Beclin-1 mRNA Research Articles

17β-Estradiol alleviates cardiac aging induced by d-galactose by downregulating the methylation of autophagy-related genes

Intrinsic cardiac aging increases cardiovascular mortality and morbidity in the elderly. Estrogen helps reduce the risk of cardiovascular disease in women, with 17β-estradiol (17β-E2) activating the autophagy pathway and inhibiting vascular aging, mainly through estrogen receptor alpha (ER α) to prevent atherosclerosis. Abnormal methylation of autophagy-related genes can impact autophagic regulation. We hypothesized that 17β-E2, specifically 17β-E2 α, downregulates the methylation of autophagy factors and delays cardiac aging. Here, we used d-galactose, 17β-E2, and ER α receptor antagonist methyl-piperidino-pyrazole (MPP) to establish different aging models in mice divided into four groups, namely negative control, D.gal, D.gal + 17β-E2, and D.gal + 17β-E2 + MPP groups. Echocardiography showed that compared with the D.gal group group, the D.gal + 17β-E2 showed substantially increased cardiac function. The level of cardiac aging markers in mice in the D.gal + 17β-E2 group was lower than that in mice in the D.gal group. Beclin1, LC3, and Atg5 mRNA and protein expression levels in mice in the D.gal + 17β-E2 group were significantly increased compared with those in the D.gal group. Additionally, Beclin1, LC3, and Atg5 methylation levels were significantly decreased in the D.gal + 17β-E2 group. All the above values of the D.gal + 17β-E2 + MPP group were between those of the D.gal and D.gal + 17β-E2 groups. The expression of Dnmt1, Dnmt2, and Dnmt3A genes was the highest in the D.gal group. In summary, our results suggest that 17β-E2, specifically 17β-E2 α, promotes autophagy by downregulating the methylation of autophagy factors, thereby inhibiting galactose-induced cardiac aging in mice. 17β-E2 may be a potential therapeutic target to mitigate the effects of cardiac aging.

ELAVL1 is transcriptionally activated by FOXC1 and promotes ferroptosis in myocardial ischemia/reperfusion injury by regulating autophagy

AimsMyocardial ischemia is the most common form of cardiovascular disease and the leading cause of morbidity and mortality. Understanding the mechanisms is very crucial for the development of effective therapy. Therefore, this study aimed to investigate the functional roles and mechanisms by which ELAVL1 regulates myocardial ischemia and reperfusion (I/R) injury.MethodsMouse myocardial I/R model and cultured myocardial cells exposed to hypoxia/reperfusion (H/R) were used in this study. Features of ferroptosis were evidenced by LDH activity, GPx4 activity, cellular iron, ROS, LPO, and GSH levels. The expression levels of autophagy markers (Beclin-1, p62, LC3), ELAVL1 and FOXC1 were measured by qRT-PCR, immunostaining and western blot. RIP assay, biotin-pull down, ChIP and dual luciferase activity assay were employed to examine the interactions of ELAVL1/Beclin-1 mRNA and FOXC1/ELAVL1 promoter. CCK-8 assay was used to examine viability of cells. TTC staining was performed to assess the myocardial I/R injury.ResultsMyocardial I/R surgery induced ferroptosis and up-regulated ELAVL1 level. Knockdown of ELAVL1 decreased ferroptosis and ameliorated I/R injury. Si-ELAVL1 repressed autophagy and inhibition of autophagy by inhibitor suppressed ferroptosis and I/R injury in myocardial cells. Increase of autophagy could reverse the effects of ELAVL1 knockdown on ferroptosis and I/R injury. ELAVL1 directly bound with and stabilized Beclin-1 mRNA. Furthermore, FOXC1 bound to ELAVL1 promoter region and activated its transcription upon H/R exposure.ConclusionFOXC1 transcriptionally activated ELAVL1 may promote ferroptosis during myocardial I/R by modulating autophagy, leading to myocardial injury. Inhibition of ELAVL1-mediated autophagic ferroptosis would be a new viewpoint in the treatment of myocardial I/R injury.

