Inhibition Of β-Site APP Cleaving Enzyme Gene To Reduce β-Amyloid Production In SK-N-SH Cells

IntroductionAlzheimer's disease(AD),which is characterized by the progressive destruction of brain function in older people,was first recognized in the early 20^th century.AD is the most common dementia in the world.It has been estimated that～5%of the population older than 65 years is affected by Alzheimer's disease.The prevalence doubles approximately every 5 years beyond age 65.And by age 85 years almost half of the human population is affected by this disease.Since then modern medicine has further increased the number of people living to old age.AD has become the third leading cause of death for adults in The United States with the number of patients estimated to increase from 4.5 million currently to 12million in 2025.In past,we have considered vascular dementia was the first dementia in China for many years.The recent research showed that Alzheimer's disease is the most dementia other than other dementias in China.Clearly,this disease is a major social and health-care problem of which there is no cure or effective treatment.'Senile plaques' and neurofibrillary degenerations were the first morphological abnormalities identified in brains of Alzheimer's disease(AD) patients.The major components of senile plaques areβ-amyloid peptides,which are derived by proteolytical processing of the amyloid precursor protein.The resultantβ-amyloid production is thought to be an early event in the pathogenesis of AD.In the amyloidogenic pathway APP is first cleaved byβ-secretase to generate a secreted form of APP(sAPP) and a 99-residue memebrane-associated fragment(C99).C99 is the substrate ofγ-secretase,and intramembrane cleavage at theγsite generates Aβand CTFγfragments.BACE1 is the main enzyme involved in neuronal Aβproduction. Because the enzymatic activity of BACE1 is a prerequisite for the generation ofβ-amyloid peptides,BACE1 appears to be a promising pharmacological target to lower the production ofβ-amyloid peptides and ameliorate the deleterious effects of the different forms ofβ-amyloid on learning and memory processes.Although the pathogenesis of AD is complex,amyloid hypothesis was proposed. According to the hypothesis,the extracellular deposition of Aβin AD brain is one of the triggers of inflammation and oxidative damage.Consequently,these can cause neurodegeneration.Prevention of the negative influence of amyloid seems an attractive therapeutic approach for Alzheimer's disease.The advantage of an amyloid therapy for AD is that it is target at correcting or preventing the pathology believed to cause the disease,Therefore,it might change the course of the disease or(if used at the appropriate time) even prevent its onset.Preventing amyloid deposition will also allow evaluation of the actual role of amyloid in Alzheimer's dementia.RNA interference(RNAi) plays an important role in diverse aspects of biology. Techniques that exploit the power of RNAi to suppress target genes have already become indispensable tools in research and may soon prove to be theraoeutically useful.In particular,the production of small interfering RNAs(siRNAs) that silence specific disease related genes could have wide-ranging therapeutic applications.One promising therapeutic role for siRNA is the silencing of genes that cause dominantly inherited disease.To reduce the production of Aβis the important therapeutic target.Now there are many way to reduce the production of Aβ.In this study,BACE1 is selected as the target genes;we inhibit the gene to observe the effects for production of Aβ.Firstly, we use RNA interfere technique to suppress or silence BACE1 gene by siRNAs,and then we would observe the expression of BACE1,and the effect for the production of Aβ42.Objective1,To study down-regulation ofβ-site amyloid precursor protein cleaving enzyme(BACE1) through siRNA interfere technique by siRNAs in SK-N-SH neuroblastoma cells.2,To detect the production of Aβ42 in neuroblastoma cells by suppressing BACE1 expression through siRNA interference.Methods1,The neuroblastoma SK-N-SH cell line,was a tumor type cell that express APP and BACE1.This cell line was provided by Shanghai Institutes or Biological Sciences,Chinese Academy Sciencer.2,Human BACE1 cDNA sequence was obtained from NCBI website.Based on the gene sequence,we commit Ambion RNA Company to design and synthesize 3 siRNA sequences for RNAi by Silencer^? pre-designed siRAN Specification system. The 3 pairs of siRNA were prepared for double-strand.3,During the process of transfection,we used Opti-MEM medium(Invitrogen) to dilute the siRNAs and siPORT NeoFX transfection reagent.To compare the effects of different concentrations of siRNAs,10 nM to 50nM siRNAs were used to transfect SK-N-SH cells.After transfection for 12-48h,we extracted total RNA from SK-N-SH cells.4,RT-PCR was used to detect the expression of BACE1 at mRNA level.5,The expression of BACE1 gene protein,β-secretase,in the SK-N-SH cells that have been treated with siRNAs was detected by Western blot.6,The extracellular level of Aβsecreted from SK-N-SH cells were measured in the conditional media samples through ELISA method. Results1,Cell culture and transfectionWe harvested SK-N-SH cells at different time after transfection using different concentration of siRNA.It has found that 40nM and 50nM siRNA concentration was more efficiency for silencing BACE1 in SK-N-SH cells.We observed that the most transfected efficiency was at 36 h and 48 h post-transfection.2,Effect of siRNA on transcript of BACE1 gene in SK-N-SH cells determined by RT-PCRThe inhibiting efficiency of BACE1 gene treated with chemistry synthesized siRNAs in SK-N-SH cells was obvious at mRNA level in the 40nM and 50nM concentrations determined by RT-PCR.And the interference efficiency was more and more obvious with time.The inhibition was statistic significant differentiation compare to control.3,The expression of BACE1(β-secretase) in SK-N-SH cells determined by Western blot after silencingA representative Western blot showed the relative level of BACE1(β-secretase) inside SK-N-SH cells while interfering for 48 h.The proteins,β-secretas was obvious reduced.In combination with the result of RT-PCR,it can be found that siRNA2 was the most effective interference for BACE1(β-secretase) in SK-N-SH cells.The interference efficiency was＞50%.Compare to the control,the reduction ofβ-secretase treated with siRNAs was statistic significant differentiation.P＜0.05.4,Effects of Aβreleased from SK-N-SH cells treated with siRNAs by ELISA assayThe results show that Aβ₄₂ secreted from SK-N-SH cells was effectively reduced by siRNA 1 and siRNA 2 following transfection.P＜0.05.However,Aβ₄₂ secreted from SK-N-SH cells were treated with siRNA3 was no significant difference to control(P＞0.05).Conclusion The results show that the endogenous expression of BACE1 in neurons could be suppressed by synthetic siRNAs.It indicates that we can suppress BACE1 gene expression by siRNAs through RNA interference technique,and further reduced the production of Aβ₄₂ significantly compared with the control.This study has shown that the inhibition or suppression BACE1 gene,could reduce the production of Aβ₄₂ in vitro through siRNAs,it is suggests that this gene can be serve as the therapeutic target gene.Significance'Senile plaques' and neurofibrillary degenerations were the first morphological abnormalities identifed in brains of Alzheimer's disease(AD) patients.Thus,the amyloid cascade hypothesis has been proposed based on the speculation that Aβ42 production plays an early and critical role in the pathogenesis of AD.This study shows that we could suppress the expression of BACE1 gene by siRNAs.The results indicate that the inhibition or suppression BACE1 gene can reduce the production of Aβ₄₂ in vitro through RNA interference technique. Proteolytic processing of APP at theβ-site by BACE1(β-secretase),the major APP proteolytic enzyme,is essential to generate Aβ.siRNA can specifically suppress BACE 1 expression,and it supports that BACE1 canbe serve as a potential therapeutic target for the treatment of AD.