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Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway.

Wan G, Xie W, Liu Z, Xu W, Lao Y, Huang N, Cui K, Liao M, He J, Jiang Y, Yang BB, Xu H, Xu N, Zhang Y - Autophagy (2013)

Bottom Line: MIR155 suppresses target-gene expression by directly interacting with their 3' untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness.Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G 1/S cell cycle arrest.Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences; Tsinghua University; Beijing, China; Key Lab in Healthy Science and Technology; Division of Life Science; Graduate School at Shenzhen; Tsinghua University; Shenzhen, China.

ABSTRACT
Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3' untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G 1/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.

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Figure 2. Overexpression of MIR155 induces autophagic activity. (A) Overexpression of MIR155 induces LC3 conversion and SQSTM1 degradation. Western blots of control (NC) and MIR155-transfected cells. (B) ImageJ densitometric analysis of the LC3-II/GAPDH or (C) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, ***P < 0.001, Student 2-tailed t test. (D) Overexpression of MIR155 increases autophagic flux. CNE or HeLa cells were transfected with MIR155 or NC, BAF was applied to the medium for 1 h at 47 h post transfection, cells were harvested for western blot. (E) ImageJ densitometric analysis of the LC3-II/GAPDH or (F) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, **P < 0.01, Student 2-tailed t test.
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Figure 2: Figure 2. Overexpression of MIR155 induces autophagic activity. (A) Overexpression of MIR155 induces LC3 conversion and SQSTM1 degradation. Western blots of control (NC) and MIR155-transfected cells. (B) ImageJ densitometric analysis of the LC3-II/GAPDH or (C) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, ***P < 0.001, Student 2-tailed t test. (D) Overexpression of MIR155 increases autophagic flux. CNE or HeLa cells were transfected with MIR155 or NC, BAF was applied to the medium for 1 h at 47 h post transfection, cells were harvested for western blot. (E) ImageJ densitometric analysis of the LC3-II/GAPDH or (F) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, **P < 0.01, Student 2-tailed t test.

Mentions: To explore the role of MIR155 in autophagy, we performed a GFP-LC3 puncta-formation assay and an LC3 conversion assay. MIR155 was transfected into CNE or HeLa cells that stably expressing GFP-LC3 fusion protein, the localization of GFP-LC3 was examined by confocal microscopy. GFP-LC3 puncta appear in the cytoplasm reflects the recruitment of LC3 proteins to autophagosomes. As shown in Figure 1B, there was a significant increase of GFP-LC3 puncta in MIR155 transfected cells. MIR203, a control miRNA that has no predicted targets in the autophagy pathway, behaved similarly to the negative control (NC) miRNA. The quantification of GFP-LC3 dots per cell confirmed that overexpression of MIR155 induced autophagosome accumulation in both CNE and HeLa cells (Fig. 1C). Next, we detected the conversion of LC3-I [cleaved, cytosolic form of MAP1LC3 (LC3)] to LC3-II (subsequently lipidated and membrane-bound form) by western blot. Consistent with the GFP-LC3 puncta formation assay, MIR155 overexpression led to a significant upregulation of lipidated LC3-II (Fig. 2A and B). Thus, both assays suggest that overexpression of MIR155 induces autophagosome accumulation.


Hypoxia-induced MIR155 is a potent autophagy inducer by targeting multiple players in the MTOR pathway.

Wan G, Xie W, Liu Z, Xu W, Lao Y, Huang N, Cui K, Liao M, He J, Jiang Y, Yang BB, Xu H, Xu N, Zhang Y - Autophagy (2013)

Figure 2. Overexpression of MIR155 induces autophagic activity. (A) Overexpression of MIR155 induces LC3 conversion and SQSTM1 degradation. Western blots of control (NC) and MIR155-transfected cells. (B) ImageJ densitometric analysis of the LC3-II/GAPDH or (C) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, ***P < 0.001, Student 2-tailed t test. (D) Overexpression of MIR155 increases autophagic flux. CNE or HeLa cells were transfected with MIR155 or NC, BAF was applied to the medium for 1 h at 47 h post transfection, cells were harvested for western blot. (E) ImageJ densitometric analysis of the LC3-II/GAPDH or (F) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, **P < 0.01, Student 2-tailed t test.
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Figure 2: Figure 2. Overexpression of MIR155 induces autophagic activity. (A) Overexpression of MIR155 induces LC3 conversion and SQSTM1 degradation. Western blots of control (NC) and MIR155-transfected cells. (B) ImageJ densitometric analysis of the LC3-II/GAPDH or (C) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, ***P < 0.001, Student 2-tailed t test. (D) Overexpression of MIR155 increases autophagic flux. CNE or HeLa cells were transfected with MIR155 or NC, BAF was applied to the medium for 1 h at 47 h post transfection, cells were harvested for western blot. (E) ImageJ densitometric analysis of the LC3-II/GAPDH or (F) SQSTM1/GAPDH ratios from immunoblots (mean ± s.d. of 4 independent experiments). *P < 0.05, **P < 0.01, Student 2-tailed t test.
Mentions: To explore the role of MIR155 in autophagy, we performed a GFP-LC3 puncta-formation assay and an LC3 conversion assay. MIR155 was transfected into CNE or HeLa cells that stably expressing GFP-LC3 fusion protein, the localization of GFP-LC3 was examined by confocal microscopy. GFP-LC3 puncta appear in the cytoplasm reflects the recruitment of LC3 proteins to autophagosomes. As shown in Figure 1B, there was a significant increase of GFP-LC3 puncta in MIR155 transfected cells. MIR203, a control miRNA that has no predicted targets in the autophagy pathway, behaved similarly to the negative control (NC) miRNA. The quantification of GFP-LC3 dots per cell confirmed that overexpression of MIR155 induced autophagosome accumulation in both CNE and HeLa cells (Fig. 1C). Next, we detected the conversion of LC3-I [cleaved, cytosolic form of MAP1LC3 (LC3)] to LC3-II (subsequently lipidated and membrane-bound form) by western blot. Consistent with the GFP-LC3 puncta formation assay, MIR155 overexpression led to a significant upregulation of lipidated LC3-II (Fig. 2A and B). Thus, both assays suggest that overexpression of MIR155 induces autophagosome accumulation.

Bottom Line: MIR155 suppresses target-gene expression by directly interacting with their 3' untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness.Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G 1/S cell cycle arrest.Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences; Tsinghua University; Beijing, China; Key Lab in Healthy Science and Technology; Division of Life Science; Graduate School at Shenzhen; Tsinghua University; Shenzhen, China.

ABSTRACT
Hypoxia activates autophagy, an evolutionarily conserved cellular catabolic process. Dysfunction in the autophagy pathway has been implicated in an increasing number of human diseases, including cancer. Hypoxia induces upregulation of a specific set of microRNAs (miRNAs) in a variety of cell types. Here, we describe hypoxia-induced MIR155 as a potent inducer of autophagy. Enforced expression of MIR155 increases autophagic activity in human nasopharyngeal cancer and cervical cancer cells. Knocking down endogenous MIR155 inhibits hypoxia-induced autophagy. We demonstrated that MIR155 targets multiple players in MTOR signaling, including RHEB, RICTOR, and RPS6KB2. MIR155 suppresses target-gene expression by directly interacting with their 3' untranslated regions (UTRs), mutations of the binding sites abolish their MIR155 responsiveness. Furthermore, by downregulating MTOR signaling, MIR155 also attenuates cell proliferation and induces G 1/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.

Show MeSH
Related in: MedlinePlus