Expression pattern of the autophagy related proteins Beclin1 and LC3B in tuberculous wound tissues

Tuberculous wound therapy is a major challenge in clinical practice, due to the protracted disease course, high recurrence rate, and an unclear pathogenesis. We explored the expression patterns of Beclin1 and LC3B in tuberculous wound tissues in human tuberculous chronic wound and normal tissues was assayed by immunohistochemistry. Rat models of tuberculous wounding were induced by the Bacillus Calmette-Guerin (BCG) method. Beclin1 and LC3B protein expression in human tuberculous wound tissues differed from that of normal skin and non-tuberculous chronic wound tissues.In rat tuberculous wound tissues, expression of Beclin1 and LC3B mRNA time-dependently changed post-infection. Abnormal fluctuation of autophagy protein in the development of tuberculosis wound could be one of the causes for the repeated occurrence and protracted disease course of the tuberculous wound.

Hsa_circ_0030042 regulates abnormal autophagy and protects atherosclerotic plaque stability by targeting eIF4A3.

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability.Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis.Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability.Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

Liver injury mediated by the UII and its receptor (UT) system is possibly associated with the activation of autophagy-related and apoptosis-resisted pathways of Kupffer cells in acute liver failure

The system of urotensin II (UII) and its receptor (UT) (or: UII/UT system) mediates hepatic immune inflamed injury in acute liver failure (ALF) with autophagy inhibition. However, it is unknown whether the system has an effect on liver autophagy in ALF. In this study, we attempted to explore hepatic autophagy response in ALF through blocking the UII/UT signal. Autophagy-related genes were examined in the liver tissues of lipopolysaccharide (LPS)/d-galactosamine (D-GalN)-induced ALF after pretreatment of UT receptor specific antagonist urantide. And then, the levels of autophagy- and apoptosis-related genes were assayed in LPS-stimulated KCs via urantide pretreatment. We found that the expressions of hepatic autophagy related genes, including Beclin-1, Atg5, Atg7, LC3 and p62 mRNA, and LC3 II and p62 protein, were significantly downregulated in LPS/D-GalN-induced ALF mice; but they were not affected by pretreatment of urantide, a special UT receptor antagonist. To probe inflammatory mechanisms of the UII/UT system, we further investigated the effect of the system on Kupffer cells (KCs), the innate immune cells in liver. We found that urantide pretreatment significantly inhibited production of inflammatory injury molecules including TRAF6 and ROS in LPS-stimulated KCs. LPS stimulation induced LC3 and p62 mRNA and LC3 II and p62 protein expression in KCs. After urantide pretreatment, LC3 and p62 mRNA and LC3 II protein were downregulated, while p62 protein was upregulated in LPS-stimulated KCs. In addition, antiapoptotic protein Bcl-2 inhibition and proapoptotic protein cleaved caspase-3 increase were observed in LPS-stimulated KCs, and the effects were enhanced after urantide pretreatment in the study. We conclude that liver injury mediated by the UII/UT system is possibly associated with the activation of autophagy-related and apoptosis-resisted pathways of KCs in ALF.

METTL14 Regulates Autophagy and Osteogenic Differentiation of BMSCs Targeting Beclin-1 via an M <sup>6</sup> A-IGF2BPs-Dependent Mechanism

