Rejuvenating the Aging Heart by Enhancing the Expression of the Cisd2 Prolongevity Gene.

Aging is the major risk factor for cardiovascular disease, which is the leading cause of mortality worldwide among aging populations. Cisd2 is a prolongevity gene that mediates lifespan in mammals. Previously, our investigations revealed that a persistently high level of Cisd2 expression in mice is able to prevent age-associated cardiac dysfunction. This study was designed to apply a genetic approach that induces cardiac-specific Cisd2 overexpression (Cisd2 icOE) at a late-life stage, namely a time point immediately preceding the onset of old age, and evaluate the translational potential of this approach. Several discoveries are pinpointed. Firstly, Cisd2 is downregulated in the aging heart. This decrease in Cisd2 leads to cardiac dysfunction and impairs electromechanical performance. Intriguingly, Cisd2 icOE prevents an exacerbation of age-associated electromechanical dysfunction. Secondly, Cisd2 icOE ameliorates cardiac fibrosis and improves the integrity of the intercalated discs, thereby reversing various structural abnormalities. Finally, Cisd2 icOE reverses the transcriptomic profile of the aging heart, changing it from an older-age pattern to a younger pattern. Intriguingly, Cisd2 icOE modulates a number of aging-related pathways, namely the sirtuin signaling, autophagy, and senescence pathways, to bring about rejuvenation of the heart as it enters old age. Our findings highlight Cisd2 as a novel molecular target for developing therapies targeting cardiac aging.

Autophagy-Related Proteins Are Differentially Expressed in Adrenal Cortical Tumor/Pheochromocytoma and Associated with Patient Prognosis.

The aim of this research was to evaluate the expression and concomitant implications of LC3A, LC3B, beclin-1, and p62, which are key components of autophagy in human adrenal gland tumors. Tissue microarray was made for 321 cases of adrenal gland tumor (adrenal cortical adenoma (ACA): 115, adrenal cortical carcinoma (ACC): 17, and pheochromocytoma (PCC): 189). Immunohistochemical staining was performed for beclin-1, p62, LC3A, and LC3B, and the results were compared with the patients' clinicopathologic parameters. LC3A, LC3B, beclin-1, and LC3B isolated single positive cells (ISPC) positivity rates were higher in PCC than in adrenal cortical tumor (ACT), whereas p62 positivity was lower in PCC than in ACT. The proportion of positive LC3B (ISPC) was higher in ACC than in ACA. In addition, the proportion of cells positive for p62 and LC3B (ISPC) was significantly higher in PCCs with a GAPP score of ≥3. In univariate Cox analysis, p62 positivity (p = 0.014) and the presence of p62 (ISPC) (p = 0.001) were associated with shorter disease-free survival in PCC. Moreover, p62 positivity was predictive of shorter overall survival (OS) in patients with PCC by multivariate analysis (relative risk, 6.240; 95% CI, 1.434-27.15; p = 0.015). Differences were found in the expression of autophagy-related proteins according to adrenal gland tumor types. Compared to ACT, the proportion of LC3A, LC3B, beclin-1, and LC3B (ISPC) positivity was higher in PCC, whereas p62 positivity was lower. Similarly, p62 positivity in PCC was associated with patient prognosis of OS.

Epigallocatechin-3-gallate protects cardiomyocytes from hypoxia-reoxygenation damage via raising autophagy related 4C expression.

Myocardial ischemia/reperfusion (I/R) injury is a serious issue during the therapy of myocardial infarction. Herein, we explored the beneficial influence of Epigallocatechin-3-gallate (EGCG) on hypoxia/reoxygenation (H/R)-stimulated cardiomyocyte H9c2 cells damage, along with possible internal molecular mechanism related autophagy related 4C (ATG4C). H9c2 cells were subjected to H/R stimulation and/or EGCG treatment. ATG4C mRNA expression was measured via q-PCR assay. ATG4C overexpression plasmid (OE-ATG4C) was transfected to arise ATG4C level. Cell viability, apoptosis, reactive oxygen species (ROS) production, ATP level were tested via CCK-8 assay, Annexin V-FITC/PI staining, DCFH-DA staining and ATP Assay Kit, respectively. Western blotting was performed to test Cleaved-caspase 3, Cleaved-caspase 9, cytochrome C, and LC3B protein levels. H/R stimulation resulted in H9c2 cell viability loss, promoted cell apoptosis, and ROS overproduction, as well as lowered ATP level in cells. EGCG treatment alleviated H/R-resulted H9c2 cell viability loss, cell apoptosis, ROS overproduction, and reduction of ATP level. Moreover, H/R stimulation reduced the ATG4C expression in H9c2 cells, while EGCG raised the ATG4C expression. Overexpression of ATG4C strengthened the beneficial influence of EGCG on H/R-stimulated H9c2 cell viability, apoptosis and ROS production. Besides, ATG4C overexpression weakened the H/R-stimulated H9c2 cell autophagy via reducing LC3B II/I expression. EGCG exerted beneficial influence on H/R-stimulated cardiomyocytes, which protected cardiomyocytes from H/R-stimulated viability loss, apoptosis, and ROS overproduction via enhancing ATG4C expression.

