Expression Of Autophagy-related Gene Research Articles

LncRNA MALAT1 suppresses monocyte-endothelial cell interactions by targeting miR-30b-5p and enhancing ATG5-mediated autophagy

BackgroundMonocyte-endothelial cell (EC) interactions are one of the earliest events in the development of atherosclerosis and play a crucial role in atherosclerotic plaque formation. Although attempts have been made to modulate this interaction, the underlying molecular signalling mechanisms remain unclear. This study aimed to investigate the role of long non-coding RNA MALAT1 in monocyte-EC interactions. MethodsThe expression of MALAT1, ICAM-1, VCAM-1, P-selectin, CCL2 and CXCL1 was evaluated in ApoE-/- mouse aortic tissues and inflamed human umbilical vein endothelial cells (HUVECs). The regulatory impact of MALAT1 on cell adhesion molecules, monocyte-EC adhesion, and autophagy was assessed. The interactions between MALAT1 and microRNAs (miRNAs) were evaluated using dual-luciferase reporter and RNA pull-down assays. ResultsMALAT1 expression decreased in ApoE-/- mouse aortic tissues and inflammatory HUVECs. MALAT1 overexpression suppressed the expression of ICAM-1, VCAM-1 and CXCL1, and reduced the migration and adhesion of monocytes to ECs. Inhibition of MALAT1 promoted cell adhesion molecule expression and monocyte-EC interactions. Mechanistically, MALAT1 binds directly to miR-30b-5p and decreases its effective expression by functioning as an endogenous sponge, thereby increasing the expression of autophagy-related gene 5 (ATG5) and stimulates endothelial autophagy. ConclusionsOur findings suggest that MALAT1 suppresses monocyte-EC interactions by targeting miR-30b-5p and enhancing ATG5-mediated endothelial autophagy. These data imply that MALAT1 may play a protective role at the early stages of the atherosclerotic process.

Metallothionein 3 Potentiates Pulmonary Artery Smooth Muscle Cell Proliferation by Promoting Zinc-MTF1-ATG5 Axis-mediated Autophagosome Formation.

Abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) is one of the critical pathological mechanisms of pulmonary hypertension (PH), and therefore is gradually being adopted as an important direction for the treatment of PH. Metallothioneins (MTs) have been reported to be associated with PH, but the underlying mechanisms are not fully understood. Here, we demonstrated that the expression level of metallothionein 3 (MT3) was significantly increased in pulmonary arterioles from PH patients and chronic hypoxia-induced rat and mouse PH models, as well as in hypoxia-treated human PASMCs. Knockdown of MT3 significantly inhibited the proliferation of human PASMCs by arresting the cell cycle in the G1 phase, while overexpression of MT3 had the opposite effect. Mechanistically, we found that MT3 increased the intracellular zinc (Zn2+) concentration to enhance the transcriptional activity of metal-regulated transcription factor 1 (MTF1), which promoted the expression of autophagy-related gene 5 (ATG5), facilitating autophagosome formation. More importantly, MT3-induced autophagy and proliferation of human PASMCs were largely prevented by knockdown of MTF1 and ATG5. Therefore, in this study, we identified MT3-Zinc-MTF1-ATG5 as a novel pathway that affects PASMC proliferation by regulating autophagosome formation, suggesting that MT3 may be a novel target for the treatment of PH.

Inhalation of ammonia promotes apoptosis and induces autophagy in hepatocytes via Bax/BCl-2 and m-TOR/ATG5/LC-3bII axes

Ammonia (NH3) is an irritating gas and atmospheric pollutant that endangers the health of humans and animals by stimulating respiratory tract's mucosa and causing liver damage. However, physiological role of ammonia gas in hepatotoxicity remains unclear. To investigate the hepatotoxic effects of inhaled ammonia gas, experiments were conducted using mouse model exposed to 100 ppm of ammonia gas for 21 days. The exposed mice exhibited signs of depression, emaciation, and reduced growth. This study revealed that inhalation of ammonia led to significant decrease in water (P < 0.0001) and food intake (P < 0.05), resulting in slower growth. Histopathological analysis showed that ammonia stress alters the microstructure of the liver by enlarging the gap between hepatic lobule and fibrosis. Moreover, ammonia-induced stress significantly reduces the expression of the anti-apoptotic protein BCl-2 (P < 0.001), while elevates the mRNA expression of the pro-apoptotic gene Bax (P < 0.001). Furthermore, ammonia inhalation significantly increases the protein expression of LC-3bII (P < 0.05) and the mRNA expression of autophagy-related gene 5 (ATG5) (P < 0.05) and p62 (P < 0.05) while remarkably decreases the mRNA expression of mammalian target of rapamycin (m-TOR) (P < 0.05). In conclusion, this study demonstrates that inhalation of ammonia gas causes liver damage and suggests autophagy happening via m-TOR/p62/LC-3bII and pro-apoptosis effect mediated by Bax/BCl-2 in the liver damage caused by ammonia inhalation. Our study provides a new perspective on ammonia-induced hepatotoxicity.

