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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

Benzylamine upregulates miR-15a/16 in a NAC-sensitive manner.Rat livers were perfused with benzylamine (0.2 mmol/l) and glucose (5 mmol/l). In the control, NAC was added with a concentration of 10 mmol/l. (a) miR-15a is significantly upregulated after 60 (p = 0.0031), 120 (p = 0.0184) and 180 minutes (p = 0.0062) of benzylamine perfusion. miR-16 is significantly upregulated after 60 minutes of benzylamine perfusion (p = 0.0245). (b) The target gene Bcl2 is significantly downregulated after 30 (p = 0.0421) and 180 minutes (p = 0.0131) of benzylamine perfusion, while Faim is significantly downregulated after 180 minutes of benzylamine perfusion (p = 0.022). (c) In presence of NAC the benzylamine-induced activation of miRNA was inhibited. (d) Bcl2, Cyclin D1 and Mcl1 are unchanged in presence of NAC, while Faim is significantly downregulated after 180 minutes of perfusion (p = 0.0468). qPCR runs were normalized according to the ΔΔCt method using β-tubulin 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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f5: Benzylamine upregulates miR-15a/16 in a NAC-sensitive manner.Rat livers were perfused with benzylamine (0.2 mmol/l) and glucose (5 mmol/l). In the control, NAC was added with a concentration of 10 mmol/l. (a) miR-15a is significantly upregulated after 60 (p = 0.0031), 120 (p = 0.0184) and 180 minutes (p = 0.0062) of benzylamine perfusion. miR-16 is significantly upregulated after 60 minutes of benzylamine perfusion (p = 0.0245). (b) The target gene Bcl2 is significantly downregulated after 30 (p = 0.0421) and 180 minutes (p = 0.0131) of benzylamine perfusion, while Faim is significantly downregulated after 180 minutes of benzylamine perfusion (p = 0.022). (c) In presence of NAC the benzylamine-induced activation of miRNA was inhibited. (d) Bcl2, Cyclin D1 and Mcl1 are unchanged in presence of NAC, while Faim is significantly downregulated after 180 minutes of perfusion (p = 0.0468). qPCR runs were normalized according to the ΔΔCt method using β-tubulin 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: In order to substantiate that ROS is the driving factor mediating the observed miRNA downregulation, rat livers were perfused with benzylamine, which triggers as a substrate of monoamine oxidase the intracellular formation of H2O2 in liver and hepatocyte shrinkage3031. Notably, the levels of miR-15a (p = 0.0031 after 60 minutes, p = 0.0184 after 120 minutes, p = 0.0062 after 180 minutes) and miR-16 (p = 0.0245 after 60 minutes) were significantly upregulated by this treatment (Fig. 5a), while Bcl2 (p = 0.0421 after 30 minutes, p = 0.0131 after 180 minutes) and Faim (p = 0.022 after 180 minutes) were significantly downregulated (Fig. 5b). On the other hand, there was a trend for Ccnd1 downregulation after 180 minutes of benzylamine perfusion (p = 0.0544), while Mcl1 expression was not affected by the treatment (Fig. 5b). In presence of the antioxidant N-acetylcysteine (NAC) the effects of benzylamine, on miRNA expression were no longer observed (5c–d). Taken together, these findings strengthen the view that ROS is required to mediate the observed response and that miR-15a, -15b and -16 are redoximiRs.


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)

Benzylamine upregulates miR-15a/16 in a NAC-sensitive manner.Rat livers were perfused with benzylamine (0.2 mmol/l) and glucose (5 mmol/l). In the control, NAC was added with a concentration of 10 mmol/l. (a) miR-15a is significantly upregulated after 60 (p = 0.0031), 120 (p = 0.0184) and 180 minutes (p = 0.0062) of benzylamine perfusion. miR-16 is significantly upregulated after 60 minutes of benzylamine perfusion (p = 0.0245). (b) The target gene Bcl2 is significantly downregulated after 30 (p = 0.0421) and 180 minutes (p = 0.0131) of benzylamine perfusion, while Faim is significantly downregulated after 180 minutes of benzylamine perfusion (p = 0.022). (c) In presence of NAC the benzylamine-induced activation of miRNA was inhibited. (d) Bcl2, Cyclin D1 and Mcl1 are unchanged in presence of NAC, while Faim is significantly downregulated after 180 minutes of perfusion (p = 0.0468). qPCR runs were normalized according to the ΔΔCt method using β-tubulin 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

f5: Benzylamine upregulates miR-15a/16 in a NAC-sensitive manner.Rat livers were perfused with benzylamine (0.2 mmol/l) and glucose (5 mmol/l). In the control, NAC was added with a concentration of 10 mmol/l. (a) miR-15a is significantly upregulated after 60 (p = 0.0031), 120 (p = 0.0184) and 180 minutes (p = 0.0062) of benzylamine perfusion. miR-16 is significantly upregulated after 60 minutes of benzylamine perfusion (p = 0.0245). (b) The target gene Bcl2 is significantly downregulated after 30 (p = 0.0421) and 180 minutes (p = 0.0131) of benzylamine perfusion, while Faim is significantly downregulated after 180 minutes of benzylamine perfusion (p = 0.022). (c) In presence of NAC the benzylamine-induced activation of miRNA was inhibited. (d) Bcl2, Cyclin D1 and Mcl1 are unchanged in presence of NAC, while Faim is significantly downregulated after 180 minutes of perfusion (p = 0.0468). qPCR runs were normalized according to the ΔΔCt method using β-tubulin 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: In order to substantiate that ROS is the driving factor mediating the observed miRNA downregulation, rat livers were perfused with benzylamine, which triggers as a substrate of monoamine oxidase the intracellular formation of H2O2 in liver and hepatocyte shrinkage3031. Notably, the levels of miR-15a (p = 0.0031 after 60 minutes, p = 0.0184 after 120 minutes, p = 0.0062 after 180 minutes) and miR-16 (p = 0.0245 after 60 minutes) were significantly upregulated by this treatment (Fig. 5a), while Bcl2 (p = 0.0421 after 30 minutes, p = 0.0131 after 180 minutes) and Faim (p = 0.022 after 180 minutes) were significantly downregulated (Fig. 5b). On the other hand, there was a trend for Ccnd1 downregulation after 180 minutes of benzylamine perfusion (p = 0.0544), while Mcl1 expression was not affected by the treatment (Fig. 5b). In presence of the antioxidant N-acetylcysteine (NAC) the effects of benzylamine, on miRNA expression were no longer observed (5c–d). Taken together, these findings strengthen the view that ROS is required to mediate the observed response and that miR-15a, -15b and -16 are redoximiRs.

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