Background: The development of osteoporosis is often accompanied by autophagy disturbance, which also caused new osteoblasts defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. We aimed to explore the biological functions of autophagy mediated by METTL14 in osteoporosis and the possibility of METTL14 as an effective target for the treatment of osteoporosis. Methods: Calcein staining and microcomputed tomography (μ-CT) were used to analyze the bone formation. immunohistochemical (IHC) staining was used to detect the expression of METTL14 and beclin-1 in bone tissues. The quantitative real time PCR (qRT-PCR) and western blotting were used to analyze the mRNA and protein expressions. Alizarin red staining (ARS) and alkaline phosphatase (ALP) staining were used to explore the osteogenic differentiation of BMSCs. The production of autophagy was determined by transmission electron microscopy (TEM) and mRFP-GFP-LC3 adenoviral. Moreover, RNA binding protein immunoprecipitation (RIP)-qPCR was used to investigate the m6A modification of beclin-1 and the binding capability of beclin-1 and IGF2BPs. Findings: Herein, we demonstrate that the expression of METTL14 is downregulated during the development of osteoporosis both in human and mice. In vivo, METTL14+/- knockdown mice exhibits elevated bone loss and impaired autophagy similar to the ovariectomized (OVX) mice, while injecting METTL14 overexpressing adenovirus delays the process of osteoporosis.In vitro, forced expression of METTL14 significantly facilitates the osteogenic differentiation ability of BMSCs through regulating the activation of autophagy by enhancing the expression of beclin-1 dependent on the m6 A modification; the opposite is true under METTL14 silencing condition. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A methylated beclin-1 mRNA and promotes its translation via mediating RNA stabilization. Interpretation: Our study reveals the METTL14-IGF2BPs-beclin-1 signaling axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14 mediated m 6 A modification in osteoporosis. Funding: This work was supported by grants from the National Natural Science Fund of China (81972117), Natural Science Foundation of Heilongjiang Province of China for excellent youth (JJ2020JQ0004), The First Affiliated Hospital of Harbin Medical University Excellent Young Talents Funding (HYD2020JQ0013), Special fund for clinical research of Wu Jieping Medical Foundation (320.6750.2020-04-50), and CAMS Innovation Fund for Medical Sciences (CIFMS, 2020-I2M-5-003). Declaration of Interest: The authors declare that they have no competing interests. Date Availability Ethical Approval: The experiments were performed under the guidance of the Experimental Animal Ethic Committee of the Harbin Medical University.

Curcumin combined with Baicalin attenuated ethanol-induced hepatitis by suppressing p38 MAPK and TSC1/ eIF-2α/ATF4 pathways in rats

Autophagy activation is one of the survival mechanisms of ethanol-induced hepatitis in mammals. Both Curcumin and Baicalin have anti-inflammatory effects, but the mechanism of their combined action in ethanol-induced hepatitis is still unclear. This study aimed to investigate whether Curcumin combined with Baicalin can resist ethanol-induced p38 MAPK-mediated hepatitis via suppressing TSC1/eIF-2α/ATF4 pathway to activate autophagy. Rats were randomly divided into Control group, EtOH group, EtOH + Curcumin group, EtOH + Baicalin group, and EtOH + Curcumin + Baicalin group. The indicators of liver function and inflammation, and the levels of p38 MAPK and autophagy-related proteins and genes were assayed. Our study found that ethanol increased serum AST, TG, T-CHO levels and liver inflammatory cytokines (TNF-α, IL-1β, INF-γ, NO and iNOS) contents, and also reduced the levels of Beclin1 mRNA and protein, LC3B mRNA and LC3BII/I ratio, but up-regulated the levels of p-p38, P62, p-TSC1, p-eIF-2α and p-ATF4 in rats. Curcumin combined with Baicalin decreased the levels of serum AST, TG, T-CHO and liver inflammatory cytokines, down-regulated the expression of p-p38, P62, p-TSC1, p-eIF-2α and p-ATF4, as well as up-regulated the expression of Beclin1 mRNA and protein, LC3B mRNA and LC3BII/I ratio in rats. Besides, combination therapy was superior to monotherapy. Our results indicated that Curcumin combined with Baicalin can improve ethanol-induced hepatitis by inhibiting p38 MAPK and TSC1/eIF-2α/ATF4 pathway, which may be a potential therapeutic strategy for ethanol-induced hepatitis in the future.

