ObjectiveTo investigate the role of p22phox and NOX5 in autophagy and apoptosis of osteoblasts induced by hypoxia.MethodsThe skull tissue of newborn rats was cut into small pieces, and the osteoblasts were separated and purified by the tissue block adherent method and the differential adherent method. The first generation cells were harvested and identified by HE staining, Alizarin red staining, alkaline phosphatase (ALP) staining, and flow cytometry. A three-gas incubator was used to prepare a hypoxia model of osteoblasts. At 0, 3, 6, 12, and 24 hours of hypoxia, the expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ were detected by Western blot, and the level of reactive oxygen species (ROS) and cell apoptosis rate were detected by flow cytometry. And the time point of the highest level of ROS was selected as the hypoxia time point for subsequent experiments. The first generation osteoblasts were divided into normal group, si-p22phox hypoxia group, and si-NOX5 hypoxia group and subjected to corresponding transfection and hypoxia treatment. The inhibition efficiency of si-p22phox and si-NOX5 were detected by RT-PCR. Then the osteoblasts were divided into normal group, si-NC hypoxia group, si-p22phox hypoxia group, and si-NOX5 hypoxia group. After transfection and hypoxia treatment, Western blot was used to detect the expressions of p22phox, NOX5, autophagy-related proteins (LC3Ⅱ/Ⅰ, Beclin), and apoptosis-related proteins (Bcl-2, Bax), and flow cytometry was used to detect the cell apoptosis rate and level of ROS. The first generation osteoblasts were divided into a hypoxia group for 12 hours (hypoxia group) and a group that simultaneously inhibited si-p22phox and si-NOX5 and hypoxia for 12 hours (inhibition+hypoxia group). The expressions of Beclin and Bax were observed by immunofluorescence staining after the corresponding treatment.ResultsAfter identification, the isolated cells were osteoblasts. After hypoxia treatment, the relative expressions of p22phox, NOX5, and LC3Ⅱ/Ⅰ proteins and the apoptosis rate of osteoblasts gradually increased (P<0.05), and the level of ROS also significantly increased (P<0.05) and reached the peak value at 12 hours. The 12-hour hypoxia model was selected for subsequent experiments. Silencing the p22phox gene did not affect the expression of NOX5, and silencing the NOX5 gene did not affect the expression of p22phox. Compared with hypoxia treatment, the relative expressions of LC3Ⅱ/Ⅰ, Beclin, and Bax proteins after inhibiting the expression of p22phox or NOX5 gene significantly decreased (P<0.05), the relative expression of Bcl-2 protein significantly increased (P<0.05), the cell apoptosis rate and level of ROS also significantly decreased (P<0.05). After silencing the expressions of p22phox and NOX5 genes at the same time, the immunofluorescence staining showed that the fluorescence of Beclin and Bax were weak.ConclusionInhibiting the expressions of p22phox and NOX5 genes can reduce the level of ROS in osteoblasts under hypoxia-induced conditions, and at the same time reduce autophagy and apoptosis, especially attenuate the excessive apoptosis of cells in the early to late stages, and strengthen the hypoxic osteoblasts proliferation.
Age-related macular degeneration is one of the major causes of blindness in the elderly. As an important pathway of cell metabolism, autophagy maintains intracellular homeostasis through the degradation and recycle of damaged organelles and macromolecules. Understanding its mechanism may promote discoveries to delay aging process, reduce the incidence of age-related diseases. In mammals, silent information regulator protein 6 (SIRT6) plays its deacetylase and ribonucleotransferase activity in multiple signaling pathways, including inhibition of cellular senescence, tumorigenesis, metabolic diseases, regulating cellular lifespan. It has a significant impact on the structure and function of tissues and organs. SIRT6 regulates intracellular autophagy mainly through the insulin-like growth factor-protein kinase B-mammalian target of rapamycin, reducing the accumulation of toxic metabolites and cellular senescence. The function of SIRT6 in age-related macular degeneration need to be combined with the genetic background, pathogenesis, clinical manifestations and other aspects of the disease, and it is expected to be further studied in subsequent studies.
