Figure 7
Figure 7. miR-34a up-regulation after TPA treatment of K562 cells is p53-independent. (A) The p53-dependent miR-34a promoter does not activate luciferase expression after TPA treatment of K562 cells. Cells were transfected with a firefly luciferase reporter vector in which luciferase expression was driven by the DB286351 miR-34a promoter (PM-34a) or the CD41 promoter (PM-CD41) or with the promoterless luciferase vector (pGL3-Basic). Samples were cotransfected with a Renilla luciferase reporter vector for normalization. Luciferase activity was measured 48 hours after TPA treatment. (B) Exogenous expression of p53 in K562 cells activates the DB286351 miR-34a promoter. K562 cells were transfected with promoterless firefly luciferase reporter vector (pGL3) or a reporter containing the miR-34a promoter (PM-34a), a Renilla luciferase reporter vector, and increasing amounts (2, 4, and 8 μg) of pCMV-p53 vector. The total amount of transfected DNA was kept constant using an empty pCMV plasmid. Luciferase activity was measured 48 hours after transfection and was normalized as described. The fold increase in luciferase activity relative to the control sample transfected with the promoterless vector is shown. The bottom panel represents the level of p53 protein in the samples analyzed herein by immunoblot. The membrane was stripped and reprobed for α-tubulin as a loading control. (C) 5′- and 3′-end RACE PCR analysis of pri-miR-34a transcripts in TPA-treated K562 cells. Agarose gel images show the 5′- and 3′-RACE PCR products obtained in K562 cells treated with TPA for 4 days. (D) Two pri-miR-34a transcripts identified by sequencing the 5′-end RACE PCR products from TPA-treated K562 cells and their location in the genome. E indicates exon; the numbers below the exons indicate their length in base pairs. Also indicated is the length of the intronic regions. For comparison, the previously described pri-miR-34a transcript, DB286351, is also shown (top). (E) The genomic region adjacent to the alternate TSS serves as the miR-34a promoter in TPA-treated K562 cells. A DNA genomic fragment comprising 1.5 kb upstream to 0.5 kb downstream of the identified alternative TSS (PM-34a-K1/2) was cloned into pGL3-basic, and the effect of TPA treatment on luciferase activity was evaluated as described.

miR-34a up-regulation after TPA treatment of K562 cells is p53-independent. (A) The p53-dependent miR-34a promoter does not activate luciferase expression after TPA treatment of K562 cells. Cells were transfected with a firefly luciferase reporter vector in which luciferase expression was driven by the DB286351 miR-34a promoter (PM-34a) or the CD41 promoter (PM-CD41) or with the promoterless luciferase vector (pGL3-Basic). Samples were cotransfected with a Renilla luciferase reporter vector for normalization. Luciferase activity was measured 48 hours after TPA treatment. (B) Exogenous expression of p53 in K562 cells activates the DB286351 miR-34a promoter. K562 cells were transfected with promoterless firefly luciferase reporter vector (pGL3) or a reporter containing the miR-34a promoter (PM-34a), a Renilla luciferase reporter vector, and increasing amounts (2, 4, and 8 μg) of pCMV-p53 vector. The total amount of transfected DNA was kept constant using an empty pCMV plasmid. Luciferase activity was measured 48 hours after transfection and was normalized as described. The fold increase in luciferase activity relative to the control sample transfected with the promoterless vector is shown. The bottom panel represents the level of p53 protein in the samples analyzed herein by immunoblot. The membrane was stripped and reprobed for α-tubulin as a loading control. (C) 5′- and 3′-end RACE PCR analysis of pri-miR-34a transcripts in TPA-treated K562 cells. Agarose gel images show the 5′- and 3′-RACE PCR products obtained in K562 cells treated with TPA for 4 days. (D) Two pri-miR-34a transcripts identified by sequencing the 5′-end RACE PCR products from TPA-treated K562 cells and their location in the genome. E indicates exon; the numbers below the exons indicate their length in base pairs. Also indicated is the length of the intronic regions. For comparison, the previously described pri-miR-34a transcript, DB286351, is also shown (top). (E) The genomic region adjacent to the alternate TSS serves as the miR-34a promoter in TPA-treated K562 cells. A DNA genomic fragment comprising 1.5 kb upstream to 0.5 kb downstream of the identified alternative TSS (PM-34a-K1/2) was cloned into pGL3-basic, and the effect of TPA treatment on luciferase activity was evaluated as described.

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