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Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus

miR-101 expression in prostate cancer cells.A, Expressions of pri-miR-101 and mature miR-101 were determined in AR positive or negative cell lines by qPCR. **, P<0.01 between two compared groups. B, LNCaP or 22Rv1 cells were transfected with AR siRNA or control siRNA (ctrl siRNA). AR knockdown effects were verified by Western blotting. Expressions of pri-miR-101 and mature miR-101 were determined by qPCR (C). *, P<0.05 versus control siRNA. D, DU145 or PC-3 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) and treated with DMSO or celastrol (CEL, 2 μM) for 24 h. Expressions of mature miR-101 were determined by qPCR. *, P<0.05 between EGFP-AR and EGFP-V transfections. E, LNCaP cells were treated with R1881 (1 nM) for 24 h after androgen starvation, as described in Fig 2C. AR protein levels were determined by Western blotting using GAPDH as a loading control. MiR-101 expressions were determined by qPCR. **, p<0.01 versus DMSO.
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pone.0140745.g004: miR-101 expression in prostate cancer cells.A, Expressions of pri-miR-101 and mature miR-101 were determined in AR positive or negative cell lines by qPCR. **, P<0.01 between two compared groups. B, LNCaP or 22Rv1 cells were transfected with AR siRNA or control siRNA (ctrl siRNA). AR knockdown effects were verified by Western blotting. Expressions of pri-miR-101 and mature miR-101 were determined by qPCR (C). *, P<0.05 versus control siRNA. D, DU145 or PC-3 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) and treated with DMSO or celastrol (CEL, 2 μM) for 24 h. Expressions of mature miR-101 were determined by qPCR. *, P<0.05 between EGFP-AR and EGFP-V transfections. E, LNCaP cells were treated with R1881 (1 nM) for 24 h after androgen starvation, as described in Fig 2C. AR protein levels were determined by Western blotting using GAPDH as a loading control. MiR-101 expressions were determined by qPCR. **, p<0.01 versus DMSO.

Mentions: AR status could affect miR-101 expression in human prostate cancer cells. Real-Time PCR revealed that the expression levels of pri-miR-101 as well as mature miR-101 were significantly higher in AR-positive LNCaP and 22Rv1 cells than that in AR-negative DU145 and PC3 cells (Fig 4A). AR was knocked down by siRNA in LNCaP (Fig 4B, left) or 22Rv1 cells (Fig 4B, right). AR knockdown resulted in decreased expressions of mature miR-101, which were comparable to the decrease of pri-miR-101 in both LNCaP and 22Rv1 cells (Fig 4C). Forced AR expression caused miR-101 levels increased in AR-negative DU145 and PC3 cells (Fig 4D). Similarly, exogenous AR also increased miR-101 expressions after celastrol treatment (Fig 4D). In LNCaP cells, endogenous AR was re-activated by R1881 after androgen deprivation. With AR activation (Fig 4E, upper), the expression levels of miR-101 were upregulated (Fig 4E). These results demonstrate that AR mainly regulates miR-101 transcription, but not maturation.


Celastrol Induces Autophagy by Targeting AR/miR-101 in Prostate Cancer Cells.

Guo J, Huang X, Wang H, Yang H - PLoS ONE (2015)

