Amyloid precursor protein (APP) has an important function in the generation of Alzheimer’s disease (AD). In our previous study, miR-193b was found to be downregulated in the hippocampi of 9-month-old APP/PS1 double-transgenic mice using microRNA (miR) array. In the present study, bioinformatic analyses showed that miR-193b was a miR that was predicted to potentially target the 3′-untranslated region (UTR) of APP. Subsequently, the function of miR-193b on APP was studied. The levels of miR-193b, exosomal miR-193b, Aβ, tau, p-tau, HCY and APOE in samples from APP/PS1 double-transgenic mice, mild cognitive impairment (MCI) and dementia of Alzheimer-type (DAT) patients, were measured. The results indicated that overexpression of miR-193b could repress the mRNA and protein expression of APP. The miR-193b inhibitor oligonucleotide induced upregulation of APP. Binding sites of miR-193b in the 3′-UTR of APP were identified by luciferase assay. MCI and DAT patients had lower exosomal miR-193b, but not total miR-193b, in the blood as compared with the controls. DAT patients had lower exosomal miR-193b levels in blood as compared with the MCI group. A decreased exosomal miR-193b expression level was additionally observed in the cerebral spinal fluid (CSF) of DAT patients. Negative correlations were found between exosomal miR-193b and Aβ42 in the CSF of DAT patients. In conclusion, these findings showed that miR-193b may function in the development of AD and exosomal miR-193b has potential as a novel, non-invasive, blood-based biomarker of MCI and DAT patients.
Alzheimer’s disease (AD) is a prominent neurodegenerative disorder characterized by progressive loss of memory and other cognitive functions. Despite considerable progress in genetics and cell biology, numerous questions remain regarding the mechanisms of neurodegeneration and the molecular and pathological components. Extracellular amyloid-β (Aβ), which is derived from the larger amyloid precursor protein (APP), is considered to be responsible for the death of neurons and dementia in Alzheimer’s disease. An increased expression of APP may increase the risk of AD (
MicroRNAs (miRs) are endogenous, short, noncoding RNAs. Mature miRs are single-stranded RNA molecules of ~20–25 nucleotides which act as important post-transcriptional regulators of gene expression by binding with their target mRNAs. They are additionally essential for normal neuronal function and survival (
Several cerebral spinal fluid (CSF) or blood-based markers, such as Aβ, tau and phosphorylated tau (p-tau) are proposed biomarkers for predicting future cognitive decline in healthy individuals, and the progression to dementia in patients who are cognitively impaired (
The design of the present study was approved by the ethics committee of Xuanwu Hospital of Capital Medical University (Beijing, China), and the written informed consents were obtained from all participants. A total of 43 MCI (23 females, 20 males, mean age 63.8±6.1) and 51 DAT patients (28 females, 23 males, mean age 64.2±6.5) were selected for this study. The CSF was drawn within 2 h following collection of blood (n=7). Age- and gender-matched control subjects were included in the experimental design. Samples were stored at −80°C until required for further analysis. The expression levels of Aβ, tau and p-tau in the plasma and CSF of the subjects were determined by ELISA kit from Cusabio (Wuhan, China). The study was approved by the Ethics Committee of Xuanwu Hospital of Capital Medical University (Beijing, China). Written informed consent was obtained from the patients’ family.
The 3, 6 and 9-month-old APP/PS1 double-transgenic mice on a C57BL/6J genetic background were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences & Comparative Medical Center (Beijing, China). All the animal protocols were approved by Ethics Committee of Xuanwu Hospital of Capital Medical University (Beijing, China). The non-transgenic mice were used as wild-type (WT) controls. Blood was taken by removing the eyeballs and CSF-like fluid was collected as previously described (
SH-SY5Y and HEK293 cell lines were purchased from Shanghai Institute of Cell Biology, China. Cells were grown in antibiotic-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. SH-SY5Y cells were transfected with 100 nM (final concentration) miR-193b mimic oligonucleotide, miR-193b inhibitor oligonucleotide or a non-specific control small interfering RNA siRNA) (GenePharma) using Lipofectamine™ 2000 reagent (Invitrogen Life Sciences, Carlsbad, CA, USA) according to the manufacturer’s instructions.
Reporter vectors containing the putative miR-193b target site from the APP 3′-UTR was synthesized with double-stranded oligos perfectly complementary to putative miR-193b target site and oligos in which the seed regions were mutated (
Total RNA from harvested cells was isolated using TRIzol™ Reagent (Invitrogen Life Sciences) according to the manufacturer’s instructions. Isolated RNA was reverse transcribed using the PrimeScript™ RT reagent (Takara Bio, Inc., Shiga, Japan). The mRNA expressions of APP was determined using SYBR® Green qPCR (Takara Bio, Inc) using a LightCycler 480 System (Roche Diagnostics, Mannheim, Germany). GAPDH was used to normalize the target genes. The PCR primer sequences were as follows: APP, forward, 5′-TTGCGAAACTCATCTTCACTGG-3′, reverse 5′-CAGTGGGCAACACACAAACTCTAC-3′; GAPDH, forward, 5′-GCACCGTCAAGGCTGAGAAC-3′, reverse 5′-TGGTGAAGACGCCAGTGGA-3′. The number of samples in each group was five.
Western blotting was performed as previously described (
The exosomes were isolated using the Total Exosome Isolation kit (Invitrogen Life Technologies) according to the manufacturer’s instructions. Briefly, the serum was centrifuged at 2,000 × g for 30 min to remove cells and debris. Following this, 400 μl clarified serum was transferred to a new tube and 0.4 volumes of the Total Exosome Isolation reagent was added. The serum/reagent solution was mixed and then incubated at 4°C for 30 min. After incubation, the samples were centrifuged at 10,000 × g for 10 min at room temperature. The supernatant was discarded and the pellet, containing the exosomes, at the bottom of the tube was resuspended in 200 μl phosphate-buffered saline (PBS).
