Bioinformatics analysis indicates that microRNA‑628‑5p overexpression may alleviate Alzheimer's disease by targeting TYROBP
- Authors:
- Published online on: December 13, 2020 https://doi.org/10.3892/mmr.2020.11781
- Article Number: 142
Abstract
Introduction
Alzheimer's disease (AD) has become a global health issue; >44 million people are currently living with AD and the global health care cost in 2010 was ~US$818 billion (1). AD is a severe neurodegenerative disease associated with progressive cognitive decline (2). The pathogenesis of AD is complex, and the precise underlying mechanism has not yet been elucidated. The amyloid hypothesis is widely accepted for the pathogenesis of AD (3). As per this hypothesis, accumulation of β-amyloid (Aβ) peptides in brain cells is the primary pathological change in AD. Therefore, drugs such as Semagacestat (4) and Tramiprosate (5) targeting inhibition of Aβ aggregation have been developed. However, no significant therapeutic effects have been observed in phase III trials of these drugs (6). Thus, identifying the precise molecular mechanisms of AD progression is necessary.
Transmembrane immune signaling adaptor TYROBP (TYROBP) is a type I transmembrane protein expressed in numerous types of immune cell, such as osteoclasts, macrophages and monocytes (7). The gene encoding TYROBP is located on the long arm of chromosome 19 at position 13.1 and contains five exons (8). TYROBP is a necessary component of activating signal transduction (9). It is typically expressed on microglial cells in the brain (10). Certain TYROBP-associated immune receptors, such as triggering receptor expressed on myeloid cells (TREM)2, signal regulatory protein β1 and complement receptor (CR)3, have been identified (10). The interaction between spleen tyrosine kinase and phosphorylated TYROBP leads to intracellular calcium mobilization and modulates immune cell function (11). In addition, TYROBP suppresses the inflammatory response by downregulating cytokine production and secretion (12). TYROBP-mediated signaling is considered to participate in regulating the expression levels of multiple genes, including TREM1 and TREM2, in the brain (13).
Modern second-generation sequencing technology can reveal genetic changes between healthy individuals and patients (14). This technique has been used in AD to identify a number of relevant genes such as TREM1 and TYROBP (15). However, owing to the large number of altered genes, a single AD-promoting gene in AD has not been identified. Bioinformatics analysis for identifying hub genes may facilitate the identification of key diagnosis biomarkers and molecular drug targets (16). Before bioinformatics analysis, exclusion of irrelevant information caused by individual differences (17) is necessary. In addition, bioinformatics analysis results need to be verified experimentally.
The present study integrated two AD profiles and analyzed the co-expression of differentially expressed genes (DEGs) in both profiles. Bioinformatics analysis was used to identify the hub genes and relevant microRNAs (miRNAs or miRs). Finally, the function of these identified miRNAs in AD was verified experimentally.
Materials and methods
Preliminary analysis of GSE113141 (18) and GSE104249 (19)
A total of two AD profiles were acquired from the Gene Expression Omnibus database (ncbi.nlm.nih.gov/gds/): GSE113141, containing information from six APP/PS1 transgenic and six normal mice, and GSE104249, containing information from three APP/PS1 transgenic and three normal mice. R software (version R 3.2.3) (20) and R studio (version R 3.5.2) (21) with the LIMMA package was used for homogenizing the raw data and identifying co-expressed DEGs.
Bioinformatics analysis
The Database for Annotation, Visualization and Integrated Discovery online tool (david.ncifcrf.gov/summary.jsp) was used for Gene Ontology (GO; http://david.ncifcrf.gov/) and Kyoto Encyclopedia of Genes and Genomes (KEGG; http://david.ncifcrf.gov/) (22) pathway analysis of all co-expressed DEGs. These genes were used as input for the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (string-db.org/cgi/input.pl) to acquire a protein-protein interaction (PPI) network, and Cytoscape (23) was used to visualize the network. The cytoHubba plugin in Cytoscape (version 3.6.1) was used to calculate the hub genes of AD. GeneMANIA (genemania.org/) was used to identify the interactions between the co-expressed genes. TargetScan (targetscan.org/) was utilized to predict the relevant miRNAs that bind the hub gene. All the predicted miRNAs were integrated with GSE138382, a miRNA gene profile associated with AD containing information from three APP/PS1 transgenic mice and three normal mice.
