Search

Aberrant upregulation and oncogenic roles of UBE2T are revealed in several cancers. However, the expression, clinical significance, and functions of UBE2T have not been explored in ovarian cancer (OC). In this study, the expression of UBE2T and its relation with clinicopathological features and prognosis of OC patients were explored by analyzing online data and experimental data. Besides, the functions of UBE2T in OC cells were investigated by in vitro experiments, including CCK-8, plate clone formation, and Transwell assays. Finally, the underlying mechanism of UBE2T associated functions in OC was analyzed. The results indicated that UBE2T was significantly upregulated in OC tissues. UBE2T expression was notably correlated with clinical features, such as primary T stage and FIGO stage in OC patients. UBE2T, acting as an independent prognostic indicator, was inversely associated with the prognosis of OC patients. The UBE2T knockdown remarkably suppressed the growth, proliferation, and invasion of OC cells, indicated by impaired cell viability, fewer cell clones, and invasive cells. Mechanistically, UBE2T depletion suppressed epithelial-mesenchymal transition (EMT), which was caused by autophagy activation due to inactivation of AKT/mTOR in OC cells with UBE2T knockdown. Collectively, our findings confirm that UBE2T upregulation predicts poor prognosis and promotes malignant progression in OC. UBE2T upregulation suppresses autophagy and subsequently boosts EMT via activating the AKT/mTOR axis, which accounts for the underlying mechanism of oncogenic roles of UBE2T in OC.

GZ17-6.02 is undergoing clinical evaluation in solid tumors and lymphoma. The present studies were performed to define its biology in renal carcinoma cells and to determine whether it interacted with axitinib to enhance tumor cell killing. GZ17-6.02 interacted in an arithmetically greater than additive fashion with axitinib to kill kidney cancer cells. GZ17-6.02 and axitinib cooperated to inactivate ERBB2, c-MET, c-KIT, c-SRC, the AMPK, STAT3, STAT5 and eIF2α and to activate PERK, ULK1 and ATG13. The drugs interacted to increase the expression of FAS-L and to decrease the levels of MCL1, BCL-XL, and HDACs 1-3. The drugs as single agents inactivated the Hippo pathway. GZ17-6.02 and axitinib interacted to enhance autophagosome formation and autophagic flux. Knock down of Beclin1, ATG5, eIF2α, toxic BH3 domain proteins or CD95/FADD significantly reduced drug combination lethality. GZ17-6.02 and axitinib increased the expression of BAK, BIM, Beclin1 and ATG5, effects blocked by knock down of eIF2α. The drugs increased phosphorylation of ULK1 S757 and ATG13 S318 and decreased the phosphorylation of mTORC1 and mTORC2, effects blocked by knock down of AMPKα. Knock down of Beclin1 or ATG5 prevented the drug combination reducing expression of HDACs 1-3 and from enhancing the expression of MHCA. Knock down of HDACs 1-3 enhanced MHCA expression. We conclude that GZ17-6.02 and axitinib interact to kill requiring ER stress signaling, autophagy and death receptor signaling. Autophagic degradation of HDACs played a key role in enhancing MHCA expression and of a potential improved response to checkpoint inhibitory immunotherapy.

Aim: To explore the function of circular RNA CUL2 (circCUL2) in colorectal cancer progression. Method: RT-PCR was carried out to detect the expression of circCUL2 in colorectal cancer tissues and cell lines. Western blot and immunofluorescence were used to determine the level of autophagy. CCK-8, clone formation assay, and EdU staining were used to assess the proliferation ability. Luciferase assay verified the relationship between miR-208a-3p and circCUL2 /PPP6C. The xenograft mouse model was used to confirm the function of circCUL2 in vivo. Results: The expression level of circCUL2 was down-regulated in colorectal cancer tissues and cell lines. Forcing expression of circCUL2 inhibited proliferation ability, induced apoptosis, and autophagy in colorectal cancer cells. Luciferase assay verified that miR-208a-3p could bind with circCUL2/PPP6C. Overexpression of circCUL2 could inhibit cancer progression via targeting the miR-208a-3p/PPP6C signal pathway. Conclusion: CircCUL2 participates in progression via the miR-208a-3p/PPP6C axis in colorectal cancer. CircCUL2 would be an underlying target for the diagnosis and therapy of colorectal cancer.

Long non-coding RNA (lncRNA) ACTA2-AS1 has been reported to play an important role in the progression of multiple human malignancies. The article aims to explore the role of ACTA2-AS1 on the cisplatin resistance of non-small cell lung cancer (NSCLC). RT-qPCR was performed to investigate the expression of ACTA2-AS1 in cisplatin-resistant NSCLC cell lines. Western blot was used to investigate the effects of ACTA2-AS1 on autophagy-related protein expression. RIP assay and RNA pull down were used to analyze the combination of ACTA2-AS1 and enhancer of zeste homolog 2 (EZH2), and CHIP was used to analyze the combination of tuberous sclerosis complex-2 (TSC2) gene promoter and Lys-27 of histone H3 (H3K27me3). In this study, ACTA2-AS1 was downregulated in cisplatin-resistant NSCLC cell lines. ACTA2-AS1 negatively regulated the cell viability and positively regulated the cell apoptosis of cisplatin-resistant NSCLC cell lines. Furthermore, our results demonstrated that ACTA2-AS1 promoted cisplatin-resistant NSCLC cells apoptosis through inhibiting autophagy. The regulation of ACTA2-AS1 to the cisplatin-resistant NSCLC cell autophagy was reversed by TSC2 increasing. Importantly, our results displayed that ACTA2-AS1 bound with EZH2, and TSC2 gene promoter combined with H3k27me3. The inhibition of ACTA2-AS1 to TSC2 expression was recused by EZH2 silencing. In conclusion, ACTA2-AS1 inhibited the cisplatin resistances of NSCLC cell lines through suppressing TSC2 expressing by recruiting EZH2 to TSC2 gene promoter.

