linked immunosorbent assay; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GSDMD = gasdermin D; H/R = hypoxia/reoxygenation; IL = interleukin; LDH = lactate dehydrogenase; LOXL1-AS1 = lysyl oxidase like 1 antisense RNA 1; NLRP3 = NOD-like receptor pyrin domain-containing protein 3; qRT-PCR = quantitative real-time polymerase chain reaction.*p<0.05; **p<0.01; ***p<0.001.

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  Figure 2 LOXL1-AS1 negatively regulated the miR-761 level as a molecular sponge. (A) Using bioinformatics to predict LOXL1-AS1 and miR-761 binding sites. (B) Using qRT-PCR to examine miR-761 knockdown and overexpression effects. (C) Using dual-luciferase to test LOXL1-AS1 and miR-761 interaction. (D) After interfering with LOXL1-AS1 in HCMs, qRT-PCR is used to monitor the miR-761 level. (E) Under H/R conditions, LOXL1-AS1 was knocked down in HCMs, and qRT-PCR was used to examine the miR-761 level. Data were presented as mean±standard deviation, and 3 separate experiments were performed in triplicate.H/R = hypoxia/reoxygenation; HCM = human cardiomyocyte; LOXL1-AS1 = lysyl oxidase like 1 antisense RNA 1; MUT = mutant type; qRT-PCR = quantitative real-time polymerase chain reaction; WT = wild-type.**p<0.01; ***p<0.001.

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  Figure 3 MiR-761 overexpression improved H/R-induced human cardiomyocyte pyroptosis. (A) Using quantitative real-time polymerase chain reaction to examine the miR-761 level. (B) Using MTT to examine cell viability. (C) Using the Release Assay Kit to detect the LDH level. (D) Using enzyme-linked immunosorbent assay to determine IL-1β and IL-18 levels. (E) Using Western blot to monitor ASC, cleaved caspase-1, GSDMD-N, NLRP3, IL-1β, and IL-18 levels. (F) Using flow cytometry to detect caspase-1 activity. (G) Using immunofluorescence to detect NLRP3 expression. Data were presented as mean±standard deviation, and 3 separate experiments were performed in triplicate.GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GSDMD = gasdermin D; HR = hypoxia/reoxygenation; IL = interleukin; LDH = lactate dehydrogenase; NLRP3 = NOD-like receptor pyrin domain-containing protein 3.*p<0.05; **p<0.01; ***p<0.001.

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  Figure 4 LOXL1-AS1 induced human cardiomyocyte pyroptosis by targeting miR-761 under H/R conditions. (A) Using quantitative real-time polymerase chain reaction to measure the miR-761 level. (B) Using MTT to detect cell viability. (C) Using the Release Assay Kit to test the LDH level. (D) Using enzyme-linked immunosorbent assay to monitor IL-1β and IL-18 levels. (E) Using Western blot to determine ASC, cleaved caspase-1, GSDMD-N, NLRP3, IL-1β, and IL-18 levels. (F) Using flow cytometry to examine caspase-1 activity. (G) Using immunofluorescence to detect NLRP3 expression. Data were presented as mean±standard deviation, and 3 separate experiments were performed in triplicate.GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GSDMD = gasdermin D; H/R = hypoxia/reoxygenation; IL = interleukin; LDH = lactate dehydrogenase; LOXL1-AS1 = lysyl oxidase like 1 antisense RNA 1; NLRP3 = NOD-like receptor pyrin domain-containing protein 3.*p<0.05; **p<0.01; ***p<0.001.

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  Figure 5 MiR-761 targeted PTEN. (A) Using bioinformatics to predict miR-761 and PTEN binding sites. (B) Using dual-luciferase to verify the miR-761 and PTEN mutually binding relationship. (C, D) Using qRT-PCR and Western blot to determine the PTEN level. (E, F) Using qRT-PCR and Western blot to monitor the PTEN level. Data were presented as mean±standard deviation, and 3 separate experiments were performed in triplicate.GAPDH = glyceraldehyde-3-phosphate dehydrogenase; H/R = hypoxia/reoxygenation; MUT = mutant type; PTEN = phosphatase and tensin homolog; qRT-PCR = quantitative real-time polymerase chain reaction; WT = wild-type.***p<0.001.

• 

  Figure 6 LOXL1-AS1 enhanced myocardial ischemia and reperfusion injury by regulating miR-761 in mice. (A, B) Using qRT-PCR to monitor LOXL1-AS1 and miR-761 levels. (C) Using HE staining to measure changes in the myocardial tissue morphology (scale bar: 50 μm). (D-F) Using the Release Assay Kit to detect LDH, CK-MB, and cTnI levels in serum. (G, H) Using qRT-PCR to determine LOXL1-AS1 and miR-761 levels. (I, J) Using qRT-PCR and Western blot to monitor the PTEN level. (K) Using ELISA to measure IL-1β and IL-18 levels. (L) Using Western blot to examine ASC, cleaved caspase-1, GSDMD-N, NLRP3, IL-1β, and IL-18 levels. (M) Immunohistochemistry detection of NLRP3 and caspase-1 expression in the myocardial tissue (scale bar: 50 μm). Data are presented as mean±standard deviation (n=5).CK-ME = creatine kinase-MB; cTnI = cardiac troponin I; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; GSDMD = gasdermin D; HE = hematoxylin and eosin; IL = interleukin; I/R = ischemia/reperfusion; LDH = lactate dehydrogenase; LOXL1-AS1 = lysyl oxidase like 1 antisense RNA 1; NLRP3 = NOD-like receptor pyrin domain-containing protein 3; PTEN = phosphatase and tensin homolog; qRT-PCR = quantitative real-time polymerase chain reaction.***p<0.001.

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