Reverse transcription-quantitative (RT-q)PCR validation
RNA (from cells, tissues and blood) was extracted using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). NanoDrop ND-1000 (Thermo Fisher Scientific, Inc.) was used to detect the integrity and concentration of the RNA samples. Total RNA was reverse-transcribed to cDNA using PrimeScript RT reagent kit with gDNA Eraser (Takara Bio, Inc.) according to the manufacturer's instructions. RT-qPCR was performed using FastStart Universal SYBR Green Master (ROX) (Roche Diagnostics) using an Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) to confirm the relative expression levels of miR-628-5p and TYROBP in mouse brain tissue. In addition, TYROBP and miR-628-5p were detected in blood samples from patients with AD. qPCR thermocycling conditions were 95°C for 5 min, followed by 40 cycles at 95°C for 10 sec, 55°C for 20 sec and 72°C for 20 sec with an extension step of 72°C for 2 min. Results were normalized to those of U6 or GAPDH and calculated using the 2−ΔΔCq (24) method. The primers were as follows: Human TYROBP forward, 5′-ACTGAGACCGAGTCGCCTTAT-3′ and reverse, 5′-ATACGGCCTCTGTGTGTTGAG-3′; moue TYROBP forward, 5′-GAGTGACACTTTCCCAAGATGC-3′ and reverse, 5′-CCTTGACCTCGGGAGACCA-3′; human GAPDH forward, 5′-CGGACCAATACGACCAAATCCG-3′ and reverse, 5′-AGCCACATCGCTCAGACACC-3′; mouse GAPDH forward, 5′-AGGTCGGTGTGAACGGATTTG-3′ and reverse, 5′-GGGGTCGTTGATGGCAACA-3′; human miR-628-5p forward, 5′-GATGCTGGATGCTGACATATTTAC-3′ and reverse, 5′-TATGGTTGTTCTGCTCTCTGTCTC-3′; and U6 forward, 5′-GCGCGTCGTGAAGCGTTC-3′ and reverse, 5′-GTGCAGGGTCCGAGGT-3′.
Participants
The present study was performed in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Changchun University of Chinese Medicine (Changchun, China; approval no. IRB:2019024421). Blood (4 ml) was obtained from median cubital vein of patients with AD or healthy controls in Department of Geriatrics or Medical Examination Center (Affiliated Hospital to Changchun University of Chinese Medicine, Changchun, China). The participants were enrolled between February 2019 and July 2019. The inclusion and exclusion criteria of AD was based on the criteria from the National Institute of Neurological and Communicative Diseases and Stroke-Alzheimer's Disease and Related Disorders Association (25). Participants were divided into two groups, AD and healthy control, based on the criteria from the National Institute of Neurological and Communicative Diseases and Stroke-Alzheimer's Disease and Related Disorders Association (25). Blood samples were collected from 20 participants, including 10 patients with AD and 10 healthy controls. The AD group comprised six females and four males (age, 71–87 years; mean age, 77.6±4.6 years). The control group comprised five females and five males (age, 73–82 years; mean age, 76.9±2.7 years). All patients agreed to the use of their samples in scientific research and provided written informed consent.
Animals
Male 6-month-old APP/PS1 (n=3) and C57/6J (normal) (n=3) mice (weight 24–28 g) were purchased from the Chinese Academy of Medical Sciences (Shanghai, China). All mice were housed in 12 h light/dark cycle conditions at a temperature of 22±1°C and a humidity of 55±2% and given food and water ad libitum. All mice were anesthetized with isoflurane (induction, 5%; maintenance, 1.5–2.5%). The hippocampus and cortex were dissected for subsequent experiments, including RT-qPCR and western blotting. Then all the mice were euthanized via cervical dislocation. Animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (26) and approved by The Changchun University of Chinese Medicine Animal Care and Use Committee (Changchun, China; approval no. KT202009096).
