Edited by: Andrei Surguchov, University of Kansas Medical Center, United States
Reviewed by: Daniel Leite Góes Gitaí, Federal University of Alagoas, Brazil; Maria Shadrina, Institute of Molecular Genetics (RAS), Russia
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Genetic studies have managed to explain many cases of familial amyotrophic lateral sclerosis (ALS) through mutations in several genes. However, the cause of a majority of sporadic cases remains unknown. Recently, epigenetics, especially miRNA studies, show some promising aspects. We aimed to evaluate the differential expression of 10 miRNAs, including miR-9, miR-338, miR-638, miR-663a, miR-124a, miR-143, miR-451a, miR-132, miR-206, and let-7b, for which some connection to ALS was shown previously in ALS culture cells, animal models or patients, and in three miRNA host genes, including
Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that typically presents in adulthood with symptoms, such as muscle weakness, atrophy and later on paralysis, leading to death within 2–3 years following diagnosis (Rowland and Shneider,
Sporadic ALS has been linked to many environmental factors, including heavy metal toxicity and exposure to pesticides, fertilizers, smoking, viral infections, physical exercise, and electromagnetic radiation (reviewed in Zufiría et al.,
Therefore, it is necessary to shift the focus to the field of epigenetics, especially miRNAs, which show some promising aspects.
miRNAs are single-stranded non-coding RNA molecules that are ~22 nucleotides in length, which act as post-transcriptional regulators of gene expression either by causing the degradation of target mRNAs or the inhibition of their translation (Pillai et al.,
miRNA pathway disruptions could be a cause or consequence of ALS pathology, which is underlain with altered RNA and protein metabolism, cytotoxicity due to faulty glutamate clearance, the inflammatory response, and neuromuscular junction impairments (Paez-Colasante et al.,
In our previous study, we determined genetic changes in 7/84 (8.3%) of Slovenian patients with a sporadic form of ALS (Vrabec et al.,
In addition, we also focused our attention on investigating the differential expression of three intragenic miRNA host genes, including
Blood samples of 84 Slovenian patients clinically diagnosed with ALS were collected at the Institute of Clinical Neurophysiology, University Medical Centre Ljubljana, Slovenia. Both genders were represented equally (42 women and 42 men), and none of the patients were related. The mean age of onset was 62 ± 11.72 years (ranging from 37 to 89 years). Sixty-two of the eighty-four (74%) patients had the spinal onset form and 22/84 (26%) had the bulbar onset form of the disease. Four patients (4.8%) had associated symptoms of frontotemporal dementia (FTD), and two patients (2.4%) had some associated symptoms of Alzheimer disease. For 68 patients we obtained information about their treatment. Thirty-one patients were treated with rulizol, and 37 patients were not yet treated. Seven patients had genetic changes (Vrabec et al.,
As a control group, 27 healthy volunteers were included. There were 14 women and 13 men, with a mean age was 56 years, ranging from 30 to 65 years. The blood samples were collected at the Institute of Clinical Neurophysiology, Division of Neurology, University Medical Centre Ljubljana, Slovenia.
This study was carried out in accordance with the recommendations of the Republic of Slovenia National Medical Ethics Committee with written informed consent from all the subjects. All the subjects gave written informed consent in accordance with the Declaration of Helsinki.
The protocol was approved by the Republic of Slovenia National Medical Ethics Committee.
For the RNA isolation, Ficoll-Paque PLUS reagent (Life Sciences, Germany) was used. Briefly, a mixture of blood and PBS buffer (1:2) was carefully layered on Ficoll. A centrifugation step created a gradient and leukocyte layer that was easily transferred into a fresh tube, and the sample was washed with PBS buffer. After washing, the pellet was resuspended in TRI reagent (Sigma-Aldrich, Germany), and further isolation was performed following the manufacturer's protocol. After separation of the water solution containing the RNA, purification was performed by miRNeasy Mini Kit (Qiagen, Germany) following the instructions for purification of total RNA, that includes long and small RNAs as are miRNAs,
For purpose of determination of an efficiency of amplification for analyzed miRNAs, initially, pools of the RNA samples from healthy adults and a pool from the ALS patients were created. After reverse transcription and serial cDNA dilution, qPCR efficiency was analyzed for each miRNA (let-7b, miR-9, miR-124a, miR-132, miR-143, miR-206, miR-338, miR-451a, miR-638, and miR-663a) and for each of the three reference genes (
Details of the ready-to-use primers for the expression analyses.
