Elevated Acute Plasma miR-124-3p Level Relates to Evolution of Larger Cortical Lesion Area after Traumatic Brain Injury

Vuokila, Niina; Gupta, Shalini; Huusko, Riina; Tohka, Jussi; Puhakka, Noora; Pitkänen, Asla

doi:10.1016/j.neuroscience.2020.02.045

Cited by 18 publications

(22 citation statements)

References 66 publications

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“…Although we did not confirm a significant decrease in miR‐124, we observed a biological effect, decreasing following AMPK activation in vitro and 3 hr following ischaemia in vivo . Increased plasma levels of miR‐124 have been reported in rat MCAO models 24 – 48 hr following occlusion and circulating levels positively correlated with tissue damage (Vuokila et al., 2020; Weng et al., 2011); disruption of brain tissue and increased blood–brain barrier permeability allowing for increased release may contribute to circulating miRNA levels not reflected in neuronal tissue. While we initially identified a decrease in miR‐124 expression following 1 hr AICAR treatment in primary neurons, neuronal miR‐124 levels were not measured in vivo until 3 hr post‐ischaemia, potentially highlighting the importance of the time point of miRNA measurement in an evolving profile.…”

Section: Discussionmentioning

confidence: 99%

AMPK‐regulated miRNA‐210‐3p is activated during ischaemic neuronal injury and modulates PI3K‐p70S6K signalling

Pfeiffer

Tomašcová

Mamrak³

et al. 2021

Journal of Neurochemistry

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Progressive neuronal injury following ischaemic stroke is associated with glutamate‐induced depolarization, energetic stress and activation of AMP‐activated protein kinase (AMPK). We here identify a molecular signature associated with neuronal AMPK activation, as a critical regulator of cellular response to energetic stress following ischaemia. We report a robust induction of microRNA miR‐210‐3p both in vitro in primary cortical neurons in response to acute AMPK activation and following ischaemic stroke in vivo. Bioinformatics and reverse phase protein array analysis of neuronal protein expression changes in vivo following administration of a miR‐210‐3p mimic revealed altered expression of phosphatase and tensin homolog (PTEN), 3‐phosphoinositide‐dependent protein kinase 1 (PDK1), ribosomal protein S6 kinase (p70S6K) and ribosomal protein S6 (RPS6) signalling in response to increasing miR‐210‐3p. In vivo, we observed a corresponding reduction in p70S6K activity following ischaemic stroke. Utilizing models of glutamate receptor over‐activation in primary neurons, we demonstrated that induction of miR‐210‐3p was accompanied by sustained suppression of p70S6K activity and that this effect was reversed by miR‐210‐3p inhibition. Collectively, these results provide new molecular insight into the regulation of cell signalling during ischaemic injury, and suggest a novel mechanism whereby AMPK regulates miR‐210‐3p to control p70S6K activity in ischaemic stroke and excitotoxic injury.

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Section: Discussionmentioning

confidence: 99%

AMPK‐regulated miRNA‐210‐3p is activated during ischaemic neuronal injury and modulates PI3K‐p70S6K signalling

Pfeiffer

Tomašcová

Mamrak³

et al. 2021

Journal of Neurochemistry

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“…Immediately after tail-vein blood sampling at 2 days post-TBI, the rats were perfusionfixed and their brain tissue was processed for histology to assess the lesion location and injury severity as described previously [30]. For this, the rats were deeply anesthetized with 5% isoflurane and decapitated.…”

Section: Sampling Of Plasma and Brain Tissuementioning

confidence: 99%

“…The 20 µL ddPCR reaction mixtures were prepared with 10 µL Bio-Rad 2× ddPCR EvaGreen Supermix (#186-4034, Bio-Rad), 1 µL miRNA PCR primers, 1 µL nuclease-free water, and 8 µL diluted cDNA template (10-fold dilution for miR-9a-3p and miR-434-3p, 5-fold dilution for miR-136-3p). Droplets were generated with 70 µL of droplet generation oil for EvaGreen (#186-4005, Bio-Rad) as described previously [30]. The thermal cycling conditions for ddPCR were as follows: 95 • C for 5 min, 40 cycles of 95 • C for 30 s, and 56 • C for 1 min, 4 • C for 5 min, 90 • C for 5 min, and then maintained at 4 • C. To obtain a clear separation between the clusters of positive and negative droplets, the fluorescence amplitude threshold was manually adjusted to 10,000 for rno-miR-9a-3p and rno-miR-434-3p, and 6000 for rno-miR-136-3p for all samples.…”

Section: Ddpcr Of Validated Mirna Candidatesmentioning

confidence: 99%

“…Animal models of TBI are crucial for biomarker identification as they provide homogenous and reproducible methods for blood biomarker discovery, avoiding several confounding factors that contribute to the heterogeneity of the TBI patient population [6,27,28]. Thus far, only three studies have investigated the levels of circulating miRNAs as biomarkers of TBI in animal models [29][30][31], and only one group has investigated the dependency of serum and brain miRNA levels on impact severity [31,32]. In those studies, however, the sensitivity and specificity of the candidate miRNAs were not assessed.…”

Section: Introductionmentioning

confidence: 99%

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Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Gupta

