Analysis of the autophagy gene expression profile of pancreatic cancer based on autophagy-related protein microtubule-associated protein 1A/1B-light chain 3

BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.

Predictive and prognostic implications of 4E-BP1, Beclin-1, and LC3for cetuximab treatment combined with chemotherapy in advanced colorectal cancer with wild-type KRAS: Analysis from real-world data

BACKGROUND Colorectal cancer(CRC) is one of the main causes of cancer-related deaths in China and around the world. Advanced CRC(ACRC) patients suffer from a low cure rate though treated with targeted therapies. The response rate is about 50% to chemotherapy and cetuximab, a monoclonal antibody targeting epidermal growth factor receptor(EGFR) and used for ACRC with wild-type KRAS. It is important to identify more predictors of cetuximab efficacy to further improve precise treatment. Autophagy, showing a key role in the cancer progression, is influenced by the EGFR pathway. Whether autophagy can predict cetuximab efficacy in ACRC is an interesting topic.AIM To investigate the effect of autophagy on the efficacy of cetuximab in colon cancer cells and ACRC patients with wild-type KRAS.METHODS ACRC patients treated with cetuximab plus chemotherapy, with detailed data and tumor tissue, at Sun Yat-sen University Cancer Center from January 1, 2005,to October 1, 2015, were studied. Expression of autophagy-related proteins[Beclin1, microtubule-associated protein 1 A/B-light chain 3(LC3), and 4 Ebinding protein 1(4 E-BP1)] was examined by Western blot in CRC cells and by immunohistochemistry in cancerous and normal tissues. The effect of autophagy on cetuximab-treated cancer cells was confirmed by MTT assay. The associations between Beclin1, LC3, and 4 E-BP1 expression in tumor tissue and the efficacy of cetuximab-based therapy were analyzed.RESULTS In CACO-2 cells exposed to cetuximab, LC3 and 4 E-BP1 were upregulated, and P62 was downregulated. Autophagosome formation was observed, and autophagy increased the efficacy of cetuximab. In 68 ACRC patients,immunohistochemistry showed that Beclin1 levels were significantly correlated with those of LC3(0.657, P < 0.001) and 4 E-BP1(0.211, P = 0.042) in ACRC tissues.LC3 was significantly overexpressed in tumor tissues compared to normal tissues(P < 0.001). In 45 patients with wild-type KRAS, the expression levels of these three proteins were not related to progression-free survival; however, the expression levels of Beclin1(P = 0.010) and 4 E-BP1(P = 0.005), pathological grade(P = 0.002), and T stage(P = 0.004) were independent prognostic factors for overall survival(OS).CONCLUSION The effect of cetuximab on colon cancer cells might be improved by autophagy.LC3 is overexpressed in tumor tissues, and Beclin1 and 4 E-BP1 could be significant predictors of OS in ACRC patients treated with cetuximab.

Association of CFH and MAP1LC3B gene polymorphisms with age-related macular degeneration in a high-altitude population

AIM: To evaluate the association of complement factor H(CFH) and microtubule-associated protein 1 light chain 3 beta(MAP1LC3B) gene polymorphisms with the risk of age-related macular degeneration(AMD) in a high-altitude population. METHODS: The study group consisted of 172 participants with symptoms of AMD who were examined and diagnosed between January 2019 and June 2020. The control group was composed of 120 healthy individuals. Each participant was required to provide two milliliters of peripheral blood for DNA extraction. Two single nucleotide polymorphisms(SNPs) of CFH(rs1061170 and rs800292) and two SNPs of MAP1LC3B(rs8044820 and rs9903) were genotyped. The genotypes and allele frequencies of the SNPs in the study and control groups were further compared using Chi-square and Fisher’s exact tests. RESULTS: In a high-altitude population, the nominally significant differences of rs800292 and rs9903’s genotype AG frequencies were observed in the AMD group(P=0.034 and 0.004, respectively). The frequencies of allele G of rs800292 and allele A of rs9903 were also significantly dif ferent in the AMD group compared to the control [(P=0.034, OR=0.70, 95%CI: 0.50-0.98) and(P=0.004, OR=1.60, 95%CI: 1.15-2.22), respectively]. No significant differences in the genotype distributions(P=0.16 and 0.40, respectively) and allele frequencies(P>0.05) of rs1061170 and rs8044820 were observed in the AMD group.CONCLUSION: Genotype AG of rs800292 may be a protective factor for AMD. Conversely, rs9903 seems to be a risk factor for AMD. Therefore, allele G of rs800292 may be a protective factor, and allele A of rs9903, a risk factor for AMD in Qinghai high-altitude population.