Stroke is the most frequent cause of disability in developed countries. A common phenomenon of stroke, cerebral ischemia, is threatening many lives worldwide. In addition, ozone treatment was previously reported to exert functions in relieving brain injury. In the current study, the therapeutic effects of ozone on cerebral ischemia are investigated. A rat model of middle cerebral artery occlusion (MCAO) was established. The brain water content was calculated by weighing brain tissues, and the 2, 3, 5-triphenyltetrazolium chloride staining was performed to measure brain infarction volume in rats. A colorimetric assay was conducted to examine expression levels of malondialdehyde, superoxide dismutase, catalase, and glutathione in the rat hippocampus. Reverse transcription quantitative polymerase-chain reaction and Western blot analyses were employed to evaluate expression levels of Beclin1, LC3B, p62, and critical factors implicated in the NF-κB signaling pathway. We found that ozone significantly improved the survival rate of MCAO model rats, reduced the cerebral water content, and decreased the neurological scores of ischemic rats. Ozone markedly reduced cerebral ischemia-induced infarction in ischemic rats. Ozone decreased MDA levels and increased SOD, catalase, and GSH levels in the hippocampus of rats. Ozone significantly inhibited autophagy by decreasing Beclin1 and LC3B expression and increasing p62 expression. The ozone inactivated the NF-κB signaling pathway by decreasing the protein levels of TLR4, p-IKKβ, p-IKBα, and p-p65. We conclude that ozone treatment alleviates the brain injury in ischemic rats by suppressing autophagy and inactivating the NF-κB signaling pathway.

This study aims to determine the effect of exogenous hydrogen sulfide (H2S) under high glucose (HG)-induced injury in endothelial progenitor cells (EPCs), and to explore the possible underlying mechanisms. Mononuclear cells were isolated from the peripheral blood of healthy volunteers by density-gradient centrifugation and identified as late EPCs by immunofluorescence and flow cytometry. EPCs were treated with high concentrations of glucose, H2S, Baf-A1, 3-MA or rapamycin. Cell proliferation, cell migration and tube formation were measured using cell counting kit-8, Transwell migration and tube formation assays, respectively. Cellular autophagy flux was detected by RFP-GFP-LC3, and Western blotting was used to examine the protein expression levels of LC3B, P62, and phosphorylated endothelial nitric oxide synthase (eNOS) at Thr495 (p-eNOSThr495). Reactive oxygen species (ROS) levels were measured using a DHE probe. H2S and rapamycin significantly reversed the inhibitory effects of HG on the proliferation, migration, and tube formation of EPCs. Moreover, H2S and rapamycin led to an increase in the number of autophagosomes accompanied by a failure in lysosomal turnover of LC3-II or p62 and p-eNOSThr495 expression and ROS production under the HG condition. However, Baf-A1 and 3-MA reversed the effects of H2S on cell behavior. Collectively, exogenous H2S ameliorated HG-induced EPC dysfunction by promoting autophagic flux and decreasing ROS production by phosphorylating eNOSThr495.

Angiogenesis plays an important role in tissue development and repair, and how to regulate angiogenesis effectively is a widely studied problem in the biomedical field. In recent years, the role of autophagy in vascular endothelial cells has attracted extensive attention. Icariin (ICA) is a traditional Chinese medicine that has been proven to have outstanding protective effects on the vascular system and to regulate cellular autophagy effectively. However, at present, it has not been reported whether ICA can affect the angiogenic ability of endothelial cells by affecting autophagy. In this study, we aimed to investigate whether ICA affects the angiogenesis capacity of EA.hy926 human vascular endothelial cells through autophagy and explain the underlying potential mechanisms. First, we determined that ICA at appropriate concentrations has the ability to promote cell migration and angiogenesis using wound healing assays and tube formation assays. Then, at the molecular level, we observed the upregulation of VEGFA, VEGFR2, ANGI, ANGII, and Tie2 mRNA and detected the upregulation of TGFβ1 protein by Western blotting. We also demonstrated that angiogenic concentrations of ICA can effectively activate autophagy. The autophagy inhibitor 3-MA significantly suppressed TGFβ1 expression and tube formation in EA.hy926 cells. Overall, we hope that our studies might help to further understand the effect of ICA on vascular endothelial cells and provide a theoretical basis for future angiogenic applications of ICA

Assessment and enhancement of liver clearance are promising strategies for protection of liver from various liver diseases. Iron-tannic nanoparticles (FTs) were previously considered as imageable autophagic enhancers with biodegradation potential. Herein, we present a new approach for utilizing Iron-tannic nanoparticles (FTs) as a tool for imaging and increasing liver clearance. Pharmacokinetic profiling suggested that FTs were initially found in blood circulation and thereafter were distributed to the liver. By using MR imaging (T1 weighted), maximum MRI signal enhancement was found to occur after 30 minutes post-injection (i.v.) and gradually decreased afterward. Decreasing MRI signal may be due to FTs metabolism by the liver. By assessing imaging-derived pharmacokinetics, we can simply determine the rate constant of liver degradation of FTs. Potentially, we might use this parameter to monitor liver function, where its clearance is of concern. Once functional implication of FTs in liver clearance was investigated, FTs were found to induce hepatocyte autophagy along with activation of lysosomes. Consequently, the hepatocytes were capable of efficiently clearing cellular debris. From these results, it is clear that FTs should be considered as a molecular tool for quantitative MRI-derived liver function assessment, and for enhancing clearance function in liver parenchyma. Hopefully, our findings will pave the way to develop new strategies for non-invasive assessment and enhancement of liver clearance.

Our previous study found that the combination of halofuginone (HF) and artemisinin (ATS) synergistically arrest colorectal cancer (CRC) cells at the G1/G0 phase of the cell cycle; however, it remains unclear whether HF-ATS induces cell death. Here we report that HF-ATS synergistically induced caspase-dependent apoptosis in CRC cells. Specifically, both in vitro and in vivo experiments showed that HF or HF-ATS induces apoptosis via activation of caspase-9 and caspase-8 while only caspase-9 is involved in ATS-induced apoptosis. Furthermore, we found HF or HF-ATS induces autophagy; ATS can't induce autophagy until caspase-9 is blocked. Further analyzing the crosstalk between autophagic and caspase activation in CRC cells, we found autophagy is essential for activation of caspase-8, and ATS switches to activate capase-8 via induction of autophagy when caspase-9 is inhibited. When apoptosis is totally blocked, HF-ATS switches to induce autophagic cell death. This scenario was then confirmed in studies of chemoresistance CRC cells with defective apoptosis. Our results indicate that HF-ATS induces cell death via interaction between apoptosis and autophagy in CRC cells. These results highlight the value of continued investigation into the potential use of this combination in cancer therapy.

