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

Suqin Gao; Lin Sun; Enji Han; Hongshun Qi; Jinbo Feng; Shunliang Xu; Wen Xia

摘要

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 ofβ-site amyloid precursor protein cleaving enzyme(BACE1) and APP genes on the production ofβ-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.

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

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