let-7b | Hs_let-7b_1 | MS00003122 | miScript, Qiagen |
miR-9 | Hs_miR-9_1 | MS00010752 | miScript, Qiagen |
miR-124a | Hs_miR-124a_1 | MS00006622 | miScript, Qiagen |
miR-132 | Hs_miR-132_1 | MS00003458 | miScript, Qiagen |
miR-143 | Hs_miR-143_1 | MS00003514 | miScript, Qiagen |
miR-206 | Hs_miR-206_1 | MS00003787 | miScript, Qiagen |
miR-338 | Hs_miR-338_1 | MS00003990 | miScript, Qiagen |
miR-451a | Hs_miR-451_1 | MS00004242 | miScript, Qiagen |
miR-638 | Hs_miR-638_4 | MS00043624 | miScript, Qiagen |
miR-663a | Hs_miR-663_3 | MS00037247 | miScript, Qiagen |
RNU6B | Hs_RNU6-2_11 | MS00033740 | miScript, Qiagen |
SCARNA17 | Hs_SCARNA17_11 | MS00014014 | miScript, Qiagen |
SNORA73A | Hs_SNORA73A_11 | MS00014021 | miScript, Qiagen |
AATK | Hs_AATK_1_SG | QT01160264 | QuantiTect, Qiagen |
C1orf61 | Hs_C1orf61_1_SG | QT01014790 | QuantiTect, Qiagen |
DNM2 | Hs_DNM2_1_SG | QT00037072 | QuantiTect, Qiagen |
GAPDH | Hs_GAPDH_1_SG | QT00079247 | QuantiTect, Qiagen |
U6 | Hs_USB1_1_SG | QT00066906 | QuantiTect, Qiagen |
Prior to qPCR, reverse transcription of 100 ng of RNA was performed using the miScript II Reverse Transcription Kit (Qiagen, Germany) with 5x miScript HiFlex buffer which enables transcription of mRNA as well as miRNA to cDNA. Inhibitor RNase (Qiagen, Germany) was also added to the reaction mixture. Reverse transcription was performed in a total volume of 10 μl according to the manufacturer's instructions.
A miScript Sybr Green PCR Kit (Qiagen, Germany) was used for all the qPCR reactions according to manufacturer's protocol in a 10 μl reaction volume. Specific primers for the miRNAs and reference genes and the 10x miScript Primer Assay (Qiagen, Germany) were used. Based on the efficiency analysis, all the ALS and control cDNA samples were diluted 1:100, and the reactions were performed on a Rotor Gene Q (Qiagen, Germany) in duplicate for each of 111 sample.
Information about the miRNA location (intragenic or intergenic) was gained through the online tools miRIAD (
qPCR was also used to identify host gene expression. For qPCR, the cDNA from miScript System was used, that was resulted from reverse transcription with HiFlex buffer, which enables reverse transcription also of mRNA using oligo-dT primers. Three genes,
To analyze qPCR data from miRNA and mRNA expression analysis, efficiency corrected model of 2−Δ
Using Human MicroRNA Disease Database (Lu et al.,
The detailed relative expressions of each miRNA analyzed are presented as the average relative expressions in the graphs (Figures
ΔCt of analyzed miRNAs and two host genes.