Stress urinary incontinence (SUI) is defined as involuntary urinary leakage happening in exertion. Nicotinamide phosphoribosyltransferase (Nampt) is seldom researched in the pathogenesis of SUI. Accordingly, the current study set out to elucidate the role of Nampt in SUI progression. Firstly, we determined Nampt expression patterns in SUI patients and rat models. In addition, fibroblasts were obtained from the anterior vaginal wall tissues of non-SUI patients and subjected to treatment with different concentrations of interleukin-1β (IL-1β), followed by quantification of Nampt expressions in fibroblasts. Subsequently, an appropriate concentration of IL-1β was selected to treat anterior vaginal wall fibroblasts. Nampt was further silenced in IL-1β-treated fibroblasts to assess the role of Nampt in autophagy and extracellular matrix (ECM) degradation. Lastly, functional rescue assays were carried out to inhibit autophagy and evaluate the role of autophagy in the mechanism of Nampt modulating IL-1β-treated fibroblast ECM degradation. It was found that Nampt was highly-expressed in SUI patients and rat models and IL-1β-treated fibroblasts. On the other hand, Nampt silencing was found to suppress ECM degradation and promote SUI fibroblast autophagy. Additionally, inhibition of autophagy attenuated the inhibitory effects of Nampt silencing on SUI fibroblast ECM degradation. Collectively, our findings revealed that Nampt was over-expressed in SUI, whereas Nampt silencing enhanced SUI fibroblast autophagy, and thereby inhibited ECM degradation.

Background: Natural killer (NK) cell-based immunotherapy is clinically limited due to insufficient tumor infiltration in solid tumors. We have previously found that the natural product rocaglamide (RocA) can enhance NK cell-mediated killing of non-small cell lung cancer (NSCLC) cells by inhibiting autophagy, and autophagic inhibition has been shown to increase NK cell tumor infiltration in melanoma. Therefore, we hypothesized that RocA could increase NK cell infiltration in NSCLC by autophagy inhibition. Methods: Flow cytometry, RNA-sequencing, real-time PCR, Western blotting analysis, and xenograft tumor model were utilized to assess the infiltration of NK cells and the underlying mechanism. Results: RocA significantly increased the infiltration of NK cells and the expressions of CCL5 and CXCL10 in NSCLC cells, which could not be reversed by the inhibitions of autophagy/ULK1, JNK and NF-κB. However, such up-regulation could be suppressed by the inhibitions of TKB1 and STING. Furthermore, RocA dramatically activated the cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling pathway, and the inhibition/depletion of STING ablated the up-regulation of CCL5 and CXCL10, NK cell infiltration, and tumor regression induced by RocA. Besides, RocA damaged mitochondrial DNA (mtDNA) and promoted the cytoplasmic release of mtDNA. The mPTP inhibitor cyclosporin A could reverse RocA-induced cytoplasmic release of mtDNA. Conclusions: RocA could promote NK cell infiltration by activating cGAS-STING signaling via targeting mtDNA, but not by inhibiting autophagy. Taken together, our current findings suggested that RocA was a potent cGAS-STING agonist and had a promising potential in cancer immunotherapy, especially in NK cell-based immunotherapy.

Background: G-protein-coupled receptor 43 (GPR43) is a posttranscriptional regulator involved in cholesterol metabolism. This study aimed to investigate the possible roles of GPR43 activation in podocyte lipotoxicity in diabetic nephropathy (DN) and explore the potential mechanisms. Methods: The experiments were conducted by using diabetic GPR43-knockout mice and a podocyte cell culture model. Lipid deposition and free cholesterol levels in kidney tissues were measured by BODIPY staining and quantitative cholesterol assays, respectively. The protein expression of GPR43, LC3II, p62, beclin1, low-density lipoprotein receptor (LDLR) and early growth response protein 1 (EGR1) in kidney tissues and podocytes was measured by real-time PCR, immunofluorescent staining and Western blotting. Results: There were increased LDL cholesterol levels in plasma and cholesterol accumulation in the kidneys of diabetic mice. However, GPR43 gene knockout inhibited these changes. An in vitro study further demonstrated that acetate treatment induced cholesterol accumulation in high glucose-stimulated podocytes, which was correlated with increased cholesterol uptake mediated by LDLR and reduced cholesterol autophagic degradation, as characterized by the inhibition of LC3 maturation, p62 degradation and autophagosome formation. Gene knockdown or pharmacological inhibition of GPR43 prevented these effects on podocytes. Furthermore, GPR43 activation increased extracellular regulated protein kinases 1/2 (ERK1/2) activity and EGR1 expression in podocytes, which resulted in an increase in cholesterol influx and autophagy inhibition. In contrast, after GPR43 deletion, these changes in podocytes were improved, as shown by the in vivo and in vitro results. Conclusion: GPR43 activation-mediated lipotoxicity contributes to podocyte injury in DN by modulating the ERK/EGR1 pathway.

Octreotide is a synthetic octapeptide of natural somatostatin. We aimed to investigate the influence of Octreotide on lipopolysaccharide (LPS)-stimulated human pulmonary epithelial cell damage. After stimulated by LPS, BEAS-2B cells were treated with various concentrations of Octreotide. CCK-8 assay and LDH kits were to evaluate cell cytotoxicity. ELISA kits were to analyze the levels of inflammatory factors. TUNEL staining was to measure cell apoptosis. Western blot assay was used to assess the expression of apoptosis-related proteins, autophagy-related proteins and AKT/mTOR signaling-related proteins. Then, 3-methyladenine (3-MA) was adopted for treating BEAS-2B cells to determine its effects on inflammation and apoptosis. Afterward, adding AKT agonist (SC79) or mTOR antagonist (rapamycin) to explore the impact of Octreotide on autophagy. Results revealed that Octreotide notably enhanced cell viability and reduced LDH activity. The levels of inflammatory factors were significantly decreased following Octreotide treatment. Additionally, Octreotide attenuated the apoptotic capacity of LPS-induced BEAS-2B cells, led to the up-regulation of Bcl-2 protein level while cut down the protein levels of Bax and cleaved caspase3. Remarkably, the expression of autophagy-related protein LC3II/I and Beclin1 was elevated after Octreotide administration. Importantly, the suppressive effects of Octreotide on the inflammation and apoptosis of LPS-induced BEAS-2B cells was abrogated by 3-MA. Further experiments suggested that Octreotide downregulated p-AKT and mTOR expression in LPS-stimulated BEAS-2B cells. SC79 addition inhibited autophagy, evidenced by downregulated LC3II/I and Beclin1 expression while rapamycin presented the opposite effects. To conclude, Octreotide activates autophagy to alleviate LPS-induced pulmonary epithelial cell injury by inhibiting the AKT/mTOR signaling.

