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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 4.MIR155 downregulates multiple players of MTOR signaling. (A) qRT-PCR analysis of mRNA expression levels of predicted targets of MIR155. CNE or (B) HeLa cells were transfected with MIR155 or NC, cells were harvested at 48 h for qRT-PCR. (mean ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (C) Western blot analysis of RHEB, RICTOR, RPS6KB2, ATG3, MTOR, phospho-MTOR (Ser2448), RPTOR, BCL2, AKT, phospho-AKT (Ser473), and GAPDH proteins in CNE or HeLa cells transfected with MIR155, MIR203, or NC. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
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Figure 4: Figure 4.MIR155 downregulates multiple players of MTOR signaling. (A) qRT-PCR analysis of mRNA expression levels of predicted targets of MIR155. CNE or (B) HeLa cells were transfected with MIR155 or NC, cells were harvested at 48 h for qRT-PCR. (mean ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (C) Western blot analysis of RHEB, RICTOR, RPS6KB2, ATG3, MTOR, phospho-MTOR (Ser2448), RPTOR, BCL2, AKT, phospho-AKT (Ser473), and GAPDH proteins in CNE or HeLa cells transfected with MIR155, MIR203, or NC. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).

Mentions: Having established the role of MIR155 in autophagy, we next wanted to identify the direct targets of MIR155. We used FindTar, a prediction algorithm designed by our laboratory, to clarify the potential targets of MIR155 in regulating autophagy.28 FindTar predicted that several upstream or downstream regulators of the MTOR signaling pathway, including RHEB, RPTOR, RICTOR, RPS6KB2, and MTOR are potential MIR155 targets. Besides, BCL2 and ATG3 were also putative MIR155 targets. We performed qRT-PCR to examine the mRNA levels of MIR155 putative targets. MIR155 but not control miRNA, led to a significant attenuation of the mRNA levels of RHEB. Other putative MIR155 target genes, however, did not show significant changes in both HeLa and CNE cells (Fig. 4A and B). Immunoblots with specific antibodies showed that the cellular levels of RHEB, RICTOR, RPS6KB2, and ATG3 proteins were decreased in MIR155-overexpressing cells (Fig. 4C). RHEB and RICTOR are reported to be positive regulators of MTORC1 and AKT; thus, we used phosphorylation site-specific antibodies to directly measure the activation of MTOR and AKT, including a phospho-Ser2448 antibody for MTOR and a phospho-Ser473 antibody for AKT. MIR155 did not significantly change the total protein levels of MTOR and AKT; however, the phosphorylation status of these 2 proteins was significantly reduced (Fig. 4C). To validate, we also checked the mRNA and protein expression levels of MIR155 targets RHEB, RICTOR, RPS6KB2, and ATG3 in CNE and HeLa cells that stably expressing MIR155 by lentiviral infection. Similar to transient expression, stable transfection of MIR155 significantly suppressed RHEB mRNA levels in both CNE and HeLa cells. The mRNA expression of RPS6KB2 was also downregulated by MIR155 in HeLa cells (Fig. S1A). Stable transfection of MIR155 suppressed the protein levels of RHEB, RPS6KB2, and RICTOR, as well as the phosphorylation of MTOR and AKT (Fig. S1B). Therefore, immunoblots from both transient and stable expression of MIR155 confirmed that MIR155 suppresses the expression of RHEB, RICTOR, RPS6KB2 and also downregulates MTOR-AKT signaling. Stable expression of MIR155 did not change the protein levels of ATG3, thus subsequent studies will concentrate on RHEB, RICTOR, and RPS6KB2.


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 4.MIR155 downregulates multiple players of MTOR signaling. (A) qRT-PCR analysis of mRNA expression levels of predicted targets of MIR155. CNE or (B) HeLa cells were transfected with MIR155 or NC, cells were harvested at 48 h for qRT-PCR. (mean ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (C) Western blot analysis of RHEB, RICTOR, RPS6KB2, ATG3, MTOR, phospho-MTOR (Ser2448), RPTOR, BCL2, AKT, phospho-AKT (Ser473), and GAPDH proteins in CNE or HeLa cells transfected with MIR155, MIR203, or NC. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
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Related In: Results  -  Collection

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Show All Figures
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Figure 4: Figure 4.MIR155 downregulates multiple players of MTOR signaling. (A) qRT-PCR analysis of mRNA expression levels of predicted targets of MIR155. CNE or (B) HeLa cells were transfected with MIR155 or NC, cells were harvested at 48 h for qRT-PCR. (mean ± s.d. of independent experiments, n = 4, **P < 0.01, ***P < 0.001, Student 2-tailed t test). (C) Western blot analysis of RHEB, RICTOR, RPS6KB2, ATG3, MTOR, phospho-MTOR (Ser2448), RPTOR, BCL2, AKT, phospho-AKT (Ser473), and GAPDH proteins in CNE or HeLa cells transfected with MIR155, MIR203, or NC. Protein ratios were calculated following ImageJ densitometric analysis. (3 independent experiments gave similar results).
Mentions: Having established the role of MIR155 in autophagy, we next wanted to identify the direct targets of MIR155. We used FindTar, a prediction algorithm designed by our laboratory, to clarify the potential targets of MIR155 in regulating autophagy.28 FindTar predicted that several upstream or downstream regulators of the MTOR signaling pathway, including RHEB, RPTOR, RICTOR, RPS6KB2, and MTOR are potential MIR155 targets. Besides, BCL2 and ATG3 were also putative MIR155 targets. We performed qRT-PCR to examine the mRNA levels of MIR155 putative targets. MIR155 but not control miRNA, led to a significant attenuation of the mRNA levels of RHEB. Other putative MIR155 target genes, however, did not show significant changes in both HeLa and CNE cells (Fig. 4A and B). Immunoblots with specific antibodies showed that the cellular levels of RHEB, RICTOR, RPS6KB2, and ATG3 proteins were decreased in MIR155-overexpressing cells (Fig. 4C). RHEB and RICTOR are reported to be positive regulators of MTORC1 and AKT; thus, we used phosphorylation site-specific antibodies to directly measure the activation of MTOR and AKT, including a phospho-Ser2448 antibody for MTOR and a phospho-Ser473 antibody for AKT. MIR155 did not significantly change the total protein levels of MTOR and AKT; however, the phosphorylation status of these 2 proteins was significantly reduced (Fig. 4C). To validate, we also checked the mRNA and protein expression levels of MIR155 targets RHEB, RICTOR, RPS6KB2, and ATG3 in CNE and HeLa cells that stably expressing MIR155 by lentiviral infection. Similar to transient expression, stable transfection of MIR155 significantly suppressed RHEB mRNA levels in both CNE and HeLa cells. The mRNA expression of RPS6KB2 was also downregulated by MIR155 in HeLa cells (Fig. S1A). Stable transfection of MIR155 suppressed the protein levels of RHEB, RPS6KB2, and RICTOR, as well as the phosphorylation of MTOR and AKT (Fig. S1B). Therefore, immunoblots from both transient and stable expression of MIR155 confirmed that MIR155 suppresses the expression of RHEB, RICTOR, RPS6KB2 and also downregulates MTOR-AKT signaling. Stable expression of MIR155 did not change the protein levels of ATG3, thus subsequent studies will concentrate on RHEB, RICTOR, and RPS6KB2.

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