Inhibition of autophagy aggravates molybdenum-induced mitochondrial dysfunction by aggravating oxidative stress in duck renal tubular epithelial cells

Excessive molybdenum (Mo) has adverse effects on animals. To elucidate the effects of autophagy on Mo-induced nephrotoxicity, the duck renal tubular epithelial cells were cultured in medium in absence and presence of (NH4)6Mo7O24.4H2O (0, 480, 720, 960 μM Mo), 3-Methyladenine (3-MA) (2.5 μM), and the combination of Mo and 3-MA for 12 h. After 12 h exposure, the MDC staining, morphologic observation, LC3 puncta, cell viability, autophagy-related genes mRNA and proteins levels, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, antioxidant indices, mitochondrial membrane potential (MMP), mitochondrial mass, mitochondrial respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) were determined. The results showed that excessive Mo exposure significantly elevated the number of autophagosome and LC3 puncta, upregulated Beclin-1, Atg5, LC3A and LC3B mRNA levels, and LC3II/LC3I and Beclin-1 protein levels, decreased mTOR, p62 and Dynein mRNA levels and p62 protein level. Besides, co-treatment with Mo and 3-MA dramatically increased LDH release, ROS level, hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents as well as cell dam age, reduced cell viability, the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), MMP, mitochondrial mass, mitochondrial RCR and OPR compared to treatment with Mo alone. Taken together, these results suggest that excessive Mo exposure can induce autophagy in duck renal tubular epithelial cells, inhibition of autophagy aggravates Mo-induced mitochondrial dysfunction by regulating oxidative stress.

The inhibitory effect of TU-100 on hepatic stellate cell activation in the tumor microenvironment.

Introduction: The tumor microenvironment is involved in acquiring tumor malignancies of colorectal liver metastasis (CRLM). We have reported that TU-100 (Daikenchuto) suppresses hepatic stellate cell (HSC) activation in obstructive jaundice. In this study, we report new findings as the direct and indirect inhibitory effects of TU-100 on cancer cell growth through the suppression of HSC activation.Materials and Methods: The HSCs (LX2) were cultured in colon cancer cells (HCT116 and HT29)-conditioned medium (CM) with or without TU-100 treatment (90, 270, 900 μg/ml). Activated HSCs (aHSCs) were detected by α-SMA and IL-6 mRNA expressions and cytokine arrays of HSC’s culture supernatants. Cancer cell growth was analyzed for proliferation and migration ability, compared with TU-100 treatment. To investigate the direct anti-tumor effect of TU-100, cancer cells were cultured in the presence of aHSC-CM and TU-100 (90, 270, 900) or aHSC-CM alone, and assessed autophagosomes, conversion to LC3-II protein, and Beclin-1 mRNA expression.Results: Colon cancer-CM significantly increased α-SMA and IL-6 mRNA expressions of aHSC. α-SMA and IL-6 mRNA expressions of aHSC, and IL-6 secretions from aHSCs were significantly decreased with TU-100 (270, 900) treatment, compared to colon cancer-CM alone. Compared with normal culture medium, aHSC-CM led to a significantly increased cell number and modified HSC-CM (TU-100; 270, 900) significantly suppressed cancer cell growth and migration. TU-100 (900) treatment induced autophagy and significantly promoted the autophagic cell death.Conclusions: TU-100 inhibited colon cancer cell malignant potential by both suppressing HSC activation and inducing directly autophagy of cancer cells.

Abnormal expression of autophagy-related proteins in immune thrombocytopenia.

Autophagy is a highly conserved protein degradation pathway that is essential for affecting some autoimmune diseases. Immune thrombocytopenia (ITP) is a common autoimmune disorder, and the complex dysregulation of cellular immunity has been observed; however, the relationship between autophagy-related proteins and immune responses in ITP remains unclear. Using real-time quantitative polymerase chain reaction (RT-PCR), the mRNA expression levels of Beclin-1, SQSTM1/p62 and LC3 were measured in the peripheral blood mononuclear cells (PBMCs) of 20 newly diagnosed patients with active ITP, 16 ITP patients in remission and 21 healthy volunteers. The stained Beclin-1 and SQSTM1/p62 proteins were also observed in the bone marrow of active ITP patients and normal controls by immunofluorescence. SQSTM1/p62 mRNA expression in PBMCs in newly diagnosed patients was significantly decreased. At the same time, Beclin-1 mRNA was increased significantly. During the remission stages, the levels of these autophagy-related proteins were comparable with those observed in healthy controls. Taken together, these results suggest that the aberrant expression of autophagy-related proteins might be involved in the pathogenesis of ITP. Further study of the autophagy pathway may provide a new strategy and direction for the treatment of ITP.