PERK is one of the transmembrane sensors of unfolded protein response (UPR) triggered by ER stress. In this study, we evaluated the role of PERK in the sensitivity of hepatocellular carcinoma (HCC) cells to high linear energy transfer (LET) carbon ions (CI). We found that CI irradiation could induce ER stress in HCC cells. On the one hand, PERK promoted autophagy via regulating ATF4 expression; on the other hand, PERK regulated p53 expression, and the latter either induced autophagy through up-regulating DRAM, or directly promoting apoptosis through the mitochondrial pathway or facilitating ferroptosis via down-regulating SLC7A11 (the extrinsic pathway), but independent of GPX4 (the intrinsic pathway). These factors jointly determined the sensitivity of HCC cells to high-LET CI radiation. Inhibiting TP53 directly increased cellular radioresistance definitely. Moreover, the death of HepG2 (TP53 wild type) cells induced by high-LET CI irradiation combined with sorafenib treatment might be caused by a mixed-type regulated cell death (RCD) including both apoptosis and ferroptosis, suggesting that apoptosis and ferroptosis are synergetic cell death modes regulated by TP53, which is one of the reasons why the sensitivity of HepG2 cells is higher than that of Hep3B (TP53 null type) and PLC/PRF5 (TP53 mutated type) cells. Therefore, our work might shed light on the potential therapeutic implication of CI radiotherapy combined with PERK targeted clinical drugs to implement personalized and precise treatment of HCCs.

Childhood asthma is the most universal chronic disease, with significant cases reported. Despite the current progress in treatment, prognosis remains poor and the existing drugs cause serious side effects. This investigation explored the mechanisms and use of miR-335-5p on childhood asthma therapy. MiR-335-5p and ATG5 expression was analyzed in clinical plasma samples through RT-qPCR. Airway smooth muscle cells (ASMCs) were cultured, and transfected with miR-335-5p mimic, miR-335-5p inhibitor, and pcDNA3.1-ATG5, or co-transfected with miR-335-5p mimic + pcDNA3.1-ATG5. Asthma cell models were constructed through TGF-β1, and animal models through ovalbumin (OVA). Monocyte-macrophage infiltration in bronchoalveolar lavage fluid (BALF) was determined by May−Grunwald−Giemsa staining, and collagen in lung tissue was assessed via Masson staining. Relationship between miR-335-5p and ATG5 was detected by dual-luciferase assay. Cell proliferation was detected by MTT assay. MiR-335-5p and ATG5 RNA expression was determined by RT-qPCR. Collagen I, collagen III, α-SMA, ATG5, LC3I/II, Beclin-1, and p62 protein expression levels in ASMCs were detected by western blot. MiR-335-5p expression was low, but ATG5 expression was high in childhood asthma. Versus OVA+ mimic NC group, the number of eosinophil and collagen in OVA+ miR-335-5p mimic group were reduced. In contrast to TGF-β1 + mimic NC group, TGF-β1 + miR-335-5p mimic group reduced inflammatory, airway fibrosis, and autophagy in ASMCs. ATG5 was miR-335-5p target. Overexpressing ATG5 significantly reversed the inhibitory effects of miR-335-5p on inflammatory response, fibrosis, and autophagy in ASMCs. Overall, the study concludes that MiR-335-5p alleviate inflammatory response, airway fibrosis, and autophagy in childhood asthma through targeted regulation of ATG5.

Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation contributes to tumor invasion and metastasis. In a previous study, we have demonstrated that EpCAM, a glycosylation protein, is associated with cell growth and metastasis in breast cancer. But the effect of EpCAM glycosylation on autophagy is not clear. the precise mechanism of regulation remains largely unknown. In this study, breast cancer cells were transfected with N-glycosylation mutation EpCAM plasmid to express deglycosylated EpCAM. The result showed that deglycosylated EpCAM promoted autophagy in breast cancer cells. We further confirmed this conclusion with the activator (Rapamycin, RAP) and inhibitor (Wortmannin) of autophagy. We also found that deglycosylated EpCAM promoted apoptosis and inhibited proliferation through activating autophagy by suppressing Akt/mTOR signaling pathway in breast cancer cells. These findings represent a novel mechanism by which deglycosylated EpCAM inhibits proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway. In conclusion, the combination of autophagy modulation and EpCAM targeted therapy is a promising therapeutic strategy in the treatment of breast cancer.

