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Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease.

Montag J, Hitt R, Opitz L, Schulz-Schaeffer WJ, Hunsmann G, Motzkus D - Mol Neurodegener (2009)

Bottom Line: Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods.Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals.In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infection Models, German Primate Center, Kellnerweg 4, 37077 Goettingen, Germany. dmotzkus@dpz.eu.

ABSTRACT
The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.

No MeSH data available.


Related in: MedlinePlus

Regulation of miRNA hsa-miR-342-3p in different prion disorders. A. Regulation of miRNAs upon BSE infection in cynomolgus macaques compared to the published miRNA profile of Scrapie infection in mice. 500 ng miRNA enriched fraction from a BSE-infected and a non-infected rhesus macaque were fluorescently labeled and applied to miRNA microarray (Probeset 1564V1, Ambion). Regulation of each miRNA was calculated from the relative fluorescence values of the infected versus the non-infected animal. Acquired miRNA regulation profile was compared to a published profile derived from Scrapie-infected mice [11]. Correlation of both regulation profiles was statistically significant (Spearman correlation, p < 0.05). Relevant miRNAs regulated above twofold in both arrays are marked by an open square. B. Regulation of miR-342-3p in samples derived from brains of patients with type 1 sCJD, type 2 sCJD and a healthy control, respectively. 5 ng miRNA were applied to qRT-PCR specific for hsa-miR-342-3p (stem-loop qRT-PCR, Applied Biosystems). Relative miRNA expression was analyzed by ΔΔCT method [27] using the small nucleolar RNA RNU48 as a housekeeping RNA and the healthy human control as a calibrator. Statistical significance was determined by student-t-test (non-parametric with Welch's correction, *** p < 0.001). The mean regulation factor ± SD of duplicates from two independent experiments are shown.
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Figure 2: Regulation of miRNA hsa-miR-342-3p in different prion disorders. A. Regulation of miRNAs upon BSE infection in cynomolgus macaques compared to the published miRNA profile of Scrapie infection in mice. 500 ng miRNA enriched fraction from a BSE-infected and a non-infected rhesus macaque were fluorescently labeled and applied to miRNA microarray (Probeset 1564V1, Ambion). Regulation of each miRNA was calculated from the relative fluorescence values of the infected versus the non-infected animal. Acquired miRNA regulation profile was compared to a published profile derived from Scrapie-infected mice [11]. Correlation of both regulation profiles was statistically significant (Spearman correlation, p < 0.05). Relevant miRNAs regulated above twofold in both arrays are marked by an open square. B. Regulation of miR-342-3p in samples derived from brains of patients with type 1 sCJD, type 2 sCJD and a healthy control, respectively. 5 ng miRNA were applied to qRT-PCR specific for hsa-miR-342-3p (stem-loop qRT-PCR, Applied Biosystems). Relative miRNA expression was analyzed by ΔΔCT method [27] using the small nucleolar RNA RNU48 as a housekeeping RNA and the healthy human control as a calibrator. Statistical significance was determined by student-t-test (non-parametric with Welch's correction, *** p < 0.001). The mean regulation factor ± SD of duplicates from two independent experiments are shown.

Mentions: Most intriguingly, Saba and colleagues examined the miRNA profile of Scrapie-infected mice [11]. In order to determine coincidences in the miRNA regulation patterns of both, Scrapie infection in mice and BSE infection in non-human primates, we correlated the miRNA expression patterns derived by miRNA-microarray. Even though several miRNAs were regulated in only one of both infection models the miRNA expression profiles showed a statistical significant correlation (Spearman, p < 0.05; Figure 2A). Among others, such as hsa-miR-191, hsa-miR-200b, hsa-miR-320 and several members of let-7 family we found that hsa-miR-342-3p was regulated at a late stage of disease in both, Scrapie-infected mice and BSE-infected macaques. It should be noted that only a very small region of the brain, i.e. basis pontis, was used for our analysis in non-human primates as compared to whole brain of Scrapie-infected mice. This may be reflected by the detected differences in regulation patterns. However, the comparable regulation of hsa-miR-342-3p may apply to prion disease in general. We concluded that upregulation of this particular miRNA may be a general marker candidate for late-stage prion disease or neurodegeneration. In this case hsa-miR-342-3p would also be upregulated in human prion disease. To strengthen our hypothesis we assessed the regulation of miR-342-3p in brains of sporadic Creutzfeldt-Jakob disease (sCJD) patients. In contrast to the experimentally induced prion disease in macaques, human sCJD occurs idiopathically. Depending on the age of onset, disease course, plaque formation, affected brain regions and biochemical properties of the prion protein aggregates human sCJD has been classified into two different types [25]. The miR-342-3p expression in the brains of a sCJD type 1 and a sCJD type 2 patient compared to a non-infected individual was analysed by qRT-PCR. MiRNA was enriched as described for simian brains and applied to stem-loop qRT-PCR for hsa-miR-342-3p. Data were normalized against the ubiquitously expressed small nucleolar RNA RNU48. Our pilot study revealed that hsa-miR-342-3p was upregulated approximately 2-fold in sCJD type 1 and 1.5-fold in sCJD type 2, respectively (Figure 2B) at a comparable level as in BSE-infected macaques.


