Table 1 Reported changes in miRNA expression in SCAs and their links with polyQ toxicity

SCA1

miR-19, −101 and −130 downregulate the ATXN1 gene

Candidate miRNAs were identified using the PicTar algorithm.

Transfection with miRNA duplexes and their specific 2′-O-methyl inhibitors followed by western blot and RT-PCR analyses. miRNAs transfected to cells either individually or collectively.

MCF7 (highly express endogenous ATXN1), HEK293T, HeLa and NIH3T3 cell lines

Eight miRNAs were chosen for further examination based on the number of target sites in the ATXN1 transcript and their neuronal expression.

Luciferase reporter assays (Promega) with vectors carrying fragments or full length human ATXN1 3′-UTRs.

HeLa cell line

miRNA levels and their expression patterns in mouse cerebella were assessed by northern blot analysis and in situ hybridization with LNA probes (Exiqon).

C57/B6 WT mouse

Cell death assays with mutant ATXN1deprived of target sites.

HEK293T cell line

miRNA expression upregulated in SCA1 patients; the increase more prominent in the cortex samples

Regulatory RNA network and TargetScan prediction algorithms.

miRCURY LNA human microRNA Array (Exiqon).

Human cerebellum and cortex, SCA1 patients and healthy controls

miR-144 slightly downregulated in SCA1 cerebellum but strongly induced in the cortex

qRT-PCR, TaqMan miRNA assays (Applied Biosystems) for miR-144 and miR-101.

Upregulated miRNAs predicted to target ATXN1, e.g., miR-101, -130a, -19a, -302

ATXN1 mRNA and protein levels analyzed with RT-PCR and western blot.

miR-144 and −101 downregulate ATXN1 expression, miR-25 does not affect ATXN1 levels

Overexpression of miRNA duplexes and miRNA inhibitors followed by western blotting.

HEK293T cell line

Luciferase assays (Promega) with full-length ATXN1-3′-UTR-hLuc reporters and miRNA duplexes or 2′-O-methyl modified masking oligos. miRs 144 and 101 tested separately and in combination.

No evidence for a statistically significant difference in miRNA expression. A trend to overexpression of miR-33-5p, −34-5p and -92a-5p

Illumina Hi-Seq 2000, small RNAs from fly heads

Drosophila models: UAS-Atxn1-82Q and UAS-Atxn1-30Q

34 miRNAs upregulated; 14 at both time points, 15 at the 4-week time point only and 5 at the 12-week time point

miRCURY LNA all species microRNA arrays (Exiqon)

Cerebellar RNA from SCA1 BO5 transgenic mice [82Q] analyzed at two time points (at 4 and 12 weeks of age)

Individual miRNAs analyzed by qRT-PCR with TaqMan assays (miRs 150, 335, 23a, 24 and 143)

12 miRNAs downregulated; 1 miRNA at both time points, 4 and 7, respectively at the 4- and 12-week time points only

miR-150 levels increased in cerebellar Purkinje neurons and slightly decreased in granule cells

In situ hybridization using dig-labeled DNA-LNA probes (Exiqon)

SCA1 BO5 transgenic mice - Purkinje neurons, granule cells

A concomitant increase in miR-150 and decrease in Rgs8 and Vegfa levels

TargetScan; Rgs8 and Vegfa identified as targets of miR-150

Quantitative PCR and immunohistochemical analyses

SCA1 cerebella, Purkinje neurons

A dose-dependent decrease in Vegfa expression induced by the increased miR-150 activity

Transient transfection with MirVana miR-150 miRNA mimics followed by qPCR and WB. Luciferase assays (Promega) with wild-type Vegfa-3′UTR and miR-150 mimic.

Mouse Neuro2A

SCA2

Upregulation of bantam and miR-12

Confocal microscopy

Drosophila, transgenic recombinant flies, wing imaginal discs cells

SCA3

bantam miRNA is a downstream modulator/suppressor of polyQ toxicity

Phenotype mutants comparison analysis, TUNEL assays, immunostaining and western blots

Drosophila dcr-1 mutants, fly eyes, HeLa cells with normal and pathogenic Ataxin-3 treated with siRNA targeting dicer mRNA

No evidence for a statistically significant difference in miRNA expression. A trend to a decrease in miR-1-3p and an increase in miR-100-5p, −33-5p and 92a-5p levels.

Illumina Hi-Seq 2000, small RNAs from fly heads

Drosophila models: UAS-Atxn3-70Q and UAS-Atxn3-19Q

miR-34b is upregulated, and miR-25, −125b, −29a are downregulated in SCA3 patients. Expression of miR-25 and -125b was associated with the course of disease.

miRbase, TargetScan and micro.org were used to search for miRNA binding sites in the human ATXN3 3′-UTR

miRCURY LNA human miRNA Array (Exiqon) (v.14.0), validation miRNA expression by qRT-PCR (Applied Biosystems)

Blood samples obtained from SCA3 patients (35) and control individuals (25)

SCA7

No evidence for a statistically significant difference in miRNA expression. A trend to an increase in miR-33-5p and -92a-5p levels and a decrease in miR-375-3p

Illumina Hi-Seq 2000, small RNAs from fly heads

Drosophila models: UAS-Atxn7-102Q and UAS-Atxn7-10Q