miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3′ UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.     

miR-376b controls starvation and mTOR inhibition-related autophagy by targeting ATG4C and BECN1

Macroautophagy (autophagy) is the major intracellular degradation pathway for long-lived proteins and organelles. It helps the cell to survive a spectrum of stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Moreover, abnormalities of autophagy play a role in major health problems including cancer and neurodegenerative diseases. Yet, mechanisms controlling autophagic activity are not fully understood. Here, we describe hsa-miR-376b (miR-376b) as a new microRNA (miRNA) regulating autophagy. We showed that miR-376b expression attenuated starvation- and rapamycin-induced autophagy in MCF-7 and Huh-7 cells. We discovered autophagy proteins ATG4C and BECN1 (Beclin 1) as cellular targets of miR-376b. Indeed, upon miRNA overexpression, both mRNA and protein levels of ATG4C and BECN1 were decreased. miR-376b target sequences were present in the 3' UTR of ATG4C and BECN1 mRNAs and introduction of mutations abolished their miR-376b responsiveness. Antagomir-mediated inactivation of the endogenous miR-376b led to an increase in ATG4C and BECN1 levels. Therefore, miR-376b controls autophagy by directly regulating intracellular levels of two key autophagy proteins, ATG4C and BECN1.     

Autophagy-associated circRNA circATG7 facilitates autophagy and promotes pancreatic cancer progression

Dysregulation of autophagy and circular RNAs (circRNAs) are involved in the pancreatic cancer (PC) progression. However, the regulatory network between circRNAs, autophagy, and PC progression remains unknown. Herein, we demonstrated that autophagy-associated circRNA circ-autophagy related 7 (circATG7) was elevated in PC tissues compared to adjacent tissues, and in PC cells treated with EBSS and hypoxia. circATG7 expression was positively associated with tumor diameter and lymph node invasion in patients with PC. circATG7 overexpression promoted PC cell proliferation, mobility, and autophagy in vitro, while circATG7 knockdown induced the opposite effects. ATG7 inhibition attenuated the effects of circATG7 on the biological functions of PC cells. CircATG7 is located in the cell cytoplasm and nucleus. Cytoplasmic circATG7 sponged miR-766-5p and decreased its expression, and increased the expression of ATG7, a target gene of miR-766-5p. Nuclear circATG7 acted as a scaffold to increase the interaction between the human antigen R protein and ATG7 mRNA and enhanced ATG mRNA stability. Furthermore, we demonstrated that circATG7 regulates PC cell proliferation and metastasis in vivo via ATG7-dependent autophagy. In conclusion, our results demonstrated that circATG7 accelerates PC progression via miR-766-5p/ATG7 and that HUR/ATG7 depends on autophagic flux. Thus, circATG7 may be a potential therapeutic target for PC.Subject terms: Pancreatic cancer, Pancreatic cancer     

miR-216b enhances the efficacy of vemurafenib by targeting Beclin-1, UVRAG and ATG5 in melanoma

Autophagy maintains cells survival in many stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Additionally, autophagic survival mechanisms are used by transformed tumor cells to inhibit cell death, limit drug effectiveness and possibly generate drug resistance. However, the mechanism of how cells utilize autophagy during drug resistance is not fully understood. Here, we demonstrate that miR-216b plays an important role in alleviating drug resistance by regulating autophagy in melanoma. We show that miR-216b attenuates autophagy by directly targeting three key autophagy genes Beclin-1, UVRAG and ATG5. Overexpression of these genes from miRNA immune cDNA constructs rescue autophagic activity in the presence of miR-216b. Antagomir-mediated inactivation of endogenous miR-216b led to an increase of Beclin-1, UVRAG, ATG5, and subsequent autophagic activity. More importantly, we have discovered that BRAF(V600E) inhibitor vemurafenib suppresses miR-216b activity, which in turn activates autophagy to generate drug resistance in both BRAFi-sensitive and -resistant cells. Strikingly, ectopic expression of miR-216b increases the efficacy of vemurafenib both in vitro and in vivo. Taken together, these data indicate that miR-216b regulates autophagy by suppressing three key autophagy genes, and enhances the antitumor activity of vemurafenib in BRAF(V600E) melanoma cells.     

MiR-135a functions as a tumor suppressor in epithelial ovarian cancer and regulates HOXA10 expression