Upregulation of miRNA hsa-miR-342-3p in experimental and idiopathic prion disease.

Montag J, Hitt R, Opitz L, Schulz-Schaeffer WJ, Hunsmann G, Motzkus D - Mol Neurodegener (2009)

Regulation of miRNA hsa-miR-342-3p in different prion disorders. A. Regulation of miRNAs upon BSE infection in cynomolgus macaques compared to the published miRNA profile of Scrapie infection in mice. 500 ng miRNA enriched fraction from a BSE-infected and a non-infected rhesus macaque were fluorescently labeled and applied to miRNA microarray (Probeset 1564V1, Ambion). Regulation of each miRNA was calculated from the relative fluorescence values of the infected versus the non-infected animal. Acquired miRNA regulation profile was compared to a published profile derived from Scrapie-infected mice [11]. Correlation of both regulation profiles was statistically significant (Spearman correlation, p < 0.05). Relevant miRNAs regulated above twofold in both arrays are marked by an open square. B. Regulation of miR-342-3p in samples derived from brains of patients with type 1 sCJD, type 2 sCJD and a healthy control, respectively. 5 ng miRNA were applied to qRT-PCR specific for hsa-miR-342-3p (stem-loop qRT-PCR, Applied Biosystems). Relative miRNA expression was analyzed by ΔΔCT method [27] using the small nucleolar RNA RNU48 as a housekeeping RNA and the healthy human control as a calibrator. Statistical significance was determined by student-t-test (non-parametric with Welch's correction, *** p < 0.001). The mean regulation factor ± SD of duplicates from two independent experiments are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Regulation of miRNA hsa-miR-342-3p in different prion disorders. A. Regulation of miRNAs upon BSE infection in cynomolgus macaques compared to the published miRNA profile of Scrapie infection in mice. 500 ng miRNA enriched fraction from a BSE-infected and a non-infected rhesus macaque were fluorescently labeled and applied to miRNA microarray (Probeset 1564V1, Ambion). Regulation of each miRNA was calculated from the relative fluorescence values of the infected versus the non-infected animal. Acquired miRNA regulation profile was compared to a published profile derived from Scrapie-infected mice [11]. Correlation of both regulation profiles was statistically significant (Spearman correlation, p < 0.05). Relevant miRNAs regulated above twofold in both arrays are marked by an open square. B. Regulation of miR-342-3p in samples derived from brains of patients with type 1 sCJD, type 2 sCJD and a healthy control, respectively. 5 ng miRNA were applied to qRT-PCR specific for hsa-miR-342-3p (stem-loop qRT-PCR, Applied Biosystems). Relative miRNA expression was analyzed by ΔΔCT method [27] using the small nucleolar RNA RNU48 as a housekeeping RNA and the healthy human control as a calibrator. Statistical significance was determined by student-t-test (non-parametric with Welch's correction, *** p < 0.001). The mean regulation factor ± SD of duplicates from two independent experiments are shown.
Mentions: Most intriguingly, Saba and colleagues examined the miRNA profile of Scrapie-infected mice [11]. In order to determine coincidences in the miRNA regulation patterns of both, Scrapie infection in mice and BSE infection in non-human primates, we correlated the miRNA expression patterns derived by miRNA-microarray. Even though several miRNAs were regulated in only one of both infection models the miRNA expression profiles showed a statistical significant correlation (Spearman, p < 0.05; Figure 2A). Among others, such as hsa-miR-191, hsa-miR-200b, hsa-miR-320 and several members of let-7 family we found that hsa-miR-342-3p was regulated at a late stage of disease in both, Scrapie-infected mice and BSE-infected macaques. It should be noted that only a very small region of the brain, i.e. basis pontis, was used for our analysis in non-human primates as compared to whole brain of Scrapie-infected mice. This may be reflected by the detected differences in regulation patterns. However, the comparable regulation of hsa-miR-342-3p may apply to prion disease in general. We concluded that upregulation of this particular miRNA may be a general marker candidate for late-stage prion disease or neurodegeneration. In this case hsa-miR-342-3p would also be upregulated in human prion disease. To strengthen our hypothesis we assessed the regulation of miR-342-3p in brains of sporadic Creutzfeldt-Jakob disease (sCJD) patients. In contrast to the experimentally induced prion disease in macaques, human sCJD occurs idiopathically. Depending on the age of onset, disease course, plaque formation, affected brain regions and biochemical properties of the prion protein aggregates human sCJD has been classified into two different types [25]. The miR-342-3p expression in the brains of a sCJD type 1 and a sCJD type 2 patient compared to a non-infected individual was analysed by qRT-PCR. MiRNA was enriched as described for simian brains and applied to stem-loop qRT-PCR for hsa-miR-342-3p. Data were normalized against the ubiquitously expressed small nucleolar RNA RNU48. Our pilot study revealed that hsa-miR-342-3p was upregulated approximately 2-fold in sCJD type 1 and 1.5-fold in sCJD type 2, respectively (Figure 2B) at a comparable level as in BSE-infected macaques.

Bottom Line: Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods.Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals.In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Infection Models, German Primate Center, Kellnerweg 4, 37077 Goettingen, Germany. dmotzkus@dpz.eu.

ABSTRACT
The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.

No MeSH data available.


Related in: MedlinePlus