Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 May 16:16:11.
doi: 10.1186/s12867-015-0034-8.

MicroRNA-19a regulates lipopolysaccharide-induced endothelial cell apoptosis through modulation of apoptosis signal-regulating kinase 1 expression

Wei-Long Jiang  1 , Yu-Feng Zhang  2 , Qing-Qing Xia  3 , Jian Zhu  4 , Xin Yu  5 , Tao Fan  6 , Feng Wang  7
Affiliations

MicroRNA-19a regulates lipopolysaccharide-induced endothelial cell apoptosis through modulation of apoptosis signal-regulating kinase 1 expression

Wei-Long Jiang et al. BMC Mol Biol. .

Abstract

Background: MicroRNAs, small non-encoding RNAs that post-transcriptionally modulate expression of their target genes, have been implicated as critical regulatory molecules in endothelial cells.

Results: In the present study, we found that overexpression of miR-19a protects endothelial cells from lipopolysaccharide (LPS)-induced apoptosis through the apoptosis signal-regulating kinase 1 (ASK1)/p38 pathway. Quantitative real-time PCR demonstrated that the expression of miR-19a in endothelial cell was markedly down-regulated by LPS stimulation. Furthermore, LPS-induced apoptosis was significantly inhibited by over-expression of miR-19a. Finally, both a luciferase reporter assay and western blot analysis showed that ASK1 is a direct target of miR-19a.

Conclusions: MiR-19a regulates ASK1 expression by targeting specific binding sites in the 3' untranslated region of ASK1 mRNA. Overexpression of miR-19a is an effective method to protect against LPS-induced apoptosis of endothelial cells.

PubMed Disclaimer

Figures

Figure 1
Figure 1
LPS down-regulates expression of miR-19a in endothelial cells. (A), HUVECs were cultured in 6-well plate, after 80% confluence, cells were treated with different concentration of LPS for 12 h. (B), HUVECs were treated with 100 ng/mL LPS for different times point as indicated. Quantitative real-time PCR showed that miR-19a expression was down-regulated, compared with that in the respective control group. Significance is indicated as *P < 0.05.
Figure 2
Figure 2
Expression of ASK1 was up-regulated during LPS-induced endothelial cell apoptosis. (A), HUVECs were cultured in 6-well plate, after 80% confluence, cells were treated with 100 ng/mL LPS for different times point as indicated. Quantitative real time PCR revealed that the expression levels of ASK1 were increased after LPS stimuli. (B), Cell lysates obtained from HUVECs were subjected to western blot. ASK1, phosphorylation of ASK1 at Thr845, and GAPDH were determined by indicated antibodies. (C), the quantitative analysis of HUVECs apoptosis was determined by ELISA assay. Significance is indicated as *P <0.05.
Figure 3
Figure 3
Expression of miR-19a in HUVECs either transduced with Ad-miR19a and transfected with miR-19a inhibitor. (A) and (B), Quantitative real time PCR revealed that the expression levels of miR-19a were increased after Ad-miR-19a transduction (A), but suppressed by miR-19a inhibitor in a dose-dependent manner (B). Significance is indicated as *P < 0.05. (C) and (D), MiR-19a regulates ASK1 expression. HUVECs were transduced with Ad-miR-19a or Ad-LacZ (50MOI) for 24 h, and then treated with 100 ng/mL LPS for another 24 h. The expression of ASK1 was determined by quantitative real time PCR (C) and western blot (D).
Figure 4
Figure 4
Over-expressing miR-19a attenuated LPS-induced Apoptosis in endothelial cells. (A), MiR-19a inhibitor induces HUVECs apoptosis. HUVECs were transduced with miR19a inhibitor or Control inhibitor for 48 h, then cell apoptosis was determined by ELISA assay. (B), HUVECs were transduced with miR19a inhibitor or Control inhibitor for 24 h, then treated with 100 ng/mL LPS for another 24 h. Cell apoptosis was determined by ELISA assay. (C), HUVECs were transduced with Ad-miR-19a or miR19a inhibitor or Ad-GFP (50MOI) for 24 h, then treated with 100 ng/mL LPS for another 24 h. Cell apoptosis was determined by ELISA assay. (D), Cell lysates obtained from HUVECs were subjected to western blot. ASK1, phosphorylation of ASK1 at Thr845, p38, phosphorylation of p38, cleaved caspase3 and GAPDH were determined by indicated antibodies.
Figure 5
Figure 5
MiR-19a suppressed expression of ASK1 through binding to its 3’UTR. (A), a representative illustration of the putative binding sites for miR-19a in human ASK1 3’UTR. (B), Luciferase assay was performed in EAhy926 cells using pGL3 reporter vector fused with either ASK1 wild-type 3’UTR or ASK1 3’UTR mutant. Over-expression of miR-19a significantly decreased the activity of luciferase gene fused with ASK1 wild-type 3’UTR, but had no effect on the activity of luciferase fused with ASK1 3’UTR mutant. (C), Detection of ASK1 expression by western blot in the whole lysates of HUVECs transduced with different dosages of AdmiR-19a. The result showed that the protein level of ASK1 was suppressed by miR-19a, however, increased by miR-19a inhibitor, in a dose-dependent manner. Significance is indicated as *P <0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Kong Y, Han JH. MicroRNA: biological and computational perspective. Genomics Proteomics Bioinformatics. 2005;3(2):62–72. - PMC - PubMed
    1. McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet. 2002;3(10):737–47. doi: 10.1038/nrg908. - DOI - PubMed
    1. Grabarek JB. RNA silencing. Adv Exp Med Biol. 2003;544:145–58. - PubMed
    1. He L, Hannon GJ. MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet. 2004;5(7):522–31. doi: 10.1038/nrg1379. - DOI - PubMed
    1. Bukhari SI, Vasquez-Rifo A, Gagne D, Paquet ER, Zetka M, Robert C, et al. The microRNA pathway controls germ cell proliferation and differentiation in C. elegans. Cell Res. 2012;22(6):1034–45. doi: 10.1038/cr.2012.31. - DOI - PMC - PubMed

Publication types

LinkOut - more resources