Effects of Lactiplantibacillus plantarum on oxidative stress, mitophagy, and NLRP3 inflammasome activation in broiler breast meat

Poultry meat has a high polyunsaturated fatty acids content, making it vulnerable to oxidative stress. Mitophagy participates in the regulation of oxidative stress and the nucleotide-binding and oligomerization domain (NOD)-like receptor family as well as pyrin domain-containing protein 3 (NLRP3) inflammasome activation. Lactiplantibacillus plantarum P8 (P8) is a probiotic strain with an antioxidant capacity. In the present study, we investigated the effects of P8 on oxidative stress, mitochondrial function, mitophagy, and NLRP3 inflammasome in the breast meat of oxidatively stressed broilers. Four hundred 1-day-old male broilers were assigned to a 2×2 factorial design with 2 P8 levels (0 or 1×108 cfu/g), either with or without dexamethasone (DEX) injection, for a 21-day experimental period. DEX was injected intraperitoneally once daily from d 16 to 21. The breast meat was collected on d 21. The results showed that P8 supplementation decreased malondialdehyde (MDA) levels, increased superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and activated the Keap1-Nrf2 pathway in DEX-injected broilers. Moreover, P8 supplementation downregulated mitochondrial DNA (mtDNA) copy number and increased the expressions of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α), silent information regulator 1 (SIRT1), mitochondrial fusion protein 1 (Mfn1), and optic atrophy protein 1 (OPA1) in DEX-treated broilers. In addition, the decreased mitophagy level in DEX-treated broilers was elevated with P8 supplementation, as reflected by the increased gene expression of autophagy-related gene 5 (ATG5), Bcl-2-interacting protein (Becline-1), Parkin, PTEN-induced kinase 1 (PINK1), light chain 3 II (LC3II)/LC31, and the protein expression of Parkin as well as decreased p62 expression. In addition, P8 supplementation inhibited NLRP3 inflammasome activation by decreasing the transcription of NLRP3, IL-18, cysteinyl aspartate-specific proteinase-1 (Caspase-1), and the expression of NLRP3 and IL-18 in DEX-treated broilers. In conclusion, dietary P8 supplementation alleviates oxidative stress, improves mitophagy, and inhibits NLRP3 inflammasome activation in the breast meat of oxidatively stressed broilers.

Protective Effect of Docetaxel Against Autophagy-Related Genes in Vitrification of Mouse Metaphase II Oocytes.

Autophagy is a conservative mechanism for cell survival as the main response of cells to stress conditions. The present study aimed to assess the effect of docetaxel on the survival, fertilization, and expression of autophagy-related genes in vitrified oocytes. The study was conducted in 2018 at the Stem Cells Technology Research Center, Shiraz University of Medical Sciences (Shiraz, Iran). Denuded oocytes were randomly selected and assigned to five groups, namely control (n=133), docetaxel (n=136), docetaxel+cryoprotectants (n=146), docetaxel+vitrification (n=138), and vitrification (n=145). The effect of vitrification on the expression of autophagy-related gene 5 (ATG5) and Beclin-1 was determined using a real-time polymerase chain reaction. Data were analyzed using SPSS software (version 26.0) and GraphPad Prism 9. Survival and fertilization rates in each experimental group were significantly reduced compared to the control group (P=0.001). After in vitro fertilization of oocytes, the 2-cell formation rate was significantly reduced in the docetaxel+vitrification and vitrification groups compared to the control and docetaxel groups (P=0.001 and P=0.001, respectively). Pre-incubation of oocytes with docetaxel reduced gene expression levels of Beclin-1 and ATG5 in the docetaxel+cryoprotectants and docetaxel+vitrification groups (P=0.001 and P=0.019, respectively). The expression level of these genes was also reduced in the docetaxel group compared to the control group (P=0.001). Incubation of mouse metaphase II oocytes with docetaxel prior to vitrification reduced the expression of autophagy-related genes and increased survival and fertilization rates compared to untreated oocytes.

Increased expression of autophagy-related gene 5 indicates poor prognosis in patients with Hepatocellular carcinoma.

