The Effect Of Long Noncoding RNA On β-amyloid Generation By Regulating BACE1Exptession

Background and objectiveAlzheimer’s disease (AD), which is the most common type of senile dementia, occurring in the senile and presenile, with cognitive impairment and behavioral impairment characterized by lesions of the central nervous system, is a chronic progressive degenerative diseases of the central nervous system. The clinical manifestation of AD are progressive memory impairment, aphasia, apraxia, agnosia, damage of visuo-spatial ability, damage of abstract thinking and calculation ability, personality and behavior change, etc. Annual report of Alzheimer’s Disease International (ADI) shows that in2010, the number of dementia patients is35.6million. This number will increase to65.7million and115.4million in2050. Among these patients, patients with AD will be a proportion of50-75%. The total expenditure for dementia patients in2010was about60.4billion dollars, about1%of total global GDP. So far, there is no specific treatment for AD, because the etiology and pathogenesis of AD are still unknown. Therefore, further study of the etiology and pathogenesis of AD, and on this basis to explore the new method to prevent and treat AD is still an urgent issue.Neuritic plaques and neurofibrillary tangles are two classical histopathological changes of AD. The amyloid cascade hypothesis was in the core position among studies on pathogenesis of AD, which believe that deposition of Aβ result in neuronal dysfunction and death. β-secretase also known as β-site APP cleaving enzyme1(BACE1), is a membrane-binding aspartate protease, a rate-limiting enzyme in produce Aβ by cleavage of APP.Noncoding RNA(ncRNA) is a kind of RNA that can not be translated into protein, but have its own biological function. Long noncoding RNA(lncRNA) are these ncRNAs which have more than400nucleotide, including long intergenic noncoding RNA(lincRNA), natural antisense transcript(NAT) and non-coding RNA repeats, etc. BACE1antisense transcript(BACE1-AS) is a kind of conservative lncRNA with a length of2KB, generated by the complementary chain of BACE1gene loci in chromosome11(11q23.3).The level of BACE1-AS in the frontal cortex, hippocampus and entorhinal cortex of AD patients was significantly increased and the level of BACE1mRNA in these area is mildly elevated, suggesting that BACE1-AS may be related to the pathogenesis of AD. Studies have shown that, BACE1-AS has a significant moderating effect on the expression of BACE1mRNA. It can bind to BACE1mRNA, enhance its stability, and promote the formation of Aβ. Aβ1-42can promote the expression of BACE1-AS in vitro, as a positive feedback in Aβ generation. Is Aβ1-42in vivo have the same effect on activation of BACE1-AS, increase BACE1mRNA level, and promotion of Aβ generation? In APPsw transgenic cells, is BACEl-AS involved in the expression of BACE1and Aβ generation? What is the mechanism of Aβ1-42increase the level of BACE1-AS expression?Based on the above background, our study first focus on the effect by exogenous Aβ1-42in vivo on BACE1-AS&BACE1expression and Aβ generation in brain of C57/BL6J mice. We also observe the effect by APPsw gene transfection in vitro on BACE1-AS&BACE1expression and Aβ generation using AD cell model, as well as the effect by siRNA suppression of BACE1-AS expression on Aβ generation. Furthermore, We also tried to study the mechanism of NF-κB mediate the promotion of BACE1-AS expression and Aβ generation by Aβ1-42, in vivo and in vitro.Contents1. The mechanism of exogenous Aβ1-42promote BACE1expression and Aβ generation mediated by BACE1-AS in cell and animal.2. The mechanism of BACE1-AS involved in BACE1expression and Aβ generation in APPsw transgenic cells.3. The mechanism of NF-κB regulate BACE1-AS expression and Aβ generation.Methods 1. To make it clear that if Aβ1-42in vivo has the same effect on activation of BACE1-AS, increase BACE1mRNA level and Aβ generation, we first observe exogenous Aβ1-42activate BACE1-AS expression in vitro, then using siRNA of BACE1-AS to interfer the expression of BACE1-AS, observe its effect on BACEl mRNA&BACE1protein expression and Aβ1-40generation. Next, we observe the expression of BACE1-AS, BACEl mRNA and Aβ1-40level in brain of C57/BL6J mouse after Aβ1-42injected directly into mouse hippocampus using the stereotactic technique.2. To make it clear that if BACE1-AS is involved in BACEl expression and Aβ generation in APPsw transgenic cells, we observe the effect of transgenic APPsw on BACE1-AS, BACE1mRNA expression and the generation of Aβ1-40and Aβ1-42using cell line20E2derived from HEK293cells stable transfected by Swedish mutation APP gene. Next, we inhibit the expression of BACE1-AS using siRNA, to observe the change on the BACE1mRNA and BACE1protein expression, and the effect on Aβ1-40and Aβ1-42generation.3. To make it clear that if NF-κB is involved in the regulation of BACE1-AS expression, We detect the phospho-NF-KB p65level using Western method in vivo by stereotactic intrahippocampal injection of Aβ1-42on mouse, in vitro by1μM Aβ1-42treatment on SH-SY5Y cells. Then, before treated by Aβ1-42, we pretreat SH-SY5Y cells with celastrol, one of the NF-κB inhibitors, in vitro, treat APPsw transgenic cells20E2with celastrol to observe the expression of BACE1-AS, BACE1mRNA and BACE1protein, as well as Aβ generation.Results1. The mechanism of exogenous Aβ1-42promotion BACE1expression and Aβ generation mediated by BACE1-AS in