Protective Effects of Curcumin Against Paclitaxel-Induced Spinal Cord and Sciatic Nerve Injuries in Rats.

Paclitaxel (PTX) is an antineoplastic agent commonly used in the treatment of solid tumors and is known to cause dose-limiting peripheral neurotoxicity. This study was performed to evaluate the protective effect of curcumin (CUR) against PTX-induced spinal cord and sciatic nerve injuries in rats. The rats were administered PTX (2mg/kg, BW) intraperitoneally for the first 5 consecutive days followed by administration of CUR (100 and 200mg/kg, BW daily in corn oil) orally for 10days. Our results showed that CUR significantly reduced mRNA expression levels of NF-κB, TNF-α, IL-6, iNOS and GFAP whereas caused an increase in levels of Nrf2, HO-1 and NQO1 in the spinal cord and sciatic nerve of PTX-induced rats. In addition, CUR suppressed the activation of apoptotic and autophagic pathways by increasing Bcl-2 and Bcl-xL, and decreasing p53, caspase-3, Apaf-1, LC3A, LC3B and beclin-1 mRNA expression levels. The results showed that CUR also maintained the spinal cord and sciatic nerve histological architecture and integrity by both LFB staining and H&E staining. Immunohistochemical expressions of 8-OHdG, caspase-3 and LC3B in the PTX-induced spinal cord tissue were decreased after administration of CUR. Taken together, our findings demonstrated that CUR has protective effects on PTX-induced spinal cord and sciatic nerve injuries in rats.

The possible effect of coenzyme Q10 and captopril on acetaminophen-induced encephalopathy in rats: possible influence on autophagy, antioxidant and Na+/K+ ATPase

IntroductionInduction of autophagy could protect against acetaminophen (APAP)-induced hepatotoxicity; however, little is known about the role of autophagy in APAP-induced encephalopathy (APAP-E). This study aimed to evaluate the effects of coenzyme Q10 (CoQ10) and captopril on APAP-E.Material and methodsForty-eight rats were randomly allotted to 4 equal groups: control, an APAP-E, coenzyme Q10-treated (CoQ10-treated), and captopril-treated groups. Behavioral tests were conducted. Serum ammonia and total antioxidant capacity (TAC) and hippocampal Na+/K+ ATPase activity were measured. The expression levels of hippocampal microtubule-associated protein light chain 3 (LC3-II) and beclin-1 mRNA were detected using quantitative polymerase chain reaction (qPCR). General histological, immunohistochemical staining for glial fibrillary acid protein (GFAP) and electron microscopy (EM) of the hippocampus were performed.ResultsIn the APAP-E group, serum ammonia was increased significantly, hippocampal LC3-II and beclin-1 mRNA were elevated insignificantly, while serum TAC and the activity of hippocampal Na+/K+ ATPase were reduced significantly compared with the control group. APAP-E rats showed remarkable degenerative changes in CA1 pyramidal neurons in the form of electron-dense cytoplasm with ill-defined nuclei and accumulation of lysosomal structure-like dense bodies. Increased immunoreactivity of astrocytes for GFAP was observed. Treatment with either CoQ10 or captopril significantly reduced ammonia levels, increased hippocampal LC3-II and beclin-1 mRNA, increased serum TAC and Na+/K+ ATPase activity, and noticeably ameliorated the hippocampal neuronal changes. EM revealed restoration of the normal structure of pyramidal neurons. These effects were more obvious in CoQ10-treated than captopril-treated rats.ConclusionsCoQ10 and captopril have neuroprotective effects on APAP-E via enhancing LC3-II, beclin-1 mRNA expression, serum TAC level and hippocampal Na+/K+ ATPase activity.

