Bioinformatic analysis of microRNA expression in Parkinson's disease
- Authors:
- Published online on: November 3, 2014 https://doi.org/10.3892/mmr.2014.2837
- Pages: 1079-1084
Abstract
Introduction
Parkinson’s disease (PD) is a disease of the nervous system, characterized by the loss of nigrostriatal dopaminergic neurons (NDN) (1). PD, also termed tremors paralysis, is a common age-associated neurodegenerative disease, which is associated with environmental and genetic factors (2,3), with a prevalence of 0.3% in the whole population and an increasing incidence in the elderly (4). The main clinical manifestation of PD includes motionless tremor, which is limited to one side of the body. Clinically, PD features motionless tremor, muscle rigidity, bradykinesia and postural instability (5). At present, the therapeutic methods used for the treatment of PD involve pharmacotherapy, gene therapy, rehabilitation and surgical treatment. In 1967, clinical studies demonstrated that oral levodopa can improve the symptoms of PD, establishing its current status in the treatment of PD. Levodopa remains the most effective drug in PD therapy (6). However, treatment with levodopa is associated with various side-effects, including a decline in efficacy following prolonged usage, switch effect and end-of-dose phenomenon (7). Based on current understanding of the neural circuits of the basal ganglia (8), PD therapy predominantly requires the use of a dopamine D1 receptor-stimulating agent (9).
microRNAs (miRNA) are small, endogenous, RNA-coding molecules, which are important in almost all biological pathways, particularly in the transcriptional regulation of gene expression. As a small molecule regulator of gene transcription and expression, miRNAs are involved in almost all important biological pathways and disease processes in the human body (10). In recent years, there have been an increasing number of studies regarding the association of miRNAs with various diseases and biological functions. miRNAs are important in cancer, inflammation and infection as well as cardiovascular, immune and degenerative diseases acting as molecular markers for screening, but also may be used as new targets for drug development and the prevention and control of severe diseases (11).
Small, gene regulating miRNAs may also be important in PD. It has previously been reported that miRNA regulates leucine repeat kinase-2 (LRRK2) which contributes to the etiology of sporadic PD (12). Jewish individuals of European and North African descent often carry a LRRK2 mutation. These mutations are strongly associated with the occurrence of PD, however, the specific mechanism remains to be elucidated.
Based on data from miRNA and mRNA double expression profiles, the present study identified a synergistic miRNA pattern in PD and constructed an miRNA network. Through gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation, the miRNA-associated pathogenesis of PD was determined. The changes in gene expression observed under pathological conditions assists in improving understanding of the pathogenesis of PD and facilitates the identification of corresponding targets for therapy. Gene expression profile analysis is a fast, high through-put detection method for miRNA expression in tissues or cells. Comparison of the differences in expression between patients and healthy controls using this method, improves knowledge of the pathogenesis and development of PD.
Materials and methods
miRNA and mRNA expression profiles of PD
The miRNA and mRNA expression profiles of PD were downloaded from the Gene Expression Omnibus and were termed GSE16658 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE16658; accessed 9th June 2013) and GSE22491 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE22491; accessed 9th June 2013), respectively (13,14). miRNA expression profiling was performed using the miRCURY LNA microRNA Array platform, which included 32 samples (19 PD samples and 13 normal control samples). The mRNA profiling was performed using the Agilent-014850 Whole Human Genome Microarray 4×44K (G4112F) platform (Agilent Technologies, Inc., Santa Clara, CA, USA), which included 18 samples (10 PD samples and eight normal control samples).
Differential expression analysis for miRNA and mRNA expression profiling
Probes of miRNA and mRNA profiling were mapped to the miRNA name and Entrez Gene ID in the miRBase, respectively. When multiple probes were mapped to one miRNA or gene, the average expression level was calculated. Log2 conversion was then performed on the expression level.
A two-tailed student’s t-test was used to analyze the differentially expressed miRNA and mRNA in the PD samples compared with the normal samples when the P-value was <0.01 following false discovery rate (FDR) adjustment.
Predicted target genes of miRNA and function and pathway enrichment analysis
Based on the seven target gene prediction algorithms PicTar, DIANA-microT, miRanda, miRBase, RNAhybrid, RNA22 and TargetScan, a set of predicted target genes of miRNA was determined. In order to reduce false positive predictions, miRNA target genes with high confidence were selected, which were predicted by at least three prediction algorithms. The miRNA target genes set with high confidence were then used for further analysis.
In order to determine which biological functions the synergistic miRNA was involved in, GO (15) was used for the miRNA regulatory target gene enrichment analysis (P<0.001). In addition, GenMAPP software (Gladstone Institute, San Francisco, CA, USA) (16) was used for KEGG pathway analysis.