let-7b | 0.65 | 3.79 | 2.32 | 3.68 | 8.69 | 5.93 |
miR-9 | 7.13 | 10.9 | 8.95 | 9.27 | 19.72 | 12.74 |
miR-124a | 7.69 | 10.11 | 8.82 | 21.40 | 28.93 | 24.50 |
miR-132 | 5.66 | 8.69 | 6.68 | 6.33 | 12.19 | 8.85 |
miR-143 | 5.39 | 13.81 | 10.12 | 5.96 | 13.73 | 9.84 |
miR-206 | 10.60 | 16.39 | 11.65 | 12.59 | 18.66 | 15.94 |
miR-338 | 3.82 | 9.50 | 7.98 | 4.41 | 14.38 | 9.90 |
miR-451a | −1.00 | 5.23 | 2.43 | 1.34 | 8.26 | 4.46 |
miR-638 | 1.16 | 4.05 | 2.76 | 4.76 | 9.02 | 6.65 |
miR-663a | 1.47 | 3.48 | 2.38 | 2.10 | 6.81 | 4.36 |
DNM2 | 6.97 | 10.56 | 8.76 | 1.63 | 7.32 | 4.86 |
AATK | 6.08 | 11.25 | 9.47 | −0.89 | 15.50 | 12.07 |
We found that all the miRNAs were significantly up-regulated (
The expression changes were statistically evaluated according to gender, the presence of mutations and disease onset, but no significant differences were detected. We only found significant differences in the expression of miR-143 by gender (
The
Using Mann–Whitney test, we have compared ΔCt of patients that were treated using rulizol, to ΔCt of patients that were not yet treated. For five miRNAs (miR-143, miR-451, miR-338, miR-638, let-7b) and for two host genes (
We observed a moderate positive correlation between miR-338 and its host gene
Using Human MicroRNA Disease Database we found that for majority of miRNAs investigated in this study there is at least one confirmed target gene in humans. For two miRNAs, namely miR-638 and miR-663a, there have been no target yet confirmed. However, the majority of confirmed target genes have been investigated in different neoplasms and none in ALS. Results are summarized in Table
List of validated target genes of investigated miRNAs according to Human MicroRNA Disease Database (Lu et al.,
miR-9 | ITGB1 | Breast cancer |
JAK, CAMTA1 | Glioblastoma | |
MMP14 | Neuroblastoma | |
ETS1, NKFB1, CDX2, CCND1 | Gastric cancer | |
miR-124a | IQGAP1 | Breast cancer |
ROCK2, EZH2, PIK3CA | Hepatocellular carcinoma | |
CDK4 | Glioma | |
CDK6, HMGA1 | Medulloblastoma | |
AR | Prostatic cancer | |
miR-132 | TMEM106B | Dementia |
miR-143 | GCK, MACC1, DNMT3A, KRAS | Colorectal cancer |
ERK5 | B-cellular lymphoma, Obesity | |
RAS | Pancreatic cancer | |
PTGS2, SERPINE1, BCL2 | Uterine cervical neoplasm | |
miR-206 | ESR1 | Breast cancer |
Notch3 | Neoplasm | |
MET | Rhabdomyosarcoma | |
CCND2 | Gastric cancer | |
miR-338 | SMO | Hepatocellular carcinoma |
CCND1 | Hepatitis B | |
miR-451a | IKBKB | Hepatocellular carcinoma |
RAB14 | Non-small cell lung cancer | |
YWHAZ | Diabetic nephropathy | |
MDR, ABCB1 | Neoplasm | |
miR-638 | / | |
miR-663a | / | |
let-7b | BCL2L1 | Hepatocellular carcinoma |
KRAS | Non-small cell lung cancer | |
IL13 | Inflammation | |
ITGB3, CCND1 | Melanoma | |
HMGA2, RAS | Neoplasm |
Heatmap of experimentally validated union pathways of investigated group of miRNAs using miRPath Database (Vlachos et al.,
Any novel findings contribute to the understanding of ALS pathogenesis. Functional studies show that miRNAs are involved in virtually all the cellular processes investigated and that the changes in their expression are closely related to the occurrence of the disease (Filipowicz et al.,
To understand the pathogenesis of ALS, it is crucial to be familiar with the signaling that occurs between the fibers of the skeletal muscles and motor neurons (Kovanda et al.,
The exosomal-mediated transfer of miRNAs is possible and was determined for miR-124a (Morel et al.,
In the leukocytes of a Chinese sALS cohort, Chen et al. identified significantly lower expression levels of several miRNAs, including hsa-miR-124, in comparison with healthy controls using a microarray (Chen et al.,
The relative expression levels of miR-124a in the peripheral blood leukocytes of sALS patients was also studied. We detected a significantly elevated expression of miR-124a in all 84 sALS samples compared to the controls regardless of the onset of the disease. Since the differential expression of miR-124a was shown in the brain and in the spinal cord of the ALS mice as well as in the leukocytes of patients with sALS, this might indicate the possible relationship between CNS and peripheral tissues and places miR-124a among the miRNAs that are worthy additional investigations in the direction toward ALS disease biomarkers and therapeutic targets, especially since an emerging critical role of microglia and astrocytes has been established in the etiology of ALS (Radford et al.,
