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Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes.

Hall JR, Messenger ZJ, Tam HW, Phillips SL, Recio L, Smart RC - Cell Death Dis (2015)

Bottom Line: Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes.We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death.We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA [2] Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, USA [3] Toxicology Program, North Carolina State University, Raleigh, NC, USA.

ABSTRACT
LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53(+/R172H) allele expressed in mouse epidermis (K5Cre(+/tg);LSLp53(+/R172H)) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

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Related in: MedlinePlus

LincRNA-p21 (Linc-p21) transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin. (a) Balb/MK2 mouse keratinocytes were exposed to UVB, collected 8 h later and immunoblot analysis conducted. (b) Balb/MK2 mouse keratinocytes were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcript levels measured. (c) Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcript levels measured at the indicated times. (d) NHEK cells were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcripts measured. (e) NHEK cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcripts measured at the indicated times. (f) SKH-1 mice (three mice per time point) were exposed to 100 or 200 mJ/cm2 UVB, epidermis was collected at 9 h and lincRNA-p21 levels measured. (g) SKH-1 mice (three mice per group) were treated with 100 mJ/cm2 and lincRNA-p21 transcripts measured at the indicated times. LincRNA-p21 levels were measured by TaqMan real-time PCR (Ct value at peak post UVB=26–29 cycles using 25 ng template for all experiments). LincRNA-p21 was normalized to β-actin. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with time 0 using student t-test
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fig1: LincRNA-p21 (Linc-p21) transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin. (a) Balb/MK2 mouse keratinocytes were exposed to UVB, collected 8 h later and immunoblot analysis conducted. (b) Balb/MK2 mouse keratinocytes were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcript levels measured. (c) Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcript levels measured at the indicated times. (d) NHEK cells were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcripts measured. (e) NHEK cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcripts measured at the indicated times. (f) SKH-1 mice (three mice per time point) were exposed to 100 or 200 mJ/cm2 UVB, epidermis was collected at 9 h and lincRNA-p21 levels measured. (g) SKH-1 mice (three mice per group) were treated with 100 mJ/cm2 and lincRNA-p21 transcripts measured at the indicated times. LincRNA-p21 levels were measured by TaqMan real-time PCR (Ct value at peak post UVB=26–29 cycles using 25 ng template for all experiments). LincRNA-p21 was normalized to β-actin. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with time 0 using student t-test

Mentions: Treatment of Balb/MK2 mouse keratinocytes with UVB radiation resulted in increased levels of p53 protein and its target gene, p21 (Figure 1a) demonstrating that Balb/MK2 keratinocytes are responsive to UVB radiation. To determine whether UVB radiation can increase lincRNA-p21 transcript levels, Balb/MK2 keratinocytes were treated with various doses of UVB radiation. UVB exposure was an potent inducer of lincRNA-p21 transcripts producing up to a ~60-fold increase in lincRNA-p21 (Figure 1b). UVB treatment increased lincRNA-p21 in a dose-dependent manner (Figure 1b). Time-course studies revealed that UVB increased lincRNA-p21 levels as early as 4 h post UVB treatment with peak transcript levels occurring at ~16 h (Figure 1c). Normal human epidermal keratinocytes (NHEK) also displayed a UVB dose-dependent induction of lincRNA-p21 (Figure 1d) and a similar time course and magnitude of induction of lincRNA-p21 (~50-fold) (Figure 1e) as mouse keratinocytes. To determine whether UVB radiation was capable of inducing lincRNA-p21 in vivo in skin (epidermis), we utilized SKH-1 hairless mice and environmentally relevant doses of UVB. SKH-1 mice are an experimental model used to study the effects of UVB in skin and are relevant to UVB-induced human SCC skin cancer as the UV-induced tumors in these mice resemble, both at the morphologic and molecular levels, the UVB-induced SCC skin cancers in humans.33 The minimal erythema dose (MED) of UVB treatment in skin is defined as the minimal dose that produces a just-perceptible erythema (redness) at 24 h. SKH-1 mice were treated with UVB doses that correspond to 0.5 (100 mJ/cm2) and 1.0 MED (200 mJ/cm2). LincRNA-p21 was highly inducible by UVB radiation in SKH-1 mouse epidermis in vivo (Figures 1f and g). Collectively, these data demonstrate that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo.


Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes.

