Autophagic Levels Research Articles

Regulation of autophagy, glucose uptake, and glycolysis under dengue virus infection.

We previously reported that dengue virus (DENV)-induced autophagy plays a promoting role in viral replication and pathogenesis both in vitro and in vivo. Although it is known that DENV infection increases glycolysis, which promotes viral replication, the role of glucose metabolism together with autophagic activity in DENV replication remains unclear. In this study, we reveal that DENV2 infection increased autophagic activity, glucose uptake, protein levels of glucose transporter-1 (GLUT1), and glycolysis rate-limiting enzyme hexokinase-2 (HK2) in cells. Furthermore, the protein levels of LC3-II and HK2 were increased in the brain tissues of the DENV2-infected suckling mice. However, DENV2 infection decreased ATP level and showed no effect on mRNA expression of HK2 and phosphofructokinase, as well as lactate production, indicating that DENV2-regulated glycolytic flux occurs at the post-transcriptional level and is lactate pathway-independent. Moreover, amiodarone-induced autophagic activity, glucose uptake, HK2 level, and viral titer were reversed by the autophagy inhibitor spautin-1 or silencing of Atg5 gene expression. Intriguingly, blocking of glycolysis, HK2 protein level, and viral titer were accordingly decreased, but autophagic activity was increased, suggesting the existence of another regulation mechanism that influences the relationship between glycolysis and autophagy. This is the first report to reveal that DENV2-induced autophagy positively regulates glycolysis and viral replication in vitro and in vivo. Our findings open a new avenue wherein metabolic modulation could be used as a target for the treatment of DENV infection.

Identification of small molecule drugs and development of a novel autophagy-related prognostic signature for kidney renal clear cell carcinoma.

Abnormal autophagic levels have been implicated in the pathogenesis of multiple cancers, however, its role in tumors is complex and has not yet been explored clearly. Hence, we aimed to explore the prognostic values of autophagy‐related genes (ARGs) for kidney renal clear cell carcinoma (KIRC). Differentially expressed ARGs and transcription factors (TFs) were identified in KIRC patients obtaining from the The Cancer Genome Atlas (TCGA) database. Then, networks between TFs and ARGs, gene ontology functional annotations and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis were conducted. Next, we performed consensus clustering, COX regression analysis and Lasso regression analysis to identify the prognostic ARGs. Finally, an individual prognostic index (PI, riskScore) was established. Based on TCGA cohort and ArrayExpress cohort, Survival analysis, ROC curve, independent prognostic analysis, and clinical correlation analysis were also performed to evaluate this PI. Based on differentially expressed ARGs, KIRC patients were successfully divided into two clusters (P = 5.916e‐04). AS for PI, it was constructed based on 11 ARGs and significantly classified KIRC patients into high‐risk group and low‐risk group in terms of OS (P = 4.885e‐15 for TCGA cohort, P = 6.366e‐03 for ArrayExpress cohort). AUC of its ROC curve reached 0.747 for TCGA cohort and 0.779 for ArrayExpress cohort. What's more, this PI was proven to be a valuable independent prognostic factor in both univariate and multivariate COX regression analysis (P < .001). Prognostic nomograms were also performed to visualize the relationship between individual predictors and survival rates in patients with KIRC. By means of connectivity map database, emetine, cephaeline and co‐dergocrine mesilate related to ARGs were found to be negatively correlated with KIRC. This study provided an effective PI for KIRC and also displayed networks between TFs and ARGs. KIRC patients were successfully divided into two clusters based on differentially expressed ARGs. Besides, small molecule drugs related to ARGs were also identified for KIRC.

Development of a pH-responsive polymersome inducing endoplasmic reticulum stress and autophagy blockade.

Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.

