Anti-inflammatory effects of microRNA-17-3p in endothelial cells stimulated by bacterial endotoxin

Abstract

MicroRNAs (miRNAs) are small, noncoding RNAs of ~ 22 nucleotides that target the 3′ untranslated region (UTR) of mRNAs to induce mRNA degradation or translational repression. The current project aimed to examine the role of miRNAs in vascular inflammation. The bacterial endotoxin, lipopolysaccharide (LPS), was used to induce inflammatory responses in human umbilical vein endothelial cells (HUVECs). Among the four miRNAs (miR-17-3p, miR-31, miR-125b and miR-146b-3p), miR-17-3p was the only one that was upregulated in HUVECs treated with LPS (10 ng/ml) for 16 hours. Subsequent studies were designed to determine its effects, and the mechanisms involved, on inflammatory reactions in endothelial cells.

In HUVECs stimulated with LPS, the release of interleukin (IL)-8 and IL-6 was increased, and the protein expressions of inhibitor of kappa B protein alpha (IκBα) [the cytoplasmic inhibitor of nuclear factor kappa B (NF-κB)] and endothelial nitric oxide synthase (eNOS) were reduced. These LPS-induced effects were smaller in HUVECs transfected with miR-17-3p agomir (which resulted in miR-17-3p overexpression) than in those transfected with its negative control. LPS did not affect the protein expression of p65 (a subunit of NF-κB) but increased its phosphorylation (and hence activation); this increase was downregulated in cells transfected with miR-17-3p agomir. These cells also expressed smaller levels of cyclooxygenase-2, intracellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin and monocyte chemoattractant protein-1 (MCP-1) in response to LPS than those transfected with the negative control; this was associated with reduced adhesion of THP-1 monocytic cells to HUVECs.

In HUVECs transfected with miR-17-3p antagomir (miR-17-3p antagonist), the inflammatory response to LPS [reflected by protein expressions of eNOS, IκBα, ICAM-1, VCAM-1 and MCP-1, and the degree of monocyte adhesion] were greater than in those transfected with the negative control. These results, thus, confirm the anti-inflammatory effects of miR-17-3p.

Using the miRNA target prediction algorithms, miRanda, MicroCosm and RNA22, the mRNA target of miR-17-3p was predicted to be NIK and IKKβ binding protein (NIBP). This prediction was validated by the micro-ribonucleoprotein immunoprecipitation study, in which high NIBP mRNA level was detected in miR-17-3p-AGO complex. Furthermore, silencing NIBP mRNA resulted in a smaller inflammatory response to LPS in HUVECs, mimicking the effects of miR-17-3p overexpression; the data thus further confirmed that NIBP was the direct target of miR-17-3p.

In mice injected with LPS (40 mg/kg, intraperitoneally), the rectal temperature was reduced; this reduction was less in mice receiving miR-17-3p agomir (1 nmol; once per day via tail vein injection for three days before LPS treatment). The increases by LPS in mRNA and protein levels of ICAM-1 and VCAM-1 in the lung were also reduced in mice receiving miR-17-3p agomir. LPS-induced macrophage recruitment to the pulmonary vascular endothelium was also reduced in mice with miR-17-3p overexpression.

In summary, miR-17-3p, the expression of which was up-regulated by LPS, inhibited the activation of NF-κB pathway in endothelial cells to reduce LPS-induced inflammatory responses, possibly by targeting NIBP. Therefore, miR-17-3p may have an anti-inflammatory role, thus making it a potential therapeutic target/agent in the management of vascular inflammation.