Reduced Expression of Autophagy Markers and Expansion of Myeloid-Derived Suppressor Cells Correlate With Poor T Cell Response in Severe COVID-19 Patients.

Widespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed ~140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with ~30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-γ production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease.

Knockdown of long non-coding RNA RMRP protects cerebral ischemia-reperfusion injury via the microRNA-613/ATG3 axis and the JAK2/STAT3 pathway.

Cerebral ischemia-reperfusion (I/R) injury can induce the mitophagy of neurons in the ischemic brain. Long non-coding RNAs (lncRNAs) play an important role in the pathogenesis of various injuries, especially in cerebral I/R injury. The purpose of this study is to investigate the molecular mechanism of lncRNA RNA component of mitochondrial RNA processing endoribonuclease (RMRP) in cerebral I/R injury. The middle cerebral artery occlusion (MCAO) mouse model was established. Neurological deficit score, pathological structure, infarcted area, neuron number, cell apoptosis, and coagulation ability of MCAO mice were evaluated. The expressions of RMRP, microRNA (miR)-613, and ATG3 in MCAO mice were detected. The binding relationships among miR-613, RMRP, and ATG3 were predicted and verified. Neuro 2A (N2a) cells were treated with oxygen-glucose deprivation/reperfusion (OGD/R) to simulate I/R injury. Cell viability and apoptosis assays were performed. The effects of miR-613, ATG3, and RMRP on I/R injury were verified by functional rescue experiments. JAK2/STAT3 phosphorylation level was detected. We found significantly upregulated RMRP and ATG3, and downregulated miR-613 expressions in MCAO mice. RMRP could escalate ATG3 mRNA expression through miR-613. RMRP knockdown promoted viability and inhibited apoptosis of OGD/R-treated N2a cells, which could be reversed by miR-613 inhibition or ATG3 overexpression. RMRP overexpression inhibited the activation of JAK2/STAT3 signaling pathway. We demonstrated that lncRNA RMRP competitively bound to miR-613, leading to the increase of ATG3 expression and the inhibition the JAK2/STAT3 pathway, thus promoting cerebral I/R injury in mice.

Targeting the MicroRNA-490-3p-ATG4B-Autophagy Axis Relieves Myocardial Injury in Ischemia Reperfusion.

We investigated the potential role of miR-490-3p in ischemia reperfusion (IR) injury. We first determined the expression of miR-490-3p and autophagy-related 4B cysteine (ATG4B) in IR. Then, to explore whether miR-490-3p would affect autophagy, apoptosis, and IR injury, we evaluated apoptosis, autophagy, and infarct size via gain- and loss-of-function experiments. Furthermore, we used adenovirus to enhance or inhibit the expression of ATG4B, and then measured autophagy, apoptosis, and IR injury. miR-490-3p was downregulated in the hearts during the process of IR, while ATG4B was upregulated. The inhibition of miR-490-3p or overexpression of ATG4B could promote the expression of LC3II, increase the autolysosomes, inhibit the expression of p62, and reduce infarct size. On all accounts, the inhibition of miR-490-3p could promote autophagy to reduce myocardial IR injury by upregulating ATG4B, a finding that provides new insights for the protective mechanism of autophagy in IR. Graphical Abstract.

Autophagy is responsible for the accumulation of proteogenic dipeptides in response to heat stress in Arabidopsis thaliana.

