Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells*☆

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Abstract

BACKGROUND:

Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein (APP) at the mRNA level. In addition, the piperlonguminine (A) and dihydropiperlonguminine (B) components (1: 0.8), which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels.

OBJECTIVE:

Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme (BACE1) and APP genes on the production of β-amyloid peptide 42 (Aβ42) in human neuroblastoma cells (SK-N-SH cells) using small interfering RNAs (siRNAs) and A/B components separated from Futokadsura stem, respectively.

DESIGN, TIME AND SETTING:

A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science & Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007.

MATERIALS:

SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R&D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG was provided by Sigma, USA.

METHODS:

The human BACE1 cDNA sequence was obtained from NCBI website (www.ncbi.nlm.nih.gov/sites/entrez). Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer® pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs (10–50 nmol/L) on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1:0.8. The A/B (1: 0.8) components were added to the SK-N-SH cells at different concentrations (13.13, 6.56, and 3.28 mg/mL).

MAIN OUTCOME MEASURES:

Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA.

RESULTS:

BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components (1: 0.8), which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components.

CONCLUSION:

Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.

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