The AMPK pathway triggers autophagy during CSF1-induced microglial activation and may be implicated in inducing neuropathic pain

The development and maintenance of neuropathic pain is now given far more attention in the clinic work. Increasing evidence has shown that colony-stimulating factor 1 (CSF1) is involved in microglial activation and may further induce pain. Here, we observed the signaling events that link the CSF1-induced microglial activated and consequences for pain processing. For the in vitro study, flow cytometry showed the microglial activity was markedly increased after CSF1 stimulation. Western blot showed the increased expression of p-PRKAA1/PRKAA1, p-AMPK/AMPK, p-ULK1/ULK1, p-S6k/S6k and LC3-II/LC3-I. QRT-PCR showed the IL-1, TNF-α and BDNF were simultaneously upregulated in the activated microglia cells, whereas the specific AMPK inhibitor compound C exhibited reverse effects in microglia. Using immunofluorescence staining and electron microscopy, we found CSF1 decreased microglial p62 expression and induced the number of autophagosomes, whereas compound C significantly exhibited the reverse effects. For the in vivo study, compared with the control and AMPK-siRNA transfection, the mice under CSF1 intrathecal injection increased CSF1 receptor and LC3 expressed in the activated spinal microglia. More importantly, qRT-PCR showed CSF1 intrathecal injection substantially upregulated BDNF and c-Fos mRNA expression as well as the ensuing neuropathic pain. Our findings demonstrated that CSF1 induced a significant upregulation of microglial activation via the AMPK signaling pathway and resulted in an increasing microglial autophagic level. An increasing CSF1 level in the central nervous system can mimic and cause pain syndromes by up-regulation of AMPK-depended autophagy, thus offering a new target for the therapy of neuropathic pain.

Autophagy Is a Potential Therapeutic Target Against Duck Tembusu Virus Infection in vivo.

Duck tembusu virus (DTMUV) is newly emerged in poultry and causes great losses to the breeding industry in China and neighboring countries. Effective antiviral strategies are still being studied. Autophagy is a cellular degradative pathway, and our lab's previous data show that autophagy promotes DTMUV replication in vitro. To study the role of autophagy further in vivo, we utilized ducks as the animal model to investigate the autophagy responses in DTMUV-targeted tissues. And also, we utilized autophagy regulators, including Rapamycin (Rapa) as the autophagy enhancer, 3-Methyladenine (3-MA) and Chloroquine (CQ) as the autophagy inhibitors, to adjust the host autophagic levels and then study the effects of autophagy on tissue damages and virus replication. As a result, we first found DTMUV infection trigged autophagy and autophagy regulator treatments regulated autophagy levels successfully in duck spleens and brains. Next, we found that autophagy inhibitors inhibited DTMUV replication and alleviated DTMUV-induced pathological symptoms, whereas the autophagy inducer treatment led to the opposite effects. And we also found that autophagic regulation was correlated with the expression of innate immune genes, including pattern recognition receptors, type I interferons, and cytokines, and caused different effects in different tissues. In summary, we demonstrated that autophagy facilitated DTMUV replication, aggravated the developments of pathological symptoms and possibly counteracts the host's innate immunity response in vivo.

Autophagy-associated circRNA circCDYL augments autophagy and promotes breast cancer progression

BackgroundAlthough both circular RNAs (circRNAs) and autophagy are associated with the function of breast cancer (BC), whether circRNAs regulate BC progression via autophagy remains unknown. In this study, we aim to explore the regulatory mechanisms and the clinical significance of autophagy-associated circRNAs in BC.MethodsAutophagy associated circRNAs were screened by circRNAs deep sequencing and validated by qRT-PCR in BC tissues with high- and low- autophagic level. The biological function of autophagy associated circRNAs were assessed by plate colony formation, cell viability, transwells, flow cytometry and orthotopic animal models. For mechanistic study, RNA immunoprecipitation, circRNAs pull-down, Dual luciferase report assay, Western Blot, Immunofluorescence and Immunohistochemical staining were performed.ResultsAn autophagy associated circRNA circCDYL was elevated by 3.2 folds in BC tissues as compared with the adjacent non-cancerous tissues, and circCDYL promoted autophagic level in BC cells via the miR-1275-ATG7/ULK1 axis; Moreover, circCDYL enhanced the malignant progression of BC cells in vitro and in vivo. Clinically, increased circCDYL in the tumor tissues and serum of BC patients was associated with higher tumor burden, shorter survival and poorer clinical response to therapy.ConclusionscircCDYL promotes BC progression via the miR-1275-ATG7/ULK1-autophagic axis and circCDYL could act as a potential prognostic and predictive molecule for breast cancer patients.

