MicroRNA, an important regulator of endothelial cell behavior in sepsis ?

Koster, T.M. (Thijs) (2015) MicroRNA, an important regulator of endothelial cell behavior in sepsis ? thesis, Medicine.

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Abstract

Background Sepsis is a clinical syndrome which is caused by infection and the subsequent systemic inflammatory response. Despite intensive treatment of the septic patient, the overall mortality of sepsis is unacceptably high, with mortality rates of 20 – 40%. Part of the pathophysiology of sepsis is a hyper inflammatory response of the immune system. Endothelial cells play a role in the inflammatory response, because they recognize pathogens such as LPS, part of the cell wall of Gram-negative bacteria. This recognition result in activation of NF-κB. NF-κB is an important regulator of inflammatory genes. Activation of NF-κB results in increased expression of pro inflammatory genes. These genes contribute to endothelial dysfunction and therefore, worsen the survival of septic patients. MiRs are endogenous short RNAs of approximately 22 nucleotides that play regulatory roles in plants and animals due to their capability to bind with mRNAs. This binding leads to translational repression of protein-coding genes and thus modulates the phenotype. The miR-24 cluster has been showed to play a role in inflammation by regulating NF-κB. The objective of this study was to define what role the miR-24 cluster in LPS stimulated endothelial cells has and if inhibition of the miR-24 cluster suppresses inflammation. Material and Methods HUVECs(P4) were stimulated with LPS 0,1μg/ml for 4,8 or 24 hours, harvested and miR expression was determined for miRs, namely miR-23b, 24, 27b, 34b, 199-3p, 199-5p, 542-3p and 542-5p. Also, HUVECs(P4) were transfected with antisense oligonucleotides to miR-23b, -24, -27b or the combination thereof(referred to as anti-miR-23b,-24, 27b, or anti-all-miR) before LPS stimulation and were harvested 4 hours after LPS stimulation. Afterwards, qPCR for different inflammatory genes was performed to determine the level of inflammation and endothelial activation. Results MiR-23 (2-fold) and miR-24(4–fold) were increased in HUVECs 4, 8 and 24 hours after LPS stimulation compared to untreated controls. MiR-27b was increased 4.5 times after 8 hours. But replication of this experiment failed. E-selectin mRNA level was twice as low in the anti-miR-27b and the anti-all-miR transfected groups compared to control. ICAM-1 mRNA level remained unchanged in transfection groups. While IL-6 mRNA level was decreased in the anti-miR-23b (1.6-fold), 27b(2.5-fold; P≤ 0.001) and anti-all group(1.6-fold), was IL-8 only decreased in the anti-miR-23b group. CCL-2 mRNA level showed a 2-fold decrease in the antisense oligonucleotides to miR-27b group and a lesser decrease in the anti-all-miR group. All groups were decreased in Ang-2 mRNA level except in anti-miR-24. However, when the control group was replaced with antisense oligonucleotides to miR-1 transfected HUVECs different results were found. Conclusion This study showed that the miR-24 cluster is increased in HUVECs after LPS stimulation. Moreover, our data suggest that inhibition of the miR-24 cluster, mainly miR-27b, results a reduction of endothelial activation after LPS stimulation. However these data could not be replicated. Inhibition of NF-κB by targeting miR-27b deserves further studies to validate these first observations

Item Type:	Thesis (Thesis)
Supervisor name:	Supervisor: and Krenning, Dr. G. Institution and department: UMCG
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:48
Last Modified:	25 Jun 2020 10:48
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/942

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