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MicroRNA-34c enhances murine male germ cell apoptosis through targeting ATF1.

Liang X, Zhou D, Wei C, Luo H, Liu J, Fu R, Cui S - PLoS ONE (2012)

Bottom Line: The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone.Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis.Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.

ABSTRACT

Background: MicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.

Methodology/principal findings: In the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3'UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.

Conclusions/significance: Our study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.

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The effects of miR-34c inhibition on Fas, Bcl-2, Bax mRNA expressions and Bcl-2/Bax ratio.(A) MiRNA inhibitor with Lipofectamine™ 2000 injected into the seminiferous tubule of 14 dpp mouse testis. 0.4% Trypan blue (10 fold more than needed for experimental concentrations and used in order to take a clear picture) was added to transfection mix. (B) Real-time PCR analysis two days after injection. (lipo: lipofectamine™; inhibitor: miR-34c inhibitor; in-NC: miRNA inhibitor nonsense control. (C) Apoptosis-related genes were tested by real-time PCR after miR-34c knockdown. (D) Bcl-2/Bax ratio increased after miR-34c inhibition. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*P<0.05, **P<0.01).
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pone-0033861-g002: The effects of miR-34c inhibition on Fas, Bcl-2, Bax mRNA expressions and Bcl-2/Bax ratio.(A) MiRNA inhibitor with Lipofectamine™ 2000 injected into the seminiferous tubule of 14 dpp mouse testis. 0.4% Trypan blue (10 fold more than needed for experimental concentrations and used in order to take a clear picture) was added to transfection mix. (B) Real-time PCR analysis two days after injection. (lipo: lipofectamine™; inhibitor: miR-34c inhibitor; in-NC: miRNA inhibitor nonsense control. (C) Apoptosis-related genes were tested by real-time PCR after miR-34c knockdown. (D) Bcl-2/Bax ratio increased after miR-34c inhibition. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*P<0.05, **P<0.01).

Mentions: In order to identify the function of miR-34c in germ cells, we initially inhibited miR-34c by transfecting a miR-34c inhibitor, which was injected into the seminiferous tubule of 14 dpp testis. 0.04% Trypan blue was added in the complex as an indicator (Fig. 2A). To measure the inhibiting efficiency, the testes (n≥6) were collected and miR-34c levels were detected by real-time PCR at 48 hours (16 dpp) after transfection. The results showed that the inhibiting efficiency was up to 85%, compared to the control (Fig. 2B).


MicroRNA-34c enhances murine male germ cell apoptosis through targeting ATF1.

Liang X, Zhou D, Wei C, Luo H, Liu J, Fu R, Cui S - PLoS ONE (2012)

The effects of miR-34c inhibition on Fas, Bcl-2, Bax mRNA expressions and Bcl-2/Bax ratio.(A) MiRNA inhibitor with Lipofectamine™ 2000 injected into the seminiferous tubule of 14 dpp mouse testis. 0.4% Trypan blue (10 fold more than needed for experimental concentrations and used in order to take a clear picture) was added to transfection mix. (B) Real-time PCR analysis two days after injection. (lipo: lipofectamine™; inhibitor: miR-34c inhibitor; in-NC: miRNA inhibitor nonsense control. (C) Apoptosis-related genes were tested by real-time PCR after miR-34c knockdown. (D) Bcl-2/Bax ratio increased after miR-34c inhibition. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*P<0.05, **P<0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316505&req=5

pone-0033861-g002: The effects of miR-34c inhibition on Fas, Bcl-2, Bax mRNA expressions and Bcl-2/Bax ratio.(A) MiRNA inhibitor with Lipofectamine™ 2000 injected into the seminiferous tubule of 14 dpp mouse testis. 0.4% Trypan blue (10 fold more than needed for experimental concentrations and used in order to take a clear picture) was added to transfection mix. (B) Real-time PCR analysis two days after injection. (lipo: lipofectamine™; inhibitor: miR-34c inhibitor; in-NC: miRNA inhibitor nonsense control. (C) Apoptosis-related genes were tested by real-time PCR after miR-34c knockdown. (D) Bcl-2/Bax ratio increased after miR-34c inhibition. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*P<0.05, **P<0.01).
Mentions: In order to identify the function of miR-34c in germ cells, we initially inhibited miR-34c by transfecting a miR-34c inhibitor, which was injected into the seminiferous tubule of 14 dpp testis. 0.04% Trypan blue was added in the complex as an indicator (Fig. 2A). To measure the inhibiting efficiency, the testes (n≥6) were collected and miR-34c levels were detected by real-time PCR at 48 hours (16 dpp) after transfection. The results showed that the inhibiting efficiency was up to 85%, compared to the control (Fig. 2B).

Bottom Line: The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone.Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis.Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agrobiotechnology, College of Biological Sciences, China Agricultural University, Beijing, People's Republic of China.

ABSTRACT

Background: MicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood.

Methodology/principal findings: In the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3'UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells.

Conclusions/significance: Our study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis.

Show MeSH
Related in: MedlinePlus