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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

qPCR validation of potential target genes of the miR-15a/b and miR-16.qPCR validation of the 18 putative targets of miR-15a, miR-15b and miR-16 detected as significantly downregulated by Affymetrix arrays in hyperosmotically perfused livers. Downregulation of Aatf, Bfar, and Ikbkb may contribute to the promotion of a proapoptotic phenotype. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 4 independent experiments. qPCR runs were normalized according to the ΔΔCt method using β-Tubulin as reference gene. The values of unstimulated controls were set arbitrarily to 100.
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f10: qPCR validation of potential target genes of the miR-15a/b and miR-16.qPCR validation of the 18 putative targets of miR-15a, miR-15b and miR-16 detected as significantly downregulated by Affymetrix arrays in hyperosmotically perfused livers. Downregulation of Aatf, Bfar, and Ikbkb may contribute to the promotion of a proapoptotic phenotype. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 4 independent experiments. qPCR runs were normalized according to the ΔΔCt method using β-Tubulin as reference gene. The values of unstimulated controls were set arbitrarily to 100.

Mentions: In order to gain insight into the molecular mechanisms down-stream of ROS-mediated regulation of gene expression, genome-wide analysis of liver transcriptomes was carried out by using microarrays. Data analysis identified that application of hyperosmotic stress affected only a small fraction of transcripts. Specifically, we found that hyperosmotic treatment significantly affected 1.3% (299) and 4.7% (1043) of the genes present on the arrays within 120 minutes and 180 minutes, respectively (Fig. 8). Of the regulated transcripts, 88 (0.4%) and 211 (1%) were up- or downregulated, respectively after 120 minutes under hyperosmotic condition, whereas 319 (1.4%) and 724 (3.3%) transcripts were up- or downregulated after 180 minutes of hyperosmotic perfusion, respectively (Fig. 8 and Fig. 9A–B). Overall, the relatively small number of genes undergoing expression alterations upon the treatment suggests that hyperosmotic stress does not trigger cascades leading to catastrophic events. In order to identify the footprint of miR-15a, -15b and -16 on gene expression in a genome wide scale, putative miR-15b and -16 targets obtained from the miRWalk algorithm (771 predicted targets) were compared with the downregulated genes at 180 minutes after hyperosmotic exposure (721 genes). As shown in Fig. 9C, a total of 18 overlapping genes was identified (see also Table 1). Notably, three of the genes included in the list, namely the apoptosis antagonizing factor (Aatf), bifunctional apoptosis regulator (Bfar) and the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (Ikbkb) are known for their anti-apoptotic activity3435363738. Importantly, the significant downregulation of Aatf, Bfar, Ikbkb and of other 12 predicted targets could be validated by using qPCR (Fig. 10). For the remaining 3 predicted targets [Leptin receptor overlapping transcript (Leprot), 2-5 oligoadenylate synthetase 1B (Oas1b) and the zinc finger protein 105 (Zfp 105)], a tendency towards downregulation could be shown. Altogether, these data indicate that the upregulation of miR-15a, -15b and -16 might repress the expression of several anti-apoptotic genes potentially impairing cell survival.


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)

qPCR validation of potential target genes of the miR-15a/b and miR-16.qPCR validation of the 18 putative targets of miR-15a, miR-15b and miR-16 detected as significantly downregulated by Affymetrix arrays in hyperosmotically perfused livers. Downregulation of Aatf, Bfar, and Ikbkb may contribute to the promotion of a proapoptotic phenotype. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 4 independent experiments. qPCR runs were normalized according to the ΔΔCt method using β-Tubulin as reference gene. The values of unstimulated controls were set arbitrarily to 100.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f10: qPCR validation of potential target genes of the miR-15a/b and miR-16.qPCR validation of the 18 putative targets of miR-15a, miR-15b and miR-16 detected as significantly downregulated by Affymetrix arrays in hyperosmotically perfused livers. Downregulation of Aatf, Bfar, and Ikbkb may contribute to the promotion of a proapoptotic phenotype. Statistical analysis was carried out by unpaired student’s t-test. Data are shown as average ± S.E.M. of 4 independent experiments. qPCR runs were normalized according to the ΔΔCt method using β-Tubulin as reference gene. The values of unstimulated controls were set arbitrarily to 100.
Mentions: In order to gain insight into the molecular mechanisms down-stream of ROS-mediated regulation of gene expression, genome-wide analysis of liver transcriptomes was carried out by using microarrays. Data analysis identified that application of hyperosmotic stress affected only a small fraction of transcripts. Specifically, we found that hyperosmotic treatment significantly affected 1.3% (299) and 4.7% (1043) of the genes present on the arrays within 120 minutes and 180 minutes, respectively (Fig. 8). Of the regulated transcripts, 88 (0.4%) and 211 (1%) were up- or downregulated, respectively after 120 minutes under hyperosmotic condition, whereas 319 (1.4%) and 724 (3.3%) transcripts were up- or downregulated after 180 minutes of hyperosmotic perfusion, respectively (Fig. 8 and Fig. 9A–B). Overall, the relatively small number of genes undergoing expression alterations upon the treatment suggests that hyperosmotic stress does not trigger cascades leading to catastrophic events. In order to identify the footprint of miR-15a, -15b and -16 on gene expression in a genome wide scale, putative miR-15b and -16 targets obtained from the miRWalk algorithm (771 predicted targets) were compared with the downregulated genes at 180 minutes after hyperosmotic exposure (721 genes). As shown in Fig. 9C, a total of 18 overlapping genes was identified (see also Table 1). Notably, three of the genes included in the list, namely the apoptosis antagonizing factor (Aatf), bifunctional apoptosis regulator (Bfar) and the inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (Ikbkb) are known for their anti-apoptotic activity3435363738. Importantly, the significant downregulation of Aatf, Bfar, Ikbkb and of other 12 predicted targets could be validated by using qPCR (Fig. 10). For the remaining 3 predicted targets [Leptin receptor overlapping transcript (Leprot), 2-5 oligoadenylate synthetase 1B (Oas1b) and the zinc finger protein 105 (Zfp 105)], a tendency towards downregulation could be shown. Altogether, these data indicate that the upregulation of miR-15a, -15b and -16 might repress the expression of several anti-apoptotic genes potentially impairing cell survival.

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