A Single Nucleotide Polymorphism In Pri-miR-146a Reduces The Expression Of MiR-146a And Is Associated With The Pathogenesis Of Alzheimer’s Disease

Background and objectAlzheimer’s disease (AD) is a progressive neurodegenerative disorder that is characterized by progressive memory loss、aphasia、out of use、agnosia、 visual spatial disorder、abstract thinking and computational power and behavioral changes and the most common form of dementia among the aging population. AD is characterized by neurodegeneration that is associated with neurofibrillary tangles and neuritic plaques. There are about 5%people in the old people that is over 65 years old.In 2006, the worldwide prevalence of AD was 26.6 million, and by 2050, AD is predicted to attain 1 in 85 people globally. At present, treatment options to AD can only be used according to its symptoms, and there are no available treatments to the prevention or to delay the disease process. However, the molecular mechanisms that underlie this relationship are not fully understood.The neuropathological alterations of AD are characterized by loss of synapses and neurons, intracellular neurofibrillary tangles and extracellular amyloid plaques formation. The classical hypothesis for the cause of AD is aberrant amyloid protein deposition of Aβ42, due to abnormal amyloid precursor protein (APP) processing. Recently, an increasing number of studies indicate that dysfunctions of neuroimmune networks also contribute to the pathogenesis of AD.The immunue system of the body is related to AD, and the congenital immune system is important to cerebral metabolism,nerve protection and restoration. If the immunue system and the inflammatory signaling pathways are activated,the body can overproduct oxygen free radicals, and the promoting inflammatory cytokines and prostaglandins will express largely,the inflammatory cascade reaction may be caused. At last the result is neurodegenerative disorder and may cause AD. Based on the above,now the pathogenesis of AD is also needed to further study.MicroRNAs (miRNAs) are a family of mature,21 to 25 nucleotide-long small noncoding and highly conserved RNA gene products that regulate gene expression by base pairing with target mRNAs at the 3’-untranslated region, leading to mRNA cleavage or translational repression. It has been suggested that miRNAs are involved in various biological processes, the dysregulation of miRNA has been implicated in the development of different pathologies, including the development of disease, cell differentiation, cell proliferation and cell death.It has been reported that the abnormal expression of miRNA is related to the occurrence,the development and prognosis of many diseases.Single-nucleotide polymorphisms (SNPs) are the most common-types of genetic variation in the human genome and contribute to human phenotypic differences. Currently, some researchers believed that the single nucleotide polymorphisms (SNPs) in miRNA or it’s binding sites were the abnormal source of genetic variation, which contributed to cancer susceptibility. There were two different nucleotides on specific position in the genome; whose frequency was no less than 1% in the population. The difference of a single nucleotide of this genomic is known as SNPs, which has a specific biology, can regulate the expression of miRNA and effect its regulatory functions, thus has significant association of the tumor development. SNPs are the minor differences in gene sequence which are present in each individual human population and fairly well be distributed throughout the human genome.Dysregulated serum miRNA events, such as the down-regulation of miR-137, miR-181c, miR-9,miR-16 and miR-29a/b in the blood of AD patients,were reported. Furthermore, prior studies pointed out that miR-146 was involved in up-regulated inflammatory signaling as associated with prion-induced neurodegeneration, rheumatoid arthritis and temporal lobe epilepsy. Particularly, the upregulation of miR-146 has also been demonstrated in human AD brain, suggesting that the misregulation of miR-146 could contribute to the inflammatory senile plaques pathology observed in AD, it has also been identified to play a crucial role in AD since aberrant expression of miR-146a has been shown in five different AD transgenic mouse models and human AD brain. Notably, miRNA-146a has been found to be particularly up-regulated in anatomical regions exhibiting AD neuropathology, while unchanged in the brain regions unaffected.However,due to the small sample sizes in the above reports, there is a lack of an extensive and genomewide analysis for the diagnostic value of miRNAs in AD patients.In this study, we sequenced pri-miR-146a in the genomic DNA of 103 AD patients. Although no previously undescribed sequence changes were detected, we noticed one normal polymorphism site was related to AD. Detected by using qRT-PCR, we proved the conclusion that the rare allele C could reduce mature miR-146a expression. More importantly, the reduction of miR-146a further led to less efficient inhibition of target genes like TLR2, which enhanced