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Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver.

Santosa D, Castoldi M, Paluschinski M, Sommerfeld A, Häussinger D - Sci Rep (2015)

Bottom Line: It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs.Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs.It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany.

ABSTRACT
microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.

No MeSH data available.


Related in: MedlinePlus

Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver.Rat livers were perfused with normoosmotic medium (305 mosmol/l), hyperosmotic medium (385 mosmol/l) or hypoosmotic medium (225 mosmol) for up to 180 min. Samples were taken at the time points indicated. (a) miR-15a is significantly upregulated after 60 and 120 minutes of hyperosmotic conditions, whereas a stable expression is observed under hypo- and normoosmotic (305 mosmol/l) conditions (* p < 0.05; **p < 0.01; ***p < 0.001). (b) miR-15b is significantly upregulated at 60 minutes of hyperosmotic perfusion, while it is stably expressed under normoosmotic and hypoosmotic conditions. (c) miR-16 is significantly upregulated under hyperosmolarity. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 5 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100. (d) CD95 was immunoprecipitated and activating CD95-tyrosine phosphorylation (P-CD95-Y) and caspase 3 cleavage were analysed by Western blot using specific antibodies. Total CD95 and γ-tubulin served as respective loading controls. Representative immunoblots from 3 independent experiments are depicted. Hyperosmolarity-induced activation of the CD95 was already detected after 30 min, whereas cleavage of caspase 3 occurred 120 min after exposure to hyperosmolarity. Normoosmotic- and hypoosmotic perfusion of rat liver do not lead to initiation of the apoptotic signaling pathway.
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f1: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver.Rat livers were perfused with normoosmotic medium (305 mosmol/l), hyperosmotic medium (385 mosmol/l) or hypoosmotic medium (225 mosmol) for up to 180 min. Samples were taken at the time points indicated. (a) miR-15a is significantly upregulated after 60 and 120 minutes of hyperosmotic conditions, whereas a stable expression is observed under hypo- and normoosmotic (305 mosmol/l) conditions (* p < 0.05; **p < 0.01; ***p < 0.001). (b) miR-15b is significantly upregulated at 60 minutes of hyperosmotic perfusion, while it is stably expressed under normoosmotic and hypoosmotic conditions. (c) miR-16 is significantly upregulated under hyperosmolarity. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 5 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100. (d) CD95 was immunoprecipitated and activating CD95-tyrosine phosphorylation (P-CD95-Y) and caspase 3 cleavage were analysed by Western blot using specific antibodies. Total CD95 and γ-tubulin served as respective loading controls. Representative immunoblots from 3 independent experiments are depicted. Hyperosmolarity-induced activation of the CD95 was already detected after 30 min, whereas cleavage of caspase 3 occurred 120 min after exposure to hyperosmolarity. Normoosmotic- and hypoosmotic perfusion of rat liver do not lead to initiation of the apoptotic signaling pathway.

Mentions: With the purpose to identify hepatic miRNAs, which are regulated in response to osmotic changes, rat livers were perfused with normo- (305 mosm/l), hyper- (385 mosm/l) or hypoosmotic (225 mosm/l) solutions. Following RNA isolation, the levels of miRNAs were measured by miQPCR as described in the Methods section. We found that the expression of miR-15a, miR-15b and miR-16 was significantly upregulated following perfusion of rat livers with hyperosmotic solution, whereas hypoosmotic perfusion had no significant effect on the expression level of these microRNAs (Fig. 1a-c). Specifically, miR-15a was found to be significantly upregulated at both 60 minutes (5.3-folds, p = 0.0002) and 120 minutes (5.3-folds; p = 0.0001) compared to the preperfusion state (defined as T0), while miR-15b (1.7-folds; p = 0.006) and miR-16 (2.1-folds; p = 0.0078) were both found significantly upregulated at 60 minutes after perfusion of the livers with hyperosmotic solution. Notably, these miRNAs belong to the miR-15/107 family, which comprises 10 paralogous miRNAs sharing the same seed sequence AGCAGC22. Altogether, these findings indicate that the expression levels of miR-15a, -15b and -16 are rapidly modulated in response to hyperosmotic stress.


Hyperosmotic stress activates the expression of members of the miR-15/107 family and induces downregulation of anti-apoptotic genes in rat liver.