Mutations in the mitochondrial genome (mtDNA) are ubiquitous in humans and can lead to a broad spectrum of disorders. However, due to the presence of multiple mtDNA molecules in the cell, co-existence of mutant and wild-type mtDNAs (termed heteroplasmy) can mask disease phenotype unless a threshold of mutant molecules is reached. Importantly, the mutant mtDNA level can change across lifespan as mtDNA segregates in an allele- and cell-specific fashion, potentially leading to disease. Segregation of mtDNA is mainly evident in hepatic cells, resulting in an age-dependent increase of mtDNA variants, including non-synonymous potentially deleterious mutations. Here we modeled mtDNA segregation using a well-established heteroplasmic mouse line with mtDNA of NZB/BINJ and C57BL/6N origin on a C57BL/6N nuclear background. This mouse line showed a pronounced age-dependent NZB mtDNA accumulation in the liver, thus leading to enhanced respiration capacity per mtDNA molecule. Remarkably, liver-specific atg7 (autophagy related 7) knockout abolished NZB mtDNA accumulat ion, resulting in close-to-neutral mtDNA segregation through development into adulthood. prkn (parkin RBR E3 ubiquitin protein ligase) knockout also partially prevented NZB mtDNA accumulation in the liver, but to a lesser extent. Hence, we propose that age-related liver mtDNA segregation is a consequence of macroautophagic clearance of the less-fit mtDNA. Considering that NZB/BINJ and C57BL/6N mtDNAs have a level of divergence comparable to that between human Eurasian and African mtDNAs, these findings have potential implications for humans, including the safe use of mitochondrial replacement therapy.Abbreviations: Apob: apolipoprotein B; Atg1: autophagy-related 1; Atg7: autophagy related 7; Atp5a1: ATP synthase, H+ transporting, mitochondrial F1 complex, alpha subunit 1; BL6: C57BL/6N mouse strain; BNIP3: BCL2/adenovirus E1B interacting protein 3; FCCP: carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mt-Atp8: mitochondrially encoded ATP synthase 8; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MT-CO2: mitochondrially encoded cytochrome c oxidase II; mt-Co3: mitochondrially encoded cytochrome c oxidase III; mt-Cytb: mitochondrially encoded cytochrome b; mtDNA: mitochondrial DNA; MUL1: mitochondrial ubiquitin ligase activator of NFKB 1; nDNA: nuclear DNA; Ndufa9: NADH:ubiquinone oxireductase subunit A9; NDUFB8: NADH:ubiquinone oxireductase subunit B8; Nnt: nicotinamide nucleotide transhydrogenase; NZB: NZB/BINJ mouse strain; OXPHOS: oxidative phosphorylation; PINK1: PTEN induced putative kinase 1; Polg2: polymerase (DNA directed), gamma 2, accessory subunit; Ppara: peroxisome proliferator activated receptor alpha; Ppia: peptidylprolyl isomerase A; Prkn: parkin RBR E3 ubiquitin protein ligase; P10: post-natal day 10; P21: post-natal day 21; P100: post-natal day 100; qPCR: quantitative polymerase chain reaction; Rpl19: ribosomal protein L19; Rps18: ribosomal protein S18; SD: standard deviation; SEM: standard error of the mean; SDHB: succinate dehydrogenase complex, subunit B, iron sulfur (Ip); SQSTM1: sequestosome 1; Ssbp1: single-stranded DNA binding protein 1; TFAM: transcription factor A, mitochondrial; Tfb1m: transcription factor B1, mitochondrial; Tfb2m: transcription factor B2, mitochondrial; TOMM20: translocase of outer mitochondrial membrane 20; UQCRC2: ubiquinol cytochrome c reductase core protein 2; WT: wild-type.

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection. Abbreviations: 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: β-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A(1); BDBV: Bundibugyo ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: Zaire ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: Sudan ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

Deubiquitination plays an important role in the regulation of the crosstalk between macroautophagy/autophagy and innate immune signaling, yet its regulatory mechanisms are not fully understood. Here we identify the deubiquitinase OTUD7B as a negative regulator of antiviral immunity by targeting IRF3 (interferon regulatory factor 3) for selective autophagic degradation. Mechanistically, OTUD7B interacts with IRF3, and activates IRF3-associated cargo receptor SQSTM1/p62 (sequestosome 1) by removing its K63-linked poly-ubiquitin chains at lysine 7 (K7) to enhance SQSTM1 oligomerization. Moreover, viral infection increased the expression of OTUD7B, which forms a negative feedback loop by promoting IRF3 degradation to balance type I interferon (IFN) signaling. Taken together, our study reveals a specific role of OTUD7B in mediating the activation of cargo receptors in a substrate-dependent manner, which could be a potential target against excessive immune responses. Abbreviations: Baf A1: bafilomycin A1; CGAS: cyclic GMP-AMP synthase; DDX58/RIG-I: DExD/H-box helicase 58; DSS: dextran sodium sulfate; DUBs: deubiquitinating enzymes; GFP: green fluorescent protein; IFN: interferon; IKKi: IKBKB/IkappaB kinase inhibitor; IRF3: interferon regulatory factor 3; ISGs: interferon-stimulated genes; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; PAMPs: pathogen-associated molecular patterns; SeV: Sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; STING1: stimulator of interferon response cGAMP interactor 1; TBK1: TANK binding kinase 1; Ub: ubiquitin; WT: wild-type; VSV: vesicular stomatitis virus.

Caffeine is among the most highly consumed substances worldwide, and it has been associated with decreased cardiovascular risk. Although caffeine has been shown to inhibit the proliferation of vascular smooth muscle cells (VSMCs), the mechanism underlying this effect is unknown. Here, we demonstrated that caffeine decreased VSMC proliferation and induced macroautophagy/autophagy in an in vivo vascular injury model of restenosis. Furthermore, we studied the effects of caffeine in primary human and mouse aortic VSMCs and immortalized mouse aortic VSMCs. Caffeine decreased cell proliferation, and induced autophagy flux via inhibition of MTOR signaling in these cells. Genetic deletion of the key autophagy gene Atg5, and the Sqstm1/p62 gene encoding a receptor protein, showed that the anti-proliferative effect by caffeine was dependent upon autophagy. Interestingly, caffeine also decreased WNT-signaling and the expression of two WNT target genes, Axin2 and Ccnd1 (cyclin D1). This effect was mediated by autophagic degradation of a key member of the WNT signaling cascade, DVL2, by caffeine to decrease WNT signaling and cell proliferation. SQSTM1/p62, MAP1LC3B-II and DVL2 were also shown to interact with each other, and the overexpression of DVL2 counteracted the inhibition of cell proliferation by caffeine. Taken together, our in vivo and in vitro findings demonstrated that caffeine reduced VSMC proliferation by inhibiting WNT signaling via stimulation of autophagy, thus reducing the vascular restenosis. Our findings suggest that caffeine and other autophagy-inducing drugs may represent novel cardiovascular therapeutic tools to protect against restenosis after angioplasty and/or stent placement.