As an critical component of the autophagic machinery, autophagy-related gene 5 (ATG5) is essential for autophagosome formation. Autophagy participates in various malignant tumor transformation and progression, but the role of ATG5 involved in hepatocellular carcinoma (HCC) remains to be illustrated. The expression of ATG5 was evaluated in 89 pairs of liver tumor and adjacent normal tissues. The correlation between ATG5 expression and clinicopathological characteristics or prognosis were evaluated. Moreover, clinical special analysis was conducted in the young patients group and the old patients group respectively, and nomograms estimating overall survival and disease-free survival were investigated. Our study demonstrated that the ATG5 level was increased in tumor specimens than adjacent normal tissues. Upregulated ATG5 level had positive association with serum α-fetoprotein (AFP). Moreover, cases with higher ATG5 level had poorer disease-free survival (DFS) and overall survival (OS) than those with lower ATG5 level. Multivariate analysis showed that overexpression of ATG5 predicts poor OS and DFS. Further analysis indicated that cases with higher ATG5 level had poorer OS and DFS in the young patients and solitary tumor number subgroup. The nomogram models suggested that it had a coherence between nomogram prediction and actual situation. Together these findings revealed that ATG5 promotes progression of HCC, making it a potentially biomarker in diagnosis and therapeutic target of hepatocellular carcinoma. This article is protected by copyright. All rights reserved.

Bisphenol A-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression by Syk activation

The widely used plasticizer bisphenol A (BPA) is known as an endocrine-disrupting chemical (EDC). Many studies have shown that BPA contributes to diseases involving immune system alterations, but the underlying mechanisms have yet to be elucidated. We previously reported that BPA at concentration of 100 μM caused human B cell death in accordance with an increase in nuclear factor (erythroid-derived 2)-like 2(Nrf2) expression. Autophagy is a cellular process that degraded and recycles cytoplasmic constituents. Here, we investigated whether BPA induces autophagy through Nrf2, which is associated with regulation of B cell death using human WiL2-NS lymphoblast B cells. Then, cell viability was assessed by various assays using trypan blue, MTT or Celltiter glo luminescent substrate and DAPI. When WiL2-NS cells were treated with BPA, cell viability was decreased and LC3 autophagy cargo protein/puncta was increased. BPA-induced autophagy was confirmed by the modification of LC3 puncta formation or autophagy flux turnover with the treatment of hydroxychloroquine(HCQ), NH4Cl and PI3K inhibitors including 3-methyladenine(3-MA), LY294002 and wortmannin. BPA treatment increased the expression of autophagy-related gene(Atg)7 and Beclin1 as well as Nrf2 induced by the production of reactive oxygen species (ROS). The inhibition of autophagy with siAtg7 or siBeclin1 and Nrf2 depletion aggravated BPA-induced cell death. BPA enhanced the bound of Nrf2 to the specific region on Beclin1 and Atg7 promoter. Spleen tyrosine kinase(Syk) activity was enhanced in response to BPA treatment. Bay61–3606, Syk inhibitor, decreased LC3 and the expression of Atg7 and Beclin1, leading to the increase of BPA-induced B cell death. The results suggest that BPA-induced autophagy ameliorates human B cell death through Nrf2-mediated regulation of Atg7 and Beclin1 expression.

Identification of an autophagy-related gene signature for predicting prognosis and immune activity in pancreatic adenocarcinoma

Adenocarcinoma of the pancreas (PAAD) is a cancerous growth that deteriorates rapidly and has a poor prognosis. Researchers are investigating autophagy in PAAD to identify a new biomarker and treatment target. An autophagy-related gene (ARG) model for overall survival (OS) was constructed using multivariate Cox regression analyses. A cohort of the Cancer Genome Atlas (TCGA)-PAAD was used as the training group as a basis for model construction. This prediction model was validated with several external datasets. To evaluate model performance, the analysis with receiver operating characteristic curves (ROC) was performed. The Human Protein Atlas (HPA) and Cancer Cell Line Encyclopedia (CCLE) were investigated to validate the effects of ARGs expression on cancer cells. Comparing the levels of immune infiltration between high-risk and low-risk groups was finished through the use of CIBERSORT. The differentially expressed genes (DEGs) between the low-/high-risk groups were analyzed further via Gene Ontology biological process (GO-BP) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, which were used to identify potential small-molecule compounds in Connectivity Map (CMap), followed by half-maximal inhibitory concentration (IC50) examination with PANC-1 cells. The risk score was finally calculated as follows: BAK1 × 0.34 + ITGA3 × 0.38 + BAG3 × 0.35 + APOL1 × 0.26–RAB24 × 0.67519. ITGA3 and RAB24 both emerged as independent prognostic factors in multivariate Cox regression. Each PAAD cohort had a significantly shorter OS in the high-risk group than in the low-risk group. The high-risk group exhibited infiltration of several immune cell types, including naive B cells (p = 0.003), plasma cells (p = 0.044), and CD8 T cells (nearly significant, p = 0.080). Higher infiltration levels of NK cells (p = 0.025), resting macrophages (p = 0.020), and mast cells (p = 0.007) were found in the high-risk group than the low-risk group. The in vitro and in vivo expression of signature ARGs was consistent in the CCLE and HPA databases. The top 3 enriched Gene Ontology biological processes (GO-BPs) were signal release, regulation of transsynaptic signaling, and modulation of chemical synaptic transmission, and the top 3 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were MAPK, cAMP, and cell adhesion molecules. Four potential small-molecule compounds (piperacetazine, vinburnine, withaferin A and hecogenin) that target ARGs were also identified. Taking the results together, our research shows that the ARG signature may serve as a useful prognostic indicator and reveal potential therapeutic targets in patients with PAAD.

