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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

Target genes Bcl2, Mcl1, Ccnd1 and Faim are downregulated by hyperosmotic stimulation in perfused rat liver.mRNA analysis of target genes of the miR-15 family under hyper- and normoosmotic conditions in perfused rat liver. mRNA levels of hyper- and normoosmotically treated samples at specific time points (30, 60, 120 and 180 minutes) were compared to unstimulated controls (T0). (a,b) Bcl2 and Ccnd1 are significantly downregulated after 120 and 180 minutes of hyperosmotic perfusion. (c) Mcl1 is upregulated under normoosmotic conditions, while this effect is attenuated under hyperosmotic conditions. (d) The anti-apoptotic molecule Faim is significantly downregulated under hyperosmotic conditions and stably expressed in the normoosmotic control. qPCR runs were normalized according to the ΔΔCt method using Gapdh as reference gene. 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.
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f2: Target genes Bcl2, Mcl1, Ccnd1 and Faim are downregulated by hyperosmotic stimulation in perfused rat liver.mRNA analysis of target genes of the miR-15 family under hyper- and normoosmotic conditions in perfused rat liver. mRNA levels of hyper- and normoosmotically treated samples at specific time points (30, 60, 120 and 180 minutes) were compared to unstimulated controls (T0). (a,b) Bcl2 and Ccnd1 are significantly downregulated after 120 and 180 minutes of hyperosmotic perfusion. (c) Mcl1 is upregulated under normoosmotic conditions, while this effect is attenuated under hyperosmotic conditions. (d) The anti-apoptotic molecule Faim is significantly downregulated under hyperosmotic conditions and stably expressed in the normoosmotic control. qPCR runs were normalized according to the ΔΔCt method using Gapdh as reference gene. 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.

Mentions: Interestingly, miR-15a, -15b and -16 are known as tumor-suppressor miRNAs as their activity directly represses the expression of anti-apoptotic genes such as Bcl2 (validated in human, mouse and rat2324), Ccnd1 (validated in human, mouse and rat25) and protein carboxyl-O-methyltransferase (Pcmt1; validated in human26). Mcl1 is indicated in the literature as validated miR-15a target in humans, however to the best of our knowledge, this interaction has never been experimentally validated and, for the purpose of this study, Mcl1 is included as miR-15a predicted target27. In order to evaluate whether the upregulation of these miRNAs results in the downregulation of their validated and predicted targets, qPCR was used to measure mRNA levels of Bcl2, Ccnd1, Mcl1 and Pcmt1 (Fig. 2 and Supplementary Figure 1). Interestingly, Bcl2 [0.4-fold after 120 minutes (p = 0.0127), 0.35-fold after 180 minutes (p = 0.0065)] and Ccnd1 [0.3-fold after 120 minutes (p = 0.0199), 0.3-fold after 180 minutes (p = 0.0227)] were found significantly downregulated by hyperosmotic stimulation (Fig. 2a-b). Whereas, Mcl1 expression shows a tendency toward downregulation compared to the control, although without reaching significance (Fig. 2c). Overall, these findings implicate that signaling pathways activated by hyperosmotic hepatocyte shrinkage downregulate the expression of anti-apoptotic genes, which are either validated (Bcl2 and Ccnd1) or predicted (Mcl1) miR-15/16 targets. Based on these data, we hypothesized that hyperosmolarity-mediated upregulation of miRNA expression could trigger or contribute to the downregulation of anti-apoptotic genes (Fig. 2).


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)

Target genes Bcl2, Mcl1, Ccnd1 and Faim are downregulated by hyperosmotic stimulation in perfused rat liver.mRNA analysis of target genes of the miR-15 family under hyper- and normoosmotic conditions in perfused rat liver. mRNA levels of hyper- and normoosmotically treated samples at specific time points (30, 60, 120 and 180 minutes) were compared to unstimulated controls (T0). (a,b) Bcl2 and Ccnd1 are significantly downregulated after 120 and 180 minutes of hyperosmotic perfusion. (c) Mcl1 is upregulated under normoosmotic conditions, while this effect is attenuated under hyperosmotic conditions. (d) The anti-apoptotic molecule Faim is significantly downregulated under hyperosmotic conditions and stably expressed in the normoosmotic control. qPCR runs were normalized according to the ΔΔCt method using Gapdh as reference gene. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4508667&req=5

f2: Target genes Bcl2, Mcl1, Ccnd1 and Faim are downregulated by hyperosmotic stimulation in perfused rat liver.mRNA analysis of target genes of the miR-15 family under hyper- and normoosmotic conditions in perfused rat liver. mRNA levels of hyper- and normoosmotically treated samples at specific time points (30, 60, 120 and 180 minutes) were compared to unstimulated controls (T0). (a,b) Bcl2 and Ccnd1 are significantly downregulated after 120 and 180 minutes of hyperosmotic perfusion. (c) Mcl1 is upregulated under normoosmotic conditions, while this effect is attenuated under hyperosmotic conditions. (d) The anti-apoptotic molecule Faim is significantly downregulated under hyperosmotic conditions and stably expressed in the normoosmotic control. qPCR runs were normalized according to the ΔΔCt method using Gapdh as reference gene. 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.
Mentions: Interestingly, miR-15a, -15b and -16 are known as tumor-suppressor miRNAs as their activity directly represses the expression of anti-apoptotic genes such as Bcl2 (validated in human, mouse and rat2324), Ccnd1 (validated in human, mouse and rat25) and protein carboxyl-O-methyltransferase (Pcmt1; validated in human26). Mcl1 is indicated in the literature as validated miR-15a target in humans, however to the best of our knowledge, this interaction has never been experimentally validated and, for the purpose of this study, Mcl1 is included as miR-15a predicted target27. In order to evaluate whether the upregulation of these miRNAs results in the downregulation of their validated and predicted targets, qPCR was used to measure mRNA levels of Bcl2, Ccnd1, Mcl1 and Pcmt1 (Fig. 2 and Supplementary Figure 1). Interestingly, Bcl2 [0.4-fold after 120 minutes (p = 0.0127), 0.35-fold after 180 minutes (p = 0.0065)] and Ccnd1 [0.3-fold after 120 minutes (p = 0.0199), 0.3-fold after 180 minutes (p = 0.0227)] were found significantly downregulated by hyperosmotic stimulation (Fig. 2a-b). Whereas, Mcl1 expression shows a tendency toward downregulation compared to the control, although without reaching significance (Fig. 2c). Overall, these findings implicate that signaling pathways activated by hyperosmotic hepatocyte shrinkage downregulate the expression of anti-apoptotic genes, which are either validated (Bcl2 and Ccnd1) or predicted (Mcl1) miR-15/16 targets. Based on these data, we hypothesized that hyperosmolarity-mediated upregulation of miRNA expression could trigger or contribute to the downregulation of anti-apoptotic genes (Fig. 2).

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