Cell culture
U251 glioblastoma cells were purchased from Institute of Biochemistry and Cell Biology and cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) with 10% FBS (cat. no. 0010; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified 5% CO2 incubator. The medium was replaced every 3 days. Mimic and inhibitor were designed by Shanghai GenePharma Co., Ltd. For transfection of miR-628-5p mimics (250 pmol; forward, 5′-AUGCUGACAUAUUUACUAGAGG-3′ and reverse, 5′-UCUAGUAAAUAUGUCAGCAUUU-3′) and inhibitor (250 pmol, 5′-CCUCUAGUAAAUAUGUCAGCAU-3′), cells were transfected with Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). In the control group, cells were transfected with Lipofectamine® 2000 alone. The time interval between transfection and subsequent experimentation was 48 h. After media was replaced with serum-free Opti-MEM (Gibco; Thermo Fisher Scientific, Inc.), the transfection complexes were added for 6 h at 37°C in a humidified 5% CO2 incubator. Then the medium was replaced with the standard culture medium (DMEM with 10% FBS) and cells harvested following incubation for 48 h at 37°C.
Western blotting
The protein was extracted using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) and quantified using a BCA assay (Thermo Fisher Scientific, Inc.). Total cell protein content (30 µg/lane) was separated by 10% SDS-PAGE (cat. no. 1200; Beijing Solarbio Science & Technology Co., Ltd.) and transferred to nitrocellulose membranes (0.45 µm; EMD Millipore). Blots were blocked using 5% non-fat milk with 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Diluted antibodies against TYROBP (cat. no. ab124834), Aβ (cat. no. ab224275)and Aβ precursor protein (APP; cat. no. ab32136; all at 1:1,000 and all from Abcam) were used to incubate the membranes overnight at 4°C, followed by incubation with secondary antibody (β-actin; 1:5,000; cat. no. bs-0061R, Bioss) at room temperature for 2.5 h. The blots were then visualized with ECL using a Series Molecular Imaging Biosystem (Azure Biosystems, Inc.). Protein expression levels were semi-quantified using ImageJ software (version 4.62; National Institutes of Health).
Dual luciferase reporter assay
293T cells were purchased from Institute of Biochemistry and Cell Biology. A total of 2×104 293T cells per well were seeded into 6-well plates for 24 h at 37°C before transfection. The wild-type (WT) 3′-UTR sequence (16–23 bp) of TYROBP that can bind to miR-628-5p or the mutant (MUT) 3′-UTR sequence was amplified and then cloned into a pGL3 vector (Promega Corporation). pGL6-TYROBP-WT, pGL6-TYROBP-MUT and miR-628-5p mimics as well as mimic controls were co-transfected into 293T cells for 24 h at 37°C using Lipofectamine® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). At 24 h post-transfection, the absorbance of the luciferase was measured at 560 nm. A dual luciferase reporter gene assay kit (Promega Corporation) was used to measure luciferase activities according to the manufacturer's protocol. Luciferase activity was normalized to Renilla luciferase.
Statistical analysis
Continuous data are expressed as the mean ± standard deviation. All experiments were performed in triplicate. One-way ANOVA followed by post hoc Tukey's test was performed to assess differences between multiple groups. All statistical analysis was performed using SPSS 22.0 software (IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
Results
Integration of GSE113141 and GSE104249
Raw data were normalized using R software and integrated (Fig. 1). The integration included information from nine APP/PS1 transgenic mice and nine normal mice. The genes co-expressed in both GSE113141 and GSE104249 were selected. There were 22 different co-expressing mRNAs in both AD profiles, including Lyz1, Clec7a, Cybb, Thbs1, Pilra, Itgb2, Tyrobp, Rac2, Cst7, Lcp1, Klk6, Wfdc17, Top2a, Lilrb4a, Lyz2, Ctse, Ptprc, Cd36, Igsf6, Mpeg1, Tnni2 and Cd52. These 22 genes were subjected to bioinformatics analysis following homogenization. These results demonstrated that these 22 genes might participate in the progress of AD.
GO analysis
Co-expression of different mRNAs for biological processes (‘immune system process’ and ‘defense response’) was observed. The cellular component was associated with ‘Extracellular space’, ‘Rough endoplasmic reticulum lumen’ and ‘Golgi cis cisterna’. The molecular function primarily corresponded to ‘lysozyme activity’, ‘Low-density lipoprotein particle binding’ and ‘Transforming growth factor β binding’ (Table I; Fig. 2). These results demonstrated that immune system process, rough endoplasmic reticulum lumen and lysozyme activity might participate in the progress of AD.
KEGG signaling pathway
All co-expressed DEG were enriched for ‘phagosome’ and ‘malaria’ (Fig. 3).
