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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 is blocked by administration of SP600125.(a–d) Addition of the JNK inhibitor SP600125 does not lead to significant changes of miRNAs under both hyperosmotic and normoosmotic conditions. qPCR runs were normalized according to the ΔΔCt method using RNU6 as reference gene. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 3 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100.
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f6: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver is blocked by administration of SP600125.(a–d) Addition of the JNK inhibitor SP600125 does not lead to significant changes of miRNAs under both hyperosmotic and normoosmotic conditions. qPCR runs were normalized according to the ΔΔCt method using RNU6 as reference gene. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 3 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100.

Mentions: NADPH oxidase-derived ROS controls the activity of several signaling pathways including JNKs, which is important for induction of CD95-mediated apoptosis2832. In order to investigate whether JNK signaling is required to mediate the observed phenotype, SP600125, a potent JNK inhibitor33, was added to the perfusion medium 30 minutes before administration of SP600125-containing hyper- and normoosmotic solutions. We found that SP600125 largely counteracted the hyperosmotic activation of miR-15a, -15b, -16 and pre-miR-15a expression (Fig. 6). Next, we evaluated the effect of SP600125 on the expression levels of miR-15a, -15b and -16 target genes. Surprisingly, we found that Bcl2, Ccnd1 and Faim were still downregulated in response to hyperosmotic exposure in combination with SP600125, whereas Mcl1 was upregulated compared to T0 under the same conditions (Fig. 7). However, addition of SP600125 in the normoosmotic control also downregulated Bcl2, Ccnd1 and Faim and upregulated Mcl1 in the livers of control perfused animals. Based on these data, it is concluded that JNK inhibition by SP600125 prevents the hyperosmolarity-mediated downregulation of Bcl2, Ccnd1, Mcl1 and Faim. Overall, these data confirm that the observed regulation of miR-15a, -15b and -16 by hyperosmolarity is down-stream to NOX and requires JNK activity.


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 is blocked by administration of SP600125.(a–d) Addition of the JNK inhibitor SP600125 does not lead to significant changes of miRNAs under both hyperosmotic and normoosmotic conditions. qPCR runs were normalized according to the ΔΔCt method using RNU6 as reference gene. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 3 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4508667&req=5

f6: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver is blocked by administration of SP600125.(a–d) Addition of the JNK inhibitor SP600125 does not lead to significant changes of miRNAs under both hyperosmotic and normoosmotic conditions. qPCR runs were normalized according to the ΔΔCt method using RNU6 as reference gene. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 3 independent experiments. The values of unstimulated controls (T0) were set arbitrarily to 100.
Mentions: NADPH oxidase-derived ROS controls the activity of several signaling pathways including JNKs, which is important for induction of CD95-mediated apoptosis2832. In order to investigate whether JNK signaling is required to mediate the observed phenotype, SP600125, a potent JNK inhibitor33, was added to the perfusion medium 30 minutes before administration of SP600125-containing hyper- and normoosmotic solutions. We found that SP600125 largely counteracted the hyperosmotic activation of miR-15a, -15b, -16 and pre-miR-15a expression (Fig. 6). Next, we evaluated the effect of SP600125 on the expression levels of miR-15a, -15b and -16 target genes. Surprisingly, we found that Bcl2, Ccnd1 and Faim were still downregulated in response to hyperosmotic exposure in combination with SP600125, whereas Mcl1 was upregulated compared to T0 under the same conditions (Fig. 7). However, addition of SP600125 in the normoosmotic control also downregulated Bcl2, Ccnd1 and Faim and upregulated Mcl1 in the livers of control perfused animals. Based on these data, it is concluded that JNK inhibition by SP600125 prevents the hyperosmolarity-mediated downregulation of Bcl2, Ccnd1, Mcl1 and Faim. Overall, these data confirm that the observed regulation of miR-15a, -15b and -16 by hyperosmolarity is down-stream to NOX and requires JNK activity.

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