Candesartan protects against d-galactose induced – Neurotoxicity and memory deficit via modulation of autophagy and oxidative stress

Purposed-galactose induces neuroinflammation and memory deficit via oxidative stress. Candesartan is an angiotensin II-receptor blocker and has proved neuroprotective properties. This study aimed to investigate the neuroprotective effect of candesartan against d-galactose induced neuroinflammation and memory deficit via autophagy. MethodsTwenty-eight male Wistar rats aged 3 months were divided into four equal groups: control (vehicle), d-gal (100 mg/kg d-galactose), cand (1 mg/kg candesartan), and cand+d-gal (100 mg/kg d-galactose & 1 mg/kg candesartan). All treatments were given orally and daily for 4 weeks. Assessment of memory was done using Morris water maze (MWM) test. Brain tissue was assessed for malondialdehyde (MDA), total thiol, catalase activity, glial fibrillary acidic protein (GFAP) and gene expression of TNF-α, GDNF-1 as well as autophagy genes (Beclin 1 and ATG 5). ResultsProphylactic treatment of candesartan in d-galactose-treated rats significantly (p < 0.001) reduced oxidative stress via reduction of MDA as well as elevation of catalase activity and total thiol levels. Additionally, candesartan prophylactic treatment significantly increased gene expression of GDNF-1 and decreased gene expression of TNF-α. Furthermore, candesartan significantly increased the expression of autophagy related gene (Beclin 1 and ATG 5) in cand+d-gal treated rats. These results were supported by the histopathological findings which showed that candesartan prevented the neuronal injury in the cerebral cortex and hippocampus and decreased GFAP positive cells of the d-galactose-treated rats. Moreover, MWM test showed that candesartan significantly improved memory deficit in cand+d-gal treated rats. ConclusionCandesartan prevents d-galactose-induced neurotoxicity and memory deficit via activating autophagy and decreasing oxidative stress. Therefore, candesartan was a good candidate for age-related neurodegenerative disorders and memory deficit.

Effects of programmed cell death induction method on somatic cell development

In this study, to analyze whether artificial regulation of apoptosis in the development of somatic cells can affect the stable growth and development of cells, 20 alpha-hydroxysteroid dehydrogenase (20α-HSD) and rapamycin were treated to induce apoptosis and autophagy in the both skin and muscle cells. Respectively, and 3-methyladenine was supplemented to inhibit cell death. Our results show that stimulation with rapamycin activated autophagy in both types of cells, but increased apoptosis more than autophagy in the case of skin cells. These results indicate that there was a difference in the expression of survival factors and cell development depending on the type of cell. In particular, in the expression of autophagy-related gene (MAP1LC3A) was higher than that of Casp-3, an apoptosis factor. Furthermore, cell development was the highest in all cell groups cultured by artificially inducing autophagy, however the lowest in the apoptosis-inhibited group. Especially, the noteworthy result of this study was that when apoptosis was induced using 20α-HSD, it was possible to induce apoptosis in both skin and muscle cells. Therefore, the main point of this study is that apoptosis induced during cell culture plays a pivotal role in cell remodeling.