Santosa D, Castoldi M, Paluschinski M, Sommerfeld A, Häussinger D - Sci Rep (2015)

Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver.Rat livers were perfused with normoosmotic medium (305 mosmol/l), hyperosmotic medium (385 mosmol/l) or hypoosmotic medium (225 mosmol) for up to 180 min. Samples were taken at the time points indicated. (a) miR-15a is significantly upregulated after 60 and 120 minutes of hyperosmotic conditions, whereas a stable expression is observed under hypo- and normoosmotic (305 mosmol/l) conditions (* p < 0.05; **p < 0.01; ***p < 0.001). (b) miR-15b is significantly upregulated at 60 minutes of hyperosmotic perfusion, while it is stably expressed under normoosmotic and hypoosmotic conditions. (c) miR-16 is significantly upregulated under hyperosmolarity. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 5 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100. (d) CD95 was immunoprecipitated and activating CD95-tyrosine phosphorylation (P-CD95-Y) and caspase 3 cleavage were analysed by Western blot using specific antibodies. Total CD95 and γ-tubulin served as respective loading controls. Representative immunoblots from 3 independent experiments are depicted. Hyperosmolarity-induced activation of the CD95 was already detected after 30 min, whereas cleavage of caspase 3 occurred 120 min after exposure to hyperosmolarity. Normoosmotic- and hypoosmotic perfusion of rat liver do not lead to initiation of the apoptotic signaling pathway.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508667&req=5

f1: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver.Rat livers were perfused with normoosmotic medium (305 mosmol/l), hyperosmotic medium (385 mosmol/l) or hypoosmotic medium (225 mosmol) for up to 180 min. Samples were taken at the time points indicated. (a) miR-15a is significantly upregulated after 60 and 120 minutes of hyperosmotic conditions, whereas a stable expression is observed under hypo- and normoosmotic (305 mosmol/l) conditions (* p < 0.05; **p < 0.01; ***p < 0.001). (b) miR-15b is significantly upregulated at 60 minutes of hyperosmotic perfusion, while it is stably expressed under normoosmotic and hypoosmotic conditions. (c) miR-16 is significantly upregulated under hyperosmolarity. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 5 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100. (d) CD95 was immunoprecipitated and activating CD95-tyrosine phosphorylation (P-CD95-Y) and caspase 3 cleavage were analysed by Western blot using specific antibodies. Total CD95 and γ-tubulin served as respective loading controls. Representative immunoblots from 3 independent experiments are depicted. Hyperosmolarity-induced activation of the CD95 was already detected after 30 min, whereas cleavage of caspase 3 occurred 120 min after exposure to hyperosmolarity. Normoosmotic- and hypoosmotic perfusion of rat liver do not lead to initiation of the apoptotic signaling pathway.
Mentions: With the purpose to identify hepatic miRNAs, which are regulated in response to osmotic changes, rat livers were perfused with normo- (305 mosm/l), hyper- (385 mosm/l) or hypoosmotic (225 mosm/l) solutions. Following RNA isolation, the levels of miRNAs were measured by miQPCR as described in the Methods section. We found that the expression of miR-15a, miR-15b and miR-16 was significantly upregulated following perfusion of rat livers with hyperosmotic solution, whereas hypoosmotic perfusion had no significant effect on the expression level of these microRNAs (Fig. 1a-c). Specifically, miR-15a was found to be significantly upregulated at both 60 minutes (5.3-folds, p = 0.0002) and 120 minutes (5.3-folds; p = 0.0001) compared to the preperfusion state (defined as T0), while miR-15b (1.7-folds; p = 0.006) and miR-16 (2.1-folds; p = 0.0078) were both found significantly upregulated at 60 minutes after perfusion of the livers with hyperosmotic solution. Notably, these miRNAs belong to the miR-15/107 family, which comprises 10 paralogous miRNAs sharing the same seed sequence AGCAGC22. Altogether, these findings indicate that the expression levels of miR-15a, -15b and -16 are rapidly modulated in response to hyperosmotic stress.

Bottom Line: It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs.Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs.It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University, Moorenstrasse 5, 40225 Düsseldorf, Germany.

ABSTRACT
microRNAs are an abundant class of small non-coding RNAs that negatively regulate gene expression. Importantly, microRNA activity has been linked to the control of cellular stress response. In the present study, we investigated whether the expression of hepatic microRNAs is affected by changes in ambient osmolarity. It is shown that hyperosmotic exposure of perfused rat liver induces a rapid upregulation of miR-15a, miR-15b and miR-16, which are members of the miR-15/107 microRNAs superfamily. It was also identified that hyperosmolarity significantly reduces the expression of anti-apoptotic genes including Bcl2, Ccnd1, Mcl1, Faim, Aatf, Bfar and Ikbkb, which are either validated or predicted targets of these microRNAs. Moreover, through the application of NOX and JNK inhibitors as well as benzylamine it is shown that the observed response is mediated by reactive oxygen species (ROS), suggesting that miR-15a, miR-15b and miR-16 are novel redoximiRs. It is concluded that the response of these three microRNAs to osmotic stress is ROS-mediated and that it might contribute to the development of a proapoptotic phenotype.

No MeSH data available.


Related in: MedlinePlus