Effects of mild moxibustion on Beclin-1/Bcl-2 expression in spinal cord of rats with cervical spondylotic radiculopathy

To observe the effects of mild moxibustion on the expression of autophagy and apoptosis factors Beclin-1, Bcl-2 mRNA and protein in spinal cord (including nerve root tissues) of cervical spondylotic radiculopathy (CSR) rats, so as to explore the analgesic mechanism of mild moxibustion at "Dazhui" (GV14) on CSR. SD rats were randomly divided into blank group, model group, mild moxibustion group and mild moxibustion+3-methyladenine(3-MA) group, with 10 rats in each group. CSR model was established by inserting the wire into the cervical nerve root. The rats in the blank group were only fed normally without any intervention.The rats in the mild moxibustion group and mild moxibustion+3-MA group were given mild moxibustion at GV14 for 10 min each time,and intraperitoneal injection of 1 mL 0.9% normal saline and 1 mL 3-MA(15 mg/kg)separately. Rats in the model group were given 0.9% normal saline every day. All the three interventions were started from the 3rd day after modeling for 7 days. The rat's behavioral reaction of gait was scored and the pain threshold of rat was measured with a pain analyzer; the expressions of Beclin-1 and Bcl-2 mRNA and protein in the spinal cord (including nerve root) were detected by fluorescence quantitative PCR and immunohistochemistry, separately. The autophagosome and ultrastructure of the spinal nerve root tissue were observed by transmission electron microscope. After modeling, the gait score was significantly increased (P<0.01) and the pain threshold significantly decreased (P<0.01) in the model group in comparison with the blank group. There was no statistical difference in Beclin-1 and Bcl-2 mRNA and protein expression between the blank and the model groups. After intervention, compared with the model group, the gait scores were significantly reduced (P<0.01), the pain threshold and the expressions of Beclin-1 and Bcl-2 mRNA and protein were significantly increased (P<0.01，P<0.05) in the mild moxibustion and mild moxibustion+3-MA groups. The improvement of the above indicators as more significant in the mild moxibustion group than that in the mild moxibustion+3-MA group (P<0.05). After modeling, the organelles in the spinal nerve root tissue cells of the model group were damaged and there were a small amount of autophagosomes. Compared with the model group, the ultrastructure of the spinal nerve root tissue cells in the mild moxibustion group were relatively complete, and the number of autophagosomes increased. Mild moxibustion at GV14 has a good analgesic effect on CSR rats, which may be related to its effects in up-regulation of Beclin-1 and Bcl-2 expressions and activation of autophagy and inhibition of apoptosis.

Autophagy dysfunction may be involved in the pathogenesis of ankylosing spondylitis.

The present study aimed to investigate the expression and significance of the mRNA of genes associated with autophagy and long non-coding RNA (lncRNA) GAS5 in peripheral blood mononuclear cells (PBMCs) of patients with ankylosing spondylitis (AS). The mRNA levels of microtubule-associated protein light chain 3 (LC3), Beclin1, autophagy-related gene (ATG)3, ATG5, ATG12, ATG 16 ligand 1 (ATG16L1) and lncRNA growth arrest-specific 5 (GAS5) in PBMCs from 60 patients with AS and 30 healthy controls (HC) were examined by reverse transcription-quantitative PCR. The correlations between the levels of LC3, Beclin1, ATG3, ATG5, ATG12 and ATG16L1 mRNA as well as lncRNA GAS5 levels with disease activity and laboratory parameters in patients with AS were determined by Spearman correlation analysis. In addition, the diagnostic value of lncRNA GAS5 for AS was explored through establishing a receiver operating characteristic (ROC) curve. The results indicated that, compared to the HCs, patients with AS had lower expression levels of LC3, ATG5, ATG12, ATG16L1 and lncRNA GAS5 in their PBMCs. Compared with those in patients with inactive AS, the levels of ATG5 and ATG12 were lower than those in patients with active AS. Of note, ATG5 and ATG12 mRNA levels were negatively correlated with disease activity indexes. lncRNA GAS5 was positively correlated with the expression of Beclin1, ATG3, ATG5, ATG12 and ATG16L1. The area under the ROC curve for the use of lncRNA GAS5 expression to diagnose AS was 0.808 with a 95% CI of 0.714-0.902. In conclusion, patients with AS had decreased expression of genes associated with autophagy and lncRNA GAS5. The extent of the reduction in ATG5 and ATG12 expression levels in patients with AS was correlated with the disease severity and activity. Furthermore, lncRNA GAS5 was a diagnostic indicator of AS.

Comparison of Autophagy mRNA Expression between Chronic Otitis Media With and Without Cholesteatoma.

Background and ObjectivesAutophagy is known to be associated with pathogen infection. However, the expression of autophagy-related proteins has not been studied in chronic otitis media without cholesteatoma (COM) or with cholesteatoma (CholeOM). This study aimed to determine whether there is a difference between COM and CholeOM in autophagy-related gene mRNA expression. Subjects and MethodsFor 47 patients with chronic otitis media, the inflammatory tissues were classified into granulation tissue (COM) or cholesteatoma (CholeOM) according to biopsy results. ResultsPI3K mRNA expression (COM vs. CholeOM, mean±SD, 0.009±0.010 vs. 0.003±0.004; p=0.004) was lower, whereas Beclin-1 mRNA expression (0.089±0.107 vs. 0.176±0.163; p=0.034) was higher in the CholeOM group. Expression of PI3K mRNA in the CholeOM group was lower than that in the COM subgroups with presence of bacteria (0.022±0.019 vs. 0.001±0.001; p=0.001), otorrhea (0.049±0.068 vs. 0.003±0.004; p=0.004), and hearing loss over 40 dB (0.083±0.130 vs. 0.003±0.004; p=0.005). ConclusionsThe data suggested that different autophagy proteins play important roles in chronic otitis media according to the presence or absence of cholesteatoma.

Decreased expression of autophagy markers in culture-positive patients with chronic otitis media.

ObjectiveAbnormal autophagy plays a role in the pathogenesis of various diseases. This study aimed to evaluate associations between the clinical manifestations of chronic otitis media (COM) and expression of autophagy markers.MethodsAssociations between presence of bacteria, otorrhea, and conductive and sensorineural hearing loss and levels of autophagy-related mRNAs were investigated in 47 patients with COM.ResultsAutophagy-related mRNAs were detected in all inflammatory tissues of COM patients. LC3-II showed the highest level of expression, followed by Beclin-1, P13KC3, Rubicon, and mTOR. Beclin-1 mRNA levels were significantly lower in culture-positive than in culture-negative patients.ConclusionAutophagy is involved in the pathogenesis of COM. The finding that expression of autophagy markers, especially Beclin-1, was lower in culture-positive than in culture-negative patients suggested that these markers are closely associated with the clinical features of COM.

Dynamic Changes of Beclin-1 in the Hippocampus of Male Mice with Vascular Dementia at Different Time Points.

This study tried to investigate the dynamic changes of Beclin-1 in the hippocampus of male mice with vascular dementia (VD) at different time points. The mouse model of VD was established by the four-vessel blocking method. Then, the VD mice were randomly divided into five groups (n = 12) according to the disease duration: the 0.1-day model group, 0.5-day model group, 1-day model group, 3-day model group, 5-day model group and 14-day model group. In addition, all surgical procedures were the same in the sham group as those in the model groups, except the mice in the sham group were not subjected to clipping. The expression of Beclin-1, LC3B, p62, Bcl-2, Bax, BACE1, GFAP, MBP and ET-1 mRNA were determined by RT-PCR; the expression of Beclin-1 was detected by Western blot and immunofluorescence; the pathological characteristics of the hippocampus were observed by haematoxylin-eosin (HE) staining; and the correlation of Beclin-1 with other VD-related proteins was analysed by Pearson's correlation. Compared with that in the sham group, the expression of Beclin-1, LC3B, Bax, BACE1, GFAP, MBP and ET-1 mRNA was increased in the VD mice at different time points (0.1day, 0.5day, 1day, 3days, 5days and 14days) (P < 0.05) and then remained relatively stable in the 0.5-day VD mice, whereas the p62 and Bcl-2 mRNA levels decreased (P < 0.05). Beclin-1 protein expression was significantly increased in the VD mice at different time points (P < 0.05). The hippocampus showed a certain degree of neuronal damage in the VD mice at different time points (P < 0.05). Additionally, certain correlations among LC3B, p62, Bcl-2, Bax, BACE1, GFAP, MBP, ET-1 and Beclin-1 were observed in this study. In conclusion, the results described above demonstrated that neuronal damage and dynamic stability of Beclin-1 expression were established in the VD male mice after 0.5day by the four-vessel blocking method.