miR-101 expression in prostate cancer cells.A, Expressions of pri-miR-101 and mature miR-101 were determined in AR positive or negative cell lines by qPCR. **, P<0.01 between two compared groups. B, LNCaP or 22Rv1 cells were transfected with AR siRNA or control siRNA (ctrl siRNA). AR knockdown effects were verified by Western blotting. Expressions of pri-miR-101 and mature miR-101 were determined by qPCR (C). *, P<0.05 versus control siRNA. D, DU145 or PC-3 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) and treated with DMSO or celastrol (CEL, 2 μM) for 24 h. Expressions of mature miR-101 were determined by qPCR. *, P<0.05 between EGFP-AR and EGFP-V transfections. E, LNCaP cells were treated with R1881 (1 nM) for 24 h after androgen starvation, as described in Fig 2C. AR protein levels were determined by Western blotting using GAPDH as a loading control. MiR-101 expressions were determined by qPCR. **, p<0.01 versus DMSO.
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pone.0140745.g004: miR-101 expression in prostate cancer cells.A, Expressions of pri-miR-101 and mature miR-101 were determined in AR positive or negative cell lines by qPCR. **, P<0.01 between two compared groups. B, LNCaP or 22Rv1 cells were transfected with AR siRNA or control siRNA (ctrl siRNA). AR knockdown effects were verified by Western blotting. Expressions of pri-miR-101 and mature miR-101 were determined by qPCR (C). *, P<0.05 versus control siRNA. D, DU145 or PC-3 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) and treated with DMSO or celastrol (CEL, 2 μM) for 24 h. Expressions of mature miR-101 were determined by qPCR. *, P<0.05 between EGFP-AR and EGFP-V transfections. E, LNCaP cells were treated with R1881 (1 nM) for 24 h after androgen starvation, as described in Fig 2C. AR protein levels were determined by Western blotting using GAPDH as a loading control. MiR-101 expressions were determined by qPCR. **, p<0.01 versus DMSO.
Mentions: AR status could affect miR-101 expression in human prostate cancer cells. Real-Time PCR revealed that the expression levels of pri-miR-101 as well as mature miR-101 were significantly higher in AR-positive LNCaP and 22Rv1 cells than that in AR-negative DU145 and PC3 cells (Fig 4A). AR was knocked down by siRNA in LNCaP (Fig 4B, left) or 22Rv1 cells (Fig 4B, right). AR knockdown resulted in decreased expressions of mature miR-101, which were comparable to the decrease of pri-miR-101 in both LNCaP and 22Rv1 cells (Fig 4C). Forced AR expression caused miR-101 levels increased in AR-negative DU145 and PC3 cells (Fig 4D). Similarly, exogenous AR also increased miR-101 expressions after celastrol treatment (Fig 4D). In LNCaP cells, endogenous AR was re-activated by R1881 after androgen deprivation. With AR activation (Fig 4E, upper), the expression levels of miR-101 were upregulated (Fig 4E). These results demonstrate that AR mainly regulates miR-101 transcription, but not maturation.

Bottom Line: In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy.Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR.Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science and Technology, Harbin Institute of Technology, Harbin, 150001, China.

ABSTRACT
Autophagy is an evolutionarily conserved process responsible for the degradation and recycling of cytoplasmic components through autolysosomes. Targeting AR axis is a standard strategy for prostate cancer treatment; however, the role of AR in autophagic processes is still not fully understood. In the present study, we found that AR played a negative role in AR degrader celastrol-induced autophagy. Knockdown of AR in AR-positive prostate cancer cells resulted in enhanced autophagy. Ectopic expression of AR in AR-negative prostate cancer cells, or gain of function of the AR signaling in AR-positive cells, led to suppression of autophagy. Since miR-101 is an inhibitor of autophagy and its expression was decreased along with AR in the process of celastrol-induced autophagy, we hypothesize that AR inhibits autophagy through transactivation of miR-101. AR binding site was defined in the upstream of miR-101 gene by luciferase reporter and ChIP assays. MiR-101 expression correlated with AR status in prostate cancer cell lines. The inhibition of celastrol-induced autophagy by AR was compromised by blocking miR-101; while transfection of miR-101 led to inhibition of celastrol-induced autophagy in spite of AR depletion. Furthermore, mutagenesis of the AR binding site in miR-101 gene led to decreased suppression of autophagy by AR. Finally, autophagy inhibition by miR-101 mimic was found to enhance the cytotoxic effect of celastrol in prostate cancer cells. Our results demonstrate that AR inhibits autophagy via transactivation of miR-101, thus combination of miR-101 mimics with celastrol may represent a promising therapeutic approach for treating prostate cancer.

No MeSH data available.


Related in: MedlinePlus