The activation of homeobox A10 (HOXA10) has been proved to be an important event in epithelial ovarian carcinogenesis, yet its regulation in epithelial ovarian cancer (EOC) is still not fully understood. Here, we aimed to reveal the mechanism that a predicted target miRNA regulates HOXA10 expression and the association of its expression with progression of EOC. Here, by using computer-assisted algorithms from PicTar, TargetScan, and miRBase, we identified that the predicted target miRNA of HOXA10 was miR-135a. MiR-135a expression in EOC tissues and controls was measured with quantitative RT-PCR. The role of miR-135a and HOXA10 in the growth and survival of several EOC cell lines was determined with several in vitro approaches. We found that miR-135a expression was downregulated in an EOC patient cohort. Also, patients with low miR-135a expression had shorter overall survival and progression-free survival durations than those with high expression. Functional analysis of three EOC-derived cell lines (SKOV-3, HEY, and OVCAR-3) demonstrated that miR-135a directly regulated HOXA10 expression by targeting its 3′-UTR. Inhibition of HOXA10 expression with miR-135a mimics and HOXA10 siRNA consistently resulted in cell apoptosis with concomitant enhancement of caspase-3, increase of p53 expression and reduction of Bcl-2 expression, and also suppressed cell growth and adhesion. These findings suggest that ubiquitous loss of miR-135a expression is a critical mechanism for the overexpression of HOXA10 in EOC cells, which is implicated in epithelial ovarian carcinogenesis. Furthermore, miR-135a may be predictive of EOC prognosis.     

Inhibition of miR-130b-3p restores autophagy and attenuates intervertebral disc degeneration through mediating ATG14 and PRKAA1

Oxidative stress-induced autophagy dysfunction is involved in the pathogenesis of intervertebral disc degeneration (IVDD). MicroRNAs (miRNAs) not only have been regarded as important regulators of IVDD but also reported to be related to autophagy. This research was aimed to explore the role of miR-130b-3p in IVDD and its regulation on autophagy mechanism. The miR-130b-3p expression in the patient’s degenerative nucleus pulposus (NP) samples and rat NP tissues was detected by qRT-PCR and FISH assay. The miR-130b-3p was knocked down or overexpressed in the human NP cells by lentivirus transfection. TBHP was used to induce oxidative stress in the human NP cells. Apoptosis, senescence, and autophagy were evaluated by flow cytometry, β-gal staining, immunofluorescence, electron microscopy, and Western blot in the miR-130b-3p knocked down human NP cells under TBHP treatment. The relationship between the miR-130b-3p and ATG14 or PRKAA1 was confirmed by luciferase assay. The siRNA transfection was used to knock down the ATG14 and PRKAA1 expression, and then the human NP cells functions were further determined. In the in vivo experiment, the IVDD rat model was constructed and an adeno-associated virus (AAV)-miR-130b-3p inhibitor was intradiscally injected. After that, MRI and histological staining were conducted to evaluate the role of miR-130b-3p inhibition in the IVDD rat model. We found that the miR-130b-3p was upregulated in the degenerative NP samples from humans and rats. Interestingly, the inhibition of miR-130b-3p rescued oxidative stress-induced dysfunction of the human NP cells, and miR-130b-3p inhibition upregulated autophagy. Mechanistically, we confirmed that the miR-130b-3p regulated the ATG14 and PRKAA1 directly and the knockdown of the ATG14 or PRKAA1 as well as the treatment of autophagy inhibitor blockaded the autophagic flux and reversed the protective effects of miR-130b-3p inhibition in the TBHP-induced human NP cells. Furthermore, the inhibition of the miR-130b-3p via AAV- miR-130b-3p injection ameliorated the IVDD in a rat model. These data demonstrated that the miR-130b-3p inhibition could upregulate the autophagic flux and alleviate the IVDD via targeting ATG14 and PRKAA1.

The translational potential of this article: The suppression of miR-130b-3p may become an effective therapeutic strategy for IVDD.

     

Identification of Ovarian Cancer Metastatic miRNAs

Serous epithelial ovarian cancer (EOC) patients often succumb to aggressive metastatic disease, yet little is known about the behavior and genetics of ovarian cancer metastasis. Here, we aim to understand how omental metastases differ from primary tumors and how these differences may influence chemotherapy. We analyzed the miRNA expression profiles of primary EOC tumors and their respective omental metastases from 9 patients using miRNA Taqman qPCR arrays. We find 17 miRNAs with differential expression in omental lesions compared to primary tumors. miR-21, miR-150, and miR-146a have low expression in most primary tumors with significantly increased expression in omental lesions, with concomitant decreased expression of predicted mRNA targets based on mRNA expression. We find that miR-150 and miR-146a mediate spheroid size. Both miR-146a and miR-150 increase the number of residual surviving cells by 2–4 fold when challenged with lethal cisplatin concentrations. These observations suggest that at least two of the miRNAs, miR-146a and miR-150, up-regulated in omental lesions, stimulate survival and increase drug tolerance. Our observations suggest that cancer cells in omental tumors express key miRNAs differently than primary tumors, and that at least some of these microRNAs may be critical regulators of the emergence of drug resistant disease.     

miR-34a Modulates Angiotensin II-Induced Myocardial Hypertrophy by Direct Inhibition of ATG9A Expression and Autophagic Activity