Role of melatonin in intestinal mucosal injury induced by restraint stress in mice

Context A growing body of evidence demonstrates that gastrointestinal motility disorder (GIMD) and gastric stress ulcers can be induced by restraint stress, while melatonin (MT) elicits anti-inflammation and antioxidant effects. Objective The present study investigated the mechanisms of MT-mediated protection effects on restraint stress-induced GIMD. Materials and methods 144 8-week-old male ICR mice were divided into four groups: control, restraint stress, restraint stress + MT and MT (positive control). 20 mg/kg MT or vehicle were intraperitoneally injected 60 min before restraint stress (10 h/day) once daily for 3 days. Biochemical parameters, intestinal mucosal integrity, tissues antioxidant ability and autophagic proteins levels were determined. Results Mice subjected to restraint stress elevated NE level by 141.41% and decreased MT content by 38.82% in plasma. Consistent with the decrease in MT level, we observed a reduction in the antioxidant ability and an increase in autophagic proteins by 14.29–46.74% in the gut, resulting in injury to intestinal mucosa which was manifested by reductions in villus height and villus height/crypt depth (V/C) ratio, number of goblet and PCAN-positive cells, and expression of tight junction protein (ZO-1, occludin and claudin-1). In contrast, MT reversed these changes caused by restraint stress and improved the intestinal mucosal injury. However, there was no significant difference between MT (positive control) and control group. Discussion and conclusion Our results suggest that MT effectively mitigates psychological stress-induced injury to intestinal mucosa, providing evidence demonstrating the potential for using MT as therapy against intestinal impairment associated with psychological stress.

MIR-190B Alleviates Cell Autophagy and Burn-Induced Skeletal Muscle Wasting via Modulating PHLPP1/Akt/FoxO3A Signaling Pathway.

Cell autophagy is an important material recycling process and is involved in regulating many vital activities under both physiological and pathological conditions. However, the mechanism of autophagy regulating burn-induced skeletal muscle wasting still needs to be elucidated. The rat burn model with 30% total body surface area and L6 cell line were used in this study. An immunofluorescence assay was used to detect autophagic levels. MicroRNA array and real-time PCR were employed to measure miR-190b levels, and its influence on PH domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) protein translation was estimated using luciferase reporter assay. The expression levels of autophagy-related proteins were analyzed by Western blot. Skeletal muscle wasting was evaluated by the ratio of tibias anterior muscle weight to body weight. Our study demonstrates that burn injury promotes expression of the autophagy-related proteins light chain 3 (LC3) and Beclin-1, suppresses expression of Akt and Forkhead box O (FoxO) 3a protein phosphorylation, and increases PHLPP1 protein level which is required for Akt dephosphorylation. miR-190b, the regulator of PHLPP1 protein translation, also significantly decreases after burn injury. Ectopic expression of miR-190b in L6 myoblast cell downregulates PHLPP1 protein expression, elevates Akt and FoxO3a phosphorylation, and subsequently reduces cell autophagy. Finally, suppressing autophagy with 3-methyladenine represses the protein expression of LC3 and Beclin-1 and mitigates burn-induced skeletal muscle wasting. Burn injury induced skeletal muscle cell autophagy and subsequently resulted in skeletal muscle wasting via regulating miR-190b/PHLPP1/Akt/FoxO3a signaling pathway.

Deficiency of apoptosis-stimulating protein 2 of p53 protects mice from acute hepatic injury induced by CCl4 via autophagy