cell and animal.1.1Exogenous Aβ1-42promote the expression of BACE1-AS, BACE1and the generation of Aβ in SH-SY5Y cells. Two hours after the administration of A β1-42(1μM) into the SH-SY5Y cells, the expression of BACE1-AS,BACE1mRNA, BACE1protein were elevated; the Aβ1-40concentration in extracellular fluid increased24hours after the administration. The above results are agreement with the reports of Faghihi MA et al. Pretreatment of BACE1-AS siRNA inhibit the promotion effect of Aβ1-42on the expression of BACE1-AS, BACE1and the generation of Aβ in SH-SY5Y cells obviously. After the pretreatment of BACE1-AS siRNA, the content of BACE1-AS. BACE1mRNA decreased, the expression of BACE1protein reduced, and the concentration of Aβ1-40in extracellular fluid diminished extremely. The results indicated that inhibition of BACE1-AS expression can inhibit the promotion effect of Aβ1-42on the expression of BACE1and the generation of Aβ in SH-SY5Y cells, which then break the post-transcriptional feed-forward mechanism involved by Aβ1-42activate BACE1-AS expression, increasing BACE1mRNA stability and generating additional Aβ1-42.1.2Exogenous Aβ1-42promote the expression of BACE1-AS, BACE1and the generation of Aβ in the brains of C57BL/6J mouse. Three days after stereotactic intrahippocampal injection of Aβ1-42, the expression of BACE1-AS and BACE1mRNA increased in the hippocampus on the injected side, and the content of A β1-40raised contrastted to the uninjected group. These reuslts indicated that exogenous Aβ1-42provided stereotactic intrahippocampal injection can promote the expression of BACE1-AS, BACE1and the generation of Aβ in the brains of C57BL/6J mouse.2. The mechanism of BACE1-AS involved in BACE1expression and Aβ generation in APPsw transgenic cells.2.1The influence on the expression of BACE1-AS, BACE1and the generation of Aβ by transfer APPsw gene into HEK293cells. The expression of BACE1-AS, BACE1mNRA increased in20E2cells, which is obtained by transfer APPsw gene into the HEK293cells, the content of the BACE1protein and the concentration of Aβ1-40, Aβ1-42all increased remarkablely contrast to the HEK293cells. These results demonstrated that the expression of BACEl-AS is excessively activated in APPsw transgenci cells.2.2The influence on the expression of BACE1mNRA, BACE1mRNA, BACEl protein, and the generation of Aβ1-40, Aβ1-42by inhibit the expression of BACE1-AS through siRNA. After the addition of BACE1-AS siRNA into the APPsw transgenic20E2cells, the experssion of BACE1-AS was inhibited, the content of mRNA and the protein of BACE1both diminished extremely, the concentration of Aβ1-40and Aβ1-42reduced remarkably. These results indicated that inhibition of BACE1-AS expression can inhibit the promotion effect of endogenous Aβ1-42on the expression of BACEl and the generation of Aβ in SH-SY5Y cells, which then break the post-transcriptional feed-forward mechanism involved in the activation of BACE1-AS, BACE1mRNA expression and A(3generating.3. The mechanism of NF-κB regulate BACE1-AS expression and Aβ generation.3.1In order to make it clear if the activation of BACE1-AS expression by exogenous Aβ1-42is mediated by NF-κB, we detected the expression of phospho-NF-κB p65, the activation form of NF-κB3days after stereotactic intrahippocampal injection of Aβ1-42in mouse, two hours after the addition of1-42(1μM) into the cultured SH-SY5Y cells. The Western bolt results indicated that the content of phospho-NF-κB p65increased extremely after the Aβ1-42treatment, and decreased when the cells were pretreated by celastrol (5μM), an inhibitor of NF-κB, which demonstrated that ectogenesis Aβ1-42can promote the activation of NF-κB in vitro, and celastrol can inhibit the Aβ1-42induced NF-κB activation. After the inhibition of Aβ1-42induced NF-κB activation, the experssion of BACE1-AS, BACE1mRNA dicreased extremely, and the content of BACE1protein reduced, the concentration of Aβ1-40in the extracellular fluid depressed. The expression of BACE1-AS and the generation of Aβ1-40can be inhibited by BACE1-AS siRNA in the ectogenesis Aβ1-42treatment SH-SY5Y cells, but the increase of phospho-NF-κB p65expression is not affected. So, we can conclude that the promotion of BACE1-AS expression and Aβ generation by exogenous Aβ1-42is mediated by NF-κ B.3.2NF-κB mediate the activation of BACEl-AS expression in APPsw transgenic cells. Western blot show that the content of phospho-NF-κB p65in APPsw transgenic20E2cells is extremely higher than that in HEK293cells, which indicated that trangenosis of APPsw gene can activate the expression of NF-κB. After the inhibition of NF-κB activation by5μ M celastrol in the20E2cells, the experssion of BACE1-AS, BACE1mRNA dicreased extremely, and the content of BACE1protein reduced, the concentration of Aβ1-40and Aβ1-42in the extracellular fluid depressed than the untreated cells. The expression of BACE1-AS and the generation of Aβ1-40, Aβ1-42can be inhibited by BACE1-AS siRNA in the20E2cells, but the increase of phospho-NF-κB p65expression is not affected. So, we can conclude that the promotion of BACE1-AS expression and Aβ generation by endogenous Aβ1-42is mediated by NF-κB. Conclusion1. The mechanism of exogenous Aβ1-42promote BACE1expression and Aβ generation is mediated by BACE1-AS in cell and animal.2. BACE1-AS is involved in the mechanism of BACE1expression and Aβ generation in APPsw transgenic cells.3. NF-κB regulate the expression of BACE1-AS and Aβ generation promoted by Aβ1-42.