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miR-541 Contributes to Microcystin-LR-Induced Reproductive Toxicity through Regulating the Expression of p15 in Mice

View Article: PubMed Central - PubMed

ABSTRACT

Microcystin-leucine arginine (MC-LR) is a harmful cyanotoxin produced by cyanobacteria. MC-LR can exert endocrine-disrupting activities in many organisms. We have previously demonstrated that MC-LR exerts both acute and chronic reproductive toxicity in male mice, resulting in a decline in sperm quality and damage to testicular structure. Moreover, we also observed extensive alterations in a panel of microRNAs in spermatogonial cells after exposure to MC-LR. In this study, we have confirmed that miR-541 was significantly increased both in GC-1 cells (in vitro) and in mouse testes (in vivo) after exposure to MC-LR. Our data support that p15 was the target gene of miR-541. Increase in miR-541 led to a reduction of p15 and murine double minute2 (MDM2), promoting the activation of p53 signaling and MC-LR-mediated cell apoptosis. Moreover, cells responded to MC-LR with reduced viability and increased apoptosis. Consistently, inhibiting miR-541 could upregulate the expression of p15 and MDM2, resulting in the downregulation of phospho-p53. Downregulation of miR-541 promoted cell viability by reducing MC-LR-induced cell apoptosis. In conclusion, we demonstrate here a crucial role for miR-541 in MC-LR-induced toxic effects on the reproductive system, in an attempt to provide a rational strategy for the diagnosis and treatment of MC-LR-induced impairment in the reproductive system.

No MeSH data available.


Related in: MedlinePlus

Effects of MC-LR (Microcystin-leucine arginine) on GC-1 cells. (A) Measurement of cell viability was carried out with the CCK-8 assay; (B) mRNA levels of miR-541 were measured with qRT-PCR in GC-1 cells after exposure to various concentrations of MC-LR for 24 h, as indicated; (C) alignment of orthologous segments of the 3′-UTR of p15, showing the conserved match to the miR-541 seed, and the seed matches (gray); (D) nucleotide sequences of murine miR-541 and the 3′-UTR of p15 region, which encompasses the miR-541 seed match region; (E) luciferase reporter assays were carried out in 293T cells following co-transfection of miR-541-mimic negative control (mimic-NC) or miR-541-mimic (mimic) together with wild-type (WT) p15 vector, mutant (MUT) p15 vector, or empty vector, as indicated. Firefly luciferase activity was normalized based on the Renilla luciferase activity; (F) The mRNA levels of p15 were measured with qRT-PCR in GC-1 cells exposed to various concentrations of MC-LR for 24 h, as indicated; (G) the protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) in GC-1 cells treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (right panels). β-actin was used as a loading control (n = 3); (H) GC-1 cells were treated with various concentrations of MC-LR for 24 h, as indicated. Cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells were revealed by flow cytometric dot plots (Q2 + Q3) and the data were summarized (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f001: Effects of MC-LR (Microcystin-leucine arginine) on GC-1 cells. (A) Measurement of cell viability was carried out with the CCK-8 assay; (B) mRNA levels of miR-541 were measured with qRT-PCR in GC-1 cells after exposure to various concentrations of MC-LR for 24 h, as indicated; (C) alignment of orthologous segments of the 3′-UTR of p15, showing the conserved match to the miR-541 seed, and the seed matches (gray); (D) nucleotide sequences of murine miR-541 and the 3′-UTR of p15 region, which encompasses the miR-541 seed match region; (E) luciferase reporter assays were carried out in 293T cells following co-transfection of miR-541-mimic negative control (mimic-NC) or miR-541-mimic (mimic) together with wild-type (WT) p15 vector, mutant (MUT) p15 vector, or empty vector, as indicated. Firefly luciferase activity was normalized based on the Renilla luciferase activity; (F) The mRNA levels of p15 were measured with qRT-PCR in GC-1 cells exposed to various concentrations of MC-LR for 24 h, as indicated; (G) the protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) in GC-1 cells treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (right panels). β-actin was used as a loading control (n = 3); (H) GC-1 cells were treated with various concentrations of MC-LR for 24 h, as indicated. Cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells were revealed by flow cytometric dot plots (Q2 + Q3) and the data were summarized (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.

