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Identification of miRNAs regulating MAPT expression and their analysis in plasma of patients with dementia

Piscopo, Paola, Grasso, Margherita, Manzini, Valeria, Zeni, Andrea, Castelluzzo, Michele, Fontana, Francesca, Talarico, Giuseppina, Castellano, Anna Elisa, Rivabene, Roberto, Crestini, Alessio, Bruno, Giuseppe, Ricci, Leonardo, Denti, Michela A.·2023·Frontiers in Molecular Neuroscience
DOI 10.3389/fnmol.2023.1127163PMC PMC10266489
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Abstract

Background: Dementia is one of the most common diseases in elderly people and hundreds of thousand new cases per year of Alzheimer’s disease (AD) are estimated. While the recent decade has seen significant advances in the development of novel biomarkers to identify dementias at their early stage, a great effort has been recently made to identify biomarkers able to improve differential diagnosis. However, only few potential candidates, mainly detectable in cerebrospinal fluid (CSF), have been described so far. Methods: We searched for miRNAs regulating MAPT translation. We employed a capture technology able to find the miRNAs directly bound to the MAPT transcript in cell lines. Afterwards, we evaluated the levels of these miRNAs in plasma samples from FTD (n = 42) and AD patients (n = 33) and relative healthy controls (HCs) (n = 42) by using qRT-PCR. Results: Firstly, we found all miRNAs that interact with the MAPT transcript. Ten miRNAs have been selected to verify their effect on Tau levels increasing or reducing miRNA levels by using cell transfections with plasmids expressing the miRNAs genes or LNA antagomiRs. Following the results obtained, miR-92a-3p, miR-320a and miR-320b were selected to analyse their levels in plasma samples of patients with FTD and AD respect to HCs. The analysis showed that the miR-92a-1-3p was under-expressed in both AD and FTD compared to HCs. Moreover, miR-320a was upregulated in FTD vs. AD patients, particularly in men when we stratified by sex. Respect to HC, the only difference is showed in men with AD who have reduced levels of this miRNA. Instead, miR-320b is up-regulated in both dementias, but only patients with FTD maintain this trend in both genders. Conclusions: Our results seem to identify miR-92a-3p and miR-320a as possible good biomarkers to discriminate AD from HC, while miR-320b to discriminate FTD from HC, particularly in males. Combining three miRNAs improves the accuracy only in females, particularly for differential diagnosis (FTD vs. AD) and to distinguish FTD from HC.