ROS-mediated BNIP3-dependent mitophagy promotes coelomocyte survival in Apostichopus japonicus in response to Vibrio splendidus infection

Organisms produce high levels of reactive oxygen species(ROS)to kill pathogens or act as signaling molecules to induce immune responses;however,excessive ROS can result in cell death.To maintain ROS balance and cell survival,mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates.In marine invertebrates,however,mitophagy and its functions remain largely unknown.In the current study,Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential(ΔΨm)and mitophagosome formation.The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide(LPS)treatment increased mitophagy flux.To explore the regulatory mechanism of mitophagy,we cloned Bcl2/adenovirus E1 B 19 kDa protein-interacting protein 3(BNIP3),a common mitophagy receptor,from sea cucumber Apostichopus japonicus(Aj BNIP3)and confirmed that Aj BNIP3 was significantly induced and accumulated in mitochondria after V.splendidus infection and LPS exposure.At the mitochondrial membrane,Aj BNIP3 interacts with microtubule-associated protein 1 light chain 3(LC3)on phagophore membranes to mediate mitophagy.After Aj BNIP3 interference,mitophagy flux decreased significantly.Furthermore,Aj BNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide(H2 O2),ROS scavengers,and ROS inhibitors.Finally,inhibition of BNIP3-mediated mitophagy by Aj BNIP3 small interfering RNA(si RNA)or high concentrations of lactate increased apoptosis and decreased coelomocyte survival.These findings highlight the essential role of Aj BNIP3 in damaged mitochondrial degradation during mitophagy.This mitophagy activity is required for coelomocyte survival in A.japonicus against V.splendidus infection.

Choline dehydrogenase interacts with SQSTM1 to activate mitophagy and promote coelomocyte survival in Apostichopus japonicus following Vibrio splendidus infection

Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes,leading to the production of excessive reactive oxygen species(ROS)and irreversible apoptotic cell death.Emerging evidence suggests that mitochondrial autophagy(mitophagy)is the most effective method for eliminating damaged mitochondria and ROS,with choline dehydrogenase(CHDH)identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3(LC3)in vertebrates.However,the functional role of CHDH in invertebrates is largely unknown.In this study,we observed a significant increase in the mRNA and protein expression levels of A.japonicus CHDH(AjCHDH)in response to V.splendidus infection and lipopolysaccharide(LPS)challenge,consistent with changes in mitophagy under the same conditions.Notably,AjCHDH was localized to the mitochondria rather than the cytosol following V.splendidus infection.Moreover,AjCHDH knockdown using si RNA transfection significantly reduced mitophagy levels,as observed through transmission electron microscopy and confocal microscopy.Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region(LIR)for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1.The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis.Furthermore,laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells.In contrast,AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria,a critical step in damaged mitochondrial degradation.Thus,AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS,followed by increased apoptosis and decreased coelomocyte survival.Collectively,these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A.japonicus following V.splendidus infection.

A novel mechanism of PHB2-mediated mitophagy participating in the development of Parkinson's disease

Endoplasmic reticulum stress and mitochondrial dysfunction play important roles in Parkinson s disease,but the regulato ry mechanism remains elusive.Prohibitin-2(PHB2)is a newly discove red autophagy receptor in the mitochondrial inner membrane,and its role in Parkinson’s disease remains unclear.Protein kinase R(PKR)-like endoplasmic reticulum kinase(PERK)is a factor that regulates cell fate during endoplasmic reticulum stress.Parkin is regulated by PERK and is a target of the unfolded protein response.It is unclear whether PERK regulates PHB2-mediated mitophagy thro ugh Parkin.In this study,we established a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced mouse model of Parkinson’s disease.We used adeno-associated virus to knockdown PHB2 expression.Our res ults showed that loss of dopaminergic neurons and motor deficits were aggravated in the MPTP-induced mouse model of Parkinson’s disease.Ove rexpression of PHB2 inhibited these abnormalities.We also established a 1-methyl-4-phenylpyridine(MPP+)-induced SH-SY5Y cell model of Parkinson’s disease.We found that ove rexpression of Parkin increased co-localization of PHB2 and microtubule-associated protein 1 light chain 3,and promoted mitophagy.In addition,MPP+regulated Parkin involvement in PHB2-mediated mitophagy through phosphorylation of PERK.These findings suggest that PHB2 participates in the development of Parkinson’s disease by intera cting with endoplasmic reticulum stress and Parkin.

