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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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AP-1 regulates miR-101 expression through the enhancer region of miR-101-2. (A) Effect of AP-1 overexpression on the luciferase activity of the various deleted versions of p(−17.4/−16.4k). HepG2 cells were co-transfected with pRL-CMV and the indicated constructs for 48 h, and then subjected to luciferase activity assay. Luciferase activity of one p(−17.4/−16.4k) transfectant was set to 1. ***P < 0.001. (B) Ectopic expression of AP-1 increased miR-101 expression. HepG2 cells were infected with equal titers of lentivirus containing pCDH or mixture of pCDH-c-Jun and pCDH-c-Fos (indicated as AP-1), followed by treatment with DMSO or 40 ng/ml TPA for 48 h. The vector controls (pCDH) treated with DMSO were used for normalization. *P < 0.05, ***P < 0.001. (C) Effects of knockdown AP-1 on the luciferase activity of p(−17.4/−16.4k). HepG2 cells were cotransfected with the indicated siRNA, p(−17.4/−16.4k) and pRL-CMV, luciferase activity was measured 24 h later. The mixture of equal amount of sic-Jun and sic-Fos was indicated as siAP-1. NC is negative control *P < 0.05. (D) Knockdown of c-Jun and c-Fos abrogated the TPA-induced miR-101 expression. HepG2 cells transfected with negative control (NC) or mixture of equal amount of sic-Jun and sic-Fos (indicated as siAP-1) were treated with or without 40 ng/ml TPA for 24 h before qPCR assay. **P < 0.01.
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Figure 4: AP-1 regulates miR-101 expression through the enhancer region of miR-101-2. (A) Effect of AP-1 overexpression on the luciferase activity of the various deleted versions of p(−17.4/−16.4k). HepG2 cells were co-transfected with pRL-CMV and the indicated constructs for 48 h, and then subjected to luciferase activity assay. Luciferase activity of one p(−17.4/−16.4k) transfectant was set to 1. ***P < 0.001. (B) Ectopic expression of AP-1 increased miR-101 expression. HepG2 cells were infected with equal titers of lentivirus containing pCDH or mixture of pCDH-c-Jun and pCDH-c-Fos (indicated as AP-1), followed by treatment with DMSO or 40 ng/ml TPA for 48 h. The vector controls (pCDH) treated with DMSO were used for normalization. *P < 0.05, ***P < 0.001. (C) Effects of knockdown AP-1 on the luciferase activity of p(−17.4/−16.4k). HepG2 cells were cotransfected with the indicated siRNA, p(−17.4/−16.4k) and pRL-CMV, luciferase activity was measured 24 h later. The mixture of equal amount of sic-Jun and sic-Fos was indicated as siAP-1. NC is negative control *P < 0.05. (D) Knockdown of c-Jun and c-Fos abrogated the TPA-induced miR-101 expression. HepG2 cells transfected with negative control (NC) or mixture of equal amount of sic-Jun and sic-Fos (indicated as siAP-1) were treated with or without 40 ng/ml TPA for 24 h before qPCR assay. **P < 0.01.

Mentions: Consistently, overexpression of c-Jun andc-Fos enhanced the activity of p(−17.4/−16.4k) enhancer reporter, which was severely attenuated when the AP-1 binding sites were deleted (Figure 4A). Remarkably, miR-101 level was increased by ectopic expression of c-Fos and c-Jun, and was further enhanced by TPA treatment (Figure 4B and Supplementary Figure S5B). On the other hand, knockdown of c-Jun or/and c-Fos led to a dramatic decrease of the transcription activity of p(−17.4/−16.4k) (Figure 4C) and TPA-induced expression of miR-101 (Figure 4D), suggesting that the expression of miR-101 depends on the activity of AP-1.


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)

AP-1 regulates miR-101 expression through the enhancer region of miR-101-2. (A) Effect of AP-1 overexpression on the luciferase activity of the various deleted versions of p(−17.4/−16.4k). HepG2 cells were co-transfected with pRL-CMV and the indicated constructs for 48 h, and then subjected to luciferase activity assay. Luciferase activity of one p(−17.4/−16.4k) transfectant was set to 1. ***P < 0.001. (B) Ectopic expression of AP-1 increased miR-101 expression. HepG2 cells were infected with equal titers of lentivirus containing pCDH or mixture of pCDH-c-Jun and pCDH-c-Fos (indicated as AP-1), followed by treatment with DMSO or 40 ng/ml TPA for 48 h. The vector controls (pCDH) treated with DMSO were used for normalization. *P < 0.05, ***P < 0.001. (C) Effects of knockdown AP-1 on the luciferase activity of p(−17.4/−16.4k). HepG2 cells were cotransfected with the indicated siRNA, p(−17.4/−16.4k) and pRL-CMV, luciferase activity was measured 24 h later. The mixture of equal amount of sic-Jun and sic-Fos was indicated as siAP-1. NC is negative control *P < 0.05. (D) Knockdown of c-Jun and c-Fos abrogated the TPA-induced miR-101 expression. HepG2 cells transfected with negative control (NC) or mixture of equal amount of sic-Jun and sic-Fos (indicated as siAP-1) were treated with or without 40 ng/ml TPA for 24 h before qPCR assay. **P < 0.01.
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Figure 4: AP-1 regulates miR-101 expression through the enhancer region of miR-101-2. (A) Effect of AP-1 overexpression on the luciferase activity of the various deleted versions of p(−17.4/−16.4k). HepG2 cells were co-transfected with pRL-CMV and the indicated constructs for 48 h, and then subjected to luciferase activity assay. Luciferase activity of one p(−17.4/−16.4k) transfectant was set to 1. ***P < 0.001. (B) Ectopic expression of AP-1 increased miR-101 expression. HepG2 cells were infected with equal titers of lentivirus containing pCDH or mixture of pCDH-c-Jun and pCDH-c-Fos (indicated as AP-1), followed by treatment with DMSO or 40 ng/ml TPA for 48 h. The vector controls (pCDH) treated with DMSO were used for normalization. *P < 0.05, ***P < 0.001. (C) Effects of knockdown AP-1 on the luciferase activity of p(−17.4/−16.4k). HepG2 cells were cotransfected with the indicated siRNA, p(−17.4/−16.4k) and pRL-CMV, luciferase activity was measured 24 h later. The mixture of equal amount of sic-Jun and sic-Fos was indicated as siAP-1. NC is negative control *P < 0.05. (D) Knockdown of c-Jun and c-Fos abrogated the TPA-induced miR-101 expression. HepG2 cells transfected with negative control (NC) or mixture of equal amount of sic-Jun and sic-Fos (indicated as siAP-1) were treated with or without 40 ng/ml TPA for 24 h before qPCR assay. **P < 0.01.
Mentions: Consistently, overexpression of c-Jun andc-Fos enhanced the activity of p(−17.4/−16.4k) enhancer reporter, which was severely attenuated when the AP-1 binding sites were deleted (Figure 4A). Remarkably, miR-101 level was increased by ectopic expression of c-Fos and c-Jun, and was further enhanced by TPA treatment (Figure 4B and Supplementary Figure S5B). On the other hand, knockdown of c-Jun or/and c-Fos led to a dramatic decrease of the transcription activity of p(−17.4/−16.4k) (Figure 4C) and TPA-induced expression of miR-101 (Figure 4D), suggesting that the expression of miR-101 depends on the activity of AP-1.

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