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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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LincRNA-p21 (linc-p21) regulates apoptosis in UVB-treated keratinocytes. (a) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 8 and 18 h after UVB exposure and immunoblot analysis for caspase 3 conducted; results shown are representative of duplicate experiments. (b) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 6, 12, 18 and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (c) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (d) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and candidate gene expression examined by Taqman RT-PCR. (e) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and immunoblot analysis conducted. (f) NHEK cells were transfected with siRNA to human lincRNA-p21 or control siRNA, 48 h later exposed to 10 mJ/cm2 UVB, collected 18 h later and lincRNA-p21 levels measured. (g) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0 and 18 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (h) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB, and candidate gene expression examined. Data are expressed in c, d, f, g and h as the mean ±S.D. N ≥3, *P<0.05 significantly different compared with siRNA control as determined by the student t-test
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fig4: LincRNA-p21 (linc-p21) regulates apoptosis in UVB-treated keratinocytes. (a) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 8 and 18 h after UVB exposure and immunoblot analysis for caspase 3 conducted; results shown are representative of duplicate experiments. (b) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 6, 12, 18 and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (c) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (d) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and candidate gene expression examined by Taqman RT-PCR. (e) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and immunoblot analysis conducted. (f) NHEK cells were transfected with siRNA to human lincRNA-p21 or control siRNA, 48 h later exposed to 10 mJ/cm2 UVB, collected 18 h later and lincRNA-p21 levels measured. (g) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0 and 18 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (h) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB, and candidate gene expression examined. Data are expressed in c, d, f, g and h as the mean ±S.D. N ≥3, *P<0.05 significantly different compared with siRNA control as determined by the student t-test

Mentions: To begin to determine the functional role of lincRNA-p21 in UVB-treated keratinocytes, we used an siRNA approach to knockdown lincRNA-p21. As shown in Figure 3a, we were able to knockdown lincRNA-p21 transcript levels in UVB-treated mouse keratinocytes by >80% using two different siRNA sequences.26 Knockdown of lincRNA-p21 had no effect on cell viability in untreated keratinocytes (Figure 3b); however, UVB-treated lincRNA-p21-depleted keratinocytes displayed an increased viability (Figure 3c). These results suggest that in UVB-treated keratinocytes, lincRNA-p21 functions to inhibit keratinocyte cell cycle progression and/or to induce keratinocyte apoptosis in response to UVB. Earlier studies showed that lincRNA-p21 has an important role in regulating p53-dependent cell cycle arrest involving p21 in doxorubicin-treated MEFs.29 Therefore, we first examined the levels of p53 and p21 in control and lincRNA-p21-depleted keratinocytes and observed that the levels of p53 and p21 were not affected by the knockdown of lincRNA-p21 (Figure 3d). These results indicate that the knockdown of lincRNA-p21 does not interfere with p53 protein levels or the regulation of p21 in response to UVB. In accord with these results, the knockdown of lincRNA-p21 had no effect on the cell cycle distribution and the DNA damage checkpoint as determined by FACS analysis (Figures 3e and f). In contrast to the lack of effect on cell proliferation and DNA damage checkpoint function, lincRNA-p21-depleted keratinocytes displayed striking decreases in UVB-induced apoptosis as determined by decreased cleaved caspase-3 levels (Figure 4a) and decreased annexin-V staining (Figure 4b). On the basis of annexin V single positive staining, there was a ~10-fold decrease in early apoptotic cells in lincRNA-p21-depleted keratinocytes at 18 h post UVB (Figure 4b). Similar results were obtained with a second lincRNA-p21 siRNA sequence (Figure 4c). These data demonstrate that lincRNA-p21 is a critical regulator of UVB-induced apoptosis and regulates at least 75% of the apoptosis induced by UVB in mouse keratinocytes (Figures 4b and c). Knockdown of lincRNA-p21 resulted in altered expression of several genes associated with apoptosis in response to UVB radiation indicating that lincRNA-p21 can both repress the expression of anti-apoptotic and activate the expression of pro-apoptotic genes in response to UVB (Figure 4d). The levels of Cdkn1a mRNA were not decreased by knockdown of lincRNA-p21 in UVB-treated keratinocytes; in fact, there was an unexpected ~two fold increase. Additionally, the protein levels of the pro-apopotic factors Noxa and Bax were significantly reduced in lincRNA-p21 knockdown cells following UVB exposure and despite changes in Stat3 mRNA, no changes in Stat3 protein were observed (Figure 4e). Next, we examined the role of lincRNA-p21 in UVB-induced apoptosis in human keratinocytes. The knockdown of human lincRNA-p21 in NHEKs (Figure 4f) resulted in decreased apoptosis as determined by annexin V staining (Figure 4g). Thus, lincRNA-p21 is a key mediator of UVB-induced apoptosis in human keratinocytes and is responsible for ~70% of UVB-induced apoptosis (Figure 4g). Knockdown of lincRNA-p21 in NHEKs resulted in the altered expression of apoptosis-related genes in response to UVB; lincRNA-p21 represses the expression of anti-apoptotic genes and activates the expression of pro-apoptotic genes in response to UVB and had no effect on CDKN1A mRNA levels (Figure 4h). These data reveal that lincRNA-p21 has a major and critical role in UVB-induced apoptotic death in human and mouse keratinocytes.