PAX5-activated lncRNA ARRDC1-AS1 accelerates the autophagy and progression of DLBCL through sponging miR-2355-5p to regulate ATG5

BackgroundDiffuse large B-cell lymphoma (DLBCL) has high cancer-related mortality. Studies have supported that lncRNAs can regulate cancer progression by affecting autophagy of cells. ARRDC1 antisense RNA 1 (ARRDC1-AS1) was found to be upregulated in DLBCL tissues in GEPIA, but it has never been detected in DLBCL. AimIn this study, we aimed to explore the regulatory mechanism of ARRDC1-AS1 in DLBCL cells. Main methodsRT-qPCR was taken to measure the expression of ARRDC1-AS1, microRNA-2355-5p (miR-2355-5p) and autophagy-related gene 5 (ATG5) in DLBCL cells. Western blot was conducted to detect protein levels. The malignant behaviors of DLBCL cells were estimated through functional assays. The molecular interactions were detected by Chromatin immunoprecipitation (ChIP), RNA pull-down, RNA immunoprecipitation (RIP) and luciferase reporter assays. ResultsWe found that ARRDC1-AS1 was upregulated in DLBCL tissues and cell lines. ARRDC1-AS1 was activated by transcription factor PAX5. Knockdown of ARRDC1-AS1 suppressed DLBCL autophagy to aggravate proliferation, repress apoptosis, and facilitate invasion and migration. Furthermore, ARRDC1-AS1 sponged miR-2355-5p to upregulate ATG5. ConclusionPresent study first showed that PAX5-activated ARRDC1-AS1 accelerates the autophagy and progression of DLBCL via sponging miR-2355-5p to regulate ATG5, revealing a novel molecular mechanism of ARRDC1-AS1 in DLBCL and suggested ARRDC1-AS1 as a potential target in DLBCL.

The role of autophagy-related gene 9B in lichen planus.

Lichen planus (LP) is an idiopathic, chronic, relapsing, inflammatory, autoimmune dermatological disease. The etiopathogenesis of LP is still unclear. Autophagy is a strictly regulated lysosomal degradation pathway that is crucial for maintaining intracellular homeostasis and normal development. The dysregulation of autophagy-associated genes was recognized to increase the susceptibility to multiple diseases, including inflammation, autoimmune disorders and cancer. Our study aimed to detect the expression of autophagy-related gene 9 b (ATG9B) in LP patients compared to normal control persons to investigate the possible role of autophagy in pathogenesis of this disease. This case-control study included 30 LP patients and 30 age-, gender-matched healthy controls. Four millimeters punch skin biopsies were obtained from LP lesions and from the controls and they were kept in lysis solution for the stability of the studied parameters and were kept frozen at -80°C till analysis of ATG9B using real-time polymerase chain reaction. The level of ATG9B in lesional skin of LP was significantly decreased compared to normal control persons (P < 0.01); also, there was a non-significant relation between ATG9B level and age, sex, duration and family history among LP patients. Limited number of patients included in our study (30 patients). Autophagy may play a role in the pathogenesis of cutaneous LP.

Effect of moxibustion on expression of autophagy related molecules in synovial tissues of rheumatoid arthritis rats

To observe the effect of moxibustion on expression of autophagy related gene(Atg), serine/threonine protein kinase-uncoordinated 51 like kinase-1 (ULK1), Beclin1 and microtubule associated proteins light chain 3 (LC3) and ultrastructure of synovium in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. Forty SD rats were randomly divided into normal control, RA model, moxibustion, cigarette-roasting and medication groups (n=8 rats in each group). The RA model was established by keeping the rats in wind, cold and wet environment for 12 h, once a day for 20 days and subcutaneous injection of Freund's adjuvant complete into the sole of the left hind paw. Moxibustion was applied to the left "Zusanli" (ST36) for 20 min, once a day for 15 days. Rats of the cigarette-roasting group was treated by ignited cigarettes instead of moxa strips. Rats of the medication group was treated by gavage of Tripterygium wilfordii polyglycoside tablet suspension (0.8 mg/100 g) once a day for 15 days. The rats' paw volume of the left hindlimb was measured by using a water-based paw plethysmometer. The synovial tissue of the left plantar joint was harvested at the end of experiments for observing changes of the ultrastructure with transmission electron microscope, and the expression of ULK1, Atg3, Atg5, and Atg12 mRNAs was detected with quantitative real-time PCR and the expression of LC3-Ⅱ and Beclin-1 proteins were detected with Western blot. Following modeling, the paw volume of the left hindlimb was significantly increased (P<0.01), while the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins of the synovial tissue were notably down-regulated in the model group relevant to the normal control group (P<0.01). The increase of the paw volume in the moxibustion and medication groups and the down-regulation of synovial Atg3, Atg12 and ULK1 mRNAs in the 3 intervention groups, and Atg5 mRNA , and LC3-Ⅱ and Beclin-1 proteins in both moxibustion and medication groups were considerably suppressed (P<0.01, P<0.05). The therapeutic effect of moxibustion was apparently superior to that of cigarette-roasting in down-regulating the paw volume, and up-regulating the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins (P<0.05, P<0.01), and notably weaker than that of medication in up-regulating Atg3 and ULK1 mRNAs (P<0.01), but was comparable to that of medication in up-regulating the expression levels of Atg5 and Atg12 mRNAs, LC3-Ⅱand Beclin-1 proteins (P＞0.05). Results of the ultrastructural observation showed an obvious injury of synovial cells, such as unclear and incomplete nuclear membrane, chromatin condensation, swollen mitochondria with broken crests, cavitation-like degeneration of cytoplasma, and appearance of autophagosomes and lysosomes in the model group, which was relatively milder in the 3 intervention groups. Moxibustion can reduce the paw edema and inflammatory injury of the plantar synovial tissue in RA rats, which may be related to its effects in up-regulating Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins to enhance the cellular autophagy. The therapeutic effect of moxibustion is obviously superior to that of cigarette-roasting and medication in relieving swelling.

