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c-Myc modulates microRNA processing via the transcriptional regulation of Drosha.

Wang X, Zhao X, Gao P, Wu M - Sci Rep (2013)

Bottom Line: A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter.Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level.Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.

ABSTRACT
The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

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Related in: MedlinePlus

c-Myc accelerates pri-miRNA processing in vitro and in vivo.(a) and (b). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in P493-6 B cells. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (c). Western blot analysis of c-Myc and Drosha expression in Tet- and Wash-treated cells. GAPDH was used as a loading control. (d) and (e). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in A549 cells treated with siRNA knockdown against c-Myc and Drosha separately and in combination. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (f). Western blot showing the siRNA gene-silencing efficiency for c-Myc and Drosha in A549 cells. Actin was used as a loading control. (g). A schematic diagram of the in vivo monitoring of pri-miRNA processing. (h). Luciferase activity was measured in A549 cells transfected with psiCHECK-pri-miR30a to monitor pri-miRNA processing in vivo. c-Myc knockdown increased the luciferase activity. The intensities were normalised by Renilla luciferase. The data represent the mean ± SD of three independent experiments. *indicates P < 0.05.
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f4: c-Myc accelerates pri-miRNA processing in vitro and in vivo.(a) and (b). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in P493-6 B cells. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (c). Western blot analysis of c-Myc and Drosha expression in Tet- and Wash-treated cells. GAPDH was used as a loading control. (d) and (e). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in A549 cells treated with siRNA knockdown against c-Myc and Drosha separately and in combination. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (f). Western blot showing the siRNA gene-silencing efficiency for c-Myc and Drosha in A549 cells. Actin was used as a loading control. (g). A schematic diagram of the in vivo monitoring of pri-miRNA processing. (h). Luciferase activity was measured in A549 cells transfected with psiCHECK-pri-miR30a to monitor pri-miRNA processing in vivo. c-Myc knockdown increased the luciferase activity. The intensities were normalised by Renilla luciferase. The data represent the mean ± SD of three independent experiments. *indicates P < 0.05.

Mentions: To understand the mechanism of c-Myc in miRNA processing, we performed an in vitro pri-miRNA processing assay by separately incubating radiolabelled pri-let7a-1 and pri-miR30a substrates with the immunoprecipitated Drosha complex from P493-6 B cells. In particular, we examined the immunoprecipitated Drosha complex from P493-6 B cells that were either treated with tetracycline (Tet) to inhibit c-Myc expression or that had been washed (Wash) to remove the tetracycline and re-induce c-Myc expression. As shown in Figure 4a, b, and c, the processing activities for both the pri-let7a-1 and pri-miR30a primary transcripts were greatly reduced when c-Myc expression was turned off (Tet) in comparison to the Mock samples. Furthermore, higher processing activity was recovered (Wash) after tetracycline was removed. Additionally, we performed a similar pri-miRNA processing assay in A549 cells in which the expression of c-Myc, Drosha, or both was silenced using siRNA. As shown in Figure 4d, e, and f, knockdown of either c-Myc, Drosha, or both greatly diminished the processing activity for both pri-let7a1 and pri-miR30a when compared to the control siRNA.


c-Myc modulates microRNA processing via the transcriptional regulation of Drosha.

Wang X, Zhao X, Gao P, Wu M - Sci Rep (2013)

c-Myc accelerates pri-miRNA processing in vitro and in vivo.(a) and (b). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in P493-6 B cells. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (c). Western blot analysis of c-Myc and Drosha expression in Tet- and Wash-treated cells. GAPDH was used as a loading control. (d) and (e). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in A549 cells treated with siRNA knockdown against c-Myc and Drosha separately and in combination. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (f). Western blot showing the siRNA gene-silencing efficiency for c-Myc and Drosha in A549 cells. Actin was used as a loading control. (g). A schematic diagram of the in vivo monitoring of pri-miRNA processing. (h). Luciferase activity was measured in A549 cells transfected with psiCHECK-pri-miR30a to monitor pri-miRNA processing in vivo. c-Myc knockdown increased the luciferase activity. The intensities were normalised by Renilla luciferase. The data represent the mean ± SD of three independent experiments. *indicates P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3672885&req=5

f4: c-Myc accelerates pri-miRNA processing in vitro and in vivo.(a) and (b). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in P493-6 B cells. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (c). Western blot analysis of c-Myc and Drosha expression in Tet- and Wash-treated cells. GAPDH was used as a loading control. (d) and (e). In vitro pri-miRNA processing assays of pri-let-7a-1 and pri-miR-30a in A549 cells treated with siRNA knockdown against c-Myc and Drosha separately and in combination. The amount of pre-miRNA resulting from pri-miRNA processing was determined by a densitometric analysis of a western blot and is indicated by Arabic numbers. (f). Western blot showing the siRNA gene-silencing efficiency for c-Myc and Drosha in A549 cells. Actin was used as a loading control. (g). A schematic diagram of the in vivo monitoring of pri-miRNA processing. (h). Luciferase activity was measured in A549 cells transfected with psiCHECK-pri-miR30a to monitor pri-miRNA processing in vivo. c-Myc knockdown increased the luciferase activity. The intensities were normalised by Renilla luciferase. The data represent the mean ± SD of three independent experiments. *indicates P < 0.05.
Mentions: To understand the mechanism of c-Myc in miRNA processing, we performed an in vitro pri-miRNA processing assay by separately incubating radiolabelled pri-let7a-1 and pri-miR30a substrates with the immunoprecipitated Drosha complex from P493-6 B cells. In particular, we examined the immunoprecipitated Drosha complex from P493-6 B cells that were either treated with tetracycline (Tet) to inhibit c-Myc expression or that had been washed (Wash) to remove the tetracycline and re-induce c-Myc expression. As shown in Figure 4a, b, and c, the processing activities for both the pri-let7a-1 and pri-miR30a primary transcripts were greatly reduced when c-Myc expression was turned off (Tet) in comparison to the Mock samples. Furthermore, higher processing activity was recovered (Wash) after tetracycline was removed. Additionally, we performed a similar pri-miRNA processing assay in A549 cells in which the expression of c-Myc, Drosha, or both was silenced using siRNA. As shown in Figure 4d, e, and f, knockdown of either c-Myc, Drosha, or both greatly diminished the processing activity for both pri-let7a1 and pri-miR30a when compared to the control siRNA.

Bottom Line: A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter.Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level.Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui, China.

ABSTRACT
The c-Myc oncogenic transcription factor is known to regulate microRNA (miRNA) expression at the transcriptional level. However, little is known about the function of c-Myc in miRNA processing. Here, we report that Drosha, one of the most important components of the miRNA processing machinery, is a c-Myc target gene. c-Myc transactivates drosha mRNA expression, thus upregulating the Drosha protein level. A chromatin immunoprecipitation (ChIP) experiment revealed that c-Myc binds directly to the E-box of the drosha promoter. Both in vitro and in vivo microRNA processing assays demonstrated that c-Myc promotes miRNA processing by upregulating the Drosha expression level. Overall, our study reveals a previously unrecognised function of c-Myc in miRNA processing and provides valuable insight into a new aspect of how c-Myc regulates microRNA expression.

Show MeSH
Related in: MedlinePlus