Total RNA in 350 μl CSF, or 200 μl serum samples was extracted using a spin column method with an miRNeasy Serum/Plasma kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Exosomal RNA was isolated and purified using a Total Exosome RNA Isolation kit (Invitrogen Life Technologies). The RNA isolated from the CSF was 530–1,500 ng/ml, 2,100–4,700 ng/ml from serum, 270–420 ng/ml from the exosome. Total RNA in hippocampus tissue from the animal models and cultured cells was extracted using a spin column method using the miRNeasy kit (Qiagen). MiRs were reverse transcribed into cDNA using the miScript II RT kit (Qiagen) in a 10 μl reaction system. MiR-193b were determined by a TaqMan qPCR method (Applied Biosystems, Foster City, CA, USA), using U6 RNA as an endogenous control.
Statistical analyses were performed using SPSS 13.0 for Windows (SPSS, Inc., Chicago, IL, USA). For normally distributed data, results are expressed as the mean ± standard deviation. The differences between groups were assessed by
A total of 34 miRs were found to be putative targets on the 3′-UTR of APP. MiR-193b was a miR that may target the 3′-UTRs of APP (
As shown in
Over-expression of miR-193b significantly reduces fluorescence from APP reporter vectors in HEK293 cells (P<0.05). These reductions were not observed when mutations were made to the 3′UTR seed regions of APP or BACE-1 were generated (
The levels of miR-193b were significantly downregulated in the hippocampi of 3, 6 and 9 month APP/PS1 transgenic mice as compared with the WT mice (P<0.05). The levels of exosomal miR-193b were significantly downregulated in the CSF-like fluid and the serum of 3, 6 and 9 month APP/PS1 transgenic mice, as compared with the WT mice (P<0.05). Levels of exosomal miR-193b in the CSF-like fluid and serum of 6 and 9 month transgenic mice were significantly lower than that of the 3 month transgenic mice, respectively (P<0.05;
Compared with the control groups, patients with MCI and DAT had lower levels of exosomal miR-193b in the serum and plasma (P<0.05). Patients with DAT had lower exosomal miR-193b levels in their serum and plasma compared with the MCI groups (P<0.05). It was additionally found that exosomal miR-193b was decreased in the CSF of patients with DAT as compared with the control group (n=7; P<0.05;
The level of exosomal miR-193b was lower in the CSF compared with serum from a given individual (P<0.05; data not shown). There was no correlation between exosomal miRNA-193b levels and the CSF and serum from a given individual (data not shown). When the cutoff value was set as the mean concentration - two standard deviations of the controls, the positive rates of exosomal miR-193b were 71.43% (5/7) and 58.82% (30/51) in the CSF and serum of patients with DAT respectively, and 58.14% (25/43) in the serum of patients with MCI.
A weak, but significant, negative correlation was found between the levels of exosomal miR-193b and Aβ42 in the CSF of patients with DAT (r =−0.442, P<0.05), as well as the control group (r =−0.503, P<0.05). MiR-193b and exosomal miR-193b had no correlation with HCY, ApoE, tau and p-tau in the serum and CSF (data not shown).
The potential benefit in the analysis of miR in the diagnosis and treatment of numerous diseases, including cancer, infection and neurodegenerative disease has been previously evaluated by numerous researchers (
Previous studies have demonstrated that miRs are stably expressed in various bodily fluids, and their unique expression patterns can serve as fingerprints of various diseases, including AD (
APP/PS-1 double-transgenic mice contained insoluble amyloid peptides at the age of 6–9 months, concomitant with the formation of amyloid plaques (
In conclusion, these findings showed that miR-193b may function in the development of AD and exosomal miR-193b has potential as a novel, noninvasive blood-based biomarkers of patients with MCI and DAT.
This study was supported by the Natural Science Foundation of China (no. 81271924) and Research Fund for the Doctoral Program of Higher Education of China (no. 20121107110001). The authors would like to thank Dr Shuang Meng of the Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China for the vector construction and fluorescence detection.
Seed region of miR-193b in the 3′-UTRs of APP. APP, amyloid precursor protein; miR, microRNA.
Effect of miR-193b and miR-193b inhibitor on the (A) mRNA and (B and C) protein expression of APP in SH-SY5Y cells. N, negative controls; NC, non-specific control. *P<0.05 as compared with the NC group. Error bars represent the standard deviation. APP, amyloid precursor protein; miR, microRNA.
Identification of the miR-193b binding site on the 3′-UTRs of amyloid precursor protein was identified. *P<0.05 as compared with the NC group. NC, non-specific control. 3′UTR, 3′ untranslated region.
Levels of miR-193b in (A) hippocampus, (B) CSF and (C) serum of amyloid precursor protein/PS1 transgenic mice. (D and E) The levels of exosomal miR-193b in the CSF and serum of APP/PS1 transgenic mice. *P<0.05 as compared with the WT group. #P<0.05 as compared with the 3-month-old transgenic group. Error bars represent the standard deviation. WT, wild type; miR, microRNA; CSF, cerebral spinal fluid.
(A) Expression levels of miR-193b in the serum from patients with MCI and DAT. (B) The levels of miR-193b in CSF from patients with DAT. (C) The levels of exosomal miR-193b in the serum from patients with MCI and DAT. (D) The levels of exosomal miR-193b in CSF from patients with DAT. *P<0.05 as compared with the control group; #P<0.05 as compared with the MCI group. Error bars represent the standard deviation. MCI, mild cognitive impairment; DAT, dementia of Alzheimer type; CSF, cerebral spinal fluid; miR, microRNA.