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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 and downregulated by administration of apocynin.(a) After addition of apocynin (20 μmol/l) miR-15a is significantly downregulated at 30 and 120 minutes of hyperosmotic treatment. miR-15a levels are stable in the normoosmotic controls. (b) miR-15b is not changed after administration of apocynin in both hyperosmotically perfused livers and controls. (c) miR-16 is significantly downregulated after addition of apocynin at 30 and 120 minutes of hyperosmotic perfusion. (d) pre-miR-15a is significantly upregulated at 60 minutes of hyperosmotic stimulation, while this effect is inhibited by apocynin. 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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f3: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver is blocked and downregulated by administration of apocynin.(a) After addition of apocynin (20 μmol/l) miR-15a is significantly downregulated at 30 and 120 minutes of hyperosmotic treatment. miR-15a levels are stable in the normoosmotic controls. (b) miR-15b is not changed after administration of apocynin in both hyperosmotically perfused livers and controls. (c) miR-16 is significantly downregulated after addition of apocynin at 30 and 120 minutes of hyperosmotic perfusion. (d) pre-miR-15a is significantly upregulated at 60 minutes of hyperosmotic stimulation, while this effect is inhibited by apocynin. 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: It was previously shown that hyperosmotic exposure of rat hepatocytes triggers a proapoptotic state through the activation of the CD95 death receptor45. This process involves ROS formation via activation of NOX as upstream event28. In order to investigate whether ROS formation is involved in the regulation of miR-15a,-15b and -16, a new set of hyper- and normoosmotic perfusion experiments using the NOX inhibitor apocynin was performed. Following RNA isolation and quality control, the expression of miR-15 family members was measured by miQPCR. The analysis identified that apocynin was able to block the hyperosmotic activation of miR-15a, -15b and -16 (Fig. 3). Specifically, statistical analysis with unpaired student’s t-test indicates that miR-15a and miR-16 were significantly downregulated, while miR-15b expression was unchanged after addition of apocynin (Fig. 3). Altogether, these data indicate that NOX activation is required to modulate the activation of miR-15a, -15b and -16. Importantly, the finding that apocynin is able to suppress miRNA activation, strongly suggests that these miRNAs may belong to the redoximiRs, a novel miRNA grouping including all the redox sensitive miRNAs29.


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 and downregulated by administration of apocynin.(a) After addition of apocynin (20 μmol/l) miR-15a is significantly downregulated at 30 and 120 minutes of hyperosmotic treatment. miR-15a levels are stable in the normoosmotic controls. (b) miR-15b is not changed after administration of apocynin in both hyperosmotically perfused livers and controls. (c) miR-16 is significantly downregulated after addition of apocynin at 30 and 120 minutes of hyperosmotic perfusion. (d) pre-miR-15a is significantly upregulated at 60 minutes of hyperosmotic stimulation, while this effect is inhibited by apocynin. 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

f3: Upregulation of miR-15a/b and miR-16 by hyperosmolarity in perfused rat liver is blocked and downregulated by administration of apocynin.(a) After addition of apocynin (20 μmol/l) miR-15a is significantly downregulated at 30 and 120 minutes of hyperosmotic treatment. miR-15a levels are stable in the normoosmotic controls. (b) miR-15b is not changed after administration of apocynin in both hyperosmotically perfused livers and controls. (c) miR-16 is significantly downregulated after addition of apocynin at 30 and 120 minutes of hyperosmotic perfusion. (d) pre-miR-15a is significantly upregulated at 60 minutes of hyperosmotic stimulation, while this effect is inhibited by apocynin. 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: It was previously shown that hyperosmotic exposure of rat hepatocytes triggers a proapoptotic state through the activation of the CD95 death receptor45. This process involves ROS formation via activation of NOX as upstream event28. In order to investigate whether ROS formation is involved in the regulation of miR-15a,-15b and -16, a new set of hyper- and normoosmotic perfusion experiments using the NOX inhibitor apocynin was performed. Following RNA isolation and quality control, the expression of miR-15 family members was measured by miQPCR. The analysis identified that apocynin was able to block the hyperosmotic activation of miR-15a, -15b and -16 (Fig. 3). Specifically, statistical analysis with unpaired student’s t-test indicates that miR-15a and miR-16 were significantly downregulated, while miR-15b expression was unchanged after addition of apocynin (Fig. 3). Altogether, these data indicate that NOX activation is required to modulate the activation of miR-15a, -15b and -16. Importantly, the finding that apocynin is able to suppress miRNA activation, strongly suggests that these miRNAs may belong to the redoximiRs, a novel miRNA grouping including all the redox sensitive miRNAs29.

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