脓毒症相关性脑病大鼠海马区神经细胞自噬

目的:探讨脓毒症相关性脑病(sepsis-associated encephalopathy,SAE)大鼠海马区神经细胞自噬现象及微管相关蛋白1轻链3(LC3)的表达。方法:盲肠结扎穿孔术(cecal ligation and puncture,CLP)建立脓毒症大鼠模型。60只30日龄健康雄性Wistar大鼠被随机分为假手术组(10只)和CLP组(50只)。CLP后12 h监测大鼠脑电图(electroencephalogram,EEG)及体感诱发电位(somatosensory evoked potential,SEP),并进行神经生物学评分。根据大鼠是否发生SAE将CLP组大鼠再分为SAE(+)组和SAE(–)组。HE染色观察大鼠海马区病理学改变;电子显微镜观察大鼠海马区神经细胞自噬的超微结构;Western印迹检测LC3-I和LC3-II蛋白的表达。结果:50只大鼠在CLP后12 h内死亡5只,存活45只大鼠中有16只出现神经行为学、EEG及SEP改变,诊断为SAE,其发病率为35.56%(16/45)。与假手术组和SAE(–)组比较,SAE(+)组大鼠在CLP后12 h时α波的频率明显减少,δ波增加,P1振幅下降,P1波和N1波潜伏期延长(P<0.05)。CLP后12 h时SAE(+)组大鼠海马区细胞明显水肿,锥体细胞明显减少,甚至溶解,细胞排列紊乱,而假手术组和SAE(–)组大鼠海马区细胞形态正常、层次清楚。透射电子显微镜观察表明:SAE(+)组大鼠海马区细胞结构紊乱,可见自噬泡、颗粒状基质和方形或长方形晶体,自噬泡内含有溶酶体等细胞器,假手术组和SAE(–)组大鼠海马区组织细胞无自噬泡形成。SAE(+)组大鼠在CLP后12 h,海马区神经细胞LC3-II/LC3-I值明显高于假手术组和SAE(–)组(P<0.05)。结论:SAE大鼠海马区神经细胞存在细胞自噬现象,LC3-II/LC3-I值明显升高。

大鼠肾脏缺血再灌注损伤后自噬的初步研究

目的研究大鼠肾脏缺血再灌注(I/R)损伤后自噬溶酶体相关蛋白随损伤时间表达的规律,检测自噬体、溶酶体的变化。方法采用肾动脉夹闭法制作大鼠肾脏I/R损伤模型,将实验动物随机分入I/R组和对照组,每组在各时间点各6只。分别于I/R后2、6、12和24h,采用Western印迹法检测肾组织中自噬相关蛋白Belinl和微管相关蛋白1轻链3(LC)的表达情况,在透射电子显微镜下观察I/R后肾组织内自噬小体的形成情况。结果 I/R组大鼠肾组织内Beclin1和LC3蛋白表达水平均自I/R后2h起开始升高,至I/R后24h达到高峰(P值均<0.05)。透射电子显微镜下见,对照组大鼠近端小管上皮细胞核膜完整,染色质结构正常,细胞质中细胞器形态正常,未见自噬小体;I/R组大鼠I/R后2h可见到损伤区域有自噬体形成,溶酶体增多,I/R后24h最为明显。结论大鼠肾I/R损伤后肾组织的自噬活性升高。

Structural insights of phosphorylated into the recognition FUNDC1 by LC3B in mitophagy

Mitophagy is an essential intracellular process that eliminates dysfunctional mitochondria and maintains cellular homeostasis. Mitophagy is regulated by the post-translational modification of mitophagy receptors. Fun14 domain-containing protein 1 （FUNDC1） was reported to be a new receptor for hypoxia-induced mitophagy in mammalian cells and interact with micro-tubule-associated protein light chain 3 beta （LC3B） through its LC3 interaction region （LIR）. Moreover, the phosphorylation modification of FUNDC1 affects its binding affinity for LC3B and regulates selective mitophagy. However, the structural basis of this regulation mechanism remains unclear. Here, we present the crystal structure of LC3B in complex with a FUNDCI LIR peptide phosphorylated at Ser17 （pS17）, demonstrating the key residues of LC3B for the specific recognition of the phosphorylated or dephosphorylated FUNDC1. Intriguingly, the side chain of LC3B Lys49 shifts remarkably and forms a hydrogen bond and electrostatic interaction with the phosphate group of FUNDC1 pS17. Alternatively, phosphorylated Tyr18 （PY18） and Ser13 （PS13） in FUNDC1 significantly obstruct their interaction with the hydrophobic pocket and Arg10 of LC3B, respectively. Structural observations are further validated by mutation and isothermal titration calorimetry （ITC） assays. Therefore, our structural and biochemical results reveal a working model for thespecific recognition of FUNDCI by LC3B and imply that the reversible phosphorylation modification of mitophagy receptors may be a switch for selective mitophagy.