Mice deficient in GHR (growth hormone receptor; ghr KO) have a dramatic lifespan extension and elevated levels of hepatic chaperone-mediated autophagy (CMA). Using quantitative proteomics to identify protein changes in purified liver lysosomes and whole liver lysates, we provide evidence that elevated CMA in ghr KO mice downregulates proteins involved in ribosomal structure, translation initiation and elongation, and nucleocytosolic acetyl-coA production. Following up on these initial proteomics findings, we used a cell culture approach to show that CMA is necessary and sufficient to regulate the abundance of ACLY and ACSS2, the two enzymes that produce nucleocytosolic (but not mitochondrial) acetyl-coA. Inhibition of CMA in NIH3T3 cells has been shown to lead to aberrant accumulation of lipid droplets. We show that this lipid droplet phenotype is rescued by knocking down ACLY or ACSS2, suggesting that CMA regulates lipid droplet formation by controlling ACLY and ACSS2. This evidence leads to a model of how constitutive activation of CMA can shape specific metabolic pathways in long-lived endocrine mutant mice. Abbreviations: CMA: chaperone-mediated autophagy; DIA: data-independent acquisition; ghr KO: growth hormone receptor knockout; GO: gene ontology; I-WAT: inguinal white adipose tissue; KFERQ: a consensus sequence resembling Lys-Phe-Glu-Arg-Gln; LAMP2A: lysosomal-associated membrane protein 2A; LC3-I: non-lipidated MAP1LC3; LC3-II: lipidated MAP1LC3; PBS: phosphate-buffered saline; PI3K: phosphoinositide 3-kinase

Osteoarthritis is a degenerative joint disease and a leading cause of adult disability. Our previous study has reported that mesenchymal stem cell-derived exosomes (MSC-Exo) mediated long non-coding RNA KLF3-AS1 improves osteoarthritis. This study aims to investigate the molecular mechanism of KLF3-AS1 in osteoarthritis. Chondrocytes were treated with IL-1β to induce chondrocyte injury, followed by MSC-Exo treatment. We found that MSC-Exo enhanced KLF3-AS1 expression in IL-1β-treated chondrocytes. IL-1β treatment reduced cell viability and enhanced apoptosis in chondrocytes. MSC-Exo-mediated KLF3-AS1 promoted cell viability and repressed apoptosis of IL-1β-treated chondrocytes. Rapamycin (autophagy activator) promoted cell viability and suppressed apoptosis of chondrocytes by activating autophagy. Moreover, KLF3-AS1 interacted with YBX1 in chondrocytes. MSC-Exo-mediated KLF3-AS1 activated PI3K/Akt/mTOR signaling pathway, which was abrogated by YBX1 silencing. MSC-Exo-mediated KLF3-AS1 repressed autophagy and apoptosis of chondrocytes by activating PI3K/Akt/mTOR signaling pathway. In conclusion, our data demonstrate that MSC-Exo-mediated KLF3-AS1 inhibits autophagy and apoptosis of IL-1β-treated chondrocyte through PI3K/Akt/mTOR signaling pathway. KLF3-AS1 activates PI3K/Akt/mTOR signaling pathway by targeting YBX1 to improve the progression of osteoarthritis. Thus, this work suggests that MSC-Exo-mediated KLF3-AS1 may be a potential therapeutic target for osteoarthritis.

The cytoplasmic accumulation and aggregates of TARDBP/TDP-43 (TAR DNA binding protein) are a pathological hallmark in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. We previously reported that the primate specific cleavage of TARDBP accounts for its cytoplasmic mislocalization in the primate brains, prompting us to further investigate how the cytoplasmic TARDBP mediates neuropathology. Here we reported that cytoplasmic mutant TARDBP reduced SQSTM1 expression selectively in the monkey brain, when compared with the mouse brain, by inducing SQSTM1 mRNA instability via its binding to the unique 3ʹUTR sequence (GU/UG)n of the primate SQSTM1 transcript. Overexpression of SQSTM1 could diminish the cytoplasmic C-terminal TARDBP accumulation in the monkey brain by augmenting macroautophagy/autophagy activity. Our findings provide additional clues for the pathogenesis of cytoplasmic TARDBP and a potential therapy for mutant TARDBP-mediated neuropathology.

Objective: To investigate the effect of NAD+ on thymus autophagy in experimental autoimmune encephalomyelitis (EAE) mice through SIRT1. Methods: Data sets GSE135511 and GSE131279 related to fatty liver in multiple sclerosis were downloaded from GEO database to search for differentially expressed genes. KEGG and GO pathway enrichment analysis was performed to construct PPI network, identify important modules and hub genes, and analyze the expression of hub genes. Animal experiments: Forty female C57BL/6 mice were randomly divided into 4 groups: control group, EAE group, NAD+ group and SIRT1 inhibitor group. The EAE model was prepared by MOG35-55 polypeptide immunoassay. The NAD+ group and SIRT1 inhibitor group were treated with NAD+ drug and fed for 4 weeks. The neurological function scores of the mice in each group were evaluated weekly. Cell experiment: The thymus tissues of wild-type mice were removed, ground and filtered into single-cell suspension, and then cultured. The thymus tissues were divided into four groups: control group, EAE group, NAD+ group and SIRT1 inhibitor group. MOG 35-55 (1μg/ mL) was given to induce EAE model in vitro. Both the NAD+ group and the SIRT1 inhibitor group were treated with NAD+. The expression of LC-3B was observed by immunofluorescence. The expressions of p-PI3K, p-AKT, p-mTOR, P62, Beclin1, LC-3B and SIRT1 were detected by WB. Results: Data sets GSE135511 and GSE131279 contained 95 up-regulated DEGs and 89 down-regulated DEGs. Enrichment analysis showed that it was mainly concentrated in PI3K-Akt-mTOR and autophagy pathway. SIRT1 was low expressed in the disease group. The EAE group, the NAD+ group and the SIRT1 inhibitor group all showed different degrees of morbidity. At week 2, there was no significant difference between the EAE group and the NAD+ group and the SIRT1 inhibitor group, but the neurological function score of the SIRT1 inhibitor group was significantly higher than that of the NAD+ group. The neurological function score of SIRT1 inhibitor group was the highest at 3 and 4 weeks. WB test showed that the expressions of P62, Beclin1, LC-3B and SIRT1 were the highest in the NAD+ group, while the expressions of p-PI3K, p-Akt and p-mTOR were the highest in the EAE group, followed by the SIRT1 inhibitor group, the NAD+ group and the control group. Cell experiment: Immunofluorescence showed that the fluorescence intensity of LC-3B in NAD+ group was the highest, followed by SIRT1 inhibitor group, EAE group, and control group. WB test showed that the expressions of P62, Beclin1, LC-3B and SIRT1 were the highest in the NAD+ group, followed by SIRT1 inhibitor group, EAE group and control group. The expressions of p-PI3K, p-AKT and p-mTOR were the highest in EAE group, followed by SIRT1 inhibitor group, NAD+ group and control group. Conclusion: NAD+ exerted a protective effect on EAE mice by inhibiting PI3K/ Akt /mTOR signaling pathway through SIRT1, and prevented EAE mice from sustained damage.