LncRNA DANCR promotes ATG7 expression to accelerate hepatocellular carcinoma cell proliferation and autophagy by sponging miR-222-3p.

LncRNA differentiation antagonizing non-protein coding RNA (DANCR) is an oncogene in various malignant cancers, including hepatocellular carcinoma (HCC). Autophagy is an intracellular self-digestion mechanism that accelerates the progression of HCC via promoting cell survival. However, the role of lncRNA DANCR in HCC, and the mechanism of lncRNA DANCR in the regulation of autophagy in HCC remains unknown. Therefore, the aims of this study are the investigation of the role of lncRNA DANCR in HCC, and the exploration of the molecular mechanism of lncRNA DANCR in regulating autophagy of HCC cells. In this study, the expression of lncRNA DANCR, miR-222-3p, and autophagy-related gene 7 (ATG7) was detected by qRT-PCR. The cell proliferation and colony formation were measured by Cell Counting Kit-8 (CCK-8) assay and colony formation assay. And the autophagic flux was evaluated by mRFP-GFP-LC3B reporter. The autophagy related proteins were analyzed by Western blotting. Besides, the relationship between lncRNA DANCR and miR-222-3p, as well as between miR-222-3p and ATG7, was determined by Dual-Luciferase reporter system. We found high expression of lncRNA DANCR and ATG7, and low expression of miR-222-3p in HCC tissues and cell lines. And lncRNA DANCR positively correlated with poor survival of HCC patients. Moreover, the knockdown of lncRNA DANCR inhibited cell proliferation and autophagy of HCC cells. And we predicted and proved that lncRNA DANCR induced cell proliferation, colony formation and autophagy by increasing ATG7 and suppressing miR-222-3p. Our study demonstrates the promoting role of lncRNA DANCR in HCC, and indicates the regulatory effects of lncRNA DANCR on regulating autophagy of HCC.

CDKL3 Targets ATG5 to Promote Carcinogenesis of Esophageal Squamous Cell Carcinoma.

ObjectiveOur previous study suggested cyclin-dependent kinase-like 3 (CDKL3) acts as a new oncogene in esophageal squamous cell carcinoma (ESCC) cell line TE-1. However, the molecular mechanisms and biological effects of CDKL3 in ESCC remain unknown. In the present study, we aimed to explore the clinical significance of CDKL3 in ESCC and how CDKL3 regulates the malignant behavior of ESCC.MethodsESCC samples were stained by immunohistochemical staining (IHC) and analyzed for the expression of CDKL3. The functions of CDKL3 on proliferation, apoptosis, migration, invasion, and colony formation were investigated by celigo assay, MTT assay, colony formation, caspase 3/7 activity analysis, transwell migration and invasion assay, respectively. A transplanted tumor model was established to study the functions of CDKL3 on the tumorigenesis of ESCC cells. Microarray analysis was utilized to identify the CDKL3-regulated genes in ESCC cells.ResultsESCC tumor tissues possessed a significantly higher expression of CDKL3 and autophagy-related gene 5 (ATG5) than matched adjacent normal tissues. The high expressions of CDKL3 were positively associated with the tumor-node-metastasis (TNM) stage and Ki67. Upregulated ATG5 expression was positively correlated with male, advanced tumor (T) stage, and TNM stage. Kaplan-Meier analysis showed that ESCC patients with higher expression of CDKL3 or ATG5 had a shorter overall survival. The worst prognosis was recognized in patients with both high manifestations of CDKL3 and ATG5. Time-dependent receiver operating characteristic (ROC) curve was established to reveal that the combination of CDKL3, ATG5, and TNM stage–based model had better prognostic accuracy than TNM stage. Moreover, CDKL3 knockdown markedly repressed cell growth and aggressivity in vitro and in vivo. Mechanistically, ATG5 was confirmed as a downstream gene involved in the pro-oncogenic function of CDKL3.ConclusionCDKL3 can be utilized as an independent poor prognostic marker in ESCC patients. Furthermore, CDKL3 may promote tumor profession, invasion, metastasis, and prohibit tumor apoptosis partly by ATG5 activation.