Cardiac hypertrophy is characterized by thickening myocardium and decreasing in heart chamber volume in response to mechanical or pathological stress, but the underlying molecular mechanisms remain to be defined. This study investigated altered miRNA expression and autophagic activity in pathogenesis of cardiac hypertrophy. A rat model of myocardial hypertrophy was used and confirmed by heart morphology, induction of cardiomyocyte autophagy, altered expression of autophagy-related ATG9A, LC3 II/I and p62 proteins, and decrease in miR-34a expression. The in vitro data showed that in hypertrophic cardiomyocytes induced by Ang II, miR-34a expression was downregulated, whereas ATG9A expression was up-regulated. Moreover, miR-34a was able to bind to ATG9A 3′-UTR, but not to the mutated 3′-UTR and inhibited ATG9A protein expression and autophagic activity. The latter was evaluated by autophagy-related LC3 II/I and p62 levels, TEM, and flow cytometry in rat cardiomyocytes. In addition, ATG9A expression induced either by treatment of rat cardiomyocytes with Ang II or ATG9A cDNA transfection upregulated autophagic activity and cardiomyocyte hypertrophy in both morphology and expression of hypertrophy-related genes (i.e., ANP and β-MHC), whereas knockdown of ATG9A expression downregulated autophagic activity and cardiomyocyte hypertrophy. However, miR-34a antagonized Ang II-stimulated myocardial hypertrophy, whereas inhibition of miR-34a expression aggravated Ang II-stimulated myocardial hypertrophy (such as cardiomyocyte hypertrophy-related ANP and β-MHC expression and cardiomyocyte morphology). This study indicates that miR-34a plays a role in regulation of Ang II-induced cardiomyocyte hypertrophy by inhibition of ATG9A expression and autophagic activity.     

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G₁/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G₁/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G₁/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.     

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G₁/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G₁/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G₁/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.     

MicroRNA-7 Inhibits Tumor Metastasis and Reverses Epithelial-Mesenchymal Transition through AKT/ERK1/2 Inactivation by Targeting EGFR in Epithelial Ovarian Cancer

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.     

AUTOPHAGY-RELATED11 Plays a Critical Role in General Autophagy- and Senescence-Induced Mitophagy in Arabidopsis

Autophagy-mediated turnover removes damaged organelles and unwanted cytoplasmic constituents and thus plays critical roles in cellular housekeeping and nutrient recycling. This “self eating” is tightly regulated by the AUTOPHAGY-RELATED1/13 (ATG1/13) kinase complex, which connects metabolic and environmental cues to the vacuolar delivery of autophagic vesicles. Here, we describe the Arabidopsis thaliana accessory proteins ATG11 and ATG101, which help link the ATG1/13 complex to autophagic membranes. ATG11 promotes vesicle delivery to the vacuole but is not essential for synthesizing the ATG12-ATG5 and ATG8-phosphatidylethanolamine adducts that are central to autophagic vesicle assembly. ATG11, ATG101, ATG1, and ATG13 colocalize with each other and with ATG8, with ATG1 tethered to ATG8 via a canonical ATG8-interacting motif. Also, the presence of ATG11 encourages starvation-induced phosphorylation of ATG1 and turnover of ATG1 and ATG13. Like other atg mutants, ATG11-deficient plants senesce prematurely and are hypersensitive to nitrogen and fixed-carbon limitations. Additionally, we discovered that the senescence-induced breakdown of mitochondria-resident proteins and mitochondrial vesicles occurs via an autophagic process requiring ATG11 and other ATG components. Together, our data indicate that ATG11 (and possibly ATG101) provides important scaffolds connecting the ATG1/13 complex to both general autophagy and selective mitophagy.     

Plasma miRNAs as Diagnostic and Prognostic Biomarkers for Ovarian Cancer

Background

Most (70%) epithelial ovarian cancers (EOCs) are diagnosed late. Non-invasive biomarkers that facilitate disease detection and predict outcome are needed. The microRNAs (miRNAs) represent a new class of biomarkers. This study was to identify and validate plasma miRNAs as biomarkers in EOC.

Methodology/Principal Findings

We evaluated plasma samples of 360 EOC patients and 200 healthy controls from two institutions. All samples were grouped into screening, training and validation sets. We scanned the circulating plasma miRNAs by TaqMan low-density array in the screening set and identified/validated miRNA markers by real-time polymerase chain reaction assay in the training set. Receiver operating characteristic and logistic regression analyses established the diagnostic miRNA panel, which were confirmed in the validation sets. We found higher plasma miR-205 and lower let-7f expression in cases than in controls. MiR-205 and let-7f together provided high diagnostic accuracy for EOC, especially in patients with stage I disease. The combination of these two miRNAs and carbohydrate antigen-125 (CA-125) further improved the accuracy of detection. MiR-483-5p expression was elevated in stages III and IV compared with in stages I and II, which was consistent with its expression pattern in tumor tissues. Furthermore, lower levels of let-7f were predictive of poor prognosis in EOC patients.

Conclusions/Significance

Our findings indicate that plasma miR-205 and let-7f are biomarkers for ovarian cancer detection that complement CA-125; let-7f may be predictive of ovarian cancer prognosis.     

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.     

MiR-216a: a link between endothelial dysfunction and autophagy

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.     

Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy

Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy.