The expression of autophagy-related proteins in peripheral blood mononuclear cells of patients with lupus nephritis is upregulated and related to kidney damage / 细胞与分子免疫学杂志

Objective To identify the relationship between nephritis activity, autophagy and inflammation in patients with SLE. Methods Western blot analysis was used to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) and P62 in peripheral blood mononuclear cells (PBMCs) of SLE patients with lupus nephritis and non-lupus nephritis patients. Tumor necrosis factor α (TNF-α) and interferon γ (IFN-γ) in the serum of SLE patients were determined by ELISA. The correlation between LC3II/LC3I ratio and SLE disease activity score (SLEDAI), urinary protein, TNF-α and IFN-γ levels was analyzed by Pearson method. Results The expression of LC3 was increased and P62 was decreased in SLE patients. TNF-α and IFN-γ were increased in the serum of SLE patients. LC3II/LC3I ratio was positively correlated with SLEDAI (r=0.4560), 24 hour urine protein (r=0.3753), IFN-γ (r=0.5685), but had no correlation with TNF-α (r=0.04 683). Conclusion Autophagy is found in PBMCs of SLE, and the autophagy is correlated with renal damage and inflammation in patients with lupus nephritis.

Variation analysis of EPG5 gene in a Vici syndrome family / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic etiology of Vici syndrome in a Chinese family.@*METHODS@#Whole exome sequencing (WES) technology was used to detect gene variants in a fetus of abnormal ultrasonic structure without abnormalities in routine chromosome karyotype analysis and SNP-array. Sanger sequencing and bioinformatics prediction were performed for the suspected variants of the fetus and parents.@*RESULTS@#The fetus and the elder sister have carried c. 2427delC (p.T809fs) and c.1886A>T (p.E629V) compound heterozygous variants of the EPG5 gene, which were respectively inherited from their mother and father. Neither variant was reported previously. According to ACMG guidelines, the c.2427delC variant was predicted as pathogenic, while the c.1886A>T variant was of uncertain significance. PolyPhen-2 and PROVEAN software indicated that c.1886A>T variant was probably damaging.@*CONCLUSION@#The c.2427delC and c.1886A>T variants of the EPG5 gene probably underlie the pathogenesis of the Vici syndrome in this family. Above finding has enriched the variational spectrum of EPG5 gene and provided a basis for genetic counseling and prenatal diagnosis for the family.

Melatonin inhibits growth and metastasis of MDA-MB-231 breast cancer cells by activating autophagy / 南方医科大学学报

OBJECTIVE@#To investigate the effects of melatonin on the growth and metastasis of MDA-MB-231 breast cancer cells and explore the mechanism.@*METHODS@#MDA-MB-231 cells were treated with 1, 3 or 5 mmol/L melatonin, and the changes in cell proliferation were examined using CCK-8 assay. Colony-forming assay and wound healing assay were used to assess the effects of melatonin treatmnent on colony-forming ability and migration of the cells. Flow cytometry and immunofluoresnce assay were employed to examine apoptosis and positive staining for autophagy-related proteins in the cells treated with 3 mmol/L melatonin. The effects of melatonin treatment alone or in combination with 3-methyladenine (3-MA) on the expressions of the proteins associated with autophagy (LC3, P62 and Beclin1), apoptosis (Bcl2 and Bax) and epithelial-mesenchymal transition (E-cadherin and Snail) were examined with Western blotting.@*RESULTS@#Melatonin treatment significantly inhibited the proliferation of breast cancer cells in a concentration- and time-dependent manner (P < 0.05), suppressed colony-forming ability and migration (P < 0.01), and promoted apoptosis of the cells (P < 0.01). Melatonin treatment alone significantly increased the expressions of Bax (P < 0.05), E-cadherin, LC3-II/LC3-I, and Beclin1 and lowered the expressions of Bcl2 (P < 0.05), Snail, P62 (P < 0.05), and Bcl2/Bax ratio (P < 0.01) in the cells, and caused enhanced positive staining of Beclin1 protein and attenuated staining of P62 protein. Compared with melatonin treatment alone, melatonin treatment combined with 3-MA significantly decreased the expressions of Beclin1 (P < 0.001), LC3-II/LC3-I (P < 0.05), Bax (P < 0.01), and E-cadherin (P < 0.001) and increased the expressions of Bcl2 (P < 0.05), Snail, and Bcl2/Bax ratio (P < 0.01).@*CONCLUSION@#Melatonin can induce autophagy of MDA-MB-231 breast cancer cells to inhibit cell proliferation and metastasis and promote cell apoptosis, and suppressing autophagy can weaken the inhibitory effect of melatonin on the growth and metastasis of breast cancer cells.

Effects of Zhongfeng capsule on autophagy related proteins expressions of brain tissue in rats with cerebral ischemia/reperfusion injury / 中国应用生理学杂志

Objective: To investigate the effects of Zhongfeng capsule on the autophagy-related proteins expression in rats with cerebral ischemia/reperfusion injury (CI/ RI), and to explore its neural protection mechanisms of the decoction. Methods: Rat middle cerebral artery ischemia/reperfusion injury model (ischemia for 2 h, reperfusion for 24 h) was prepared by the improved line plug method. Sixty male SD rats were randomly divided into sham operation group, model group, butylphthalide group(0.054 g/kg), Zhongfeng capsule high-dose groups (1.08 g/kg), Zhongfeng capsule middle-dose groups (0.54 g/kg), Zhongfeng capsule low-dose groups (0.27 g/kg), with 10 rats in each group. Rats were treated with Zhongfeng capsule by gavage once a day for 10 days. The rats were sacrificed and the brain tissue was obtained after the experiment in each group. Score neurological deficit was evaluated after 24 h of the last intervention in rat of each group. The pathological changes of brain tissue were observed by HE staining. The serum levels of estradiol (E2) and follicle stimulating hormone (FSH) were determined by ELISA. The expressions of key genes and proteins of PI3K/Akt/Beclin1 signaling pathway in brain tissue were detected by qRT-PCR and Western blot respectively. Results: Compared with the sham operation group, the body weight and protein expressions of p-PI3k and p-Akt in brain tissue of rats were decreased significantly in the model group, while the brain index, neurological deficit score, gene and protein expressions of Beclin1 and LC3 were increased markedly in the model group(P＜0.05 or P＜0.01). In the model group, nerve cells of brain tissue were loosely packed, interstitial edema, triangular in shape, nuclear pyknosis and dark-blue staining were observed. Compared with the model group, the body weight of rats was increased obviously, the neurological deficit score was decreased significantly and the pathological injury of brain tissue was alleviated evidently in high-dose of Zhongfeng capsule group (P＜0.05). The brain index, the gene and protein expressions of Beclin1 and LC3 were decreased apparently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01), while the expressions of p-PI3k and p-Akt in brain tissue were increased evidently in Zhongfeng capsule treatment groups(P＜0.05 or P＜0.01). Conclusion: Zhongfeng capsule can inhibit autophagy and improve brain neurons lesion of CIRI rats, the mechanism may be related to regulate the expression of Beclin1 and LC3 in PI3K/Akt/Beclin1 signaling pathway.

Overexpression of autophagy-related gene 3 promotes autophagy and inhibits salinomycin-induced apoptosis in breast cancer MCF-7 cells / 南方医科大学学报

OBJECTIVE@#To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms.@*METHODS@#We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor).@*RESULTS@#In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis.@*CONCLUSIONS@#ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.

Sodium valprovate suppresses autophagy in SH-SY5Y cells activating miR-34c-5p/ATG4B signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effect of sodium valproate (VPA) on activation of miR-34c-5p/ATG4B signaling pathway and autophagy in SH-SY5Y cells.@*METHODS@#Routinely cultured SH-SY5Y cells were treated with VPA at different doses for 24 h, and the changes in the mRNA levels of ATG4B and miR-34c-5p and the protein expression of ATG4B were assessed using qRTPCR and immunoblotting, respectively. The effect of transfection with a plasmid containing ATG4B promoter on the promoter activity of ATG4B in VPA-treated SH-SY5Y cells was assessed using the reporter gene assay. The stability of ATG4B mRNA was analyzed with qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with the transcription inhibitor actinomycin D. The expression level of miR-34c-5p was detected using qPCR in SH-SY5Y cells treated with VPA alone or with VPA combined with miR-34c-5p mimics or antagonist, and the role of miR-34c-5p in VPA-induced ATG4B down-regulation was evaluated. The changes in the level of autophagy were evaluated by detecting LC3-Ⅱ expression in the cells after treatment with VPA or VPA combined with miR-34c-5p antagonist.@*RESULTS@#VPA dose-dependently down-regulated the expression of ATG4B at both the mRNA and protein levels in SH-SY5Y cells. VPA treatment did not significantly affect the promoter activity of ATG4B, but obviously lowered the mRNA stability of ATG4B in SH-SY5Y cells. VPA treatment up-regulated the expression of miR-34c-5p, and the miR-34c-5p antagonist reversed VPA-induced down-regulation of ATG4B in SH-SY5Y cells. VPA also down-regulated the expression level of LC3-Ⅱ in SH-SY5Y cells.@*CONCLUSIONS@#VPA suppresses autophagy in SH-SY5Y cells possibly via activating miR-34c-5p/ATG4B signaling pathway.

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

Correlation of the autophagosome gene ATG16L1 polymorphism and inflammatory bowel disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To understand the relationship between the susceptibility to inflammatory bowel disease (IBD) and ATG16L1 gene single nucleotide polymorphism (SNP) site, rs2241880.</p><p><b>METHODS</b>Peripheral blood samples were collected from 80 IBD patients (including 40 with Crohn's disease and 40 with ulcerative colitis) and 50 healthy controls, and the genomic DNA was extracted from the white blood cells. Specific primers were designed according to the target gene sequence for PCR amplification of the target gene fragment, and the PCR products were purified followed by sequence analysis of the target region of ATG16L1 gene. The results of the sequence analysis were compared with the BenBank data to analyze the relationship between the allele gene polymorphisms and the susceptibility to Crohn's disease.</p><p><b>RESULTS</b>No significant differences were noted in the ATG16L1 gene SNP site rs2241880 polymorphisms among the patients with Crohn's disease, ulcerative colitis and the control subjects (Chi(2)=4.94, P=0.293).</p><p><b>CONCLUSION</b>ATG16L1 gene polymorphisms in the SNP site rs2241880 are not found to correlate to the susceptibility to Crohn's disease as reported in literature. The SNP site associated with Crohn's disease susceptibility identified in foreign populations does not seem to be identical with that in Chinese population.</p>