Our results showed an elevated relative expression of let-7b in the leukocytes of the Slovenian sALS patients studied. The dysregulation of let-7b was, until now, determined in connection with ALS in TDP-43 knockdown in culture cells, where the authors showed that the removal of TDP-43 from the cell nucleus caused specific downregulation of let-7b that could further influence the expression of other potential transcripts involved in neurodegeneration and synapse formation (Buratti et al.,
In a study by Freischmidt et al. they observed significant differences in the relative levels of let-7b, miR-143 and miR-132 in the serum of sALS patients compared to the mean expression of the healthy controls. They found that the mean relative expression of all three miRNAs significantly decreased in their sALS cohort (Freischmidt et al.,
We observed the differential expression of intergenic miR-451a and miR-663a. Using a microarray strategy, De Felice et al. evaluated the expression of miRNAs in the leukocytes of 8 sALS patients and 12 unaffected healthy controls. They identified seven miRNAs, including miR-451a, that were down-regulated across different gender groups and in all the tested sALS samples (De Felice et al.,
Changes in the expression of miR-663a have been studied following TDP-43 knockdown in culture cells, where miR-663a up-regulation was observed (Buratti et al.,
Three of the investigated miRNAs, miR-9, miR-338-3p, and miR-638, were intragenic. The expression of miR-9 was previously studied in p.Gly93Ala-SOD1 mice, and its significant increase was detected in whole brain at late stage disease compared to Wt-SOD1 control brains as well as in manually dissected brainstem motor nuclei and primary motor cortex (Marcuzzo et al.,
De Felice et al. reported over-expression of miR-338-3p in blood leukocytes as well as in cerebrospinal fluid, serum, and spinal cord from sALS patients (De Felice et al.,
miR-638 was up-regulated in all 84 samples in our sALS cohort. In one previous study, leukocytes from 8 patients with sporadic ALS and from 12 unaffected healthy controls were analyzed using a microarray analysis. The differential expression of miR-638 was detected and was down-regulated in eight analyzed sALS samples (De Felice et al.,
To further elucidate the functional role of investigated miRNAs we performed
The differential expression of three miRNA host genes, including
The protein encoded by the
It was shown that Cdk5-LMTK1-Rab11A pathway is a regulatory mechanism of dendrite development and axon outgrowth (Takano et al.,
The significant up-regulation of
It is also known that ALS shares genetic characteristics with other complex diseases, including Charcot-Marie-Tooth (CMT4J); namely, mutations in
Furthermore, hereditary spastic paraplegia (HSP) is another complex disease that exhibits some genetic overlap with ALS through mutations in
Thus, dysfunctional or dysregulated
In conclusion, we detected the differential expression of 10 miRNAs involved in the ALS pathology in the leukocyte samples of patients affected with the sporadic form of ALS. Seven of these miRNAs have not been previously investigated in peripheral blood leukocytes. We observed significant aberrant dysregulation across our patient cohort for miR-124a, miR-206, miR-9, let-7b, and miR-638. Since we did not use neurological controls in this study we cannot conclude that the revealed differences in expression of investigated miRNAs are specific for ALS. Nevertheless, the group of these five miRNAs is worth of additional research in leukocytes of larger cohorts from different populations in order to verify their potential association to ALS disease. The detected significant up-regulation of
MR-G: Substantially contributed to the conception and design of the work and wrote the paper; KV and EB: Substantially contributed to the acquisition, analysis, and interpretation of the data; BK, LD, LL, and JZ: Substantially contributed to the acquisition and interpretation of the data; BR and DG: Substantially contributed to the conception and design of the study. All the authors contributed in critically revising the manuscript for important intellectual content and gave the final approval of the version to be published. All the authors agree to be accountable for the content of the work.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
This research was supported by the Slovenian research agency (ARRS—Javna Agencija za Raziskovalno Dejavnost RS)—ARRS Research Program P3-054 and the ARRS Ph.D. thesis grant to KV. Additionally, we thank all of the patients who were willing to cooperate in this research and made it possible.