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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.


The effects of miR-541 on GC-1 cells. (A,B) Six hours after GC-1 cells were transfected with miR-541-inhibitor (inhibitor), miR-541-inhibitor negative control (inhibitor-NC), miR-541-mimic (mimic), or miR-541-mimic negative control (mimic-NC), cells were incubated with 500 nM MC-LR for 24 h, followed by measurement of the mRNA levels of miR-541 (A) and p15 (B) with qRT-PCR; (C) Six hours after GC-1 cells were transfected with mimic-NC, mimic, inhibitor-NC, or inhibitor, cells were incubated with or without 500 nM MC-LR for 24 h. The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. Protein expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) GC-1 cells were transfected with mimic, mimic-NC, inhibitor, or inhibitor-NC, followed by treatment with 500 nM MC-LR for 24 h. The viability of GC-1 cells was revealed by FDA-PI staining. Uptake of FDA (green) indicates live cells, while uptake of propidium iodide (PI; red) indicates late apoptotic and dead cells. The percentages of FDA-/PI+ positive cells were quantified (right panels; n = 3); (E) GC-1 cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells (Q2 + Q3) were examined with flow cytometry. The percentage of apoptotic cells was calculated and summarized (lower panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f002: The effects of miR-541 on GC-1 cells. (A,B) Six hours after GC-1 cells were transfected with miR-541-inhibitor (inhibitor), miR-541-inhibitor negative control (inhibitor-NC), miR-541-mimic (mimic), or miR-541-mimic negative control (mimic-NC), cells were incubated with 500 nM MC-LR for 24 h, followed by measurement of the mRNA levels of miR-541 (A) and p15 (B) with qRT-PCR; (C) Six hours after GC-1 cells were transfected with mimic-NC, mimic, inhibitor-NC, or inhibitor, cells were incubated with or without 500 nM MC-LR for 24 h. The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. Protein expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) GC-1 cells were transfected with mimic, mimic-NC, inhibitor, or inhibitor-NC, followed by treatment with 500 nM MC-LR for 24 h. The viability of GC-1 cells was revealed by FDA-PI staining. Uptake of FDA (green) indicates live cells, while uptake of propidium iodide (PI; red) indicates late apoptotic and dead cells. The percentages of FDA-/PI+ positive cells were quantified (right panels; n = 3); (E) GC-1 cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells (Q2 + Q3) were examined with flow cytometry. The percentage of apoptotic cells was calculated and summarized (lower panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.

Mentions: To examine the regulatory effects of miR-541 and p15 on cellular functions, we transfected GC-1 cells with miR-541-inhibitor, miR-541-mimic, and their negative controls followed by exposure to 500 nM MC-LR for 24 h. miR-541 was decreased in cells transfected with miR-541-inhibitor and highly increased in cells transfected with miR-541-mimic (Figure 2A). The expression of p15 mRNA remained unchanged following transfection with either miR-541-mimic or the inhibitor (Figure 2B). We next measured the effect of modulation of miR-541 on the protein levels of p15 and a group of associated regulatory proteins. Upregulating miR-541 suppressed the expression of p15, MDM2 (murine double minute2) and phospho-MDM2, while it increased the level of phospho-p53 (Figure 2C). Exactly the opposite trends were observed if intracellular miR-541 was suppressed. Furthermore, forced expression of miR-541 promoted MC-LR-induced cell apoptosis, whereas inhibiting miR-541 suppressed apoptosis (Figure 2D,E).


miR-541 Contributes to Microcystin-LR-Induced Reproductive Toxicity through Regulating the Expression of p15 in Mice
The effects of miR-541 on GC-1 cells. (A,B) Six hours after GC-1 cells were transfected with miR-541-inhibitor (inhibitor), miR-541-inhibitor negative control (inhibitor-NC), miR-541-mimic (mimic), or miR-541-mimic negative control (mimic-NC), cells were incubated with 500 nM MC-LR for 24 h, followed by measurement of the mRNA levels of miR-541 (A) and p15 (B) with qRT-PCR; (C) Six hours after GC-1 cells were transfected with mimic-NC, mimic, inhibitor-NC, or inhibitor, cells were incubated with or without 500 nM MC-LR for 24 h. The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. Protein expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) GC-1 cells were transfected with mimic, mimic-NC, inhibitor, or inhibitor-NC, followed by treatment with 500 nM MC-LR for 24 h. The viability of GC-1 cells was revealed by FDA-PI staining. Uptake of FDA (green) indicates live cells, while uptake of propidium iodide (PI; red) indicates late apoptotic and dead cells. The percentages of FDA-/PI+ positive cells were quantified (right panels; n = 3); (E) GC-1 cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells (Q2 + Q3) were examined with flow cytometry. The percentage of apoptotic cells was calculated and summarized (lower panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
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toxins-08-00260-f002: The effects of miR-541 on GC-1 cells. (A,B) Six hours after GC-1 cells were transfected with miR-541-inhibitor (inhibitor), miR-541-inhibitor negative control (inhibitor-NC), miR-541-mimic (mimic), or miR-541-mimic negative control (mimic-NC), cells were incubated with 500 nM MC-LR for 24 h, followed by measurement of the mRNA levels of miR-541 (A) and p15 (B) with qRT-PCR; (C) Six hours after GC-1 cells were transfected with mimic-NC, mimic, inhibitor-NC, or inhibitor, cells were incubated with or without 500 nM MC-LR for 24 h. The protein levels of p15, murine double minute2 (MDM2), phospho-MDM2 (p-MDM2), p53, and phospho-p53 (p-p53) were measured by Western blot. Protein expression levels were quantified with ImageJ (lower panels; n = 3). β-actin was used as a loading control; (D) GC-1 cells were transfected with mimic, mimic-NC, inhibitor, or inhibitor-NC, followed by treatment with 500 nM MC-LR for 24 h. The viability of GC-1 cells was revealed by FDA-PI staining. Uptake of FDA (green) indicates live cells, while uptake of propidium iodide (PI; red) indicates late apoptotic and dead cells. The percentages of FDA-/PI+ positive cells were quantified (right panels; n = 3); (E) GC-1 cells were stained with FITC-coupled Annexin V and propidium iodide (PI). Both early and late apoptotic cells (Q2 + Q3) were examined with flow cytometry. The percentage of apoptotic cells was calculated and summarized (lower panels; n = 3). Results are expressed as means ± S.D. * p < 0.05.
Mentions: To examine the regulatory effects of miR-541 and p15 on cellular functions, we transfected GC-1 cells with miR-541-inhibitor, miR-541-mimic, and their negative controls followed by exposure to 500 nM MC-LR for 24 h. miR-541 was decreased in cells transfected with miR-541-inhibitor and highly increased in cells transfected with miR-541-mimic (Figure 2A). The expression of p15 mRNA remained unchanged following transfection with either miR-541-mimic or the inhibitor (Figure 2B). We next measured the effect of modulation of miR-541 on the protein levels of p15 and a group of associated regulatory proteins. Upregulating miR-541 suppressed the expression of p15, MDM2 (murine double minute2) and phospho-MDM2, while it increased the level of phospho-p53 (Figure 2C). Exactly the opposite trends were observed if intracellular miR-541 was suppressed. Furthermore, forced expression of miR-541 promoted MC-LR-induced cell apoptosis, whereas inhibiting miR-541 suppressed apoptosis (Figure 2D,E).

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.