Cloning, Expression Analysis, 20-Hydroxyecdysone Induction, and RNA Interference Study of Autophagy-Related Gene 8 from Heortia vitessoides Moore.

Autophagy is a highly conserved and regulated process in eukaryotic cells and remodels cytoplasm, recovers essential nutrients, and disposes of unwanted cytoplasmic components. Autophagy-related gene (ATG) 8, identified in Heortia vitessoides Moore, which is an oligophagous pest of Aquilaria sinensis (Lour.), was characterized (HvATG8). Multiple sequence alignment showed that HvATG8 possesses highly conserved domain structures. Stage- and tissue-specific expressions indicated that HvATG8 is highly expressed in prepupal, pupal, and adult stages and in the midgut of larvae and abdomen of adults. Lack of function of HvATG8 by RNA interference resulted in a significant decrease in survival rate and an increase in abnormal or nonviable phenotypes in H. vitessoides. Transition rate from larval to pupal stages was 33.0% and from pupal to adult stages was 15.0% after injection. Reduction of ATG8 expression reduced survival of H. vitessoides. Therefore, HvATG8 possibly plays a key role in normal growth stage of H. vitessoides. HvATG8 suppression downregulates HvATG3 expression, suggesting that the two genes are interconnected. Further, HvATG8 expression increased by 20-hydroxyecdysone treatment, starvation, and extreme temperature exposure. Starvation also altered expression of other ATGs in H. vitessoide. This study may be used to guide research on molecular mechanisms of autophagy in insects.

Cannabinoid receptor 2 activation alleviates septic lung injury by promoting autophagy via inhibition of inflammatory mediator release

Septic lung injury is one of main causes of high mortality in severe patients. Inhibition of excessive inflammatory response is considered as an effective strategy for septic lung injury. Previous studies have shown that cannabinoid receptor 2 (CB2), a G protein-coupled receptor, play an important role in immunosuppression. Whether CB2 can be used as a therapeutic target for septic lung injury is unclear. The aim of this study is to explore the role of CB2 in sepsis and its potential mechanism. In this study, treatment with HU308, a specific agonist of CB2, could reduce lung pathological injury, decrease the level of inflammatory cytokines and strengthen the expression of autophagy-related gene after cecal ligation puncture (CLP)-induced sepsis in mice. Similar results were obtained in RAW264.7 macrophages after LPS treatment. Furthermore, the effect of HU308 could be blocked by autophagy blocker 3-MA in vivo and in vitro. These results suggest that CB2 serves as a protective target for septic lung injury by decreasing inflammatory factors, which is associated with the enhancement of autophagy.

Expression of autophagy related gene 5 and cyclin E in coronary heart disease and its clinical significance.

To explore the expression of autophagy related genes 5 (ATG5) and cyclin E in coronary heart disease (CHD) and its clinical significance. From April 2018 to August 2018, 80 patients diagnosed with CHD in the Second Xiangya Hospital, Central South University were selected as an observation group, and another 80 healthy subjects were selected as a control group. The expression of ATG5 and cyclin E mRNA in nucleate cells and the plasma protein in the 2 groups were detected and analyzed. The model of macrophage-derived foam cells induced by oxidized low density lipoprotein (ox-LDL) was used to simulate atherosclerosis. The proliferation of macrophage- derived foam cells and the protein levels of ATG5 and cyclin E induced by ox-LDL at different concentrations were examined. Compared with the control group, the levels of ATG5 mRNA and protein in the blood in the observation group were decreased, and the cyclin E mRNA and protein levels were increased, there were statistically difference (both P<0.05). Receiver operating characteristic (ROC) curve showed that the area under curve (AUC) of ATG5 mRNA, cyclin E mRNA, ATG5 protein and cyclin E protein were 0.739, 0.780, 0.671 and 0.807, respectively. Pearson analysis showed that the ATG5 mRNA was negatively correlated with the cyclin E mRNA (r=-0.734, P<0.05),while the plasma ATG5 protein was negatively correlated with the plasma cyclin E protein (r=-0.746, P<0.05). Macrophage-derived foam cell model induced by ox-LDL showed that the proliferation of foam cells and the expression levels of cyclin E protein were increased in a concentration and time-dependent manner, and the expression levels of ATG5 protein were decreased in a concentration-dependent manner. The levels of ATG5 mRNA and protein are lowly expressed while the levels of cyclin E mRNA and protein are highly expressed in the patients with CHD.The ATG5 protein levels are lowly expressed in ox-LDL-treated macrophage-derived foam cells while the cyclin E protein levels are highly expressed in ox-LDL-treated macrophage-derived foam cells. Based on these observations, we conclude that ATG5 inhibits the degradation of the cyclin E and promotes the proliferation of macrophages, involving in the occurrence and development of CHD.