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) regulates apoptosis in UVB-treated keratinocytes. (a) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 8 and 18 h after UVB exposure and immunoblot analysis for caspase 3 conducted; results shown are representative of duplicate experiments. (b) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 6, 12, 18 and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (c) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (d) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and candidate gene expression examined by Taqman RT-PCR. (e) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and immunoblot analysis conducted. (f) NHEK cells were transfected with siRNA to human lincRNA-p21 or control siRNA, 48 h later exposed to 10 mJ/cm2 UVB, collected 18 h later and lincRNA-p21 levels measured. (g) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0 and 18 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (h) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB, and candidate gene expression examined. Data are expressed in c, d, f, g and h as the mean ±S.D. N ≥3, *P<0.05 significantly different compared with siRNA control as determined by the student t-test
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fig4: LincRNA-p21 (linc-p21) regulates apoptosis in UVB-treated keratinocytes. (a) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 8 and 18 h after UVB exposure and immunoblot analysis for caspase 3 conducted; results shown are representative of duplicate experiments. (b) Control and lincRNA-p21 #1 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0, 6, 12, 18 and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (c) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and 24 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (d) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and candidate gene expression examined by Taqman RT-PCR. (e) Control and lincRNA-p21 #1 and #2 knockdown Balb/MK2 cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB and immunoblot analysis conducted. (f) NHEK cells were transfected with siRNA to human lincRNA-p21 or control siRNA, 48 h later exposed to 10 mJ/cm2 UVB, collected 18 h later and lincRNA-p21 levels measured. (g) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 UVB (48 h post transfection) and collected at 0 and 18 h after UVB. Cells were stained with annexin-V and PI and analyzed by FACS. Similar results were obtained in three independent experiments. (h) Control and lincRNA-p21 knockdown NHEK cells were exposed to 10 mJ/cm2 and collected at 18 h post UVB, and candidate gene expression examined. Data are expressed in c, d, f, g and h as the mean ±S.D. N ≥3, *P<0.05 significantly different compared with siRNA control as determined by the student t-test
Mentions: To begin to determine the functional role of lincRNA-p21 in UVB-treated keratinocytes, we used an siRNA approach to knockdown lincRNA-p21. As shown in Figure 3a, we were able to knockdown lincRNA-p21 transcript levels in UVB-treated mouse keratinocytes by >80% using two different siRNA sequences.26 Knockdown of lincRNA-p21 had no effect on cell viability in untreated keratinocytes (Figure 3b); however, UVB-treated lincRNA-p21-depleted keratinocytes displayed an increased viability (Figure 3c). These results suggest that in UVB-treated keratinocytes, lincRNA-p21 functions to inhibit keratinocyte cell cycle progression and/or to induce keratinocyte apoptosis in response to UVB. Earlier studies showed that lincRNA-p21 has an important role in regulating p53-dependent cell cycle arrest involving p21 in doxorubicin-treated MEFs.29 Therefore, we first examined the levels of p53 and p21 in control and lincRNA-p21-depleted keratinocytes and observed that the levels of p53 and p21 were not affected by the knockdown of lincRNA-p21 (Figure 3d). These results indicate that the knockdown of lincRNA-p21 does not interfere with p53 protein levels or the regulation of p21 in response to UVB. In accord with these results, the knockdown of lincRNA-p21 had no effect on the cell cycle distribution and the DNA damage checkpoint as determined by FACS analysis (Figures 3e and f). In contrast to the lack of effect on cell proliferation and DNA damage checkpoint function, lincRNA-p21-depleted keratinocytes displayed striking decreases in UVB-induced apoptosis as determined by decreased cleaved caspase-3 levels (Figure 4a) and decreased annexin-V staining (Figure 4b). On the basis of annexin V single positive staining, there was a ~10-fold decrease in early apoptotic cells in lincRNA-p21-depleted keratinocytes at 18 h post UVB (Figure 4b). Similar results were obtained with a second lincRNA-p21 siRNA sequence (Figure 4c). These data demonstrate that lincRNA-p21 is a critical regulator of UVB-induced apoptosis and regulates at least 75% of the apoptosis induced by UVB in mouse keratinocytes (Figures 4b and c). Knockdown of lincRNA-p21 resulted in altered expression of several genes associated with apoptosis in response to UVB radiation indicating that lincRNA-p21 can both repress the expression of anti-apoptotic and activate the expression of pro-apoptotic genes in response to UVB (Figure 4d). The levels of Cdkn1a mRNA were not decreased by knockdown of lincRNA-p21 in UVB-treated keratinocytes; in fact, there was an unexpected ~two fold increase. Additionally, the protein levels of the pro-apopotic factors Noxa and Bax were significantly reduced in lincRNA-p21 knockdown cells following UVB exposure and despite changes in Stat3 mRNA, no changes in Stat3 protein were observed (Figure 4e). Next, we examined the role of lincRNA-p21 in UVB-induced apoptosis in human keratinocytes. The knockdown of human lincRNA-p21 in NHEKs (Figure 4f) resulted in decreased apoptosis as determined by annexin V staining (Figure 4g). Thus, lincRNA-p21 is a key mediator of UVB-induced apoptosis in human keratinocytes and is responsible for ~70% of UVB-induced apoptosis (Figure 4g). Knockdown of lincRNA-p21 in NHEKs resulted in the altered expression of apoptosis-related genes in response to UVB; lincRNA-p21 represses the expression of anti-apoptotic genes and activates the expression of pro-apoptotic genes in response to UVB and had no effect on CDKN1A mRNA levels (Figure 4h). These data reveal that lincRNA-p21 has a major and critical role in UVB-induced apoptotic death in human and mouse keratinocytes.

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