BackgroundApoptosis-stimulating protein 2 of p53 (ASPP2) has a variety of biological functions, and is involved in cellular apoptosis, autophagy and inflammatory reaction. However, the role of ASPP2 in acute hepatic injury remains unclear. MethodsWe established an animal model of acute hepatic injury by intraperitoneal injection of CCl4. The expression profile of ASPP2 was measured in wild type (ASPP2+/+) mice with acute hepatic injury induced by CCl4. Hepatic pathological changes and liver function, apoptosis, inflammation and autophagic levels were measured in ASPP2+/+and ASPP2 haploid deletion (ASPP2+/−) mice with acute hepatic injury, respectively. After 3-methyladenine (3-MA) treatment, indicators of hepatic injury were observed in ASPP2+/+and ASPP2+/- mice with CCl4 injection. ResultsDuring the development of acute hepatic injury, ASPP2 expression significantly upregulated at 24 h and 48 h after CCl4 injection. ASPP2 haplotype deletion protected against acute hepatic injury, and this was mainly reflected in decreased ALT and AST levels, less hepatic tissue hemorrhage and necrosis, and reduced cellular inflammation and apoptosis in ASPP2+/− mice compared with ASPP2+/+ mice with acute hepatic injury. ASPP2 haploid deletion activates autophagy in mice with acute hepatic injury, and protects mice from acute hepatic injury via the autophagic signal pathway. ConclusionASPP2 haplotype deletion protected mice against acute hepatic injury through autophagy activation, which inhibited inflammation and apoptosis in acute hepatic injury.

Abstract 524: Activated Fms-like Tyrosine Kinase 3 Receptor Prevents Ventricular Remodeling Induced by Ang Ii Through the Attenuation of Autophagy

Aim: FMS-like tyrosine kinase 3 (Flt3) is a receptor tyrosine kinase. This study was to determine whether activation Flt3 could regulate autophagy ameliorate AngII-induced heart cardiac remodeling and to elucidate the mechanisms of action. Methods: In vivo cardiac remodeling was induced in male C57BL/6 mice by implanting subcutaneously osmotic minipumps releasing Angiotensin II (Ang II) for 28 days (1100ng/kg/min). Flt3 ligand (FL) was administered intraperitoneally (5μg/mouse/2days). Heart hypertrophy, fibrosis and function were evaluated based on echocardiography, histological and biochemical measurements. In vitro, hypertrophy was induced by Ang II on adenovirus vector-mediated overexpression Flt3 receptor in H9c2 cells, and NRCMs. Crystal violet staining, flow cytometry and TUNEL were used for in vitro experiments. The levels of signaling proteins were measured using western blotting, while the expression of the relevant genes was analyzed using real-time qRT-PCR. Results: Echocardiography demonstrated that Ang II induced marked damaged heart function, while cardiac Flt3 receptor activation significantly decrease left ventricular internal dimension at end-diastole (LVIDd), left ventricular posterior wall thickness at end-diastole (LVPWd), and increase ejection fraction (EF%) and fractional shortening (FS%). FL-treated mice showed a significant attenuation of cardiac hypertrophy, ratios of heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL), as well as cardiac fibrosis and apoptosis. Additionally, the autophagic levels in cardiomyocytes of FL-treated mice were distinctly decreased as evidenced by expression of LC3 in immunohistochemical staining and western blotting. In H9c2 cells with adenovirus vector-mediated overexpression of Flt3 and neonatal rat cardiomyocytes, FL treatment significantly decrease the cell size, apoptosis and autophagy induced by Ang II. Additionally, using rapamycin or metformin, respectively canceled FL protective effects. Conclusion: Flt3 activation suppresses Ang II-induced cardiac hypertrophy and apoptosis via AMPK /mTOR/ FOXO3a autophagy signaling pathway. These results provide evidence supporting Flt3 as a novel therapeutic target in cardiac remodeling.

