Previous studies have found that the delay between the onset of ALS symptoms and diagnosis ranged up to 15.6 months [29]. Also, the patients with sporadic forms of the disease took relatively longer to diagnose than other patients[44]. Delays in diagnosis can affect the outcome as well as the survival of ALS patients, therefore, the discovery of an accurate and sensitive biomarker is critical to current ALS research. Since blood sampling has the advantages of easy handling and low-cost multiplex assays, biomarkers in the blood are our primary consideration for research. Besides, on the basis that 40% of lncRNAs are expressed in the human brain, it is a more promising potential biomarker for neurodegenerative diseases.
LncRNAs have been validated in recent studies of various disease pathways to be relevant to many biomolecular functions [17, 21]. In this study, we have profiled lncRNAs in SALS patients as well as in healthy controls by microarray to expand our knowledge of molecular alterations in the transcriptome and obtain new data on their dysregulation. In the top 30 dysregulated lncRNAs, we validated 6 of them have differential expression in peripheral leukocytes of SALS comparing with healthy controls. We observed these 6 lncRNA transcripts have legible down-regulation (lnc-CNTN4-2:1, lnc-NR3C2-8:1, lnc-ABCA12-3:1, lnc-DYRK2-7:1, lnc-POTEM-4:7 and XIST in male cases) in SALS cases. Total 3 lncRNAs (lnc-CNTN4-2:1, lnc-NR3C2-8:1 and XIST in male cases) showed the same trend of regulation but the differences are not expressed to the same extent in SALS and PD according to the P-values. Therefore, we believe that lnc-ABCA12-3:1, lnc-DYRK2-7:1 and lnc-POTEM-4:7 can be considered as SALS-specific peripheral blood biomarkers, while the other lncRNAs may serve as peripheral blood biomarkers for neurodegenerative diseases. It is also worth mentioning that other studies have shown that the use of peripheral blood for ALS-associated biomarker screening is a good foundation to pave the way for ALS-associated RNA function studies [31]. It is still unfortunate that for most of the remaining lncRNA sequences that overlap highly or completely with the mRNA sequences, we are currently unable to have a more suitable method to validate their expression in clinical samples.
Substantial evidence reveals that RNA as a key regulator associated with other ALS relative proteins plays an important role in ALS relevant RNA metabolism as well as in the transcription of other proteins[8, 9, 45, 46]. Among these disease related RNAs, two main RNA families can be distinguished, coding RNAs and non-coding RNAs. They are both associated with RNA metabolism and can produce cellular defects that can be the cause of ALS. Alteration of numerous lncRNAs has been described in different types of motor neuron diseases, however only very few of them have been subjected to in-depth studies of functions related to disease mechanisms [47]. The paraspeckle, a distinct cellular feature of ALS, was revealed that its formation connected with lncRNA NEAT1_2 which was reported association with ALS [32, 48]. This lncRNA has been shown directly bound to TDP-43 and FUS, which are the proteins enriched in paraspeckles [32]. This is one of the most in-depth studies targeting the mechanism of lncRNA in ALS, while our data will provide more evidence and ideas for studies on lncRNA as a biomarker in ALS and related mechanisms.
What has attracted the attention of researchers in recent years is the variation and significance of its interaction with miRNAs or mRNAs and the networks they associate with various diseases [49–51]. Through bioinformatics analysis, we found that a significant proportion of lncRNAs with differential expression in microarray and peripheral blood leukocyte screens were associated with various signaling pathways and transcription factors. In the GO component analysis, we obtained results similar to those in the literature, with differentially expressed lncRNAs being most associated with the messenger ribonucleoprotein complex in cellular components which is closely related to mRNA. In GO molecular function and biological process analysis, the items most associated with differentially expressed lncRNAs: 3-oxoacid CoA-transferase and cellular ketone body metabolic process are both strongly associated with fatty acid metabolism, and their deficiency is expressed in the development of multiple diseases[52–55]. Also, some findings suggest that unsaturated fatty acid metabolism is significantly dysregulated in the brains of patients with neurodegenerative diseases [56, 57]. Furthermore, among the predicted results of lncRNA-associated coding gene and transcription factor, SNCA (encoding alpha-synuclein), FBXW11 (encoding F-box family member) and BRCA1 (DNA repair-associated) may be associated with neurodegenerative diseases [58–60]. Therefore, we believe that the results of this bioinformatic analysis of lncRNAs are consistent with other clinical findings and can be used in the future as an idea to study the mechanism and treatment of lncRNAs related to ALS.
LncRNAs can regulate their molecular functions as well as those of other molecules in the cytoplasm or nucleus through a variety of mechanisms [21]. Thus, for differential expressed lncRNAs it is also necessary to perform intracellular localization experiments in multiple cell lines. The intracellular localization of lncRNAs is highly relating to their association with other molecules which is similar to mRNA [61]. XIST, a lncRNA named X-inactive specific transcript, has been intensively expounded in multiple studies in recent years. The intracellular localization of XIST has been confirmed in the nucleus [62]. Correlation of XIST with functions and mechanisms studied in the nucleus has also been confirmed: association with silencing transcription, inducing chromatin formation and diacylation [63, 64]. In this trial XIST was downregulated in the peripheral blood leukocytes of male SALS and PD patients, thus more studies on the mechanisms and functions of neurodegenerative diseases associated with XIST need to be demonstrated as soon as possible [65]. The other lncRNAs reported in this study serve as novel biomarkers that need to be predicted and validated for their ALS-related mechanisms and functions based on their respective intracellular localization. Most lncRNAs were validated for their association with mRNAs or miRNAs after they were verified for anomalous change in expression in the presence of disease. Numerous results illustrate that lncRNA-mRNA-miRNA pathway studies can indeed provide conjecture and basis for our understanding of the functional mechanisms of lncRNAs [49, 50, 66–68]. However, only a few ceRNA interactions have been studied concerning neurodegenerative diseases. Nevertheless, we are still interested in lncRNA-miRNA interactions in neurodegenerative diseases and will continue to better understand this mechanism in subsequent studies. Besides, our bioinformatics data indicates the possible relation between dysregulated lncRNAs and their target genes or relative transcription factors.