PPI network and hub genes
Interaction analysis using the STRING database demonstrated 22 nodes with 69 edges. These nodes and edges were utilized by Cytoscape for pictorial depiction of the network. The five hub genes predicted by CytoHubba analysis were those encoding TYROBP (score=14), cytochrome b-245 β chain (score=13), integrin sub-unit β 2 (score=12), protein tyrosine phosphatase receptor type C (score=12) and lysozyme 2 (score 11) (Fig. 4). GeneMANIA database also demonstrated that TYROBP served a central role in ‘physical interactions’, ‘pathway’, ‘predicted’ and ‘co-expression’ networks (Fig. 5A-D, respectively). These results demonstrated that the function of TYROBP acted as a hub gene in AD and required further investigation.
TargetScan prediction
TargetScan (version 7.2; www.targetscan.org/vert_72) was used to predict miRNAs that target TYROBP (Table SI). In total, there were 77 miRNAs having binding sites for TYROBP. Subsequent integration of GSE138382 with the 77 predicted miRNAs demonstrated only one miRNA, miR-628-5p, that was downregulated in GSE138382; this was also predicted to target TYROBP. These results demonstrated miR-628-5p/TYROBP may be used as an AD biomarker and molecular drug candidate.
High expression levels of TYROBP in APP/PS1 mice
RT-qPCR verification was performed using samples taken from mice (three APP/PS1 and three normal) and patients with AD. Analysis using RT-qPCR indicated that TYROBP was increased in APP/PS1 mice (Fig. 6A). Low miR-628-5p expression levels were detected in blood from patients with AD (Fig. 6A and B), which was consistent with GSE147232 (a miRNA microarray profile in the plasma of patients with mild cognitive impairment due to AD). Western blot analysis also confirmed that TYROBP, APP and Aβ were more highly expressed in APP/PS1 mice compared with normal mice (Fig. 6C and D). These results demonstrated that high expression of TYROBP fulfilled important roles in AD.
Increased miR-628-5p expression levels inhibit TYROBP in vitro
The transfection efficiency of miR-628-5p was confirmed by RT-qPCR. U251 cells transfected with miR-628-5p mimic demonstrated increased expression levels of miR-628-5p (Fig. 6E), whereas those transfected with miR-628-5p inhibitor exhibited upregulated TYROBP, compared with the control. Western blot analysis demonstrated that decreased expression of miR-628-5p resulted in the upregulation of TYROBP, APP and Aβ compared with control group (Fig. 6F). These results demonstrated that high expression of miR-628-5p could inhibit TYROBP in U251 cells.
TYROBP targeting by miR-628-5p
Dual-luciferase reporter analysis demonstrated that co-transfection with miR-628-5p mimics significantly decreased the luciferase activity of the wild-type TYROBP reporter compared with that in the normal or mimic inhibitor samples and the mutant reporter (Fig. 7). These results demonstrated that miR-628-5p had binding sites with TYROBP and could inhibit translation of TYROBP in AD.
Discussion
AD has become one of the worldwide disorders involving central nervous system neurodegeneration and cognitive decline (27). However, its detailed molecular pathogenesis remains unclear. Although a number of hypotheses such as the cholinergic hypothesis (28), amyloid hypothesis (29) and tau hypothesis (30) have been proposed, the amyloid hypothesis is the most widely accepted (29). A number of drugs, including Semagacestat (4) and Tramiprosate (5) have been developed to inhibit Aβ; however, results have been unsatisfactory. For example, Semagacestat failed phase III clinical trials for AD treatment (4). Thus, further studies on the molecular pathogenesis of AD and novel drug targets are necessary.
Second-generation sequencing technology provides a fast method to screen DEGs between normal and impaired tissue such as brain, liver and kidney (14). Differential expression levels of both coding and non-coding RNAs may provide novel biomarkers for diagnosis and treatment targets (31). However, the abundance of DEGs obtained by this technique may mask core pathogenic genes. In addition, ‘junk’ information caused by individual differences (17) may be included among sequencing results. Therefore, normalization of raw data and integration analysis are necessary to provide reliable data for bioinformatics analysis.
The present GO analysis indicated that biological processes of AD were associated with immune and inflammatory responses. Neuro-inflammation has been reported to be involved in the progress of AD (32). Microglia in the brain produce pro-inflammatory cytokines to activate the inflammatory response. Moreover, NLR pyrin family domain containing 3 (NLRP3) has been reported to be activated by Aβ (33); activated NLRP3 can enhance AD progression via the chronic inflammatory response. In addition, the complement system is a family of key proteins that regulate the immune response and is considered to be the first line of homeostasis maintenance (34). Members of the complement system have also been found to have high expression levels in patients with AD. For example, C1q protein expression levels, associated with the classical complement pathway, are upregulated following activation by Aβ, and cause neuronal atrophy (35). Another study indicated that inhibiting C1q decrease synapse loss in mice (36).