Identification of synergistic miRNAs
Each miRNA set with high confidence of target genes was obtained and an accumulative hyper-geometric distribution test was performed to locate miRNAs sharing the same target genes. These miRNAs were defined as synergistic miRNAs. P<0.01 was considered to indicate a statistically significant difference following multiple FDR adjustment.
Results
Differential expression analysis
For expression analysis, 200 significantly differentially expressed miRNAs were identified in the PD samples compared with the normal samples. In the mRNA expression profile analysis, 2,966 differentially expressed mRNAs were identified.
Identification of synergistic miRNA and functional annotation for target genes
A total of 3,860 abnormal miRNA interactions were identified, which regulated at least one common target gene. A total of 1,502 miRNA interactions (P≤0.01) were identified based on super geometric distribution algorithm, including 147 miRNAs. These significantly synergistic miRNA pairs were involved in the regulation of 304 abnormally expressed genes.
In order to determine the biological functions that PD-associated disordered miRNAs were involved in, 304 abnormal target genes were used to perform GO function enrichment. A total of 74 GO biological processes were significantly enriched, including the biosynthetic process, the cellular biosynthetic process, the cellular component assembly involved in morphogenesis, mitogen-activated protein kinase (MAPK) signaling, the myometrial relaxation and contraction pathways and calcium regulation in the cardiac cell (Table I). In addition, 304 target genes were significantly enriched in eight KEGG pathways (Table II).
Table IIKyoto Encyclopedia of Genes and Genomes pathway annotation of synergistic microRNA regulatory targeted genes. |
Synergistic miRNA network construction
The synergistic miRNA was used to construct a synergistic miRNA network associated with PD (Fig. 1). The miRNAs in this network comprised all the abnormal molecules in the disease process and are important gene regulatory factors. Therefore, the miRNAs in the network may be beneficial to uncover the mechanism of PD. Through binding to their target genes, miRNAs control post-transcriptional translation or directly degrade the mRNA target genes. Degree represents the number of interaction partners and the node with the highest degree is essential for the stabilization of the network (17). As shown in Table III, a high degree was observed in miR-627, miR-634, miR-514, miR-563 and miR-613, which may be associated with the pathogenesis of PD.
Discussion
PD is a common neurological degenerative disease in elderly individuals. It is well established that miRNA is involved in adjusting the target genes involved in cell proliferation, differentiation, apoptosis and extensive biological processes (18). In addition, miRNA is also important in the differentiation of stem cells (19,20). Dysfunction of miRNA may affect the development of the nervous system, which causes diseases, including Alzheimer’s disease and PD (21). In the present study, the expression profiles of miRNA and mRNA were used to identify disordered miRNA. A total of 304 abnormal miRNA regulatory target genes were identified. GO function and KEGG pathway analysis demonstrated that miRNA regulatory target genes were enriched in several biosynthetic processes and pathways, among which cell apoptosis (22,23), the MAPK signal pathway (24), calcium ion regulation (25) and insulin signals (26) have been associated with the development or the treatment process and response to DNA damage in PD.
In the present study, a total of 1,502 synergistic miRNA interactions were identified and an miRNA synergistic network was constructed. Within this network, miR-7 has been demonstrated to regulate critical genes in the nervous system and to be involved the processes of PD (14,27). In the synergistic network constructed in the present study, miR-7 and another six miRNAs regulated common target genes. In addition, through binding to the 3′UTR of amyloid precursor protein (APP), miR-147 regulated the level of APP expression and, therefore, affected the risk of developing Alzheimer’s disease and PD (28). The miRNA network demonstrated that miR-627, miR-634, miR-514, miR-563 and miR-613 exhibited a high degree. miR-627, miR-634 and miR-514 have been investigated in human colorectal cancer cells (29), acute lymphoblastic leukemia (30) and ovarian cancer (31). miR-563 has been reported to be differentially expressed in Alzheimer’s disease samples (32) and targets the nuclear liver X receptor, which is important in the metabolism and homeostasis of cholesterol, bile acids, lipids and steroid hormones (33). However, to the best of our knowledge, no studies have investigated the association between these miRNAs and PD or other nerve-associated diseases. Therefore, the present study demonstrated for the first time, to the best of our knowledge, that miR-627, miR-634, miR-514, miR-563 and miR-613 are associated with PD. Further studies are required in order to confirm these results.
Using the expression profile data, the entire genomic expression situation of PD was examined. This revealed differentially expressed miRNAs and mRNAs. In addition, miRNAs were obtained and a synergistic miRNA network was constructed. From this, miR-627, miR-634, miR-514, miR-563 and miR-613 were obtained, which were newly reported in the present study. However, further studies are required to confirm these results.
Acknowledgements
This study was supported by grants from The Major Project of Biomedical Research of the Science and Technology Commission of Shanghai Municipality (grant no. 10411954400) and The National Natural Science Foundation of China General Program (grant no. 81171296).
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