Ciszek

Heiskanen

et al. 2021

IJMS

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Noninvasive, affordable circulating biomarkers for difficult-to-diagnose mild traumatic brain injury (mTBI) are an unmet medical need. Although blood microRNA (miRNA) levels are reportedly altered after traumatic brain injury (TBI), their diagnostic potential for mTBI remains inconclusive. We hypothesized that acutely altered plasma miRNAs could serve as diagnostic biomarkers both in the lateral fluid percussion injury (FPI) model and clinical mTBI. We performed plasma small RNA-sequencing from adult male Sprague–Dawley rats (n = 31) at 2 days post-TBI, followed by polymerase chain reaction (PCR)-based validation of selected candidates. miR-9a-3p, miR-136-3p, and miR-434-3p were identified as the most promising candidates at 2 days after lateral FPI. Digital droplet PCR (ddPCR) revealed 4.2-, 2.8-, and 4.6-fold elevations in miR-9a-3p, miR-136-3p, and miR-434-3p levels (p < 0.01 for all), respectively, distinguishing rats with mTBI from naïve rats with 100% sensitivity and specificity. DdPCR further identified a subpopulation of mTBI patients with plasma miR-9-3p (n = 7/15) and miR-136-3p (n = 5/15) levels higher than one standard deviation above the control mean at <2 days postinjury. In sTBI patients, plasma miR-9-3p levels were 6.5- and 9.2-fold in comparison to the mTBI and control groups, respectively. Thus, plasma miR-9-3p and miR-136-3p were identified as promising biomarker candidates for mTBI requiring further evaluation in a larger patient population.

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“…For ddPCR, 1.33 μl of un-normalized and normalized cDNA templates from the TaqMan RT reaction (without carrier RNA, same samples as used for RT-qPCR) were added to a 20-μl reaction mixture containing 10 μl Bio-Rad 2x ddPCR Supermix for probes (#186-3010, Bio-Rad), 1 μl 20x miR-23a-3p PCR primer, and 7.67 μl nuclease-free water. DdPCR reaction was performed as described previously 31 . For each sample, the reaction was performed in duplicate, and the mean of miR-23a-3p copies/20-μl PCR reaction from the two replicates was calculated and used for representation in the figures (with automatic amplitude threshold).…”

Section: Rt-qpcr Of the Spike-in Unisp6 With Mircury Chemistrymentioning

confidence: 99%

Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization

Gupta

Ndode-Ekane

Puhakka

et al. 2020

Sci Rep

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Quantification of plasma microRNAs (miRNAs) as non-invasive disease biomarkers is subject to multiple technical variabilities. This study aimed to develop an optimized protocol for miRNA quantification from rodent plasma. We hypothesized that a fixed small RNA concentration input for reverse transcription (RT) reaction will provide better miRNA quantification than a fixed RNA volume input. For this, tail-vein plasma was collected from 30 naïve, adult male Sprague-Dawley rats. Plasma hemolysis was measured with NanoDrop-1000 and Denovix DS-11 spectrophotometers. Plasma was then pooled, and RNA was extracted from 50-μl, 100-μl or 200-μl pool aliquots. Small RNA concentration was measured with Qubit miRNA assay. A fixed RNA volume (un-normalized) or a fixed small RNA concentration was used for RT (concentration-normalized). The method was setup with miR-23a-3p and validated with miR-103a-3p and miR-451a. Hemolysis measurements from Denovix and NanoDrop strongly correlated. Qubit revealed increased small RNA concentrations with increasing starting plasma volumes. With concentration-normalization, miRNA levels from 100-µl and 200-µl plasma volume groups mostly normalized to the level of the 50-µl in ddPCR. Our results indicate that miRNA quantification with ddPCR should be performed with small RNA concentration-normalization to minimize variations in eluted RNA concentrations occuring during RNA extraction. MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides long) that regulate the expression of protein-coding genes through translational repression 1. Dysregulation of miRNA levels is observed in many disease conditions, both in the affected tissue and the circulation 2-5. Circulating serum/plasma miRNAs are extremely stable under appropriate sampling and storage conditions 6-10 , and are a potential source of non-invasive diagnostic, prognostic, and predictive biomarkers of disease states 11,12. Despite the stability of circulating plasma miRNAs, there are several challenges associated with their reliable and reproducible quantification as biomarkers. First, hemolysis in plasma impairs the detection of circulating cell-free miRNAs 6,9,13. Second, the total amount of small RNA in the plasma is usually low, making it challenging to obtain accurate concentration measurements using common methods like the NanoDrop or Agilent Bioanalyzer 14. Third, there are no endogenous normalizer miRNAs that can be used as an internal standard 15. Some attempts have been made to overcome these challenges. For example, multiple methods are proposed for detecting plasma hemolysis 16-18. The need to carefully report the protocols used for blood collection, centrifugation, storage, and handling of plasma has been emphasized, because these factors profoundly affect circulating miRNA levels, and the harmonization of these techniques is important for standardizing plasma biomarker research across laboratories 18-22. The addition of carrier RNAs and synthetic spike-ins during RNA extraction and reverse transcription-quantitative polym...

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Elevated Acute Plasma miR-124-3p Level Relates to Evolution of Larger Cortical Lesion Area after Traumatic Brain Injury

Abstract: Elevated acute plasma miR-124-3p level relates to evolution of larger cortical lesion area after traumatic brain injury,

Cited by 18 publications

References 66 publications

AMPK‐regulated miRNA‐210‐3p is activated during ischaemic neuronal injury and modulates PI3K‐p70S6K signalling

AMPK‐regulated miRNA‐210‐3p is activated during ischaemic neuronal injury and modulates PI3K‐p70S6K signalling

Plasma miR-9-3p and miR-136-3p as Potential Novel Diagnostic Biomarkers for Experimental and Human Mild Traumatic Brain Injury

Droplet digital polymerase chain reaction-based quantification of circulating microRNAs using small RNA concentration normalization

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