microglia participated inflammation.And at last it could result in AD.Methods1. Collected specimensA total of 103 patients with AD and 206 healthy controls were recruited from the hospital of the author.. All recruited people were Han population. All of the patients are independent of unrelated han population. Normal control group randomly selected from the hospital health examination center. And no obvious clinical symptoms. Matching the corresponding control according to age. We collected their blood samples. The study was approved by Ethics Committee of the hospital of the author,and informed consent was obtained from all participants.2. Cell cultureRAW264.7, HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum,100 IU/ml penicillin and 10 mg/mL streptomycin. All cells were maintained at 37℃ under an atmosphere of 5% CO2.3. RNA extraction and the detection of miR-146aTotal RNA from samples and cell lines were obtained with the TRIzol isolation reagent, and the total RNA were reverse transcribed to synthesize cDNA. qRT-PCR method was used to quantify mature miR-146a and pri-miR-146a.4. vector constructionObtaining the mutants of clips by PCR, gel extraction, enzyme digestion the mutants of clips and vector, and then connection, conversion and pick the cloning, then identified by PCR and enzyme digestion.5. Transient miRNA-146a transfectionHEK293T cell was chosen to do the transfection experiments which had the lowest expression of miR-146a. HEK293T cells in 12-well plates were transfected with miR146a precursor (miR-146a mimic) or scrambled pre-miR negative control (miR-146a mimic NC), then measured the influence of miR-146a to the HEK293T cell.6. Dual luciferase assayFor luciferase reporter assays, HEK293T cells were seeded in 48-well plates. MiR-146a mimic/inhibitor and luciferase reporter vectors were co-transfected by using lipofectamine 2000. Two days later, cells were harvested and assayed with the Dual-Luciferase Assay. Each treatment was performed in triplicate in three independent experiments. The results were expressed as relative luciferase activity.7.Western blottingThe proteins were separated by electrophoresis, and the proteins in the gels were blotted onto PVDF membranes by electrophoretic transfer. The membrane was incubated with monoclonal antibody. The specific proteinantibody complex was detected by using horseradish peroxidase conjugated goat anti rabbit or rabbit anti-mouse IgG. Detection by the chemiluminescence reaction was carried using the ECL kit. The β-actin signal was used as a loading control.8. ELISAUsing the kit of ELISA to detect the level of TNF-a in the cell supernate. 9. Statistical analysisBiostatistical analyses were performed using SPSS 16.0.Student’s t-test, one-way ANOVA, or x2-test was using to test the difference among groups. Pearson’s x2-test was used to compare the the basic situation between the case group and the control group. P< 0.05 was considered statistically significant.Results1.The rare allele C of rs2910164 is significantly increased in ADTo wander, if there were some relations between nucleotides variant of pre-miR-146 and the pathogenesis of AD, we scanned pre-miR-146a coding region in 103 AD patients and 206 healthy controls by Taqman. we noticed the rare allele C was abnormally increased in the blood of AD2.The C allele can reduce miR-146a expression in vitro and in vivoDetecting mature miR-146a amount in different genotype pri-miR-146a expression vectors transient transfected HEK293T cells by qRT-PCR. As expected, C allele reduced mature miR-146a expression to 32% relative to G allele. To understand whether this SNP affect the expression of miR-146a in vivo; we detected miR-146a expression level in the serum of Chinese-Han people between GG and CC genotype by qRT-PCR, the expression of miR-146 was used as the internal normalization control. Our data demonstrated that the miR-146a level of CC genotype in AD patients was significantly down-regulated.3.The expression of TLR2 is repressed by miR-146aTo validate whether TLR2 be the target gene of miR-146a, the full length of 828bp segment of TLR23’-UTR was cloned into the downstream of the firefly luciferase reporter gene in the pGL3 control vector (designated as pGL3-TLR2) for the dual luciferase assay. HEK293T cells were co-transfected with pGL3-TLR2 and miR-146a mimics or inhibitor. Compared with the miRNA control, the luciferase activity was significantly suppressed by the miR-146a.4.The rare allele C in pri-miR-146a up-regulates RAW264.7 cells TNF-α production when Aβ42 stimulatedWe detected the biological function of rare allele C in this cell line. RAW264.7 cells were co-transfected with different allele pri-miR-146a, As shown, compared with the cells transfected with empty vector, the supernatant TNF-α amount raised 71.1% in the cells transfected with pri-miR-146a-C when they were stimulated by Aβ42.Conclusions1. There is nucleotides variant of pre-miR-146 in ADs;2. MiR-146a nucleotides variant rs2910164 C allele is related to the risk of suffering ADs;3. MiR-146a plays an important role in the occurrence of ADs.