Abstract LB-307: Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis

Abstract Introduction: Autophagy plays a critical role in cancer initiation and progression. CircRNAs were recently identified as new molecules in regulation of cancer cell biology. However, it remains unclear whether circRNAs are involved in the process of autophagy and regulate cancer biology via autophagy. Methods: We performed circRNAs microarray to screen autophagy-associated circRNAs in hypoxia-induced autophagy model and qRT-PCR was used to verify the expression level of candidate circRNAs after autophagy induction in both hypoxia and starvation induced autophagy models. RNase R digestion assay and Poly A detective experiment were used to verify the circular characteristic of circRNA candidates. The proliferative effects of knocking down and overexpressing circRNA in breast cancer cells were tested by colony formation and cell counting assays. Western Blot was used to test the protein level of LC3 and ATG7 in cancer cells. Ago2 RIP assay was used to test the miRNA sponge potential of circRNA candidates. CHiRP assays, miRNA pull down assays and dual-luciferase reporter system assays were used to verify the miRNA sponge of circRNA candidate. The location and expression levels of AUACA1 and ATG7 in human breast tissue was detected by ISH and IHC respectively. Survival rates and curves in a breast cancer cohort of 109 patients were determined by the Kaplan-Meier method and the comparison of survival differences was evaluated by using the log-rank test. Results: In our study, we found that breast cancer cells after autophagy induction displayed specific circRNA profiles. Among the altered circRNAs, upregulation of AUACA1 (named Autophagy Associated CircRNA 1) was most obvious in both hypoxia and starvation-induced autophagy. Moreover, AUACA1 upregulation was parallel to the degree of autophagy in breast cancer cells, while inhibition of autophagy did not affect the level of the AUACA1. Rnase R digestion and Poly A detective experiment showed that character of AUACA1 was circular rather than linear. Nuclear plasma separation assay and in-situ hybridization showed that AUACA1 mostly located in the cytoplasm. Knocking down of AUACA1 significantly inhibited the proliferation of breast cancer cells, but did not affect cell apoptosis and migration. Knocking down AUACA1 decreased autophagic level by detecting LC3-II. Proliferative assay demonstrated that AUACA1 promoted proliferation via autophagy in MDA-MB-231. Ago2 RIP assay significantly enriched AUACA1, indicating AUACA1 may worked as miRNA sponge. CHiRP and miRNAs microarray proved that AUACA1 worked as miR-1275 sponge. MiRNA pull down assay and dual-luciferase reporter system assays also verified that AUACA1 worked as miR-1275 sponge. MiR-1275 affected ATG7 expression at both RNA and protein level after transfecting miR-1275 mimics in MDA-MB-231. In addition, clinical data indicated that AUACA1 expression level was positively correlated with ATG7 and Ki-67 index. High expression of AUACA1 was correlated with high tumour burden and poor disease-free survival in 109 breast cancer patients. Conclusion: Our findings indicate that AUACA1 is an autophagy–associated circRNA and plays a critical role in promoting the proliferation of breast cancer cell via miR- 1275/ATG7/autophagic axis. AUACA1 may be a potential prognostic biomarker for breast cancer. Citation Format: Gehao Liang, Yun Ling, Zihao Liu, Maryam Mehrpour, Ahmed Hamai, Zhenluan Tian, Luyuan Tan, Wenjing Zhong, Wanyi Lin, Patrice Codogno, Erwei Song, Chang Gong. Autophagy-associated circRNA AUACA1 mediates the proliferation of breast cancer cells via a miR-1275/ATG7/autophagic flux axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-307.

&lt;p&gt;Identification and validation of an individualized autophagy-clinical prognostic index in bladder cancer patients&lt;/p&gt;

Purpose: Autophagy is a major catabolic system by which eukaryotic cells undergo self-degradation of damaged, defective, or unwanted intracellular components. An abnormal autophagic level is implicated in the pathogenesis of multiple diseases, including cancers. The aim of this study is to explore the prognostic value of autophagy in bladder cancer (BC), which is a major cause of cancer-related death globally.Patients and methods: First, 27 differentially expressed autophagy-related genes (ARGs) were identified in BC patients based on The Cancer Genome Atlas (TCGA) database. Functional enrichment analyses hinted that autophagy may act in a tumor-suppressive role in the initiation of BC. Then, the Cox proportional hazard regression model were employed to identify three key prognostic ARGs (JUN, MYC, and ITGA3), which were related with overall survival (OS) significantly in BC. The three genes represented important clinical significance and prognostic value in BC. Then a prognostic index (PI) was constructed.Results: The PI was constructed based on the three genes, and significantly stratified BC patients into high- and low-risk groups in terms of OS (HR=1.610, 95% CI=1.200–2.160, P=0.002). PI remained as an independent prognostic factor in multivariate analyses (HR=2.355, 95% CI=1.483–3.739, P<0.001). When integrated with clinical characteristics of age and stage, an autophagy-clinical prognostic index (ACPI) was finally validated, which had improved performance in predicting OS of BC patients (HR=2.669, 95% CI=1.986–3.587, P<0.001). The ACPI was confirmed in datasets of GSE13507 (HR=7.389, 95% CI=3.645–14.980, P<0.001) and GSE31684 (HR=1.665, 95% CI=0.872–3.179, P=0.122).Conclusion: This study provides a potential prognostic signature for predicting prognosis of BC patients and molecular insights of autophagy in BC.