Proteogenic dipeptides are intermediates of proteolysis as well as an emerging class of small-molecule regulators with diverse and often dipeptide-specific functions. Herein, prompted by differential accumulation of dipeptides in a high-density Arabidopsis thaliana time-course stress experiment, we decided to pursue an identity of the proteolytic pathway responsible for the buildup of dipeptides under heat conditions. By querying dipeptide accumulation versus available transcript data, autophagy emerged as a top hit. To examine whether autophagy indeed contributes to the accumulation of dipeptides measured in response to heat stress, we characterized the loss-of-function mutants of crucial autophagy proteins to test whether interfering with autophagy would affect dipeptide accumulation in response to the heat treatment. This was indeed the case. This work implicates the involvement of autophagy in the accumulation of proteogenic dipeptides in response to heat stress in Arabidopsis.

miR-708 and miR-335-3p Inhibit the Apoptosis of Retinal Ganglion Cells Through Suppressing Autophagy.

This study aimed to clarify the regulation role of miR-708 and miR-335-3p in retinal ganglion cell (RGC) autophagy and apoptosis in glaucoma. Chronic glaucoma mice were established by laser photocoagulation. RGCs were isolated and transfected with a series of plasmids and the cultured in 60 mmHg pressure. miR-335-3p, miR-708, and ATG3 mRNA expressions were detected by qRT-PCR. Protein levels of ATG3, autophagy-related protein LC3, and p62 were detected by Western blot. The apoptosis of RGCs was detected by flow cytometry. The regulation role of miR-335-3p/miR-708 in ATG3 was confirmed by the dual-luciferase reporter gene. The expressions of several miRNAs were measured in retinal tissues from chronic glaucoma mice and RGCs under pressure conditions, and results showed that both miR-335-3p and miR-708 were down-regulated. Besides, the inhibition of miR-708 and miR-335-3p induced the apoptosis of RGCs through promoting autophagy. Also, miR-708 and miR-335-3p could bind to ATG3 and targeted regulated ATG3. Furthermore, the interference with miR-708/miR-335-3p induced RGC apoptosis by up-regulating ATG3 to promote autophagy. In general, the down-regulation of miR-708 and miR-335-3p contributed to the apoptosis of RGCs through promoting autophagy in glaucoma.

Autophagy related 4B, upregulated by HIF-1α, attenuates the sensitivity to cisplatin in nasopharyngeal carcinoma cells.

OBJECTIVE: Increasing evidence has shown that autophagy related proteins and hypoxia-inducible factor-1α (HIF-1α) are both involved in the malignant progress of nasopharyngeal carcinoma (NPC), and HIF-1α plays an emerging role in the chemosensitivity of NPC cells. However, it is still unknown whether autophagy related proteins are associated with HIF-1α in regulating the chemosensitivity of NPC cells. MATERIALS AND METHODS: Quantitative Real-time PCR (qPCR) was applied to determine mRNA levels of HIF-1α and the autophagy related proteins, such as ATG3, ATG4B, ATG5, Beclin1, ATG7, ATG10, ATG12 and ATG16L1. Western blot was applied to determine protein levels of HIF-1α, ATG4B and cleaved Caspase-3. Cell viability and death were investigated by cell counting kit-8 and trypan blue exclusion assay. In addition, Caspase-3 activity was detected to reflect apoptosis. Furthermore, Luciferase reporter assay was applied to explore the mechanism by which HIF-1α transcriptionally upregulated ATG4B expression. RESULTS: Our study reveals that HIF-1α increased ATG4B expression in NPC cells, and in turn upregulated the cisplatin (DDP)-induced protective autophagy, resulting in enhanced killing effect of DDP to NPC cells. In mechanism, reporter assay showed that HIF-1α upregulated ATG4B expression by activating its gene promoter region. The binding site (-225 to -216) was required for HIF-1α-induced increase of ATG4B gene promoter activity. CONCLUSIONS: These results indicate that HIF-1α elevates ATG4B via promoting its transcription, which alleviates the sensitivity of DDP in NPC cells through enhancing protective autophagy, suggesting that ATG4B, upregulated by HIF-1α, may be a novel target for DDP sensitization in the treatment of NPC in clinic.

MicroRNA-375 exacerbates knee osteoarthritis through repressing chondrocyte autophagy by targeting ATG2B.