Autophagy is a programmed cell degradation process that is involved in a variety of physiological and pathological processes including malignant tumors. Abnormal induction of autophagy plays a key role in the development of hepatocellular carcinoma (HCC). We established a prognosis prediction model for hepatocellular carcinoma based on autophagy related genes. Two hundred and four differentially expressed autophagy related genes and basic information and clinical characteristics of 377 registered hepatocellular carcinoma patients were retrieved from the cancer genome atlas database. Cox risk regression analysis was used to identify autophagy-related genes associated with survival, and a prognostic model was constructed based on this. A total of 64 differentially expressed autophagy related genes were identified in hepatocellular carcinoma patients. Five risk factors related to the prognosis of hepatocellular carcinoma patients were determined by univariate and multivariate Cox regression analysis, including TMEM74, BIRC5, SQSTM1, CAPN10 and HSPB8. Age, gender, tumor grade and stage, and risk score were included as variables in multivariate Cox regression analysis. The results showed that risk score was an independent prognostic risk factor for patients with hepatocellular carcinoma (HR = 1.475, 95% CI = 1.280–1.699, P < 0.001). In addition, the area under the curve of the prognostic risk model was 0.739, indicating that the model had a high accuracy in predicting the prognosis of hepatocellular carcinoma. The results suggest that the new prognostic risk model for hepatocellular carcinoma, established by combining the molecular characteristics and clinical parameters of patients, can effectively predict the prognosis of patients.
Objective To explore the relationship between Beclin-1 and the development of pancreatic ductal adenocarcinoma (PDAC). Methods ① Twenty-five PDAC specimens and 20 matched adjacent normal pancreatic tissues were obtained after radical surgery between April 2009 and November 2009 in West China Hospital of Sichuan University. Beclin-1 mRNA and protein expressions were examined by using real-time PCR and immunohistochemistry, respectively. Correlations between expressions of Beclin-1 protein with clinical data of PDAC patients were evaluated. ② PDAC cells were divided into 2 groups, cells of transfection group were transfected with PLenO-WPI-Beclin-1 vector, and cells of non-transfection group didn’t transfected with PLenO-WPI-Beclin-1 vector. Expressions levels of Beclin-1 mRNA in the 2 groups were detected by real-time PCR at 24 hours and 48 hours after transfection. ③ PDAC cells were divided into 3 groups, cells of transfection group were transfected with PLenO-WPI-Beclin-1 vector, cells of empty vector group transfected with PLenO-WPI, cells of blank control group didn’t accepted any vector. OD value was detected by MTT once a day during 1–7 days after transfection. Results ① Expression levels of Beclin-1 mRNA and its protein were significantly lower in PDAC tissue than those of adjacent normal pancreatic tissues (P<0.05). Increased Beclin-1 expression was associated with early TNM stage of Ⅰ and Ⅱ(P<0.05) and negative distant metastasis (P=0.011). ② At the same time point of 24 hours and 48 hours after transfection, the expression levels of Beclin-1 mRNA were higher in transfection group than those of non-transfection group (P<0.05). ③ MTT assay showed that PANC-1 cell proliferation ability was lower in the transfection group compared to the blank control group and empty vector groups in vitro on day 4–7 after transfection (P<0.05), but there was no significant in the cell proliferation ability among the 3 groups on day 1, 2, and 3 (P>0.05). Conclusions Down regulation of Beclin-1 and autophagy inhibition play an important role in the tumorigenesis and development of PDAC. Activating autophagy via overexpression of Beclin-1 may be a potential treatment for some PDACs and warrants further investigation.
ObjectiveTo investigate the expression of autophagy-related genes and proteins in the lung tissues of patients with non-small cell lung cancer (NSCLC).MethodsPulmonary tissues were obtained from the surgically resected lung tissues of patients with NSCLC who were clinical diagnosed. The lung cancer tissues were derived from the pathologically diagnosed NSCLC and the normal tissues were from lung tissues 5 cm away from the lung lesions (29 cases in the lung cancer group and 32 cases in the normal group). The expression of autophagy-related proteins ATG5, LC3B, and p62 in lung tissues were measured by Western blot, and mRNA expression of ATG5 and p62 in the lung tissues were measured by real-time PCR.ResultsWestern blot analysis showed that the expression of ATG5 and p62 in lung cancer group were significantly higher than those in normal group (P<0.05). However, the expression of LC3B in lung cancer group was significantly lower than that in normal group (P<0.05). Real-time PCR analysis found that the mRNA expression of ATG5 and p62 in lung cancer group were significantly higher than those in normal group (P<0.05). The expression of ATG5, LC3B and p62 had no relationship with gender, age, smoking history, tumor location, tumor size, clinicopathological classification, differentiation or TNM stage. The expression of ATG5 had statistical significance in lymph node metastasis (P<0.05), but there was no difference for LC3B or p62 in lymph node metastasis (P>0.05).ConclusionsAutophagy plays a role in the tumorigenesis of lung cancer. If it’s possible to regulate and control autophagy-related genes and proteins effectively, it may supply new insights or targets into treatment for lung cancer patients.