Simvastatin aggravates impaired autophagic flux in NSC34-hSOD1G93A cells through inhibition of geranylgeranyl pyrophosphate synthesis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by selective loss of motor neurons. Statins are widely used as cholesterol-lowering drugs and significantly reduce the risk of cardiovascular and cerebrovascular diseases. Increasing evidence indicates the protective effects of statins against certain neurodegenerative diseases. However, in ALS, many studies have found that statins might accelerate disease progression and shorten survival, although the exact mechanism is unclear. In the present study, we investigated the effect of simvastatin on NSC34cells stably transfected with the G93A mutation in human SOD1 (NSC34-hSOD1G93A cells), a recognized in vitro model of ALS. Our results showed that simvastatin caused a decrease in cell viability and the accumulation of autophagic vacuoles with elevated levels of LC3 II/I and P62 in NSC34-hSOD1G93A cells. Conversely, these outcomes were completely reversed by co-incubation with mevalonate, farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) but not cholesterol. In addition, inhibition of geranylgeranyl transferase I by GGTI-286 led to similar alterations in cell viability and autophagic marker levels. These results indicated that the cytotoxic effect of simvastatin on NSC34-hSOD1G93A cells might be due to the aggravation of autophagic flux impairment through the inhibition of GGPP synthesis.

Mitofusin 2 Participates in Mitophagy and Mitochondrial Fusion Against Angiotensin II-Induced Cardiomyocyte Injury.

BackgroundMitochondrial dynamics play a critical role in mitochondrial function. The mitofusin 2 (MFN2) gene encodes a mitochondrial membrane protein that participates in mitochondrial fusion to maintain and operate the mitochondrial network. Moreover, MFN2 is essential for mitophagy. In Ang II-induced cardiac remodeling, the combined effects of MFN2-mediated mitochondrial fusion and mitophagy are unclear. This study was designed to explore a novel strategy for preventing cardiomyocyte injury via modulation of mitochondrial dynamics.MethodsWe studied the function of MFN2 in mitochondrial fusion and mitophagy in Ang II-stimulated cardiomyocyte injury. Cardiomyocyte injury experiments, including reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and apoptosis rate of cardiomyocytes were performed. The mitochondrial morphology in cardiomyocytes was examined via transmission electron microscopy (TEM) and confocal microscopy. Autophagic levels in response to Ang II were examined by immunoblotting of autophagy-related proteins. Moreover, PINK1/MFN2/Parkin pathway-related proteins were examined.ResultsWith stimulation by Ang II, MFN2 expression was progressively reduced. MFN2 deficiency impaired mitochondrial quality, resulting in exacerbated mitochondrial damage induced by Ang II. The Ang II-induced increases in ROS production and apoptosis rate were alleviated by MFN2 overexpression. Moreover, MFN2 alleviated the Ang II-induced reduction in MMP. MFN2 promoted mitochondrial fusion, and MFN2 promoted Parkin translocation and phosphorylation, leading to mitochondrial autophagy. The effects of MFN2 overexpression were reversed by autophagy inhibitors.ConclusionMitofusin 2 promotes Parkin translocation and phosphorylation, leading to mitophagy to clear damaged mitochondria. However, the beneficial effects of MFN2 were reversed by autophagy inhibitors. Additionally, MFN2 participates in mitochondrial fusion to maintain mitochondrial quality. Thus, MFN2 participated in mitophagy and mitochondrial fusion against Ang II-induced cardiomyocyte injury.

