The molecular complexity of Alzheimer’s pathophysiology is bound to several molecular factors including multiple pathways and mechanisms. Besides, to date, there has been no reliable evidence that any cure for AD, which has existed as a health problem for many years. Yet, it might be predicted thanks to some molecular clue prior to reaching late AD level, thereby can be developed the quality of life in patients with AD. Hereby, in the complexity of molecular machinery, intercellular shuttled molecules may provide insight in enlightening the mechanism of AD (Rastogi et al. 2021). To contribute to the scope of that issue, the current paper explores the plasma exosomal non-coding RNAs including circRNA and miRNA expression profiles in AD patients by screening experimentally validated miRNA-target interaction databases.
In the present study, common 19 DEmiRs and 34 circRNAs in both AD and MCI groups were found in the plasma exosome compared to the healthy group based on NGS from ArrayExpress dataset. Theoretically, assuming that exosomes are secreted from AD brain tissue into blood, it was discerned that 15 of the commonly upregulated circRNAs in Table 2 were of neuronal origin by screening the circBase database, and were regulated by some downregulated miRNAs (see Fig. 5). Furthermore, we interrogated the circRNA genes in the Single-cell Atlas of the Entorhinal Cortex in Human Alzheimer’s Disease database (Grubman et al. 2019) to assign which cell type expressed the upregulated circRNAs, and 26 out of 34 circRNA genes were found in the dataset (Supplementary Fig.1).
The priority in AD physiology approaches is to focus on the dysfunction and misregulation of beta-amyloid and tau protein (Weller and Budson 2018; Rastogi et al. 2021). However, little is known about AD and it is not clear what factors cause AD pathology. In this context, 10 AD-associated genes mentioned in Table 4 with an enrichment score of 1.69 were regulated by hsa-mir-185, hsa-mir-3177-3p, hsa-mir-374a, hsa-mir-409, hsa-mir-4446-5p, hsa-mir-485, hsa-mir-493-3p, hsa-mir-504, hsa-mir-5698 in the DEmiRs (Supplementary Fig.3). In the sub-network, VEGFA, IGF1R and GSK3B genes are also related to Phosphoinositide 3-kinases-Akt (PI3K-Akt) signaling pathway and regulated by hsa-mir-504, hsa-mir-5698, and hsa-mir-374a-5p respectively.
CCND1, CCND2, CDK6, CDKN1A, MDM2 and CCNE2 are common regulated target genes of upregulated miRNAs in both PI3K-Akt and p53 signaling pathway, and CCNE2 from these genes is a hub gene that downregulated by hsa-mir-196a-5p, hsa-mir-205-5p and hsa-let-7c-5p. As related to AD, in the biological process of negative regulation of apoptosis, CCND2, CDKN1A, IGF1R, MDM2, MYC and PRLR genes in PI3K-Akt were regulated by hsa-mir-615-5p, hsa-mir-615-3p, hsa-mir-196a-5p, hsa-let-7c-5p in the downregulated DEmiRs. We uncovered the genes of CDKN1A, IGF1R, MDM2, and MYC as hub genes in the network of target genes of downregulated miRNA. This finding was unexpected and suggests that the same genes may be found in both networks of up/down regulated DEmiRs, as previous studies have found that a gene is regulated by more than one miRNA in several pathologic conditions (Catalanotto et al. 2016). The hub genes of CCND1, CCNE2, and CDK6 in PI3K-Akt pathway are commonly associated with regulation of cell cycle and cell division process. Besides, occurring evidence demonstrates that one of the complicated roles of circRNAs in biological functions is regulating transcription (Kristensen et al. 2019). Among target genes of upregulated miRNAs, 17 genes are involved in the negative regulation of transcription from RNA polymerase II promoter, while 12 genes from circRNAs are associated with the positive regulation of transcription in the DNA-templated. On the other hand, hsa-mir-615-3p, hsa-mir-205-5p, hsa-mir-196a-5p, hsa-let-7c-5p regulated XBP1 (Wolter et al. 2014), TUBB, PSMD8, CSNK2A1 and APP (Helwak et al. 2013), WIPI1 and NRAS (Johnson et al. 2005), DVL3 (Memczak et al. 2013), CALM1 (Kishore et al. 2011) in the main pathway of AD (Supplementary Fig.4). These miRNAs are the mature form of hsa-mir-196a-1, hsa-mir-205, hsa-mir-615, and hsa-let-7c into plasma exosomal statistically significant found in the upregulated DEmiRs in AD group compared to healthy group (see Table 1).