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Figure 1 MAPT mRNA miR-CATCH. (A) Schematic representation of miR-CATCH (1) active mRNA:miRISC complexes are cross-linked using formaldehyde fixation; (2) cells are lysed; (3) capture oligonucleotide probes complexed with metal beads are hybridized to the target sequence in the mRNA of interest; (4) target mRNAs and the miRISC complexes bound to them are pulled down using magnetic separation; (5) the unbound non-target mRNAs (in grey) are washed away resulting in the enrichment of target mRNA:miRISCs complexes; (6) cross-links are reversed and capture oligonucleotides removed enabling measurement of the enrichment of the target MAPT mRNA and of the captured miRNAs (compared to a scramble capture oligo). (B) In the red call-out, the accessible, partially single-stranded region chosen as target for the capture oligonucleotide is shown. The region is located between bases 1855 and 1875 of transcript variant 9 and is present in all variants. (C) miR-CATCH was performed in Kelly cells using capture oligonucleotide (Capture; n = 3) or scrambled control oligonucleotide (Scrambled; n = 3). (D) Identification of MAPT mRNA-binding miRNAs. Heat Map showing the miRNAs significantly enriched in MAPT-miR-CATCHed samples. Data are reported as Fold Change (FC = 2-Ct). The ten miRNAs selected for further validation are shown in bold. (E) Enrichment of miR-190b, miR-146a-5p, let7a-5p, let7e-5p, miR-320b, miR-320a, miR-320c, miR-320d, miR-92a-3p, and miR-1260a. **** p < 0.0001.
Figure 1 MAPT mRNA miR-CATCH. (A) Schematic representation of miR-CATCH (1) active mRNA:miRISC complexes are cross-linked using formaldehyde fixation; (2) cells are lysed; (3) capture oligonucleotide probes complexed with metal beads are hybridized to the target sequence in the mRNA of interest; (4) target mRNAs and the miRISC complexes bound to them are pulled down using magnetic separation; (5) the unbound non-target mRNAs (in grey) are washed away resulting in the enrichment of target mRNA:miRISCs complexes; (6) cross-links are reversed and capture oligonucleotides removed enabling measurement of the enrichment of the target MAPT mRNA and of the captured miRNAs (compared to a scramble capture oligo). (B) In the red call-out, the accessible, partially single-stranded region chosen as target for the capture oligonucleotide is shown. The region is located between bases 1855 and 1875 of transcript variant 9 and is present in all variants. (C) miR-CATCH was performed in Kelly cells using capture oligonucleotide (Capture; n = 3) or scrambled control oligonucleotide (Scrambled; n = 3). (D) Identification of MAPT mRNA-binding miRNAs. Heat Map showing the miRNAs significantly enriched in MAPT-miR-CATCHed samples. Data are reported as Fold Change (FC = 2-Ct). The ten miRNAs selected for further validation are shown in bold. (E) Enrichment of miR-190b, miR-146a-5p, let7a-5p, let7e-5p, miR-320b, miR-320a, miR-320c, miR-320d, miR-92a-3p, and miR-1260a. **** p < 0.0001.CC BY · PubMed Central
Figure 2 miRNA overexpression and inhibition effects on Tau levels in Kelly cells. (A) Effects of the overexpression of miR-92a-3p, miR-320a, miR-320b, miR-320c, miR-320d, miR-1260a, miR-190b, let7a-5p, and let7e-5p on Tau protein levels in Kelly cells. Densitometric analysis from three independent experiments is shown in the lower panel. Data are shown as mean ± S.D. and are compared by t-test for negative control miR (C: Control) versus each miR of interest (value of p: *p < 0.05; ****p < 0.0001). (B) Effects of the inhibition of miR-320a, miR-320b and miR-92a-3p on Tau protein levels in Kelly cells. Densitometric analysis from three independent experiments is shown in the lower panel. Data are shown as mean ± S.D. and are compared by t-test for LNA inhibitor negative control (Control LNA) versus each miR inhibitor of interest (value of p: *p < 0.05; **p < 0.01; ****p < 0.0001). (C) Predicted binding sites of miR-320a, miR-320b and miR-92a-3p on MAPT mRNA. The miRNAs’ target sequences in MAPT mRNA and their hybridization minimum free energy (mfe) were computed with RNAHybrid. CDS: Coding Sequence; 3’UTR: 3’ Untranslated Region.
Figure 2 miRNA overexpression and inhibition effects on Tau levels in Kelly cells. (A) Effects of the overexpression of miR-92a-3p, miR-320a, miR-320b, miR-320c, miR-320d, miR-1260a, miR-190b, let7a-5p, and let7e-5p on Tau protein levels in Kelly cells. Densitometric analysis from three independent experiments is shown in the lower panel. Data are shown as mean ± S.D. and are compared by t-test for negative control miR (C: Control) versus each miR of interest (value of p: *p < 0.05; ****p < 0.0001). (B) Effects of the inhibition of miR-320a, miR-320b and miR-92a-3p on Tau protein levels in Kelly cells. Densitometric analysis from three independent experiments is shown in the lower panel. Data are shown as mean ± S.D. and are compared by t-test for LNA inhibitor negative control (Control LNA) versus each miR inhibitor of interest (value of p: *p < 0.05; **p < 0.01; ****p < 0.0001). (C) Predicted binding sites of miR-320a, miR-320b and miR-92a-3p on MAPT mRNA. The miRNAs’ target sequences in MAPT mRNA and their hybridization minimum free energy (mfe) were computed with RNAHybrid. CDS: Coding Sequence; 3’UTR: 3’ Untranslated Region.CC BY · PubMed Central
Figure 3 Scatter plots of miRNAs expression levels (2-ΔCt) among CT, FTD and AD in the whole population and in males and females. The data are represented as the mean ± SEM. CT, Controls; FTD, Frontotemporal Dementia; AD, Alzheimer’s Disease; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.
Figure 3 Scatter plots of miRNAs expression levels (2-ΔCt) among CT, FTD and AD in the whole population and in males and females. The data are represented as the mean ± SEM. CT, Controls; FTD, Frontotemporal Dementia; AD, Alzheimer’s Disease; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.CC BY · PubMed Central
Figure 4 Receiver operating characteristic curve of differentially expressed miR-92a-3p, miR-320a and miR-320b for CT versus FTD, CT versus AD and FTD versus AD in the whole population and in males and females. CT, Controls; FTD, Frontotemporal Dementia; AD, Alzheimer’s Disease; AUC, Area Under Curve.
Figure 4 Receiver operating characteristic curve of differentially expressed miR-92a-3p, miR-320a and miR-320b for CT versus FTD, CT versus AD and FTD versus AD in the whole population and in males and females. CT, Controls; FTD, Frontotemporal Dementia; AD, Alzheimer’s Disease; AUC, Area Under Curve.CC BY · PubMed Central

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Authors
Piscopo, P., Grasso, M., Manzini, V., Zeni, A., Castelluzzo, M., Fontana, F., Talarico, G., Castellano, A. E., Rivabene, R., Crestini, A., Bruno, G., Ricci, L., & Denti, M. A.
Venue
Frontiers in Molecular Neuroscience
Publisher
Frontiers Media SA
Published
2023-05-31
Volume
16
ISSN
1662-5099
DOI
10.3389/fnmol.2023.1127163
PMC
PMC10266489
Cited by
15
References
4
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
University of Trento (I85H20000200005)

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