HMGB1 mediated autophagy protects glioblastoma cells from carbon-ion beam irradiation injury

The present study investigated autophagy changes and the expression of HMGB1 in human glioblastoma cells, responding to carbon-ion beam irradiation (35 keV/μm, 80.55 MeV/u). U251 cells were irradiated with carbon-ion beams and cell proliferation was measured by counting the number of living cells. The expression of Light Chain 3 beta (LC3B), Beclin 1, high-mobility-group box 1 (HMGB1), pro-form caspase-3 and Cellular FLICE-like inhibitory protein (c-FLIP) was analyzed by western blotting. Caspase enzyme activity was determined via a caspase cleavage based florescent substrate commercial Kit. Living cell counting demonstrated a time- and dose-dependent cell death in U251 cells. The expression of LC3B and Beclin 1 revealed that, a high level of autophagy was induced 24 h after irradiation with 1 Gy carbon ions and then decreased in a time- and dose-dependent manner. The expression of the whole HMGB1 showed a well correlation with the dynamic autophagic level. Cytoplasmic HMGB1 maintained autophagy was concluded. Enzyme-Linked Immuno Sorbent Assay (ELISA) measurement found that, HMGB1 was released into the extracellular space in a time- and dose-dependent manner. Lower intracellular HMGB1 levels correlated with decreased autophagy as measured by the expression of LC3B. Decreased expression of pro-form caspase-3 and c-FLIP as well as the increased caspase enzyme activity indicated that apoptosis was induced by carbon-ion beam irradiation. Inhibition of HMGB1 release from the area of intracellular to that of extracellular significantly increased cell survival. In summary, carbon-ion beam irradiation could elevate autophagy and HMGB1 expression efficiently, which would protect the cells from programmed cell death via inducing autophagy. Apoptosis as measured by expression of caspase activities increased as the dose increased, which was accompanied with decreased levels of LC3B and HMGB1.

Investigation of the effects of zoledronic acid in breast cancer stem cell line (mcf7) for autophagic gene level

Amaç: Zoledronik asit (ZA) uygulanmış meme kanser kök hücrelerinde (KKH), kanser kök hücresi ve otofaji ile ilişkili gen ekspresyonlarındaki farklılıkları incelemek. Gereç ve Yöntem: CD44yüksek/CD24yüksek yüzey belirteçleri pozitif olan MCF-7 meme kanseri hücre hattından KKH’ler akış sitometri metodu ile izole edilmiştir. ZA tedavisi uygulanmış ve uygulanmamış olan KKH gruplarının toplam RNA izolasyonu için cDNA sentezlenmiştir. Son olarak 41 adet otofaji için başlıca ilişkili olabilecek genlerin ekspresyon seviyeleri qRT-PCR analizi ile incelenmiştir. Bulgular: ZA tedavisi uygulanan grup tedavi uygulanmayan grup ile karşılaştırıldığında TGM2, TNF, HSPA8 genlerinin azalmış regüle olduğu ve HSP90AA1 geninin artmış regüle olduğu görülmüştür. Sonuç: Otofaji ile ilişkili genlerin ekspresyon profillerinde ZA tedavisi uygulanan ve uygulanmayan gruplar arasında anlamlı farklar bulunmuştur. Bu bulgular, KKH’lerine karşı hedef olabilecek daha etkili tedavi ajanları geliştirilebilmesi için önemli katkılar sağlayabilir.

Enhanced Autophagy Contributes to Reduced Viral Infection in Black Flying Fox Cells.

Bats are increasingly implicated as hosts of highly pathogenic viruses. The underlying virus–host interactions and cellular mechanisms that promote co-existence remain ill-defined, but physiological traits such as flight and longevity are proposed to drive these adaptations. Autophagy is a cellular homeostatic process that regulates ageing, metabolism, and intrinsic immune defense. We quantified basal and stimulated autophagic responses in black flying fox cells, and demonstrated that although black flying fox cells are susceptible to Australian bat lyssavirus (ABLV) infection, viral replication is dampened in these bat cells. Black flying fox cells tolerated prolonged ABLV infection with less cell death relative to comparable human cells, suggesting post-entry mechanisms interference with virus replication. An elevated basal autophagic level was observed and autophagy was induced in response to high virus doses. Pharmacological stimulation of the autophagy pathway reduced virus replication, indicating autophagy acts as an anti-viral mechanism. Enhancement of basal and virus-induced autophagy in bat cells connects related reports that long-lived species possess homeostatic processes that dampen oxidative stress and macromolecule damage. Exemplifying the potential that evolved cellular homeostatic adaptations like autophagy may secondarily act as anti-viral mechanisms, enabling bats to serve as natural hosts to an assortment of pathogenic viruses. Furthermore, our data suggest autophagy-inducing drugs may provide a novel therapeutic strategy for combating lyssavirus infection.

