Dataset Information

MiR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells.

ABSTRACT: Mineralocorticoids trigger a profibrotic process in the kidney. In mouse cortical collecting duct cells, the present study addressed two main questions: 1) what are microRNAs (miRNAs) and their target genes that are changed by aldosterone? and 2) what do miRNAs, in response to aldosterone, regulate regarding signaling pathways related to fibrosis? A microarray chip assay was done in cells in the absence or presence of aldosterone treatment (10-6 M; 3 days). The candidate miRNAs were identified by the criteria of >30% of fold change among the significantly changed miRNAs ( P < 0.05). Twenty-nine miRNAs were upregulated (>1.3-fold), and 27 miRNAs were downregulated (<0.7-fold). Putative target genes of identified miRNAs were associated with 74 Kyoto Encyclopedia of Genes and Genomes pathways. Among them, the wingless-related integration site (Wnt) signaling pathway was highly ranked, where 15 mature miRNAs were observed. These miRNAs were further analyzed by real-time quantitative PCR, and among them, miR-130b-3p, miR-34c-5p, and miR-146a-5p were selected. Through the identification of putative target genes of these three miRNAs, mRNA and protein expression of the Ca2+/calmodulin-dependent protein kinase type II ?-chain ( Camk2b) gene (a target gene of miR-34c-5p) were found to be increased significantly in aldosterone-treated cells, where fibronectin (FN) and ?-smooth muscle actin were induced. When CaMKII? small interfering RNA or the miR-34c-5p mimic was transfected, aldosterone-induced FN expression was significantly attenuated, along with reduced CaMKII? protein expression. A luciferase reporter assay revealed a decrease of CaMKII? translation in cells transfected with miRNA mimics of miR-34c-5p. In conclusion, aldosterone-induced downregulation of miR-34c-5p in the Wnt signaling pathway and a consequent increase of CaMKII? expression are likely to be involved in aldosterone-induced fibrosis.

SUBMITTER: Park EJ

PROVIDER: S-EPMC6048446 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells.

Park Eui-Jung EJ Jung Hyun Jun HJ Choi Hyo-Jung HJ Cho Jeong-In JI Park Hye-Jeong HJ Kwon Tae-Hwan TH

American journal of physiology. Renal physiology 20171025 3

Mineralocorticoids trigger a profibrotic process in the kidney. In mouse cortical collecting duct cells, the present study addressed two main questions: 1) what are microRNAs (miRNAs) and their target genes that are changed by aldosterone? and 2) what do miRNAs, in response to aldosterone, regulate regarding signaling pathways related to fibrosis? A microarray chip assay was done in cells in the absence or presence of aldosterone treatment (10<sup>-6</sup> M; 3 days). The candidate miRNAs were i ...[more]

PMID: 29070573

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Project description:BACKGROUND:Duchenne (DMD) and Becker (BMD) muscular dystrophies are caused by mutations in the DMD gene coding for dystrophin, a protein being part of a large sarcolemmal protein scaffold that includes the neuronal nitric oxide synthase (nNOS). The nNOS was shown to play critical roles in a variety of muscle functions and alterations of its expression and location in dystrophic muscle fiber leads to an increase of the muscle fatigability. We previously revealed a decrease of nNOS expression in BMD patients all presenting a deletion of exons 45 to 55 in the DMD gene (BMDd45-55), impacting the nNOS binding site of dystrophin. Since several studies showed deregulation of microRNAs (miRNAs) in dystrophinopathies, we focused on miRNAs that could target nNOS in dystrophic context. METHODS:By a screening of 617 miRNAs in BMDd45-55 muscular biopsies using TLDA and an in silico study to determine which one could target nNOS, we selected four miRNAs. In order to select those that targeted a sequence of 3'UTR of NOS1, we performed luciferase gene reporter assay in HEK393T cells. Finally, expression of candidate miRNAs was modulated in control and DMD human myoblasts (DMDd45-52) to study their ability to target nNOS. RESULTS:TLDA assay and the in silico study allowed us to select four miRNAs overexpressed in muscle biopsies of BMDd45-55 compared to controls. Among them, only the overexpression of miR-31, miR-708, and miR-34c led to a decrease of luciferase activity in an NOS1-3'UTR-luciferase assay, confirming their interaction with the NOS1-3'UTR. The effect of these three miRNAs was investigated on control and DMDd45-52 myoblasts. First, we showed a decrease of nNOS expression when miR-708 or miR-34c were overexpressed in control myoblasts. We then confirmed that DMDd45-52 cells displayed an endogenous increased of miR-31, miR-708, and miR-34c and a decreased of nNOS expression, the same characteristics observed in BMDd45-55 biopsies. In DMDd45-52 cells, we demonstrated that the inhibition of miR-708 and miR-34c increased nNOS expression, confirming that both miRNAs can modulate nNOS expression in human myoblasts. CONCLUSION:These results strongly suggest that miR-708 and miR-34c, overexpressed in dystrophic context, are new actors involved in the regulation of nNOS expression in dystrophic muscle.

Dataset Information

MiR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells.

Publications

miR-34c-5p and CaMKII are involved in aldosterone-induced fibrosis in kidney collecting duct cells.

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