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N-ethylmaleimide‑sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells.

Zhou Y, Yang SX, Yue YN, Wei XF, Liu Y - Mol Med Rep (2016)

Bottom Line: In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05).The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249).These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N‑ethylmaleimide sensitive factor (NSF) and Weibel‑Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N‑terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF‑shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF‑shRNA. Therefore NSF‑siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

No MeSH data available.


Related in: MedlinePlus

Protein expression levels of NSF. (A) Western blot analysis of NSF protein. (B) Relative gray values. Lane 1, experimental group 0 h; 2, experimental group 24 h; 3, experimental group 48 h; 4, experimental group 72 h; 5, negative control 0 h; 6, negative control 24 h; 7, negative control 48 h; 8, negative control 72 h; 9, blank control 0 h; 10, blank control 24 h; 11, blank control 48 h; 12, blank control 72 h. GAPDH served as the internal control. NSF, N-ethylmaleimide sensitive factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05 compared with the negative control.
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f3-mmr-14-02-1061: Protein expression levels of NSF. (A) Western blot analysis of NSF protein. (B) Relative gray values. Lane 1, experimental group 0 h; 2, experimental group 24 h; 3, experimental group 48 h; 4, experimental group 72 h; 5, negative control 0 h; 6, negative control 24 h; 7, negative control 48 h; 8, negative control 72 h; 9, blank control 0 h; 10, blank control 24 h; 11, blank control 48 h; 12, blank control 72 h. GAPDH served as the internal control. NSF, N-ethylmaleimide sensitive factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05 compared with the negative control.

Mentions: The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), and the decreased protein expression level of NSF in the experimental group occurred in a time-dependent manner. No changes were observed in the protein levels of NSF and GAPDH in the blank and negative control groups, and no statistical differences were observed between the blank and negative control groups (P=0.249; Fig. 3).


N-ethylmaleimide‑sensitive factor siRNA inhibits the release of Weibel-Palade bodies in endothelial cells.

Zhou Y, Yang SX, Yue YN, Wei XF, Liu Y - Mol Med Rep (2016)

Protein expression levels of NSF. (A) Western blot analysis of NSF protein. (B) Relative gray values. Lane 1, experimental group 0 h; 2, experimental group 24 h; 3, experimental group 48 h; 4, experimental group 72 h; 5, negative control 0 h; 6, negative control 24 h; 7, negative control 48 h; 8, negative control 72 h; 9, blank control 0 h; 10, blank control 24 h; 11, blank control 48 h; 12, blank control 72 h. GAPDH served as the internal control. NSF, N-ethylmaleimide sensitive factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05 compared with the negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4940057&req=5

f3-mmr-14-02-1061: Protein expression levels of NSF. (A) Western blot analysis of NSF protein. (B) Relative gray values. Lane 1, experimental group 0 h; 2, experimental group 24 h; 3, experimental group 48 h; 4, experimental group 72 h; 5, negative control 0 h; 6, negative control 24 h; 7, negative control 48 h; 8, negative control 72 h; 9, blank control 0 h; 10, blank control 24 h; 11, blank control 48 h; 12, blank control 72 h. GAPDH served as the internal control. NSF, N-ethylmaleimide sensitive factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05 compared with the negative control.
Mentions: The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), and the decreased protein expression level of NSF in the experimental group occurred in a time-dependent manner. No changes were observed in the protein levels of NSF and GAPDH in the blank and negative control groups, and no statistical differences were observed between the blank and negative control groups (P=0.249; Fig. 3).

Bottom Line: In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05).The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249).These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038, P.R. China.

ABSTRACT
The aim of the present study was to examine the effect of small interfering RNA (siRNA) methods on the expression of N‑ethylmaleimide sensitive factor (NSF) and Weibel‑Palade body (WPB) release in endothelial cells. A small hairpin RNA (shRNA), mediated with an adenovirus vector, was designed to target the N‑terminal functional area of NSF. Subsequently, viruses were transfected into human aortic endothelial cells. The mRNA and protein expression levels of NSF were detected using reverse transcription‑quantitative polymerase chain reaction and Western blot analyses, respectively, and the release of WPBs in the endothelial cells was examined using immunofluorescence. The mRNA expression of NSF in the endothelial cells, which were transfected with the adenoviruses carrying the NSF‑shRNA was significantly decreased, compared with the negative control group (P=0.035) and blank control group (P=0.02). In addition, the mRNA expression of NSF was gradually decreased as duration increased; there were marked differences between the 24, 48 and 72 h groups (P<0.05). The protein expression of NSF was significantly decreased in the experimental group, compared with the negative control group (P=0.004) and blank control group (P=0.031), however, no difference was observed between the negative control and blank control groups (P=0.249). The immunofluorescence staining showed that the release of WPBs in the endothelial cells induced with thrombin was inhibited markedly following transfection with the virus carrying the NSF‑shRNA. Therefore NSF‑siRNA inhibited the mRNA and protein expression levels of NSF, and inhibited the release of WPBs in endothelial cells induced with thrombin. These results suggested that NSF-siRNA may be valuable for preventing and treating atherosclerosis and acute coronary syndrome.

No MeSH data available.


Related in: MedlinePlus