MicroRNA-137 inhibits autophagy and chemosensitizes pancreatic cancer cells by targeting ATG5.

Autophagy play an important role in tumor chemotherapy resistance. It has been reported that miR-137 expression was reducedand involved in the regulation of sensitivity of PC cells to chemotherapy. However, little is known about the underlying molecular mechanisms. In this study, we hypothesized that miR-137 might sensitize PC cells to chemotherapy thought regulating cell autophagy. Cell survival was determined with MTT assay. Apoptotic cells were assessed with flow cytometric analysis. Fluorescence intensity of GFP-LC3 and RFP-GFP-LC3 were examined with immunofluorescence analysis to determine the autophagy and autophagic flux level. Western blotting assay was used to determine protein expression levels of LC3II/LC3I, P62, FUNDC1 and ATG5. mRNA expression level of miR-137 was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Dual-luciferase reporter assay was used to evaluate the directly binding of miR-137 with its targets. Xenograft model was setup to evaluate tumor growth. The results showed that doxorubicin (Dox) induced autophagy but downregulated the expression level of miR-137 in pancreatic cancer (PC) cells. In turn, overexpression of miR-137 enhanced the effect of Dox on decreasing cell survival, inducing cell apoptosis and inhibiting autophagy rather than influencing autophagic flux in PC cells. Further mechanistic study identified that ATG5 was a direct target of miR-137. Moreover, overexpression of ATG5 dramatically reversed the promotion of apoptosis and inhibition of autophagy mediated by higher expression level of miR-137. We also demonstrated that miR-137 sensitized PANC-1 cells to Dox through inhibiting ATG5 and autophagy in vivo. Our findings demonstrated for the first time that miR-137 was able to promote sensitivity of PC cells to chemotherapy via inhibition of autophagy mediated by ATG5. Therefore, miR-137 may act as a potential therapeutic target for pancreatic cancer.

Vitamin D protects podocytes from autoantibodies induced injury in lupus nephritis by reducing aberrant autophagy

SubjectThe aim of this study was to investigate whether vitamin D plays a protective role in podocyte injury induced by autoantibodies purified from the serum of patients with lupus nephritis (LN) via reducing aberrant autophagy.MethodsAutophagic activities of renal tissues of patients with LN were evaluated under transmission electronic microscope (TEM). Immunoglobulin G (IgG) from patients with LN was purified to induce human podocyte injury, and the role of vitamin D in injury was observed. Podocytes were observed under TEM, autophagic activity was evaluated by western blot analysis and quantitative real-time polymerase chain reaction, and mRFP-GFP-LC3B adenovirus was infected into human podocytes in vitro.ResultsSignificantly higher autophagic levels were observed in patients with LN (P <0.05), and apparently greater autophagic levels in podocytes were shown (P <0.05). Among different classifications of LN, class V (n = 5), III + V (n = 5), and IV + V (n = 5) gained higher autophagic levels than class III (n = 5) and IV (n = 5). Induced autophagy, which was evident by increased LC3B-II and Beclin 1 level, caused consumption of p62, more autophagosomes observed under TEM, and more LC3B dots observed under confocal microscope in the IgG group, along with decreased nephrin expression, which suggests podocyte injury. Reduction of autophagy as well as alleviated podocyte injury was observed in the IgG+ vitamin D group.ConclusionThis study demonstrates that vitamin D plays a protective role in podocyte injury induced by autoantibodies from patients with LN and appears to be a novel therapy target in LN.

A dual-mode nanoprobe for evaluation of the autophagy level affected by photothermal therapy.

A novel dual-mode nanoprobe (Apt@MNPS) was created for the detection of autophagy-related miRNAs to monitor the autophagic level and study the effect of PTT on autophagy. Interestingly, using our developed probe, PTT was found to be able to activate the autophagy by down regulation of miR-18a* and miR-4802, which in turn restricted the PTT efficiency for cancer.