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A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells.

Liu JJ, Lin XJ, Yang XJ, Zhou L, He S, Zhuang SM, Yang J - Nucleic Acids Res. (2014)

Bottom Line: Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription.These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling.Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P.R. China.

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Direct interaction of AP-1 with the enhancer region of miR-101-2 gene in vivo and in vitro. (A) AP-1 interacts with the enhancer region of miR-101-2 gene in vivo. A scheme of the amplicons is shown at the top. AP-1 binding sites are depicted as short vertical lines and the arrows show the primer set used. (B) Knockdown of c-Jun or c-Fos abrogated the interaction of AP-1 with the enhancer region of miR-101-2. In (A) and (B), the promoter region of MMP1 including AP-1 biding site was used as positive control, and genomic region in the upstream of miR-101-2 lacking AP-1 binding sites was used as negative control. (C) EMSA verified the interaction of nuclear proteins with Site A (canonical AP-1 binding sites) and Site B (non-canonical AP-1 binding sites) sequences of the miR-101-2 enhancer. (D) Antibody-supershift assay identified c-Jun and c-Fos as potential nuclear proteins interacting with Site A and B sequences. In (C) and (D), the arrow indicates the DNA–protein complexes.
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Figure 3: Direct interaction of AP-1 with the enhancer region of miR-101-2 gene in vivo and in vitro. (A) AP-1 interacts with the enhancer region of miR-101-2 gene in vivo. A scheme of the amplicons is shown at the top. AP-1 binding sites are depicted as short vertical lines and the arrows show the primer set used. (B) Knockdown of c-Jun or c-Fos abrogated the interaction of AP-1 with the enhancer region of miR-101-2. In (A) and (B), the promoter region of MMP1 including AP-1 biding site was used as positive control, and genomic region in the upstream of miR-101-2 lacking AP-1 binding sites was used as negative control. (C) EMSA verified the interaction of nuclear proteins with Site A (canonical AP-1 binding sites) and Site B (non-canonical AP-1 binding sites) sequences of the miR-101-2 enhancer. (D) Antibody-supershift assay identified c-Jun and c-Fos as potential nuclear proteins interacting with Site A and B sequences. In (C) and (D), the arrow indicates the DNA–protein complexes.

Mentions: To examine the binding of c-Jun and c-Fos to the enhancer of miR-101-2, ChIP assays were performed in TPA-treated HepG2 cells. A specific band of the expected size was amplified from either the individual or combination of c-Jun and c-Fos antibody-precipitated DNA, using primers that covered the AP-1 binding sites in the −17.4 to −16.4 k enhancer of miR-101-2 (Figure 3A). Whereas knockdown of either c-Fos or c-Jun (Supplementary Figure S5A) markedly reduced the antibody-precipitated DNAs (Figure 3B), suggesting the interaction between AP-1 and the miR-101 enhancer in vivo. The direct interaction between c-Jun/c-Fos and canonical (Site A)/non-canonical (Site B) AP-1 binding sites was further verified by EMSA and antibody-supershift assays (Figure 3C and D).


A novel AP-1/miR-101 regulatory feedback loop and its implication in the migration and invasion of hepatoma cells.

Liu JJ, Lin XJ, Yang XJ, Zhou L, He S, Zhuang SM, Yang J - Nucleic Acids Res. (2014)

Direct interaction of AP-1 with the enhancer region of miR-101-2 gene in vivo and in vitro. (A) AP-1 interacts with the enhancer region of miR-101-2 gene in vivo. A scheme of the amplicons is shown at the top. AP-1 binding sites are depicted as short vertical lines and the arrows show the primer set used. (B) Knockdown of c-Jun or c-Fos abrogated the interaction of AP-1 with the enhancer region of miR-101-2. In (A) and (B), the promoter region of MMP1 including AP-1 biding site was used as positive control, and genomic region in the upstream of miR-101-2 lacking AP-1 binding sites was used as negative control. (C) EMSA verified the interaction of nuclear proteins with Site A (canonical AP-1 binding sites) and Site B (non-canonical AP-1 binding sites) sequences of the miR-101-2 enhancer. (D) Antibody-supershift assay identified c-Jun and c-Fos as potential nuclear proteins interacting with Site A and B sequences. In (C) and (D), the arrow indicates the DNA–protein complexes.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231742&req=5

Figure 3: Direct interaction of AP-1 with the enhancer region of miR-101-2 gene in vivo and in vitro. (A) AP-1 interacts with the enhancer region of miR-101-2 gene in vivo. A scheme of the amplicons is shown at the top. AP-1 binding sites are depicted as short vertical lines and the arrows show the primer set used. (B) Knockdown of c-Jun or c-Fos abrogated the interaction of AP-1 with the enhancer region of miR-101-2. In (A) and (B), the promoter region of MMP1 including AP-1 biding site was used as positive control, and genomic region in the upstream of miR-101-2 lacking AP-1 binding sites was used as negative control. (C) EMSA verified the interaction of nuclear proteins with Site A (canonical AP-1 binding sites) and Site B (non-canonical AP-1 binding sites) sequences of the miR-101-2 enhancer. (D) Antibody-supershift assay identified c-Jun and c-Fos as potential nuclear proteins interacting with Site A and B sequences. In (C) and (D), the arrow indicates the DNA–protein complexes.
Mentions: To examine the binding of c-Jun and c-Fos to the enhancer of miR-101-2, ChIP assays were performed in TPA-treated HepG2 cells. A specific band of the expected size was amplified from either the individual or combination of c-Jun and c-Fos antibody-precipitated DNA, using primers that covered the AP-1 binding sites in the −17.4 to −16.4 k enhancer of miR-101-2 (Figure 3A). Whereas knockdown of either c-Fos or c-Jun (Supplementary Figure S5A) markedly reduced the antibody-precipitated DNAs (Figure 3B), suggesting the interaction between AP-1 and the miR-101 enhancer in vivo. The direct interaction between c-Jun/c-Fos and canonical (Site A)/non-canonical (Site B) AP-1 binding sites was further verified by EMSA and antibody-supershift assays (Figure 3C and D).

Bottom Line: Furthermore, reintroduction of miR-101 efficiently suppressed the AP-1 activity and pri-miR-101-2 transcription.These data thus suggest a novel AP-1/miR-101 regulatory circuitry, that is, AP-1 promotes the transcription of miR-101, whereas the expression of miR-101 reduces the level of ERK2 and c-Fos and thereby attenuates the AP-1 signaling.Further investigation disclosed that the AP-1 activator TPA-induced MMP9 activity and the TPA-promoted migration and invasion of hepatoma cells were significantly attenuated by miR-101 but were enhanced by miR-101 inhibitor.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Gene Engineering of the Ministry of Education, State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, P.R. China.

Show MeSH
Related in: MedlinePlus