Mentions: We treated GC-1 cells with various concentrations of MC-LR for 24 h. Cell viability was significantly decreased at an MC-LR concentration of 500 nM or above (Figure 1A). The expression of miR-541 was significantly upregulated after exposure to MC-LR at these concentrations (Figure 1B).


miR-541 Contributes to Microcystin-LR-Induced Reproductive Toxicity through Regulating the Expression of p15 in Mice
Effects of MC-LR (Microcystin-leucine arginine) on GC-1 cells. (A) Measurement of cell viability was carried out with the CCK-8 assay; (B) mRNA levels of miR-541 were measured with qRT-PCR in GC-1 cells after exposure to various concentrations of MC-LR for 24 h, as indicated; (C) alignment of orthologous segments of the 3′-UTR of p15, showing the conserved match to the miR-541 seed, and the seed matches (gray); (D) nucleotide sequences of murine miR-541 and the 3′-UTR of p15 region, which encompasses the miR-541 seed match region; (E) luciferase reporter assays were carried out in 293T cells following co-transfection of miR-541-mimic negative control (mimic-NC) or miR-541-mimic (mimic) together with wild-type (WT) p15 vector, mutant (MUT) p15 vector, or empty vector, as indicated. Firefly luciferase activity was normalized based on the Renilla luciferase activity; (F) The mRNA levels of p15 were measured with qRT-PCR in GC-1 cells exposed to various concentrations of MC-LR for 24 h, as indicated; (G) the protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) in GC-1 cells treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (right panels). β-actin was used as a loading control (n = 3); (H) GC-1 cells were treated with various concentrations of MC-LR for 24 h, as indicated. Cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells were revealed by flow cytometric dot plots (Q2 + Q3) and the data were summarized (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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Related In: Results  -  Collection

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toxins-08-00260-f001: Effects of MC-LR (Microcystin-leucine arginine) on GC-1 cells. (A) Measurement of cell viability was carried out with the CCK-8 assay; (B) mRNA levels of miR-541 were measured with qRT-PCR in GC-1 cells after exposure to various concentrations of MC-LR for 24 h, as indicated; (C) alignment of orthologous segments of the 3′-UTR of p15, showing the conserved match to the miR-541 seed, and the seed matches (gray); (D) nucleotide sequences of murine miR-541 and the 3′-UTR of p15 region, which encompasses the miR-541 seed match region; (E) luciferase reporter assays were carried out in 293T cells following co-transfection of miR-541-mimic negative control (mimic-NC) or miR-541-mimic (mimic) together with wild-type (WT) p15 vector, mutant (MUT) p15 vector, or empty vector, as indicated. Firefly luciferase activity was normalized based on the Renilla luciferase activity; (F) The mRNA levels of p15 were measured with qRT-PCR in GC-1 cells exposed to various concentrations of MC-LR for 24 h, as indicated; (G) the protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) in GC-1 cells treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (right panels). β-actin was used as a loading control (n = 3); (H) GC-1 cells were treated with various concentrations of MC-LR for 24 h, as indicated. Cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells were revealed by flow cytometric dot plots (Q2 + Q3) and the data were summarized (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
Mentions: We treated GC-1 cells with various concentrations of MC-LR for 24 h. Cell viability was significantly decreased at an MC-LR concentration of 500 nM or above (Figure 1A). The expression of miR-541 was significantly upregulated after exposure to MC-LR at these concentrations (Figure 1B).

View Article: PubMed Central - PubMed

ABSTRACT

Microcystin-leucine arginine (MC-LR) is a harmful cyanotoxin produced by cyanobacteria. MC-LR can exert endocrine-disrupting activities in many organisms. We have previously demonstrated that MC-LR exerts both acute and chronic reproductive toxicity in male mice, resulting in a decline in sperm quality and damage to testicular structure. Moreover, we also observed extensive alterations in a panel of microRNAs in spermatogonial cells after exposure to MC-LR. In this study, we have confirmed that miR-541 was significantly increased both in GC-1 cells (in vitro) and in mouse testes (in vivo) after exposure to MC-LR. Our data support that p15 was the target gene of miR-541. Increase in miR-541 led to a reduction of p15 and murine double minute2 (MDM2), promoting the activation of p53 signaling and MC-LR-mediated cell apoptosis. Moreover, cells responded to MC-LR with reduced viability and increased apoptosis. Consistently, inhibiting miR-541 could upregulate the expression of p15 and MDM2, resulting in the downregulation of phospho-p53. Downregulation of miR-541 promoted cell viability by reducing MC-LR-induced cell apoptosis. In conclusion, we demonstrate here a crucial role for miR-541 in MC-LR-induced toxic effects on the reproductive system, in an attempt to provide a rational strategy for the diagnosis and treatment of MC-LR-induced impairment in the reproductive system.

No MeSH data available.


Related in: MedlinePlus