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

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 G₁/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.     

MicroRNAs Enhance Keratinocyte Proliferative Capacity in a Stem Cell-Enriched Epithelium

MicroRNAs are critical regulators of stem cell behavior. The miR-103/107 family is preferentially expressed in the stem cell-enriched corneal limbal epithelium and plays an important role in coordinating several intrinsic characteristics of limbal epithelial stem cells. To elucidate further the mechanisms by which miRs-103/107 function in regulating limbal epithelial stem cells, we investigate the global effects of miRs-103/107 on gene expression in an unbiased manner. Using antagomirs-103/107, we knocked down endogenous miRs-103/107 in keratinocytes and conducted an mRNA profiling study. We show that miRs-103/107 target mitogen-activated protein kinase kinase kinase 7 (MAP3K7) and thereby negatively regulate the p38/AP-1 pathway, which directs epithelial cells towards a differentiated state. Pharmacological inhibition of p38 increases holoclone colony formation, a measure of proliferative capacity. This suggests that the negative regulation of p38 by miRs-103/107 contributes to enhanced proliferative capacity, which is a hallmark of stem cells. Since miRs-103/107 also promote increased holoclone colony formation by regulating JNK activation through non-canonical Wnt signaling, we believe that this microRNA family preserves “stemness” by mediating the crosstalk between the Wnt/JNK and MAP3K7/p38/AP-1 pathways.     

MIR181A regulates starvation- and rapamycin-induced autophagy through targeting of ATG5

Macroautophagy (autophagy herein) is a cellular catabolic mechanism activated in response to stress conditions including starvation, hypoxia and misfolded protein accumulation. Abnormalities in autophagy were associated with pathologies including cancer and neurodegenerative diseases. Hence, elucidation of the signaling pathways controlling autophagy is of utmost importance. Recently we and others described microRNAs (miRNAs) as novel and potent modulators of the autophagic activity. Here, we describe MIR181A (hsa-miR-181a-1) as a new autophagy-regulating miRNA. We showed that overexpression of MIR181A resulted in the attenuation of starvation- and rapamycin-induced autophagy in MCF-7, Huh-7 and K562 cells. Moreover, antagomir-mediated inactivation of endogenous miRNA activity stimulated autophagy. We identified ATG5 as an MIR181A target. Indeed, ATG5 cellular levels were decreased in cells upon MIR181A overexpression and increased following the introduction of antagomirs. More importantly, overexpression of ATG5 from a miRNA-insensitive cDNA construct rescued autophagic activity in the presence of MIR181A. We also showed that the ATG5 3′ UTR contained functional MIR181A responsive sequences sensitive to point mutations. Therefore, MIR181A is a novel and important regulator of autophagy and ATG5 is a rate-limiting miRNA target in this effect.     

Vmp1 Regulates PtdIns3P Signaling During Autophagosome Formation in Dictyostelium discoideum

Generation and turnover of phosphatidylinositol 3‐phosphate (PtdIns3P) signaling is essential for autophagosome formation and other membrane traffic processes. In both Dictyostelium discoideum and mammalian cells, autophagosomes are formed from specialized regions of the endoplasmic reticulum (ER), called omegasomes, which are enriched in the signaling lipid PtdIns3P. Vacuole membrane protein 1 (Vmp1) is a multispanning membrane protein localized at the ER that is required for autophagosome formation. There are conflicting reports in the literature as to whether Vmp1 is strictly required or not for autophagy‐related PtdIns3P signaling and its hierarchical relationship with Atg1 and PI3K. We have now addressed these questions in the Dictyostelium model. We show that Dictyostelium cells lacking Vmp1 have elevated and aberrant PtdIns3P signaling on the ER, resulting in an increased and persistent recruitment of Atg18 and other autophagic proteins. This indicates that Vmp1 is not strictly essential for the generation of PtdIns3P signaling but rather suggests a role in the correct turnover or modulation of this signaling. Of interest, these PtdIns3P‐enriched regions of the ER surround ubiquitinated protein aggregates but are unable to form functional autophagosomes. vmp1 null cells also have additional defects in macropinocytosis and growth, which are not shared by other autophagy mutants. Remarkably, we show that these defects and also the aberrant PtdIns3P distribution are largely suppressed by the concomitant loss of Atg1, indicating that aberrant autophagic signaling on the ER inhibits macropinocytosis. These results suggest that Atg1 functions upstream of Vmp1 in this signaling pathway and demonstrates a previously unappreciated link between abnormal autophagy signaling and macropinocytosis.      

LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

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

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 G₁/S cell cycle arrest. Collectively, these data present a new role for MIR155 as a key regulator of autophagy via dysregulation of MTOR pathway.     

MIR125B1 represses the degradation of the PML-RARA oncoprotein by an autophagy-lysosomal pathway in acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) is characterized by the t(15;17)-associated PML-RARA fusion gene. We have previously found that MIR125B1 is highly expressed in patients with APL and may be associated with disease pathogenesis; however, the mechanism by which MIR125B1 exerts its oncogenic potential has not been fully elucidated. Here, we demonstrated that MIR125B1 abundance correlates with the PML-RARA status. MIR125B1 overexpression enhanced PML-RARA expression and inhibited the ATRA-induced degradation of the PML-RARA oncoprotein. RNA-seq analysis revealed a direct link between the PML-RARA degradation pathway and MIR125B1-arrested differentiation. We further demonstrated that the MIR125B1-mediated blockade of PML-RARA proteolysis was regulated via an autophagy-lysosomal pathway, contributing to the inhibition of APL differentiation. Furthermore, we identified DRAM2 (DNA-damage regulated autophagy modulator 2), a critical regulator of autophagy, as a novel target that was at least partly responsible for the function of MIR125B1 involved in autophagy. Importantly, the knockdown phenotypes for DRAM2 are similar to the effects of overexpressing MIR125B1 as impairment of PML-RARA degradation, inhibition of autophagy, and myeloid cell differentiation arrest. These effects of MIR125B1 and its target DRAM2 were further confirmed in an APL mouse model. Thus, MIR125B1 dysregulation may interfere with the effectiveness of ATRA-mediated differentiation through an autophagy-dependent pathway, representing a novel potential APL therapeutic target.     

A novel tumor-promoting mechanism of IL6 and the therapeutic efficacy of tocilizumab: Hypoxia-induced IL6 is a potent autophagy initiator in glioblastoma via the p-STAT3-MIR155-3p-CREBRF pathway

Hypoxia induces protective autophagy in glioblastoma cells and new therapeutic avenues that target this process may improve the outcome for glioblastoma patients. Recent studies have suggested that the autophagic process is upregulated in glioblastomas in response to extensive hypoxia. Hypoxia also induces the upregulation of a specific set of proteins and microRNAs (miRNAs) in a variety of cell types. IL6 (interleukin 6), an inflammatory autocrine and paracrine cytokine that is overexpressed in glioblastoma, has been reported to be a biomarker for poor prognosis because of its tumor-promoting effects. Here, we describe a novel tumor-promoting mechanism of IL6, whereby hypoxia-induced IL6 acts as a potent initiator of autophagy in glioblastoma via the phosphorylated (p)-STAT3-MIR155-3p pathway. IL6 and p-STAT3 levels correlated with the abundance of autophagic cells and HIF1A levels in human glioma tissues and with the grade of human glioma, whereas inhibition of exogenous or endogenous IL6 repressed autophagy in glioblastoma cells in vitro. Knockdown of endogenous MIR155-3p inhibited IL6-induced autophagy, and enforced expression of MIR155-3p restored the anti-autophagic activity of IL6 inhibitors. We show that the hypoxia-IL6-p-STAT3-MIR155-3p-CREBRF-CREB3-ATG5 pathway plays a central role in malignant glioma progression, with blockade of the IL6 receptor by tocilizumab demonstrating a certain level of therapeutic efficacy in a xenograft model in vivo, especially in combination with temozolomide. Moreover, tocilizumab inhibits autophagy by promoting tumor apoptosis. Collectively, our findings provide new insight into the molecular mechanisms underlying hypoxia-induced glioma cell autophagy and point toward a possible efficacious adjuvant therapy for glioblastoma patients.     

