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Journal Article Epublish 15 citations

PubMed: 37587410 DOI: 10.1186/s11658-023-00482-5

MiR-15b-5p and PCSK9 inhibition reduces lipopolysaccharide-induced endothelial dysfunction by targeting SIRT4.

Cell Mol Biol Lett 28(1):66

Authors: Elisa Martino, Nunzia D'Onofrio, Anna Balestrieri, Luigi Mele, Celestino Sardu, Raffaele Marfella, Giuseppe Campanile, Maria Luisa Balestrieri

Affiliations

  • Department of Precision Medicine, University of Campania Luigi Vanvitelli, Via L. De Crecchio 7, 80138, Naples, Italy.
  • Department of Precision Medicine, University of Campania Luigi Vanvitelli, Via L. De Crecchio 7, 80138, Naples, Italy.
  • Food Safety Department, Istituto Zooprofilattico Sperimentale del Mezzogiorno, Via Salute 2, 80055, Portici, Italy.
  • Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Via Luciano Armanni 5, 80138, Naples, Italy.
  • Department of Advanced Clinical and Surgical Sciences, University of Campania Luigi Vanvitelli, Piazza Miraglia, 80138, Naples, Italy.
  • Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Via F. Delpino 1, 80137, Naples, Italy.

Abstract

BACKGROUND

Endothelial dysfunction and deregulated microRNAs (miRNAs) participate in the development of sepsis and are associated with septic organ failure and death. Here, we explored the role of miR-15b-5p on inflammatory pathways in lipopolysaccharide (LPS)-treated human endothelial cells, HUVEC and TeloHAEC.

METHODS

The miR-15b-5p levels were evaluated in LPS-stimulated HUVEC and TeloHAEC cells by quantitative real-time PCR (qRT-PCR). Functional experiments using cell counting kit-8 (CCK-8), transfection with antagomir, and enzyme-linked immunosorbent assays (ELISA) were conducted, along with investigation of pyroptosis, apoptosis, autophagy, and mitochondrial reactive oxygen species (ROS) by cytofluorometric analysis and verified by fluorescence microscopy. Sirtuin 4 (SIRT4) levels were detected by ELISA and immunoblotting, while proprotein convertase subtilisin-kexin type 9 (PCSK9) expression was determined by flow cytometry (FACS) and immunofluorescence analyses. Dual-luciferase reporter evaluation was performed to confirm the miR-15b-5p-SIRT4 interaction.

RESULTS

The results showed a correlation among miR-15b-5p, PCSK9, and SIRT4 levels in septic HUVEC and TeloHAEC. Inhibition of miR-15b-5p upregulated SIRT4 content, alleviated sepsis-related inflammatory pathways, attenuated mitochondrial stress, and prevented apoptosis, pyroptosis, and autophagic mechanisms. Finally, a PCSK9 inhibitor (i-PCSK9) was used to analyze the involvement of PCSK9 in septic endothelial injury. i-PCSK9 treatment increased SIRT4 protein levels, opposed the septic inflammatory cascade leading to pyroptosis and autophagy, and strengthened the protective role of miR-15b-5p inhibition. Increased luciferase signal validated the miR-15b-5p-SIRT4 binding.

CONCLUSIONS

Our in vitro findings suggested the miR-15b-5p-SIRT4 axis as a suitable target for LPS-induced inflammatory pathways occurring in sepsis, and provide additional knowledge on the beneficial effect of i-PCSK9 in preventing vascular damage by targeting SIRT4.

MeSH

Sepsis, Molecular Biology, Biophysics, Physiology, Apoptosis, Cells, Luciferases, Enzyme-Linked Immunosorbent Assay, Lipopolysaccharides, Micrornas, Autophagy, Real-Time Polymerase Chain Reaction, Pyroptosis

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