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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

Validation of the functions of miR-541 in mouse testes. Mice received miR-541-inhibitor (inhibitor), miR-541-mimic (mimic), miR-541-inhibitor negative control (inhibitor-NC), or miR-541-mimic negative control (mimic-NC) via efferent duct injection, with or without intraperitoneal administration of 15 μg/kg body weight MC-LR. The mRNA levels of miR-541 (A) and p15 (B) were measured with qRT-PCR; (C) The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) The expression of p15, MDM2, p-MDM2, p53, and p-p53 in testes was observed by immunohistochemistry. Positive cells are marked by an arrow; (E) The testicular tissue structure was determined by H&E staining. The arrow indicates the loss of spermatogenic cells; (F) Fluorescent images of testicular tissues following double staining for TUNEL (yellow) and the nuclear stain DAPI (blue) are shown. The percentages of TUNEL positive cells were quantified (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f005: Validation of the functions of miR-541 in mouse testes. Mice received miR-541-inhibitor (inhibitor), miR-541-mimic (mimic), miR-541-inhibitor negative control (inhibitor-NC), or miR-541-mimic negative control (mimic-NC) via efferent duct injection, with or without intraperitoneal administration of 15 μg/kg body weight MC-LR. The mRNA levels of miR-541 (A) and p15 (B) were measured with qRT-PCR; (C) The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) The expression of p15, MDM2, p-MDM2, p53, and p-p53 in testes was observed by immunohistochemistry. Positive cells are marked by an arrow; (E) The testicular tissue structure was determined by H&E staining. The arrow indicates the loss of spermatogenic cells; (F) Fluorescent images of testicular tissues following double staining for TUNEL (yellow) and the nuclear stain DAPI (blue) are shown. The percentages of TUNEL positive cells were quantified (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.

Mentions: Constructs containing miR-541-mimic, miR-541-inhibitor, and their negative controls were delivered through the efferent ducts. We found that the mimic increased the expression of miR-541, whereas the inhibitor suppressed the expression of miR-541. Interestingly, miR-541-inhibitor was potent in antagonizing MC-LR-mediated upregulation of miR-541 (Figure 5A). However, mRNA expression of p15 was not modulated by the regulation of miR-541, which was consistent with our in vitro data (Figure 5B). We further investigated the effect of manipulated miR-541 expression on the protein levels of p15, MDM2, phospho-MDM2, p53 and phospho-p53 either with or without exposure to MC-LR by Western blot. Upregulating the expression of miR-541 suppressed the levels of p15, MDM2, and phospho-MDM2 while it increased the level of phospho-p53 (Figure 5C). Suppression of miR-541 was correlated with upregulation of p15, MDM2 and phospho-MDM2, but downregulation of phospho-p53 in the testicular tissue irrespective of prior exposure of the mice to MC-LR (Figure 5C). These results were also supported by immunohistochemical staining (Figure 5D). Furthermore, inhibiting miR-541 effectively restored testicular structures, mature sperm counts, and testicular cell viability following exposure to MC-LR (Figure 5E,F).


miR-541 Contributes to Microcystin-LR-Induced Reproductive Toxicity through Regulating the Expression of p15 in Mice
Validation of the functions of miR-541 in mouse testes. Mice received miR-541-inhibitor (inhibitor), miR-541-mimic (mimic), miR-541-inhibitor negative control (inhibitor-NC), or miR-541-mimic negative control (mimic-NC) via efferent duct injection, with or without intraperitoneal administration of 15 μg/kg body weight MC-LR. The mRNA levels of miR-541 (A) and p15 (B) were measured with qRT-PCR; (C) The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) The expression of p15, MDM2, p-MDM2, p53, and p-p53 in testes was observed by immunohistochemistry. Positive cells are marked by an arrow; (E) The testicular tissue structure was determined by H&E staining. The arrow indicates the loss of spermatogenic cells; (F) Fluorescent images of testicular tissues following double staining for TUNEL (yellow) and the nuclear stain DAPI (blue) are shown. The percentages of TUNEL positive cells were quantified (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f005: Validation of the functions of miR-541 in mouse testes. Mice received miR-541-inhibitor (inhibitor), miR-541-mimic (mimic), miR-541-inhibitor negative control (inhibitor-NC), or miR-541-mimic negative control (mimic-NC) via efferent duct injection, with or without intraperitoneal administration of 15 μg/kg body weight MC-LR. The mRNA levels of miR-541 (A) and p15 (B) were measured with qRT-PCR; (C) The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. The expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) The expression of p15, MDM2, p-MDM2, p53, and p-p53 in testes was observed by immunohistochemistry. Positive cells are marked by an arrow; (E) The testicular tissue structure was determined by H&E staining. The arrow indicates the loss of spermatogenic cells; (F) Fluorescent images of testicular tissues following double staining for TUNEL (yellow) and the nuclear stain DAPI (blue) are shown. The percentages of TUNEL positive cells were quantified (right panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
Mentions: Constructs containing miR-541-mimic, miR-541-inhibitor, and their negative controls were delivered through the efferent ducts. We found that the mimic increased the expression of miR-541, whereas the inhibitor suppressed the expression of miR-541. Interestingly, miR-541-inhibitor was potent in antagonizing MC-LR-mediated upregulation of miR-541 (Figure 5A). However, mRNA expression of p15 was not modulated by the regulation of miR-541, which was consistent with our in vitro data (Figure 5B). We further investigated the effect of manipulated miR-541 expression on the protein levels of p15, MDM2, phospho-MDM2, p53 and phospho-p53 either with or without exposure to MC-LR by Western blot. Upregulating the expression of miR-541 suppressed the levels of p15, MDM2, and phospho-MDM2 while it increased the level of phospho-p53 (Figure 5C). Suppression of miR-541 was correlated with upregulation of p15, MDM2 and phospho-MDM2, but downregulation of phospho-p53 in the testicular tissue irrespective of prior exposure of the mice to MC-LR (Figure 5C). These results were also supported by immunohistochemical staining (Figure 5D). Furthermore, inhibiting miR-541 effectively restored testicular structures, mature sperm counts, and testicular cell viability following exposure to MC-LR (Figure 5E,F).

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