Hypoxia inducible factor-1 (HIF-1) is the main transcription factor and the core regulator for cells to adapt to hypoxia, and oxygen homeostasis is achieved by controlling and utilizing oxygen delivery. Autophagy and apoptosis play an important role in determining cell fate and maintaining cell homeostasis. In recent years, it has been found that the dynamic change of HIF-1 expression plays a key role in the hypoxic adaptive response of cardiomyocytes. The regulation of HIF-1 on autophagy and apoptosis of hypoxic cardiomyocytes determines the survival of cardiomyocytes, which is of great significance for the prognosis of ischemic heart disease.
The neuroretinal injuries of diabetic retinopathy (DR) include retinal neuronal damage and reactive gliosis, both of which are induced by hyperglycemia and presented as early features of DR. They promote to develop mutually and accelerate the progression of DR. The molecular mechanisms study of neuronal damage mainly focuses on the alterations of extracellular environment and related signaling pathways, include inflammation, oxidative stress, endoplasmic reticulum stress, the formation of advanced glycation end products, glutamate toxicity and so on. These alterations mainly result in neuronal apoptosis and autophagy. The damaged neurons activate the glial cells with apparent changes in morphology, cell counts and the level of intracellular protein expression. In non-proliferative DR, glial cells are moderately hypertrophic and slightly increased in numbers. In proliferative DR, there is a significant rise in glial cell number with enhanced level of inflammatory factors and vascular active substances which lead a further neuronal damage. Signaling pathways of extracellular signal-regulated kinase 1/2, c-Fos and p38 mitogen-activated protein kinase are associated with their activation. Researches on the molecular mechanisms and signaling pathways of the DR will promote controlling the DR progression at the cellular level.
ObjectiveTo systematically evaluate relationship between expression of autophagy-related protein Beclin-1 in gastric cancer and its clinicopathologic features and its clinical significances.MethodsThe researches on the expression and significance of Beclin-1 protein in the gastric tumor tissues published from the database establishment to June 1, 2018 in the Cochrane Library, Springer Link, Web of Science, Embase, PubMed, CNKI, Wanfang, VIP, and other databases were searched. Two researchers independently screened and evaluated the literatures, extracted the relevant data, and conducted the meta-analysis using the Review Manager 5.3 and Stata 15.0 software.ResultsFinally, 10 articles were included, and there were 1 402 patients with gastric cancer. The meta-analysis showed that the positive rate of Beclin1 protein expression in the gastric cancer tissues was significantly lower than that in the non-gastric cancer tissues [OR=0.30, 95% CI (0.13, 0.72), P=0.007], which in the patients with TNM stage Ⅲ/Ⅳ and distant metastatic gastric cancer were significantly lower than those in the patients with stage Ⅰ/Ⅱ [OR=1.82, 95% CI (1.03, 3.20), P=0.04] and without distant metastasis [OR=0.36, 95% CI (0.20, 0.63), P=0.000 4], which were not associated with the gender, age, tumor size, lymph node metastasis, serosa invasion, and tumor differentiation degree of gastric cancer patients (P>0.05). For the studies of existed heterogeneity, further the subgroup analysis showed that the positive expression rate of Beclin-1 protein in the gastric cancer tissues was significantly lower than that in the non-gastric cancer tissues [OR=0.19, 95% CI (0.13, 0.29), P<0.000 01], which in the patients with lymph node metastasis, invasion of serosa, and poorly differentiated gastric cancer were significantly lower than those in the non-lymph node metastasis [OR=0.35, 95% CI (0.22, 0.57), P<0.000 1], non-invasion of serosa [OR=0.56, 95% CI (0.33, 0.94), P=0.03], and moderately/highly differentiated gastric cancer tissues [OR=0.29, 95% CI (0.20, 0.43), P<0.000 01].ConclusionsLow expression of Beclin-1 in gastric cancer tissues is related to stage and distant metastasis of gastric cancer. It is suggested that it might not only be an important cause of gastric cancer, but also play a regulatory role in progress of gastric cancer.
Autophagy is a lysosome dependent, conservative material degradation process, which exists in all eukaryotic cells and plays import roles in many pathophysiology process. Erectile dysfunction (ED) is a common male disease with multiple etiology. In recent years, more and more evidences have demonstrated that autophagy has a close relation to ED, therefore, we combine previous study to classify ED by hypoxia, aging, diabetes and other causes, and review the advances of autophagy in ED.