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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 3. Knockdown of endogenous MIR155 inhibits hypoxia-induced autophagy. (A) Inhibition of MIR155 in hypoxia suppresses GFP-LC3 translocation. HeLa or CNE cells stably expressing GFP-LC3 were transfected with LNA-NC or LNA-155 and exposed to 1% oxygen. The number of GFP-LC3 puncta was quantified. (mean ± s.d. of independent experiments, n = 4, *P < 0.05,**P < 0.01, Wilcoxon rank sum test). (B) Representative images from the quantification are shown. Scale bar: 10 μm. (C) Knockdown of endogenous MIR155 inhibits autophagy. HeLa or CNE cells transfected with LNA-NC or LNA-155 were cultured in 1% oxygen for 36 h, cells were harvested for western blot. Protein ratio of SQSTM1/GAPDH was calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
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Figure 3: Figure 3. Knockdown of endogenous MIR155 inhibits hypoxia-induced autophagy. (A) Inhibition of MIR155 in hypoxia suppresses GFP-LC3 translocation. HeLa or CNE cells stably expressing GFP-LC3 were transfected with LNA-NC or LNA-155 and exposed to 1% oxygen. The number of GFP-LC3 puncta was quantified. (mean ± s.d. of independent experiments, n = 4, *P < 0.05,**P < 0.01, Wilcoxon rank sum test). (B) Representative images from the quantification are shown. Scale bar: 10 μm. (C) Knockdown of endogenous MIR155 inhibits autophagy. HeLa or CNE cells transfected with LNA-NC or LNA-155 were cultured in 1% oxygen for 36 h, cells were harvested for western blot. Protein ratio of SQSTM1/GAPDH was calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).

Mentions: To document the physiological relevance of MIR155 on autophagy, we inhibited the expression of endogenous MIR155 and repeated the above validation assays in both CNE and HeLa cells. LNA-derived MIR155 inhibitor was used to inhibit the high level of endogenous MIR155 during hypoxia treatment. Hypoxia-induced GFP-LC3 puncta accumulation was dramatically suppressed by LNA-155 in both HeLa and CNE cells (Fig. 3A and B). Compared with LNA-NC control, SQSTM1 degradation during hypoxia treatment was also reduced upon LNA-155 transfection, reflecting a decrease of autophagic activity (Fig. 3C). Hence, these results demonstrate the physiological relevance of endogenous MIR155 on regulating autophagy process during hypoxia treatment.


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 3. Knockdown of endogenous MIR155 inhibits hypoxia-induced autophagy. (A) Inhibition of MIR155 in hypoxia suppresses GFP-LC3 translocation. HeLa or CNE cells stably expressing GFP-LC3 were transfected with LNA-NC or LNA-155 and exposed to 1% oxygen. The number of GFP-LC3 puncta was quantified. (mean ± s.d. of independent experiments, n = 4, *P < 0.05,**P < 0.01, Wilcoxon rank sum test). (B) Representative images from the quantification are shown. Scale bar: 10 μm. (C) Knockdown of endogenous MIR155 inhibits autophagy. HeLa or CNE cells transfected with LNA-NC or LNA-155 were cultured in 1% oxygen for 36 h, cells were harvested for western blot. Protein ratio of SQSTM1/GAPDH was calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
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Related In: Results  -  Collection

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Figure 3: Figure 3. Knockdown of endogenous MIR155 inhibits hypoxia-induced autophagy. (A) Inhibition of MIR155 in hypoxia suppresses GFP-LC3 translocation. HeLa or CNE cells stably expressing GFP-LC3 were transfected with LNA-NC or LNA-155 and exposed to 1% oxygen. The number of GFP-LC3 puncta was quantified. (mean ± s.d. of independent experiments, n = 4, *P < 0.05,**P < 0.01, Wilcoxon rank sum test). (B) Representative images from the quantification are shown. Scale bar: 10 μm. (C) Knockdown of endogenous MIR155 inhibits autophagy. HeLa or CNE cells transfected with LNA-NC or LNA-155 were cultured in 1% oxygen for 36 h, cells were harvested for western blot. Protein ratio of SQSTM1/GAPDH was calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
Mentions: To document the physiological relevance of MIR155 on autophagy, we inhibited the expression of endogenous MIR155 and repeated the above validation assays in both CNE and HeLa cells. LNA-derived MIR155 inhibitor was used to inhibit the high level of endogenous MIR155 during hypoxia treatment. Hypoxia-induced GFP-LC3 puncta accumulation was dramatically suppressed by LNA-155 in both HeLa and CNE cells (Fig. 3A and B). Compared with LNA-NC control, SQSTM1 degradation during hypoxia treatment was also reduced upon LNA-155 transfection, reflecting a decrease of autophagic activity (Fig. 3C). Hence, these results demonstrate the physiological relevance of endogenous MIR155 on regulating autophagy process during hypoxia treatment.

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