Limits...
ΔNp63 intronic miR-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation.

Kim KH, Cho EG, Yu SJ, Kang H, Kim YJ, Kim SH, Lee TR - Nucleic Acids Res. (2015)

Bottom Line: We found that miR-944 is highly expressed in keratinocytes, in a manner that is concordant with that of ΔNp63 mRNA, but the regulation of miR-944 expression under various conditions did not correspond with that of ΔNp63.Our results indicated that miR-944 biogenesis is dependent on ΔNp63 protein, even though it is generated from ΔNp63 mRNA-independent transcripts.Our findings suggested that miR-944, as an intronic miRNA and a direct target of ΔNp63, contributes to the function of ΔNp63 in the induction of epidermal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Bioscience Research Division, R&D Unit, AmorePacific Corporation, Yongin-si, Gyeonggi-do 446-729, Republic of Koreaf.

Show MeSH

Related in: MedlinePlus

TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4551945&req=5

Figure 5: TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test.

Mentions: ΔNp63 expression is gradually reduced in the epidermis during differentiation. However, miR-944 expression is not decreased, even though it is the target of ΔNp63. This raised the question of whether miR-944 biogenesis is sustained during differentiation. Interestingly, we found that ΔNp63-binding to the MIR944 promoter is maintained during the differentiation of keratinocytes (Figure 5A); additionally, mutation of the ΔNp63-binding site blocked the differentiation-induced increase in the activation of the MIR944 promoter (Figure 5B).


ΔNp63 intronic miR-944 is implicated in the ΔNp63-mediated induction of epidermal differentiation.

Kim KH, Cho EG, Yu SJ, Kang H, Kim YJ, Kim SH, Lee TR - Nucleic Acids Res. (2015)

TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4551945&req=5

Figure 5: TFAP2A and TFAP2C functionally support the ΔNp63-mediated activity of the MIR944 promoter. (A) ChIP-qPCR results for binding of ΔNp63 to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. (B) Keratinocytes were transfected with a MIR944 promoter-luciferase construct or a p63-binding site #3 mutant of the MIR944 promoter-luciferase construct. After 5 days of incubation in high-calcium medium, differentiated keratinocytes were lysed, and luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (C) ChIP-qPCR results for binding of TFAP2A (left panel) and TFAP2C (right panel) to the MIR944 promoter in undifferentiated and differentiated keratinocytes. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus Ca treatment 0 day, unpaired Student's t-test. (D) The effect of TFAP2A or TFAP2C overexpression on the ΔNp63-mediated activities of the MIR944 promoter was examined. WM266-4 melanoma cells were transfected with a MIR944 promoter-luciferase construct along with the indicated expression vectors. After 1 day of incubation, the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05, unpaired Student's t-test. (E) Reduction in the expression of miR-944 with TFAP2A or TFAP2C depletion in keratinocytes. Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. After 2 days, RNA was extracted, and expression levels were analyzed using RT-qPCR. TFAP2A and TFAP2C mRNA levels were normalized to RPLP0 expression, and miR-944 expression was normalized to that of RNU48 small RNA. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test. (F) Keratinocytes were transfected with 50 nM siRNA against TFAP2A (si-TFAP2A), TFAP2C (si-TFAP2C), or si-NC. The following day, keratinocytes were transfected with a MIR944 promoter-luciferase construct. After 2 days of incubation, keratinocytes were lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activities were normalized to β-galactosidase activities. Data represent the means ± SD of triplicate biological samples and are representative of three different experiments. *P < 0.05 versus si-NC, unpaired Student's t-test.
Mentions: ΔNp63 expression is gradually reduced in the epidermis during differentiation. However, miR-944 expression is not decreased, even though it is the target of ΔNp63. This raised the question of whether miR-944 biogenesis is sustained during differentiation. Interestingly, we found that ΔNp63-binding to the MIR944 promoter is maintained during the differentiation of keratinocytes (Figure 5A); additionally, mutation of the ΔNp63-binding site blocked the differentiation-induced increase in the activation of the MIR944 promoter (Figure 5B).

Bottom Line: We found that miR-944 is highly expressed in keratinocytes, in a manner that is concordant with that of ΔNp63 mRNA, but the regulation of miR-944 expression under various conditions did not correspond with that of ΔNp63.Our results indicated that miR-944 biogenesis is dependent on ΔNp63 protein, even though it is generated from ΔNp63 mRNA-independent transcripts.Our findings suggested that miR-944, as an intronic miRNA and a direct target of ΔNp63, contributes to the function of ΔNp63 in the induction of epidermal differentiation.

View Article: PubMed Central - PubMed

Affiliation: Bioscience Research Division, R&D Unit, AmorePacific Corporation, Yongin-si, Gyeonggi-do 446-729, Republic of Koreaf.

Show MeSH
Related in: MedlinePlus