Dysregulation of autophagy-related lncRNAs in peripheral blood of coronary artery disease patients

Coronary artery disease (CAD) as a major cause of death has been associated with dysregulation of several processes among them is autophagy. In the current study, we assessed expression of autophagy related gene 5 (ATG5) and three ATG5-associated long non-coding RNAs (lncRNAs Chast, HULC and DICER1-AS1) in the peripheral blood of patients with premature CAD and healthy subjects. Expression levels of ATG5, Chast, HULC and DICER1-AS1 were significantly lower in peripheral blood of CAD cases compared with healthy subjects. Receiver Operating Characteristic (ROC) curve analysis showed that HULC and DICER1-AS1 can properly differentiate CAD patients from healthy subjects (area under curve (AUC) values of 0.90 and 0.87, respectively). Expression levels of ATG5 and Chast were inversely correlated with FBS levels (r = −0.41, P < 0.0001 and r = −0.38, P < 0.0001 respectively) but no other biochemical factors. Expression of DICER1-AS1 was inversely correlated with FBS (r = −0.54, P < 0.0001), TG (r = −0.29, P < 0.0001) and TG/HDL ratio (r = −0.27, P < 0.0001). Expression of HULC was inversely correlated with age (r = −0.24, P < 0.0001), FBS (r = −0.62, P < 0.0001) and TG (r = −0.31, P < 0.0001). There were significant pairwise correlations between expression levels of all genes. The most robust correlations were detected ATG5 and Chast (r = 0.81, P < 0.0001) and between DICER1-AS1 and HULC (r = 0.75, P < 0.0001). The current study further verified associations between dysregulation of autophagy and CAD. Moreover, our results indicate appropriateness of two autophagy-related lncRNAs for differentiation of CAD status.

Long Noncoding RNA KCNQ1OT1 Promotes the Progression of Non-Small Cell Lung Cancer via Regulating miR-204-5p/ATG3 Axis.

PurposeNon-small cell lung cancer (NSCLC) is the first leading cause of cancer-related death globally. Long noncoding RNA KCNQ1 overlapping transcript 1 (KCNQ1OT1) was involved in the progression of multiple cancers by sponging target miRNA. We aimed to explore the pathological mechanism of KCNQ1OT1 in NSCLC progression.MethodsThe expression of KCNQ1OT1, miR-204-5p and autophagy-related gene 3 (ATG3) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometry assay were conducted for the detection of cell proliferation and apoptosis, respectively. Western blot assay was performed to examine the protein levels of B-cell lymphoma-2 (BCL-2), BCL2-Associated X (Bax), cleaved caspase-3, cleaved caspase-9 and LC3Ⅱ/LC3Ⅰ and P62. The interaction between miR-204-5p and KCNQ1OT1 or ATG3 was validated by dual-luciferase reporter system and RNA immunoprecipitation (RIP) assay. Murine xenograft assay was conducted to explore the function of KCNQ1OT1 in vivo. Immunohistochemistry (IHC) staining assay was used for the analysis of ki67-positive cell percentage.ResultsThe expression of KCNQ1OT1 and ATG3 was up-regulated whereas miR-204-5p was down-regulated in NSCLC tumors and cells. MiR-204-5p was inversely correlated with KCNQ1OT1 or ATG3. In addition, KCNQ1OT1 knockdown facilitated apoptosis, inhibited autophagy and proliferation of NSCLC cells in vitro and blocked tumor growth in vivo. However, the miR-204-5p inhibitor reversed the effects. More importantly, ATG3 was a target gene of miR-204-5p and ATG3 overexpression restored the effect of miR-204-5p on NSCLC cell progression.ConclusionKCNQ1OT1 promotes cell proliferation and autophagy and inhibits cell apoptosis via regulating miR-204-5p/ATG3 axis, providing a promising target for NSCLC therapy.