It is well known that there is a contrary relationship between the expression of miRNA and the expression of the target gene. Considering this view, KEGG pathway enrichment analysis displayed that the targets of downregulated were enhanced in pathways related to PI3K-Akt, p53 signaling pathway, cellular senescence, insulin signaling, and mTOR signaling pathway while targets of downregulated solely were enriched PI3K-Akt and p53 signaling pathway. Involving in apoptosis and cell senescence, for the 14 downregulated DEmiRs, hsa-mir-185-5p and hsa-mir-374a-5p had the most target genes that number is eight (Supplementary Fig.5). From downregulated DEmiRs, hsa-mir-485-5p is regulated TP53 in cell senescence pathway (see Fig. 5) and noteworthily the some neuronal circRNAs in Table 2. BCL2L11 and BBC3 genes in apoptosis pathway associated to response to ER stress, BCL2L11 also related cellular response to beta-amyloid, and AKT1 in negative regulation of autophagy, which is highest degree hub gene in MCC model, were regulated by commonly downregulated hsa-mir-185-3p, hsa-mir-185-5p, whilst hub genes of ACTB and MAPK1 in tau protein binding were regulated by hsa-mir-5698. More importantly, in our statistical results, we merely found the hsa-mir-5698 from DEmiRs is downregulated in AD group despite being upregulated in MCI group compared to healthy group. Interestingly, from these circRNAs, a remarkable correlation was discerned between hub gene TP53 and UBN1in cellular senescence pathway, since UBN1 takes a role as a novel regulator of aging and resides in the formation of aging-related heterochromatin foci (SAHF), which suppresses the expression of proliferation-promoting genes (Banumathy et al. 2009). Moreover, although UBN1 is predominantly localized in the nucleoplasm, our output suggests that it is the most statistically significant of the DEcircRs in plasma exosomes (Adj.P.Val =1.35E-17).
The hub genes of AKT1, GSK3B and IGF1R in the insulin signaling, mTOR and PI3K-Akt signaling pathway were regulated by commonly hsa-mir-185-3p, hsa-mir-185-5p, and hsa-mir-409-3p. Very recently, previous studies have revealed that exosomal hsa-miR-185-5p is that acts on APP dysregulation (Ding et al. 2022) and neurofibrillary pathology (Sabaie et al. 2022) by discovering reduced amounts of hsa-miR-185-5p in AD patients’ plasma samples. As mentioned before, among several AD types related to 10 genes, AKT1 and GSK3B in three pathways are commonly activated in the negative regulation of neuron death, IGF1R and GSK3B in the mTOR pathway are taken part in the cellular response to beta-amyloid. Likewise, by involving in the PI3K-Akt signaling pathway, BCL2L11, CREB3L2, HSP90B1, and THBS1 are associated with the biological process of response to ER stress, HSP90AA1, HSP90AB1 and GSK3B related to positive regulation of tau protein kinase activity, and BCL2L11, GSK3B, and IGF1R take place in the cellular response to beta-amyloid. Overall, possible target genes of downregulated DEmiRs related to mentioned pathways in Table 3-4 represent in supplementary Fig.5.
Briefly, the comprehensive structure of the study is to investigate the plasma exosomal circRNAs and miRNAs expression patterns performed the functional enrichment analysis correlated to Alzheimer’s Disease. In this manner, based on the statistically significant miRNA and circRNA network, it was determined that most of the target genes were associated with the PI3K-Akt pathway and that they were mostly regulated by hsa-mir-615-3p, hsa-mir-196a-5p, hsa-let-7c-5p hsa-let-205-5p, hsa-mir-185-3p, hsa-mir-185-5p, hsa-mir-374a-5p, hsa-mir-374a-3p. From these micro RNAs, has-mir-615-3p (Liu et al. 2022), mir-196 and mir-185 (Zeng et al. 2021), and mir-374a (Bian et al. 2019) are consistent with those of other studies with AD, and in this study, they were found in the plasma exosomes. Mir-let-7c and mir-205 were not associated with AD before to the best of our knowledge and might be a novel biomarker candidate. However, there are some limitations to scientifically influencing the results of the study. Firstly, the reader should bear in mind that the study was based on prediction in part of DEmiRs and circRNAs analysis by bioinformatic tools, so it needs to be validated by further experimental investigations. Secondly, due to the lack of adequate data in the databases, all DEmiRs and circRNAs could not be integrated into associated with AD. Third, the sample size of the NGS employed here was not large sufficiently, exclusively comprising 9 tissue samples.