Following bioinformatics analysis, TYROBP was predicted as a hub gene for AD. High expression levels of TYROBP in AD have previously been reported (37), which is consistent with previous results from second generation sequencing (15). However, previous research has focused on Aβ42, τ and TREM2 instead of TYROBP (38). Consistent with the present GO analysis, TYROBP serves as a core molecule in the immune system, particularly as the adapter for TREM2 and CRs, which control signal transduction (39). TREM2 and CR3 have been demonstrated to regulate the pathogenesis of AD (39). In addition, TYROBP inhibits inflammatory responses via downregulation of microglia-mediated cytokine production (40). Based on the amyloid hypothesis, a number of drugs have been developed to inhibit Aβ. TYROBP may downregulate Aβ and clear apoptotic neurons by enhancing microglial phagocytic activity (41). To the best of our knowledge, the present study is the first to use bioinformatics analysis to predict TYROBP as a hub gene in AD. TYROBP may be a novel AD diagnosis biomarker and treatment target.
Previous studies have reported the function of TYROBP in AD (42,43). Although the detailed mechanism underlying the cognitive decline of AD remains unknown, a partial loss of the colony stimulating factor 1 receptor (CSF1R)/TYROBP signaling pathway has been reported in neurological phenotypes (44). The immune response is reported to serve an important role during AD progression (45). TYROBP may mediate immune cell phagocytosis by regulating CSF1R and by interfering with macrophage proliferation, leading to white matter disease, which is clinically characterized by behavioral, cognitive and motor abnormalities (46). In addition, Haure-Mirande et al (41) suggested that TYROBP slows, arrests or prevents the development of sporadic late-onset AD (LOAD). The complement system is activated in AD (41). Deletion of TYROBP in a tauopathy mouse model downregulates C1q expression levels, leading to elevated learning behavior (retention tests) and synaptic function (47). Therefore, decreased expression levels of complement by TYROBP may be beneficial during AD pathology by improving learning behavior and synaptic function. Sekiya et al (38) found that glial expression of TYROBP promotes τ-mediated neurodegeneration and affects LOAD; furthermore, TYROBP was identified as a potential target gene by comparing human AD profiles, which is similar to the results of the present study.
miRNAs are a class of non-coding RNA molecule that inhibit translation or cause degradation of target mRNAs (48). To date, miR-628-5p has not been reported to participate in AD. Previously, miR-628-5p has been reported to inhibit glioblastoma cell proliferation and migration by targeting high mobility group box 3 (49); it can also decrease the proliferation of prostate (50) and epithelial ovarian cancer cells (51) by targeting fibroblast growth factor receptor 2 and inhibiting glioma proliferation by binding DEAD-box helicase 59 (52). Therefore, miR-628-5p may serve as a suppressor miRNA targets TYROBP, highlighting the potential of TYROBP as a novel therapeutic target for AD.
To the best of our knowledge, the present study is the first to use bioinformatics analysis and experimental confirmation to predict that miR-628-5p may inhibit the expression of AD-associated proteins. miR-628-5p/TYROBP may serve as a novel molecular diagnostic marker and candidate therapeutic target for AD.
Supplementary Material
Supporting Data
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
ML and LY conceived and designed the study. SL, CM, XW and YS performed the experiments and collected the data. ML, HL and ZG analyzed and interpreted the data. ML performed experiments and drafted the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All experiments involving animals were approved by The Changchun University of Chinese Medicine Animal Care and Use Committee. RNA extraction, RNA reverse transcription and RT-qPCR validation involving patients' blood was approved by the Ethics Committee of Changchun University of Chinese Medicine.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Glossary
Abbreviations
Abbreviations:
BP |
biological processes |
CC |
cellular component |
DEGs |
differentially expressed genes |
GO |
Gene Ontology |
KEGG |
Kyoto Encyclopedia of Genes and Genomes |
MF |
molecular function |
PPI |
protein-protein interaction |
RT-q |
reverse transcription-quantitative |
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