Hall JR, Messenger ZJ, Tam HW, Phillips SL, Recio L, Smart RC - Cell Death Dis (2015)

LincRNA-p21 (Linc-p21) transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin. (a) Balb/MK2 mouse keratinocytes were exposed to UVB, collected 8 h later and immunoblot analysis conducted. (b) Balb/MK2 mouse keratinocytes were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcript levels measured. (c) Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcript levels measured at the indicated times. (d) NHEK cells were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcripts measured. (e) NHEK cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcripts measured at the indicated times. (f) SKH-1 mice (three mice per time point) were exposed to 100 or 200 mJ/cm2 UVB, epidermis was collected at 9 h and lincRNA-p21 levels measured. (g) SKH-1 mice (three mice per group) were treated with 100 mJ/cm2 and lincRNA-p21 transcripts measured at the indicated times. LincRNA-p21 levels were measured by TaqMan real-time PCR (Ct value at peak post UVB=26–29 cycles using 25 ng template for all experiments). LincRNA-p21 was normalized to β-actin. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with time 0 using student t-test
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4385943&req=5

fig1: LincRNA-p21 (Linc-p21) transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin. (a) Balb/MK2 mouse keratinocytes were exposed to UVB, collected 8 h later and immunoblot analysis conducted. (b) Balb/MK2 mouse keratinocytes were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcript levels measured. (c) Balb/MK2 cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcript levels measured at the indicated times. (d) NHEK cells were exposed to the indicated doses of UVB, collected 16 h later and lincRNA-p21 transcripts measured. (e) NHEK cells were exposed to 10 mJ/cm2 UVB and lincRNA-p21 transcripts measured at the indicated times. (f) SKH-1 mice (three mice per time point) were exposed to 100 or 200 mJ/cm2 UVB, epidermis was collected at 9 h and lincRNA-p21 levels measured. (g) SKH-1 mice (three mice per group) were treated with 100 mJ/cm2 and lincRNA-p21 transcripts measured at the indicated times. LincRNA-p21 levels were measured by TaqMan real-time PCR (Ct value at peak post UVB=26–29 cycles using 25 ng template for all experiments). LincRNA-p21 was normalized to β-actin. Data are expressed as the mean ±S.D. N≥3, *P <0.05 significantly different compared with time 0 using student t-test
Mentions: Treatment of Balb/MK2 mouse keratinocytes with UVB radiation resulted in increased levels of p53 protein and its target gene, p21 (Figure 1a) demonstrating that Balb/MK2 keratinocytes are responsive to UVB radiation. To determine whether UVB radiation can increase lincRNA-p21 transcript levels, Balb/MK2 keratinocytes were treated with various doses of UVB radiation. UVB exposure was an potent inducer of lincRNA-p21 transcripts producing up to a ~60-fold increase in lincRNA-p21 (Figure 1b). UVB treatment increased lincRNA-p21 in a dose-dependent manner (Figure 1b). Time-course studies revealed that UVB increased lincRNA-p21 levels as early as 4 h post UVB treatment with peak transcript levels occurring at ~16 h (Figure 1c). Normal human epidermal keratinocytes (NHEK) also displayed a UVB dose-dependent induction of lincRNA-p21 (Figure 1d) and a similar time course and magnitude of induction of lincRNA-p21 (~50-fold) (Figure 1e) as mouse keratinocytes. To determine whether UVB radiation was capable of inducing lincRNA-p21 in vivo in skin (epidermis), we utilized SKH-1 hairless mice and environmentally relevant doses of UVB. SKH-1 mice are an experimental model used to study the effects of UVB in skin and are relevant to UVB-induced human SCC skin cancer as the UV-induced tumors in these mice resemble, both at the morphologic and molecular levels, the UVB-induced SCC skin cancers in humans.33 The minimal erythema dose (MED) of UVB treatment in skin is defined as the minimal dose that produces a just-perceptible erythema (redness) at 24 h. SKH-1 mice were treated with UVB doses that correspond to 0.5 (100 mJ/cm2) and 1.0 MED (200 mJ/cm2). LincRNA-p21 was highly inducible by UVB radiation in SKH-1 mouse epidermis in vivo (Figures 1f and g). Collectively, these data demonstrate that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo.

Bottom Line: Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes.We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death.We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biological Sciences, North Carolina State University, Raleigh, NC, USA [2] Center for Human Health and the Environment, North Carolina State University, Raleigh, NC, USA [3] Toxicology Program, North Carolina State University, Raleigh, NC, USA.

ABSTRACT
LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53(+/R172H) allele expressed in mouse epidermis (K5Cre(+/tg);LSLp53(+/R172H)) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

Show MeSH
Related in: MedlinePlus