The Effects Of Aβ25-35 On The Expressions Of C9ORF72 Gene And Protein In Vitro

Background and ObjectiveAlzheimer disease(AD)is the most common type of dementia in elders,which has become the severe medical issue of modern society with the aged tendency of population,characterized by the highest morbidity rate in neurological degenerative diseases.AD has two special pathological features including the formation of senile plaques(SPs)which consist of amyloid protein deposition and neurofibrillarytangles(NFTs)which may be as a result of excessive phosphorylation of tau proteins.Furthermore,most of studies have demonstrated that Amyloid β-protein(Aβ)playing a toxic role in neurons is universally regarded as the dominant sector of senile plaques.Chromosome 9 open reading frame(C9ORF72)located on chromosome 9 encodes twelve exons,however,the corresponding proteins have remained unclear.In addition,it has been clarified that the repeated expansion of GGGGCC in non-coding region exists in neurological degenerative diseases and is the main reason that leads to amyotrophic lateral sclerosis(ALS)and frontotemporal dementia(FTD).What’s more,the further studies have indicated that Alzheimer disease may be also responsible for C9ORF72 expansion mutation.Based on the above-mentioned references,our study aims at exploring the Aβ25-35-induced apoptotic effect on PC12 cells and its influences on the expressions of C9ORF72 whole genes and C9ORF72 proteins.Materials and MethodsAfter having using different concentrations of Aβ25-35(0 μM,10 μM,20 μM and 40 μM)to cultivate PC12 cells for 48 hours which were divided into four groups,we examined the situation of cell apoptosis by using AnnexinV/PI method.Quantative Real-time PCR(qRT-PCR)was performed to detect the expression of C9ORF72 whole genes in different groups and Western blotting was carried out to present the expression of C9ORF72 protein.Results(1)Apoptosis detection of PC12 cells: Compared with control group in the observation of scatter diagram,with the increase of Aβ25-35 concentration,both the number of apoptotic PC12 cells and the apoptotic rate of PC12 cells in right upper quadrant were elevated in each Aβ25-35 group(10 μM,20 μM and 40 μM).(2)The expression of C9ORF72 gene: 2-ΔΔCt values of control group,10 μM group,20 μM group and 40 μM group were 0.651±0.016,0.674±0.123,1.000±0.075 and 2.839±0.199,respectively,which showed that the more increasing concentration of Aβ25-35,the higher level mRNA presented and 20 μM group as well as 40 μM group displayed tremendously statistically significant difference compared with control group(all P<0.01).(3)The expression of C9ORF72 protein: According to graph of Western blotting,all groups had the expressions of C9ORF72 proteins.The levels of expressions of C9ORF72 proteins in control group,10 μM group,20 μM group and 40 μM group were 0.400±0.015,0.430±0.015,0.520±0.025 and 0.920±0.053,respectively,which showed that the levels of C9ORF72 protein expression were positively associated with the concentration of Aβ25-35,and 20 μM group as well as 40 μM group displayed tremendously statistically significant difference compared with control group(all P<0.01).Conclusions(1)Aβ25-35 has an apoptosic effect on PC12 cells.(2)Aβ25-35 is responsible for the high expressions of C9ORF72 gene and protein,which may be the one of the pathogenic machansim of Alzheimer disease.