Autophagy is an important cellular mechanism for maintaining cellular homeostasis, and its impairment correlates highly with age and age-related diseases. Retinal pigment epithelial (RPE) cells of the eye represent a crucial model for studying autophagy, as RPE functions and integrity are highly dependent on an efficient autophagic process. Cysteinyl leukotriene receptor 1 (CysLTR1) acts in immunoregulation and cellular stress responses and is a potential regulator of basal and adaptive autophagy. As basal autophagy is a dynamic process, the aim of this study was to define the role of CysLTR1 in autophagy regulation in a chronobiologic context using the ARPE-19 human RPE cell line. Effects of CysLTR1 inhibition on basal autophagic activity were analyzed at inactive/low and high lysosomal degradation activity with the antagonists zafirlukast (ZTK) and montelukast (MTK) at a dosage of 100 nM for 3 hours. Abundances of the autophagy markers LC3-II and SQSTM1 and LC3B particles were analyzed in the absence and presence of lysosomal inhibitors using western blot analysis and immunofluorescence microscopy. CysLTR1 antagonization revealed a biphasic effect of CysLTR1 on autophagosome formation and lysosomal degradation that depended on the autophagic activity of cells at treatment initiation. ZTK and MTK affected lysosomal degradation, but only ZTK regulated autophagosome formation. In addition, dexamethasone treatment and serum shock induced autophagy, which was repressed by CysLTR1 antagonization. As a newly identified autophagy modulator, CysLTR1 appears to be a key player in the chronobiological regulation of basal autophagy and adaptive autophagy in RPE cells.

We study whether microRNA miR-30a inhibits the autophagy through transforming growth factor (TGF)-β/Smad4 to generate cisplatin (DDP) resistance in ovarian cancer cells. The expression of miR-30a, Smad4, and TGF-β was detected in the serum of ovarian cancer patients and DDP-resistant cell lines (A2780) by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search and western blotting were used to demonstrate the downstream target of miR-30a in ovarian cancer cells. Cell viability was measured with CCK8 assay. Apoptosis and autophagy of ovarian cancer cells were analyzed by flow cytometry and transmission electron microscopy, and the expressions of Beclin1 and LC3II protein were detected by western blotting. Expression of miR-30a was significantly decreased, while expressions of TGF-β and Smad4 mRNA were increased in serum of ovarian cancer patients after DDP chemotherapy as well as in DDP-resistant cells. Activation of autophagy contributed to DDP-resistance cells. Moreover, Bioinformatics software predicted Smad4 to be a target of miR-30a. Overexpression of miR-30a decreased the expression of Smad4 and TGF-β. Additionally, miR-30a-overexpressing inhibited DDP-induce autophagy and promoted DDP-resistant cell apoptosis. In conclusion, miR-30a mediates DDP resistance in ovarian cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.

Lung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer with poor prognosis. This study aimed to explore the role of KDM2B in the development of LUSC. The results of this study demonstrated that KDM2B was upregulated in LUSC tissues and cell lines. Knockdown of KDM2B reduced cell viability and colony forming ability in SK-MES-1 and NCI-H520 cells. KDM2B inhibition reduced glucose consumption, lactate production, ATP level, and also downregulated the expression of LDHA and GLUT1. KDM2B knockdown decreased the protein expression of LC3-I and p62, and increased LC3-II and Beclin-1. Furthermore, KDM2B silencing inhibited the phosphorylation of AKT, mTOR and P70S6K. KDM2B knockdown led to reduced tumor size in mouse model. In conclusion, KDM2B is upregulated in LUSC tissues and cell lines. KDM2B silencing inhibits glycolysis and promotes autophagy through inactivation of the PI3K/Akt/mTOR signaling pathway.

Glaucoma, characterized with progressive degeneration of retinal ganglion cells (RGCs), is the second frequently leading cause of sight loss in the word after cataract. Baicalin plays a protective role in age-related macular degeneration, retinopathy of prematurity, branch retinal vein occlusion, and ischemia-induced neurodegeneration in the retina. The present study aimed to investigate the role of baicalin in glaucoma. RGCs were stimulated with N-methyl-D-aspartate (NMDA) to mimic the in vitro model of glaucoma. A mouse model of glaucoma induced by chronic elevated intraocular pressure was also established. The apoptosis, oxidative stress, and autophagy of RGCs were detected by flow cytometry analysis, 2,7-dichlorodihydrofluorescein diacetate staining, and Western blotting, respectively. Retinal pathological changes were exhibited by hemotoxylin and eosin staining. Baicalin restrained the NMDA-induced cell apoptosis, autophagy, and oxidative stress of RGCs by activating the PI3K/AKT signaling in vitro. The elevated intraocular pressure-induced pathological changes in retinas of glaucoma mice were attenuated by baicalin. Moreover, the number of RGCs was significantly decreased in glaucoma mice, and then increased by baicalin treatment. Baicalin also inhibited autophagy and activated PI3K/AKT signaling in vivo. In conclusion, baicalin suppresses glaucoma pathogenesis by regulating the PI3K/AKT signaling in vitro and in vivo.