MicroRNA expression profiling of Leishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

Leishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.     

MicroRNA targets autophagy in pancreatic cancer cells during cancer therapy

The therapeutic outcome of pancreatic cancer is generally poor due to the inherent or acquired resistance of cancer cells to treatment. Pancreatic cancer cells have higher basal autophagy levels than other cancer cell types, which may correlate with their nonresponsiveness to the available cancer therapy. Therefore, understanding the mechanisms behind autophagy activation in pancreatic cancer cells may ultimately improve therapeutic outcomes. Here we demonstrated that MIR23B is a potent inhibitor of autophagy. MIR23B targets the 3′UTR of the autophagy-related gene ATG12, thereby decreasing autophagic activity and ultimately promoting radiation-induced pancreatic cancer cell death. Thus, our study clarified some of the underlying molecular mechanisms of activated autophagy in response to cancer therapy in pancreatic cancer.     

Autophagy and Akt promote survival in glioma

Signaling through phosphatidylinositol 3-kinase (PtdIns3K)-Akt-mTOR is frequently activated in cancers including glioblastoma multiforme (GBM), where this kinase network regulates survival. It is thus surprising that inhibitors of these pathways induce minimal cell death in glioma. We showed that the dual PtdIns3K-mTOR inhibitor PI-103 induces autophagy in therapy-resistant, PTEN-mutant glioma, with blockade of mTOR complex 1 (mTORC1) and complex 2 (mTORC2) contributing independently to autophagy. Inhibition of autophagosome maturation synergizes with PI-103 to induce apoptosis through the Bax-dependent intrinsic mitochondrial pathway, indicating that PI-103 induces autophagy as a survival pathway in this setting. Not all inhibitors of PtdIns3K-Akt-mTOR signaling synergize with inhibitors of autophagy. The allosteric mTORC1 inhibitor rapamycin fails to induce apoptosis in conjunction with blockade of autophagy, due to feedback-activation of Akt. Apoptosis in the setting of rapamycin therapy requires concurrent inhibition of both autophagy and of PtdIns3K-Akt. Moreover, the clinical PtdIns3K-mTOR inhibitor NVP-BEZ235 cooperates with the clinical lysosomotropic autophagy inhibitor chloroquine to induce apoptosis in PTEN-mutant glioma xenografts in vivo, offering a therapeutic approach translatable to patients.     

AMPKα promotes basal autophagy induction in Dictyostelium discoideum

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5′-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα⁻ cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα⁻ cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα⁻ cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK–ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, β-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα⁻ cells exhibit enhanced poly-ubiquitinated protein levels and allied large ubiquitin-positive protein aggregates. Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.     

Ceramide signaling targets the PP2A-like protein phosphatase Sit4p to impair vacuolar function,vesicular trafficking and autophagy in Isc1p deficient cells

The vacuoles play important roles in cellular homeostasis and their functions include the digestion of cytoplasmic material and organelles derived from autophagy. Conserved nutrient signaling pathways regulate vacuolar function and autophagy, ensuring normal cell and organismal development and aging. Recent evidence implicates sphingolipids in the modulation of these processes, but the impact of ceramide signaling on vacuolar dynamics and autophagy remains largely unknown. Here, we show that yeast cells lacking Isc1p, an orthologue of mammalian neutral sphingomyelinase type 2, exhibit vacuolar fragmentation and dysfunctions, namely decreased Pep4p-mediated proteolysis and V-ATPase activity, which impairs vacuolar acidification. Moreover, these phenotypes are suppressed by downregulation of the ceramide-activated protein phosphatase Sit4p. The isc1Δ cells also exhibit defective Cvt and vesicular trafficking in a Sit4p-dependent manner, ultimately contributing to a reduced autophagic flux. Importantly, these phenotypes are also suppressed by downregulation of the nutrient signaling kinase TORC1, which is known to inhibit Sit4p and autophagy, or Sch9p. These results support a model in which Sit4p functions downstream of Isc1p in a TORC1-independent, ceramide-dependent signaling branch that impairs vacuolar function and vesicular trafficking, leading to autophagic defects in yeast.