Preliminary Study of hsa-mir-626 Change in the Cerebrospinal Fluid in Parkinson's Disease
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.310102
Source of Support: None, Conflict of Interest: None
Keywords: Biomarker, cerebrospinal fluid, hsa-miR-626, Parkinson's disease
Parkinson's disease (PD) is a common neurodegenerative disease, mainly leading to progressive movement disorders. Diagnosis is mostly based on clinical symptoms and response to levodopa, but by then, massive irreversible neuronal death would have already occurred, and effective therapeutic intervention is difficult to achieve. Compared with symptoms, which appear at relatively late stages in the pathogenesis, the underlying molecular changes begin much earlier, and interest has been focused on the role of the noncoding RNAs in the past 10 years.
microRNAs (miRNAs) are small noncoding RNAs comprising 21–23 nucleotides that regulate gene expression at post-transcriptional level by either repressing translation or inducing degradation of their complementary mRNAs., A variety of miRNAs have been reported to regulate diverse biological and pathological processes, including cell differentiation, proliferation, apoptosis, metabolism, and tumor growth, invasion, neurodegeneration., With technological advances in small RNA profiling in recent years, miRNA changes in the serum and other body fluids are being used as tumor diagnosis protein marker,, such as miR-24, miR-125b, and miR-214. In addition, circulating miRNA change was observed in the cerebrospinal fluid (CSF) of Alzheimer's disease (AD) and primary central nervous system (CNS) lymphoma patients. These advances in the miRNA field have shown that miRNAs could function as powerful biomarkers in human diseases, especially the neurodegenerative ones. In this study, we hypothesized that miR-626 could be a useful CSF-based marker for the detection of PD, as our unpublished data and previous study have showed that miR-626 is a possible target for LRRK2, which is involved in the pathogenesis of PD. To understand whether the expression of hsa-miR-626 may be misregulated in PD, real-time polymerase chain reaction (qRT-PCR) using CSF samples from PD patients, AD patients, and controls was performed.
In total, 15 patients with PD, 11 with AD, and 16 controls with other neurologic disorders were recruited from The Second Xiangya Hospital from 2011 to 2014 in accordance with China law and ethical guidelines, and informed consent was obtained from patients and controls before sample collection. As clinical diagnosis is uncertain in some patients with extrapyramidal movement disorders, we selected only those patients in whom the diagnosis of PD was clinically reliable. The 15 sporadic PD cases in our study were diagnosed by two experienced neurologists who have been studying PD for years and met the UK Brain Bank criteria; 11 AD cases met NINCS-ADRDA criteria for probable or definite AD. All of them were on their first visit to the hospital and have not had any treatment before.
CSF samples without blood contamination were obtained from all the subjects through lumbar puncture. After that, 2-mL CSF samples were individually centrifuged (500 g, 10 minutes, room temperature) within 60 minutes of collection to remove cells and debris. The samples were then stored at − 80°C until further processing.
RNA extraction from CSF
Total RNA was extracted using a mirVana RNA isolation kit (Ambion) according to the manufacturer's instructions. Briefly, 2 mL of CSF was diluted with an equal volume of mirVana PARIS 2× denaturing solution and incubated for 5 minutes on ice. Subsequently, equal volumes of acid/phenol/chloroform (Ambion) were added, and samples were centrifuged at 10,000 g for 5 minutes. Next, glycogen was added to aqueous phases, and mixed with 1.25 volumes of 100% ethanol. After passage through a mirVana PARIS column and washing, RNA was recovered in 30 μL of elution buffer. The total RNA concentration was determined by measuring with a NanoDrop ND-3300 Fluorospectrometer (Thermo Scientific).
Reverse-transcription reactions were performed with 20 μL of total RNA solution using Omniscript RT Kit (Qiagen; 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes). qRT-PCR was performed with 7900HT Fast Real-Time PCR System (Applied Biosystems). Sequences of miR-626 are listed at http://www.mirbase.org/. Forward primers used for quantitative PCR were AGCTGTCTGAAAATGTCTT, while reverse primers came with the assay kit itself. TaqMan miRNA assays (Applied Biosystems) to quantify miR-626 levels were applied according to the manufacturer's instructions. In total, 40 cycles each of 15 seconds at 95°C and 60 seconds at 60°C were performed. To calculate the expression of miRNA relative to endogenous control, the comparative ΔΔCt values were calculated for hsa-miR-626. The amount of target miRNA was normalized relative to the amount of miR-24. Every sample processing was repeated three times.
Results are presented as means ± standard deviations (SDs). The Mann–Whitney U test was used to determine the significance of intergroup differences. SPSS v. 15.0 (SPSS, Chicago, IL, USA) was used for the analyses, and two-tailed P values <0.05 were considered significant.
Our study included 15 PD patients (9 men and 6 women) aged 55–82 years (median, 70.6 ± 12.1 years), 11 AD patients (7 men and 4 women) aged 62–84 years (median, 72.1 ± 10.8 years), and 16 controls (11 men and 5 women) aged 53–84 years (median, 70.2 ± 15.8 years). There was no significant difference in age and duration between the patient samples and the controls [Table 1].
Detection of hsa-miR-626 in the CSF of patients by qRT-PCR
We used TaqMan qRT-PCR assays for the detection of hsa-miR-626 in CSF samples collected from all patients. This method has been successfully applied in the study of the CSF of AD and primary CNS lymphoma patients. Finally, we found that the expression of hsa-miR-626 was distinctly different between groups [Figure 1]. For hsa-miR-626, the mean expression level was significantly reduced in the CSF of patients with PD (1.675 ± 1.956) compared with controls (3.384 ± 2.150) and AD patients (2.982 ± 1.991; P < 0.05).
