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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

Toxic effects of MC-LR on testicular tissue. Mice were intraperitoneally injected with various concentrations of MC-LR. (A) The impact of MC-LR on testicular tissue structural changes was determined by H&E staining. The arrows indicate the loss of spermatogenic cells; (B) Fluorescent images of testicular tissues following double staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (yellow) and the nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI) (blue) are shown. The percentages of TUNEL positive cells were quantified (lower panels; n = 3); (C) The mRNA levels of caspase 3, cytochrome c (cyt c), bcl-2-associated x (bax), protein phosphatase 2A (PP2A), and B-cell lymphoma-2 (bcl-2) were measured with qRT-PCR in testicular tissues exposed to various concentrations of MC-LR, as indicated; (D) The protein levels of caspase 3, cyt c, bax, PP2A, phospho-PP2A (p-PP2A), bcl-2, and phospho-bcl-2 (p-bcl-2) in testicular tissue treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (E) Caspase 3, bax, and p-bcl-2 in mouse testicular tissue were revealed by immunohistochemistry. Positive cells are marked by an arrow. Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f003: Toxic effects of MC-LR on testicular tissue. Mice were intraperitoneally injected with various concentrations of MC-LR. (A) The impact of MC-LR on testicular tissue structural changes was determined by H&E staining. The arrows indicate the loss of spermatogenic cells; (B) Fluorescent images of testicular tissues following double staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (yellow) and the nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI) (blue) are shown. The percentages of TUNEL positive cells were quantified (lower panels; n = 3); (C) The mRNA levels of caspase 3, cytochrome c (cyt c), bcl-2-associated x (bax), protein phosphatase 2A (PP2A), and B-cell lymphoma-2 (bcl-2) were measured with qRT-PCR in testicular tissues exposed to various concentrations of MC-LR, as indicated; (D) The protein levels of caspase 3, cyt c, bax, PP2A, phospho-PP2A (p-PP2A), bcl-2, and phospho-bcl-2 (p-bcl-2) in testicular tissue treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (E) Caspase 3, bax, and p-bcl-2 in mouse testicular tissue were revealed by immunohistochemistry. Positive cells are marked by an arrow. Results are expressed as means ± S.D. * p < 0.05.

Mentions: Two weeks after exposure to MC-LR, mouse testicular tissue structure was disrupted, as evidenced by H&E staining. The testicular structure of mice that received 7.5 μg/kg MC-LR altered modestly. In contrast, the spermatogenic epithelium became loosened in its organization and disordered spermatogenic cell arrangement was pronounced upon treatment with 15 μg/kg and 30 μg/kg MC-LR (Figure 3A).


miR-541 Contributes to Microcystin-LR-Induced Reproductive Toxicity through Regulating the Expression of p15 in Mice
Toxic effects of MC-LR on testicular tissue. Mice were intraperitoneally injected with various concentrations of MC-LR. (A) The impact of MC-LR on testicular tissue structural changes was determined by H&E staining. The arrows indicate the loss of spermatogenic cells; (B) Fluorescent images of testicular tissues following double staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (yellow) and the nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI) (blue) are shown. The percentages of TUNEL positive cells were quantified (lower panels; n = 3); (C) The mRNA levels of caspase 3, cytochrome c (cyt c), bcl-2-associated x (bax), protein phosphatase 2A (PP2A), and B-cell lymphoma-2 (bcl-2) were measured with qRT-PCR in testicular tissues exposed to various concentrations of MC-LR, as indicated; (D) The protein levels of caspase 3, cyt c, bax, PP2A, phospho-PP2A (p-PP2A), bcl-2, and phospho-bcl-2 (p-bcl-2) in testicular tissue treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (E) Caspase 3, bax, and p-bcl-2 in mouse testicular tissue were revealed by immunohistochemistry. Positive cells are marked by an arrow. 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-f003: Toxic effects of MC-LR on testicular tissue. Mice were intraperitoneally injected with various concentrations of MC-LR. (A) The impact of MC-LR on testicular tissue structural changes was determined by H&E staining. The arrows indicate the loss of spermatogenic cells; (B) Fluorescent images of testicular tissues following double staining for transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (yellow) and the nuclear stain 4′, 6-diamidino-2-phenylindole (DAPI) (blue) are shown. The percentages of TUNEL positive cells were quantified (lower panels; n = 3); (C) The mRNA levels of caspase 3, cytochrome c (cyt c), bcl-2-associated x (bax), protein phosphatase 2A (PP2A), and B-cell lymphoma-2 (bcl-2) were measured with qRT-PCR in testicular tissues exposed to various concentrations of MC-LR, as indicated; (D) The protein levels of caspase 3, cyt c, bax, PP2A, phospho-PP2A (p-PP2A), bcl-2, and phospho-bcl-2 (p-bcl-2) in testicular tissue treated with various concentrations of MC-LR were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (E) Caspase 3, bax, and p-bcl-2 in mouse testicular tissue were revealed by immunohistochemistry. Positive cells are marked by an arrow. Results are expressed as means ± S.D. * p < 0.05.
Mentions: Two weeks after exposure to MC-LR, mouse testicular tissue structure was disrupted, as evidenced by H&E staining. The testicular structure of mice that received 7.5 μg/kg MC-LR altered modestly. In contrast, the spermatogenic epithelium became loosened in its organization and disordered spermatogenic cell arrangement was pronounced upon treatment with 15 μg/kg and 30 μg/kg MC-LR (Figure 3A).

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