OBJECTIVE: This study aimed to explore the underlying mechanism of miR-375 in exacerbating osteoarthritis (OA). RESULTS: MiR-375 expression were upregulated in OA cartilage tissues, whereas ATG2B expression was decreased. MiR-375 targeted ATG2B 3' UTR and inhibited its expression in the chondrocytes, and then suppressed autophagy and promoted endoplasmic reticulum stress (ERs). The apoptosis rate of chondrocytes was increased after being transfected with miR-375 mimics. In vivo results further verified that inhibition of miR-375 could relieve OA-related symptoms. CONCLUSION: miR-375 can inhibit the expression of ATG2B in chondrocytes, suppress autophagy and promote the ERs. It suggests that miR-375 could be considered to be a key therapy target for OA. METHODS: Differential expression analyses for mRNA and miRNA microarray datasets from ArrayExpress were performed. MiR-375 and ATG2B expressions in cartilage tissues were detected by qRT-PCR. Dual luciferase assay was applied to verify the targeting relationship between ATG2B and miR-375. In vitro, the role of miR-375 on chondrocyte autophagy and ERs was investigated by western blot and immunofluorescence. The apoptotic rate was quantified by flow cytometry. In vivo, OA mice model was established, HE and Safranin O and Fast Green staining, as well as the OARSI and modified Mankin scores, were applied to measure the OA cartilage damage severity.

Expression of WIPI2B counteracts age-related decline in autophagosome biogenesis in neurons.

Autophagy defects are implicated in multiple late-onset neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Alzheimer's, Huntington's, and Parkinson's diseases. Since aging is the most common shared risk factor in neurodegeneration, we assessed rates of autophagy in mammalian neurons during aging. We identified a significant decrease in the rate of constitutive autophagosome biogenesis during aging and observed pronounced morphological defects in autophagosomes in neurons from aged mice. While early stages of autophagosome formation were unaffected, we detected the frequent production of stalled LC3B-negative isolation membranes in neurons from aged mice. These stalled structures recruited the majority of the autophagy machinery, but failed to develop into LC3B-positive autophagosomes. Importantly, ectopically expressing WIPI2B effectively restored autophagosome biogenesis in aged neurons. This rescue is dependent on the phosphorylation state of WIPI2B at the isolation membrane, suggesting a novel therapeutic target in age-associated neurodegeneration.

Down-regulation of exosomal microRNA-224-3p derived from bone marrow-derived mesenchymal stem cells potentiates angiogenesis in traumatic osteonecrosis of the femoral head.

Traumatic osteonecrosis of the femoral head (ONFH) is a condition leading to the collapse of the femoral head, and the primary treatment is a total hip replacement, which has a poor prognosis. The current study was conducted with the aim of investigating the role of exosomes from bone marrow-derived mesenchymal stem cells (BM-MSCs) carrying microRNA-224-3p (miR-224-3p) in traumatic ONFH. Initially, a microarray analysis was performed to screen the differentially expressed genes and miRs associated with traumatic ONFH. Patients with traumatic and nontraumatic ONFH were enrolled, and HUVECs were obtained. The BM-MSCs-derived exosomes were purified and characterized, after which HUVECs were cocultured with exosomes. The functional role of miR-224-3p in traumatic ONFH was determined using ectopic expression, depletion, and reporter assay experiments. Endothelial cell proliferation, migration, invasion abilities, and angiogenesis were evaluated. Based on microarray analysis, miR-224-3p was found to be down-regulated, whereas focal adhesion kinase family interacting protein of 200 kDa (FIP200) was up-regulated in ONFH. Traumatic ONFH exosomes resulted in the up-regulation of FIP200 and down-regulation of miR-224-3p. FIP200 was confirmed to be a target gene of miR-224-3p. Exosomes were internalized by vascular endothelial cells. The down-regulation of exosomal miR-224-3p was observed to promote endothelial cell proliferation, migration, invasion abilities, angiogenesis, and FIP200 expression. In addition, FIP200 overexpression promoted angiogenesis. In summary, the results highly indicated that lower miR-224-3p levels in exosomes derived from BM-MSCs promote angiogenesis of traumatic ONFH by up-regulating FIP200. The present study provides a potential strategy for the treatment of traumatic ONFH.-Xu, H.-J., Liao, W., Liu, X.-Z., Hu, J., Zou, W.-Z., Ning, Y., Yang, Y., Li, Z.-H. Down-regulation of exosomal microRNA-224-3p derived from bone marrow-derived mesenchymal stem cells potentiates angiogenesis in traumatic osteonecrosis of the femoral head.

STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

RNA Binding Protein HuR Promotes Autophagosome Formation by Regulating Expression of Autophagy-Related Proteins 5, 12, and 16 in Human Hepatocellular Carcinoma Cells.