m TOR signaling in liver regeneration: Rapamycin combined with growth factor treatment

AIM: To investigate the effects of mammalian target of rapamycin(mT OR) inhibition on liver regeneration and autophagy in a surgical resection model.METHODS: C57BL/6 mice were subjected to a 70% partial hepatectomy(PH) and treated intraperitoneally every 24 h with a combination of the m TOR inhibitor rapamycin(2.5 mg/kg per day) and the steroid dexamethasone(2.0 mg/kg per day) in phosphate bufferedsaline(PBS) or with PBS alone as vehicle control. In the immunosuppressant group, part of the group was treated subcutaneously 4 h prior to and 24 h after PH with a combination of human recombinant interleukin 6(IL-6; 500 μg/kg per day) and hepatocyte growth factor(HGF; 100 μg/kg per day) in PBS. Animals were sacrificed 2, 3 or 5 d after PH and liver tissue and blood were collected for further analysis. Immunohistochemical staining for 5-Bromo-2'-deoxyuridine(Brd U) was used to quantify hepatocyte proliferation. Western blotting was used to detect hepatic microtubule-associated protein 1 light chain 3(LC3)-Ⅱ protein expression as a marker for autophagy. Hepatic gene expression levels of proliferation-, inflammation- and angiogenesisrelated genes were examined by real-time reverse transcription-polymerase chain reaction and serum bilirubin and transaminase levels were analyzed at the clinical chemical core facility of the Erasmus MC-University Medical Center.RESULTS: m TOR inhibition significantly suppressed regeneration, shown by decreased hepatocyte proliferation(2% vs 12% Brd U positive hepatocyte nuclei at day 2, P < 0.01; 0.8% vs 1.4% at day 5, P = 0.02) and liver weight reconstitution(63% vs 76% of initial total liver weight at day 3, P = 0.04), and furthermore increased serum transaminase levels(aspartate aminotransferase 641 U/L vs 185 U/L at day 2, P = 0.02). Expression of the autophagy marker LC3-Ⅱ, which was reduced during normal liver regeneration, increased after mT OR inhibition(46% increase at day 2, P = 0.04). Hepatic gene expression showed an increased inflammation-related response [tumor necrosis factor(TNF)-α 3.2-fold upregulation at day 2, P = 0.03; IL-1Ra 6.0-fold upregulation at day 2 and 42.3-fold upregulation at day 5, P < 0.01] and a reduced expression of cell cycle progression and angiogenesis-related factors(HGF 40% reduction at day 2; vascular endothelial growth factor receptor 2 50% reduction at days 2 and 5; angiopoietin 1 60% reduction at day 2, all P ≤ 0.01). Treatmentwith the regeneration stimulating cytokine IL-6 and growth factor HGF could overcome the inhibitory effect on liver weight(75% of initial total liver weight at day 3, P = 0.02 vs immunosuppression alone and P = 0.90 vs controls) and partially reversed gene expression changes caused by rapamycin(TNF-α and IL-1Ra levels at day 2 were restored to control levels). However, no significant changes in hepatocyte proliferation, serum injury markers or autophagy were found.CONCLUSION: mT OR inhibition severely impairs liver regeneration and increases autophagy after PH. These effects are partly reversed by stimulation of the IL-6 and HGF pathways.

肠道病毒71型诱导人恶性胶质瘤细胞U251自噬的研究

目的初步研究肠道病毒71型（EV71）诱导人恶性胶质瘤细胞U251的自噬现象及其对微管相关蛋白1轻链3（LC3）表达的影响。方法RPMI1640培养U251细胞24h后随机分为实验组和对照组，实验组按感染复数（MOI）值为1加入EV71，EV71感染12h后采用单丹磺酰戊二胺（MDC）染色标志细胞自噬泡，荧光显微镜观察自噬泡的形成。EV71感染24h后细胞免疫荧光检测LC3，荧光显微镜观察LC3在U251细胞内的分布。EV71感染2、4、8、12、24、48h采用Westernblot检On．0LC3-I和LC3-II蛋白的表达，并对LC3-II蛋白进行半定量分析。结果白噬泡经过MDC染色后，荧光显微镜观察发现自噬泡呈点状结构。与对照组比较，实验组在EV71感染12h，U251细胞内自噬泡明显增多；EV71感染24h，U251细胞皱缩、变小、形态不规则，LC3蛋白表达明显增多，主要分布在细胞质和细胞核周围；EV71感染4h后，LC3-II蛋白表达开始增加，明显高于对照组（P〈0．01）。结论EV71能有效诱导U251细胞自噬，发挥其溶瘤作用。