miR-30e-3p in natural killer cell-derived exosomes inhibits the proliferation and invasion of human esophageal squamous carcinoma cells / 细胞与分子免疫学杂志

Objective To investigate the effects of natural killer (NK)-cell-derived miR-30e-3p-containing exosomes (Exo) on esophageal squamous cell carcinoma (ESCC) cell proliferation, apoptosis and invasion. Methods NK cells were isolated and amplified from the peripheral blood of healthy donors, and NK cell-derived Exo was isolated and identified, which were further co-cultured with NEC cells and were randomly grouped into Exo1 and Exo2 groups. Transmission electron microscopy (TEM) was used to observe the morphology and size of exosomes. Western blot analysis was used to detect the expression levels of exosome markers apoptosis related gene 2- interacting protein X(ALIX), tumor susceptibility gene 101(TSG101), CD81 and calnexin. The NC plasmids, mimics and inhibitors of miR030e-3p were respectively delivered into the NK cells, and the corresponding NK cells-derived Exo were co-cultured with NEC cells, which were divided into NC, Exo, mimic and inhibitor groups. CCK-8 assay was used to evaluate cell proliferation, flow cytometry was conducted to determine cell cycle, annexin V-FITC/PI double staining was employed to detect cell apoptosis, and TranswellTM assay was performed to detect cell invasion abilities. Real-time quantitative PCR was used to detect the expression of miR-23b, miR-422a, miR-133b, miR-124, miR-30e-3p and miR-99a in NCE cells and exosomes. Results The percentages of CD56+CD3+ cells and CD56+CD16+ cells in NK cells were (0.071±0.008)% and (90.6±10.6)%, respectively. Exosome isolated from NK cells ranged from 30 nm to 150 nm, and was positive for ALIX, TSG101 and CD81, while negative for calnexin. NK cell-derived Exos inhibited the proliferation, reduced the proportion of S-phase cells and the number of invaded cells of NEC cells, and promoted the apoptosis and the proportion of G1 phase cells. Overexpression of miR-30E-3p in NK cell-derived exosome inhibited the proliferation and invasion of NEC cells, and blocked cell cycle and promoted apoptosis, while knockdown miR-30e-3p in NK cell-derived exosomes did the opposite. Conclusion miR-30e-3p in NK cell-derived exosomes can inhibit the proliferation and invasion of ESCC cells, block their cell cycle and induce their apoptosis.

Expressions of CCAAT/enhancer-binding Protein Homologous Protein and Calnexin in the Hippocampus of a Mouse Model of Mesial Temporal Lobe Epilepsy / 中国医学科学院学报

Objective To explore the temporal and spatial distribution of CCAAT/enhancer-binding protein homologous protein (CHOP) and calnexin (CNX) in the dentate gyrus of mesial temporal lobe epilepsy (mTLE) mouse model. Methods We used kainic acid (KA) to induce acute phase (12 h and 24 h) mTLE mouse models and performed Western blotting and immunofluorescence to detect the different expressions and distribution pattern of CHOP and CNX in CA3 of the hippocampus. Results Compared with the controls,the expressions of CHOP(F=1.136,P=0.4069) and CNX (F=2.378,P=0.2087) did not increase in CA3 of hippocampus 12 h following KA injection in the acute phase of mTLE mouse models,whereas the expressions in CA1 and CA3 of hippocampus 24 h after injection were significantly higher (F=8.510,P=0.0362;F=6.968,P=0.0497,respectively). As shown by immunofluorescence analysis,CHOP was expressed mainly in CA3 of hippocampus 12 h after KA injection,and increased in CA1 and CA3 24 h after KA administration. Compared with the controls,the expressions of CHOP(F=24.480,P=0.0057) and CNX (F=7.149,P=0.0478) were significantly higher 24 h after KA injection.Conclusions The expression of CHOP increases along with the progression of seizures,indicating the increased level of endoplasmic reticulum stress. An increasing number of CNX,which serves as molecular chaperone,may be needed to facilitate the unfolded protein to complete the folding process.