Autophagy is a process of lysosomal self-degradation of cellular components by forming autophagosomes. Autophagosome formation is an essential process in autophagy and is fine-tuned by various autophagy-related gene (ATG) products, including ATG5, ATG12, and ATG16. Although several reports have shown that numerous factors affect multiple levels of gene regulation to orchestrate cellular autophagy, the detailed mechanism of autophagosome formation still needs further investigation. In this study, we demonstrate that the RNA binding protein HuR (human antigen R) performs an essential function in autophagosome formation. We observe that HuR silencing leads to inhibition of autophagosome formation and autophagic flux in liver cells. Ribonucleoprotein immunoprecipitation (RIP) assay allows the identification of ATG5, ATG12, and ATG16 mRNAs as the direct targets of HuR. We further show that HuR mediates the translation of ATG5, ATG12, and ATG16 mRNAs by binding to their 3' untranslated regions (UTRs). In addition, we show that HuR expression positively correlates with the levels of ATG5 and ATG12 in hepatocellular carcinoma (HCC) cells. Collectively, our results suggest that HuR functions as a pivotal regulator of autophagosome formation by enhancing the translation of ATG5, ATG12, and ATG16 mRNAs and that augmented expression of HuR and ATGs may participate in the malfunction of autophagy in HCC cells.

Autophagy related markers (Beclin-1 and ATG4B) are strongly expressed in Wilms' tumor and correlate with favorable histology.

BACKGROUND: Wilms' tumor treatment has achieved great success in the last decade. Nevertheless, some cases still fail to respond to the current multimodality therapy. These cases fall mainly in the unfavorable histology group with very few belonging to the favorable histology group. In recent years, autophagy manipulation whether inhibition or stimulation has been shown to affect cancer cell behavior and has emerged as a novel mechanism to improve cancer cell response to currently used therapeutic regimens. OBJECTIVE: The current study aimed to investigate the expression of autophagy related markers (ATG4B and Beclin1) in WT, its association with the different clinic-pathological parameters and its impact on patient survival. METHODS: Twenty-one formalin fixed paraffin embedded (FFPE) WT specimens were immunohistochemically stained using autophagy related markers; Beclin-1 and ATG4B. All clinical, radiological and follow up data were retrieved from the patient records. RESULTS: All specimens showed positive expression of both Beclin-1 and ATG4B. The staining score for Beclin1 varied between 50 and 300, and its expression was significantly associated with favorable histology (p=0.007). Similarly, ATG4B expression was significantly higher in favorable histology tumors compared to unfavorable histology (p=0.046). A statistically significant positive correlation between Beclin-1 and ATG4B expression was observed. The cumulative disease-free survival in patients with favorable histology was significantly higher compared to patients with unfavorable histology (p=0.0027). CONCLUSIONS: Beclin-1 and ATG4B expression were both found to be statistically significant discriminators of survival. Collectively these findings suggest that the expression of autophagy-related markers is associated with a favorable histology and could predict better survival in these patients.

RETRACTED: Long noncoding RNA RMRP upregulation aggravates myocardial ischemia-reperfusion injury by sponging miR-206 to target ATG3 expression.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Features appear similar within the panel "Hypoxia" from Figure 1B, as well as to features from the panel "Hypoxia" of Figure 3C. Also, a section of panel "Hypoxia+pcDNA3.1" from Figure 3D appear similar to sections of the panels "Hypoxia+shNC" and "Hypoxia+sh-RMRP". A section of the "Control" panel of Figure 3D appears similar to sections of panels from Figures 5E-F of the article published by Shenfa Zhuang, Fengxian Liu and Pingping Wu in the Journal of Cellular Biochemistry 120 (2019) 13392-13402 https://doi.org/10.1002/jcb.28614 and Figure 5G of the article published by Yonghui Zhang, Jing Fang, Hongmeng Zhao, Yue Yu, Xuchen Cao and Bin Zhang in the Journal of Cellular Biochemistry 120 (2019) 5097-5107 https://doi.org/10.1002/jcb.27786. Another section of the "Control" panel of Figure 3D appears similar to a section of the panel "miR-1469 inhibitor" from Figure 5F of the article published by the Journal of Cellular Biochemistry 120 (2019) 5097. Given the comments of Dr Elisabeth Bik regarding this article "This paper belongs to a set of over 400 papers (as per February 2020) that share very similar Western blots with tadpole-like shaped bands, the same background pattern, and striking similarities in title structures, paper layout, bar graph design, and - in a subset - flow cytometry panels", the journal requested the authors to provide the raw data. However, the authors were not able to fulfil this request.