Cross talk between alcohol-induced oxidative stress and HCV replication.

Alcohol consumption exacerbates the pathogenesis of hepatitis C virus (HCV) infection and aggravates disease consequences in alcohol-abusing patients. Although the exact reasons by which alcohol consumption affects several cellular pathways in liver cells are not clear, they might be partially attributed to the ability of alcohol to further suppress the innate immunity, modulation of autophagy and also its relationship with reactive oxygen species (ROS) generation. To evaluate these issues, Huh7 cells harboring HCV replicon and Cytochrome p450 (CYP2E1) plasmid were exposed to ethanol and mRNA expression of Beclin-1, interferon-stimulated gene15 (ISG15) genesand HCV NS5B for two different times were relatively quantitated. ROS was determined by flow cytometry. The results showed that alcohol treatment in a short time caused an increase in HCV NS5B and Beclin-1 mRNA and decreased ISG 15 mRNA. Long-lasting alcohol treatment increased ROS production in Huh-7 cells and HCV replication was reduced. In conclusion, acute alcohol treatment might contribute to increase HCV replication by interference in innate immunity and induction of autophagy. Chronic alcohol treatment caused oxidative stress, which disrupts autophagy and thereby increased the rate of Huh7 cell injury.

Activation of Autophagy in Human Uterine Myometrium During Labor

Objective: The purpose of this study was to analyze the autophagy of the human uterine myometrium during the labor. Methods: We collected uterine myometrium strips from term, singleton, nulliparous healthy women undergoing cesarean delivery before labor (nonlabor group, n = 10) or during normal labor (in-labor group, n = 10) without rupturing of membrane. The indications for cesarean delivery were breech presentation or maternal request. Transmission electron microscopy was used to observe autophagosomes. Reverse transcriptase polymerase chain reaction, immunofluorescence, and Western blot were used to quantify the messenger RNA (mRNA) and protein level of the autophagy markers LC3B, P62, and Beclin-1 in the uterine muscle strips. Results: There were no differences between both groups in maternal age, body mass index, gestational week, neonatal weight, operative bleeding, and postpartum bleeding. Transmission electron micrographs showed that autophagosomes existed in myometrial tissue in both groups. There were more autophagosomes in the in-labor group than in the nonlabor group, and the difference had significance. The in-labor group had significantly greater LC3B mRNA expression but significantly lower P62 mRNA expression compared with the nonlabor group. Semiquantitative immunofluorescence in uterine myometrial cells in the in-labor group showed increased LC3B puncta formation and greater Beclin-1 expression but reduced P62 puncta formation compared with the nonlabor group. The ratio of LC3BII/I proteins was significantly higher, but P62 protein was significantly lower in the in-labor group compared with the nonlabor group. The Beclin-1 mRNA and protein expressions were not significantly different between the 2 groups. Conclusion: Autophagy was activated in human uterine myometrium during labor and might play an important role in maintaining uterine contraction function.

Lentiviral-Mediated Beclin-1 Overexpression Inhibits Cell Proliferation and Induces Autophagy of Human Esophageal Carcinoma Eca109 Cell Xenograft in Nude Mice.

Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.