Abnormal levels of autophagy have been implicated in the pathogenesis of multiple diseases, including cancer. However, little is known about the role of autophagy-related genes (ARGs) in low-grade gliomas (LGG). Accordingly, the aims of this study were to assess the prognostic values of ARGs and to establish a genetic signature for LGG prognosis. Expression profile data from patients with and without primary LGG were obtained from The Cancer Genome Atlas (TCGA) and Genome Tissue Expression databases, respectively, and consensus clustering was used to identify clusters of patients with distinct prognoses. Nineteen differentially expressed ARGs were selected with threshold values of FDR < 0.05 and |log2 fold change (FC)| ≥ 2, and functional analysis revealed that these genes were associated with autophagy processes as expected. An autophagy-related signature was established using a Cox regression model of six ARGs that separated patients from TCGA training cohort into high- and low-risk groups. Univariate and multivariate Cox regression analysis indicated that the signature-based risk score was an independent prognostic factor. The signature was successfully validated using the TCGA testing, TCGA entire, and Chinese Glioma Genome Atlas cohorts. Stratified analyses demonstrated that the signature was associated with clinical features and prognosis, and gene set enrichment analysis revealed that autophagy- and cancer-related pathways were more enriched in high-risk patients than in low-risk patients. The prognostic value and expression of the six signature-related genes were also investigated. Thus, the present study constructed and validated an autophagy-related prognostic signature that could optimize individualized survival prediction in LGG patients.

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

This study investigated the role of miRNA-144 (miR-144) targeting of the long noncoding DNAJC3-AS1 in regulating breast cancer chemosensitivity. Real-time quantitative polymerase chain reaction was employed to detect the levels of miR-144 in different drug-resistant cells. MTT assays were used to measure the proliferation of cells in different treatment groups. The apoptosis rate of transfected cells was detected by flow cytometry. Western blotting was used to detect levels of DNAJC3-AS1 protein and of autophagy-related proteins. A double luciferase report experiment was performed to evaluate the targeting effect of miR-144 on DNAJC3-AS1. The level of miR-144 was significantly downregulated in MCF-7 doxorubicin-resistant cells. Upregulated expression of miR-144 increased the doxorubicin sensitivity of drug-resistant cells and the rate of apoptosis. DNAJC3-AS1 was the direct target of miR-144; overexpression of DNAJC3-AS1 significantly rescued the apoptosis induced by miR-144 and reversed the inhibition of autophagy by miR-144. Overexpression of miR-144 can reduce drug resistance in breast cancer cells by inhibiting autophagy or targeting DNAJC3-AS1 for downregulation. miR-144/DNAJC3-AS1 provide a new target for reducing drug resistance in breast cancer.

We aim to evaluate the efficacies of combination therapy with low-frequency ultrasound-stimulated microbubbles (USMB) and radiofrequency ablation (RFA) on suppressing the proliferation of pancreatic cancer cell and treating Panc02 subcutaneous xenograft mice. The proliferation of HPDE6-C7 and Panc02 cells after the treatment of USMB and RFA alone or combination were evaluated by CCK-8 assay. Scratch test was performed to assess the cell migration capability. Panc02-bearing mice were received 14-day treatment of USMB and RFA alone or combination. Tumor size and survival rate were recorded once two days. The serum levels of immune-related factors and changes of apoptosis- and autophagy-related factors were detected by ELISA and western blotting methods. As a result, CKK-8 assays revealed significant inhibition on Panc02 cell proliferation in combination therapy with USMB and RFA relative to other groups (all p

Endoplasmic reticulum stress (ERS)-mediated autophagy is indispensable for modulation of replication and pathogenesis of numerous mammalian viruses. We have previously shown that classical swine fever virus (CSFV) infection induces ERS-mediated autophagy for maintaining viral replication both in vivo and in vitro, however, the underlying mechanism remains unclarified. Here we found that CSFV infection activates the PERK pathway-dependent complete autophagy to promote viral replication in cultured PK-15 and 3D4/2 cells. Likewise, our results also suggested the essential roles of the IRE1/GRP78-mediated complete autophagy in CSFV replication in vitro. Furthermore, we suggested that CSFV infection induces activation of the PERK and IRE1 pathway for potential immunoregulation via promoting transcription of proinflammatory cytokine (IFN-γ and TNF-α) genes in the CSFV-infected cells. Finally, pharmacological treatment of PERK- or IRE1-pathway regulators, and the corresponding SiRNAs interventions did not affect the viabilities of the cells, excluding the potential interference elicited by altered cell viabilities. Taken together, our results suggest that CSFV infection induces complete autophagy through activation of the PERK and IRE1 pathway to facilitate viral replication in cultured cells, and modulation of proinflammatory cytokines may be a potential mechanism involved in this event. Our findings will open new horizons for molecular mechanisms of sustainable replication and pathogenesis of CSFV, and lay a theoretical foundation for the development of ERS-autophagy-targeting therapeutic strategies for clinical control of CSF.

Lung cancer is one of the leading causes of human death, and the 5-year survival rate for lung cancer patients remains a relative low level. Pinocembrin (Pino) was reported to play an important role in the inhibition of cancer development, so this study was designed to explore the role of pino in lung cancer. A549 cells were treated with different concentration of Pino (25, 50, 100, 150 and 200 µM) for 24, 48 and 72, respectively to detect cell viability by Cell counting kit-8 (CCK-8) assay. Then, the proliferation, apoptosis and autophagy of A549 cells under pino exposure were detected using colony formation, TUNEL and immunofluorescence staining, respectively. Western blot was used to analyze proliferation-, apoptosis-, and autophagy-related proteins. To measure the effects of pino on cell autophagy, the above-mentioned functional assays were conducted again in A549 cells treated with pino and 20 µM autophagy activator rapamycin (RAPA). Declined trends in cell viability, proliferation, and autophagy were found in A549 cells treated with increasing doses of pino, by contrast with those without any treatment. Additionally, the apoptosis of A549 cells was enhanced upon pino exposure, accompanied by elevated caspase3 activity. However, RAPA reversed the anti-proliferative, anti-autophagic and pro-apoptotic properties of pino in A549 cells. In conclusion, this paper is the first to verify that pino suppresses the proliferation and enhances the apoptosis of lung cancer cells by restraining autophagy, indicating that pino has potential therapeutic effects on the treatment of lung cancer.