Physostigmine Restores Impaired Autophagy in the Rat Hippocampus after Surgery Stress and LPS Treatment.

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.

WIPI1, BAG1, and PEX3 Autophagy-Related Genes Are Relevant Melanoma Markers.

ROS and oxidative stress may promote autophagy; on the other hand, autophagy may help reduce oxidative damages. According to the known interplay of ROS, autophagy, and melanoma onset, we hypothesized that autophagy-related genes (ARGs) may represent useful melanoma biomarkers. We therefore analyzed the gene and protein expression of 222 ARGs in human melanoma samples, from 5 independent expression databases (overall 572 patients). Gene expression was first evaluated in the GEO database. Forty-two genes showed extremely high ability to discriminate melanoma from nevi (63 samples) according to ROC (AUC ≥ 0.85) and Mann-Whitney (p < 0.0001) analyses. The 9 genes never related to melanoma before were then in silico validated in the IST online database. BAG1, CHMP2B, PEX3, and WIPI1 confirmed a strong differential gene expression, in 355 samples. A second-round validation performed on the Human Protein Atlas database showed strong differential protein expression for BAG1, PEX3, and WIPI1 in melanoma vs control samples, according to the image analysis of 80 human histological sections. WIPI1 gene expression also showed a significant prognostic value (p < 0.0001) according to 102 melanoma patients' survival data. We finally addressed in Oncomine database whether WIPI1 overexpression is melanoma-specific. Within more than 20 cancer types, the most relevant WIPI1 expression change (p = 0.00002; fold change = 3.1) was observed in melanoma. Molecular/functional relationships of the investigated molecules with melanoma and their molecular/functional network were analyzed via Chilibot software, STRING analysis, and gene ontology enrichment analysis. We conclude that WIPI1 (AUC = 0.99), BAG1 (AUC = 1), and PEX3 (AUC = 0.93) are relevant novel melanoma markers at both gene and protein levels.

HSF1 upregulates ATG4B expression and enhances epirubicin-induced protective autophagy in hepatocellular carcinoma cells.

Considerable evidences have shown that both heat shock transcription factor 1 (HSF1) and autophagy can attenuate the sensitivity of hepatocellular carcinoma (HCC) cells to chemotherapeutic reagents. However, it is still little known whether HSF1 is associated with autophagy in regulating the chemosensitivity of HCC cells. In this study, we for the first time demonstrated that HSF1 markedly attenuated the killing effect of epirubicin (EPI) to HCC cells via enhancing the EPI-induced protective autophagy. Mechanistically, HSF1 upregulated autophagy related 4B (ATG4B) in HCC cells, which enhanced the EPI-triggered protective autophagy. Reporter assay showed that HSF1 increased the transcriptional activity of ATG4B gene promoter, and chromatin immunoprecipitation assay verified that HSF1 bound to the site (-1429 to -1417) in ATG4B gene promoter region. The experiments in nude mice showed that knockdown of HSF1 or ATG4B strengthened the anti-HCC effect of EPI in vivo. Collectively, these results revealed that HSF1 elevates ATG4B via promoting its transcription, which alleviates the sensitivity of EPI in HCC cells through enhancing protective autophagy, suggesting that the "HSF1/ATG4B/protective autophagy" pathway may be a novel target for developing sensitizing strategy to HCC chemotherapy.

Silencing of NRAGE induces autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells.

NRAGE has been reported to be overexpressed in cancer cells, especially in lung cancer cells. To determine the role of NRAGE in non-small-cell lung cancer cells, we investigated the effects of NRAGE on autophagy in non-small-cell lung cancer cells. Human A549 and H1299 cells were transfected with NRAGE-specific small-interfering RNA. The Cell Counting Kit-8 and plate clone assay showed that downregulation of NRAGE could induce the proliferation in A549 and H1299 cells. In addition, our data suggested that downregulation of NRAGE enhances autophagosome formation by immunofluorescence. We found that knockdown of NRAGE induced autophagy, together with downregulation of P62 and upregulation of LC3-II protein. Furthermore, to elucidate the mechanism of NRAGE in suppressing autophagy, the protein expressions of AMPK, Ulk1, and Atg13 were assessed. Collectively, these results demonstrate the effective anti-autophagic of NRAGE in non-small-cell lung cancer cells through AMPK/Ulk1/Atg13 autophagy signaling pathways. Therefore, NRAGE could be used as a potential therapeutic target for lung cancer.