As a small noncoding RNA, miRNAs inhibit gene expression post-transcriptionally through repressing translation of target mRNA or degrading target mRNA and are involved in multiple disease-related pathways. With the recent rapid acceleration in the field of miRNA research, the potential predictive and diagnostic uses of miRNAs have also attracted significant attention. A lot of miRNAs, such as miR-18a-5p, miR-21, and miR-155, miR-216, have been identified as potential circulating biomarkers of carcinoma, although none of them has been put into clinical practice. In addition, miRNA expression changes are associated with the neurodegenerative disease process, either directly or as part of feedback circuits.,
As the second most common neurodegenerative disorder, PD places a burden on the health of individuals because it results in progressive impairment of motor function. Although advancements have been made in understanding the genetic and molecular basis of PD, the clinical diagnosis remains difficult. Mounting evidence supports that, because of high compensatory potential of the brain, symptoms of neurodegenerative diseases, such as AD, PD, and Huntington disease (HD), usually occur 10–20 years after the beginning of the pathology. So, searching for new diagnostic biomarkers in asymptomatic stage, years before massive death of neurons, is undoubtedly imperative. The characteristic of an ideal biomarker is being simple, objective, sensitive, and specific, and circulating miRNAs are promising diagnostic biomarkers in PD.
In 2012, Sok et al. identified three miRNAs differently expressed in the plasma of PD patients by significance analysis of microarrays (SAM), which were subsequently confirmed by qRT-PCR. In their study, miR-626 demonstrated high specificity (100%) and could provide a potential biomarker to assist diagnosis. They reported the first plasma-based circulating miRNA biomarkers for PD. Subsequent studies have revealed other miRNAs, such as miR-133b, and miR-34b/c, involved in the pathogenesis of PD. Although using plasma or serum is indeed a minimally invasive approach, similar pathologic changes in many other organs may present with the same blood features. CSF is a colorless and clear fluid that flows within the ventricles and subarachnoid space of the brain and spinal cord. In contrast to plasma or serum, CSF communicates with the extracellular space of the brain directly and can reflect the brain pathological processes more accurately. Secondly, the amount of miRNA secreted from other organs into the CSF is very limited owing to the blood–brain barrier. Moreover, miRNAs in the CSF have clear biological activity and show remarkable stability. Above all, CSF is an attractive source of biomarkers. In 2008, Cogswell et al. discovered AD-specific miRNA changes in the CSF. In recent studies, disease-altered miRNA expression in the CSF was also found in various CNS disorders, including primary CNS lymphoma, multiple sclerosis, and glioblastoma.
Besides, our unpublished data and previous study have showed that miR-626 is a possible target for LRRK2, which is related to the pathogenesis of PD. Therefore, we detected the levels of hsa-miR-626 in the CSF from PD patients in this study.
Given the overlapping clinical phenotypes and pathological characteristics among these neurodegenerative diseases, such as cognitive impairment, neuronal loss, and protein accumulation, controls should include other neurodegenerative diseases, rather than healthy persons only. To investigate the correlation of miR-626 and PD, we chose AD, the most common neurodegenerative disorder, as control and identified that the expression of miR-626 was indeed altered in the CSF in PD patients when compared with AD patients and controls (P < 0.05). In view of this result, we hypothesized that miR-626 could be a useful CSF-based marker for the detection of PD. Moreover, it can be used for discriminating PD from AD.
Normalization is an important step for accurate quantification of RNA levels with qRT-PCR. For miRNA, circulating in extracellular body fluids, including those detected in the CSF, no consensus internal controls have been established yet. miR-24 has been reported as an appropriate miRNA control for normalization purposes. In this study, miR-24 was chosen as the normalizing control, which is similar to the findings reported for serum and CSF.,
Although change of hsa-miR-626 in the CSF of PD patients was identified in our study, we must admit that it was just a preliminary study, and further studies including hundreds of patients and controls should be conducted. It is well known that PD is characterized by progressive degeneration of dopaminergic neurons and formation of Lewy bodies, mainly in the substantia nigra pars compacta (SNpc)., Whether hsa-miR-626 plays direct or indirect roles in the pathogenesis of PD remains to be elucidated. More studies are necessary to further clarify which pathway hsa-miR-626 takes part in, as many miRNAs may be involved in inflammation and immunological response.,, Additionally, it still needs to be determined whether hsa-miR-626 expression changes are consistent with the time-dependent features of PD pathology.
Obviously, the ultimate goal is to provide a sensitive, specific, and objective detection of PD neuropathological changes before its clinical manifestation. Despite increasing number of publications on the diagnostic value of circulating miRNAs, their use in screening for neurodegenerative diseases is still in its early stage of development. Whether hsa-miR-626 can be used in PD diagnosis, alone or in combination with other PD biomarkers, warrants further investigation.
Furthermore, sample size in the present study was small. More samples and PD patients with different clinical features and severity undergoing such studies separately are essential. Our further research would be focusing on this.
This work was supported by the State Key Laboratory of Medical Genetics and by grants from the Major State Basic Research Development Program of China (973 Program; 2011CB510000, 2011CB510001); the National Natural Science Foundation of China (81000542, 81200870, 30900469, 81430023, 81130021, 81371405, 81361120404); the Science and Technology Program of Hunan Province (2014TT2014), and SRFDP (20120162120079).
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.