Association of defective HLA-I expression with antigen processing machinery and their association with clinicopathological characteristics in Kazak patients with esophageal cancer / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has been confirmed that defective expression of human leukocyte antigen class I (HLA-I) molecules can contribute to the immune evasion of cancer cells in some types of cancer. The aim of this study was to examine the expression of HLA class I antigen and the antigen-processing machinery (APM) components in esophageal squamous cell carcinoma (ESCC) and their role in high risk human papillomavirus (HPV) infection, and to analyze their association with histopathological characteristics in the Kazak ethnic group.</p><p><b>METHODS</b>A total of 50 formalin-fixed, paraffin-embedded ESCC lesions were collected from the First Affiliated Hospital of Xinjiang Medical University, China. The expression levels of HLA-I antigen and APM components were determined by immunohistochemistry; the HPV DNA were detected using polymerase chain reaction (PCR).</p><p><b>RESULTS</b>A high frequency of down-regulation or loss of expression of HLA and APM components were found in esophageal cancer in Kazak people. HLA-I, TAP1, CNX, LMP7, Erp57, Tapasin and ERAP1 were down-regulated in 68%, 44%, 48%, 40%, 52%, 32% and 20% of ESCC lesions then, respectively. The loss of expression of HLA-I antigen was significantly correlated with part of the APM components and positively correlated with high risk HPV16 infection. TAP1, CNX, LMP7, Erp57 and Tapasin loss were significantly associated with tumor grading, lymph node metastasis and depth of invasion (P < 0.05).</p><p><b>CONCLUSION</b>Our results suggest that APM component defects are a mechanism underlying HLA-I antigen down-regulation in ESCC lesions, and indicate that the loss expression of HLA-I and APM components will become an important marker of ESCC and analysis of HLA-I and APM component expression can provide useful prognostic information for patients with ESCC from the Kazak ethnic group.</p>

Clonagem, caracterização e expressão heteróloga de uma calnexina homóloga do fungo patogênico Paracoccidioides brasiliensis / Cloning, characterization and heterologue expression of a calnexin homologue from the pathogenic fungus Paracoccidioides brasiliensis

Descrevemos neste trabalho a clonagem do cDNA de, que codifica uma calnexina homóloga do retículo endoplasmático de Paracoccdiioides brasiliensis. Esta chaperone reconhece especificamente glicoproteínas monoglicosiladas no retículo endoplasmático, sendo assim um componente essencial para o processo de dobramento de glicoproteínas nascentes que serão secretadas. A fase aberta de leitura da PbCnx foi encontrada num fragmento de 1.701 pares de bases (pb) que codifica uma proteína de 567 resíduos de aminoácidos, com massa estimada em 62.680 Da. A seqüência deduzida de aminoácidos compartilha identidade com as sequências descritas das calnexinas de Aspergillus fumigatus, Aspergillus niger e apresenta vinte potenciais sítios de fosforilação e dois de glicosilação. Além disso, esta seqüência deduzida de aminoácidos apresenta um extraordinário grau de conservação, especialmente numa região central, denominada domínio central altamente conservado (hcd) que contém, como uma 'marca', sequências repetidas KPEDWD; estas sequências são consideradas sítios ligantes de cálcio de alta afinidade e são encontradas em todos os membros da família das calnexinas e calreticulinas. A análise das hibridações por Southem e Northem blots, mostraram que a proteína é codificada por um único gene ou gene de poucas cópias. O cDNA que codifica PbCnx foi expresso como proteína recombinante em Escherlchia colí. A proteína recombinante purificada foi reconhecida por soros de paciente com paracoccidioidomicose (forma crônica). Foi gerado também um soro policlonal monoespecífico, a partir da proteína recombinante de fusão, utilizado para a localização celular da proteína, através de microscopia confocal. O padrão de marcação observado por imunofluorescência mostra a proteína intensamente distribuída na região citosólica da célula. Além disso, este soro policlonal reconheceu especificamente a calnexina nativa através de ensaios de immunoblotting do extrato total de células leveduriformes de P. brasilíensis.

Age-related decline in expression of calnexin

Aging is accompanied by the changes in the cells that decrease their capacity to respond to various forms of stress. Cells are known to respond to stresses through expression of stress- response proteins, heat-shock proteins composed of molecular chaperones. Recent studies suggest that chaperone level and stress-induced chaperone expression could decrease with aging. The aim of the present study is to identify chaperones that show a significant change in protein expression with aging. We used an in vitro aging model system of human diploid fibroblasts (HDF). Proteome analysis of HDF showed that endoplasmic reticulum (ER) chaperone, calnexin, significantly decreased with aging. Oxidative stress-induced expression of calnexin also attenuated in old HDF compared to young cells. These findings suggest calnexin decreases with aging and might contribute to a cytoprotection in a variety of human age-related diseases.