Serum exosomal proteomics analysis of lung adenocarcinoma to discover new tumor markers.

BACKGROUND: Among the most aggressive and rapidly lethal types of lung cancer, lung adenocarcinoma is the most common type. Exosomes, as a hot area, play an influential role in cancer. By using proteomics analysis, we aimed to identify potential markers of lung adenocarcinoma in serum. METHODS: In our study, we used the ultracentrifugation method to isolate serum exosomes. The Liquid chromatography-mass spectrometry (LC-MS) and bioinformatics analysis were used to identify potential serum exosomal proteins with altered expression among patients with advanced lung adenocarcinoma, early lung adenocarcinoma, and healthy controls. A western blot (WB) was performed to confirm the above differential expression levels in a separate serum sample-isolated exosome, and immunohistochemistry (IHC) staining was conducted to detect expression levels of the above differential proteins of serum exosomes in lung adenocarcinoma tissues and adjacent tissues. Furthermore, we compared different expression models of the above differential proteins in serum and exosomes. RESULT: According to the ITGAM (Integrin alpha M chain) and CLU (Clusterin) were differentially expressed in serum exosomes among different groups as well as tumor tissues and adjacent tissues. ITGAM was significantly and specifically enriched in exosomes. As compared to serum, CLU did not appear to be significantly enriched in exosomes. ITGAM and CLU were identified as serum exosomal protein markers of lung adenocarcinoma. CONCLUSIONS: This study can provide novel ideas and a research basis for targeting lung adenocarcinoma treatment as a preliminary study.

lncCRLA Enhanced Chemoresistance in Lung Adenocarcinoma That Underwent EpithelialMesenchymal Transition.

EMT confers increased metastatic potential and the resistance to chemotherapies to cancer cells. However, the precise mechanisms of EMT-related chemotherapy resistance remain unclear. c-Src-mediated caspase 8 phosphorylation essential for EMT in lung adenocarcinoma cell lines preferentially occurs in cells with the mesenchymal phenotype, resulting in chemoresistance to cisplatin plus paclitaxel in patients with resectable lung adenocarcinoma and a significantly worse 5-year PFS. Cisplatin killed lung adenocarcinoma cells regardless of caspase 8. Paclitaxel-triggered necroptosis in lung adenocarcinoma cells was dependent on the phosphorylation or deficiency of caspase 8, during which FADD interacted with RIPK1 to activate the RIPK1/RIPK3/MLKL signaling axis. Accompanied with c-Src-mediated caspase 8 phosphorylation to trigger EMT, a novel lncRNA named lncCRLA was markedly upregulated and inhibited RIPK1-induced necroptosis by impairing RIPK1RIPK3 interaction via binding to the intermediate domain of RIPK1. Dasatinib mitigated c-Src-mediated phosphorylation of caspase 8-induced EMT and enhanced necroptosis in mesenchymal-like lung adenocarcinoma cells treated with paclitaxel, while c-FLIP knockdown predominantly sensitized the mesenchymal-like lung adenocarcinoma cells to paclitaxel+dasatinib. c-Srccaspase 8 interaction initiates EMT and chemoresistance via caspase 8 phosphorylation and lncCRLA expression, to which the dasatinib/paclitaxel liposome+siFLIP regimen was lethal.

Prognostic Hub Genes in the Immune Microenvironment of Lung Adenocarcinoma by Estimation.

BACKGROUND: The mortality of lung adenocarcinoma (LUAD) is high. Recent studies have found that the degree of immune infiltration and stromal cells in the tumour microenvironment or tumours makes a significant contribution to prognosis. METHODS: During the study, we screened differentially expressed genes (DEGs) of the TCGA database for prognostic genes in the LUAD immune microenvironment. Furthermore, immune and stromal cells were quantified using the ESTIMATE algorithm. To study the effects of immune and stromal cell-associated genes on the prognosis of LUAD, LUAD patients were divided into high and low groups according to their immune/stromal scores. The obtained scores were found to be related to the phenotype and survival rate of LUAD patients. By selecting DEGs with high expression in immune and stromal cells, we performed functional enrichment analysis and found that most genes are associated with pathways of cancer, stimulus response and MAPK signaling. The functions and enriched pathways of LUAD prognostic genes were shown by a protein-protein interaction (PPI) network. Nonetheless, an external database was used to validate the prognostic genes from the TCGA. RESULTS: Prognostic genes were listed according to their expression position and protein function. CONCLUSION: We provided new targets for immunotherapy of LUAD, which further provide basic knowledge for future clinical research.

Integrative bioinformatics analysis the clinical value of KMT5A in different subtypes of lung cancer.

To combat cancer disease, The Cancer Genome Atlas (TCGA) collects a large amount of information through high-throughput genome analysis technology. At present, there are only few treatments available for lung squamous cell carcinoma (LUSC). Although lysine methyltransferase 5A (KMT5A) is considered as a new biomarker for LUSC, its functionalities needs to be confirmed clinically in lung adenocarcinoma (LUAD). Therefore, in the current investigation we obtained the expression data of KMT5A in LUSC and LUAD by TCGA database. This study evaluated the prognostic value of KMT5A expression in LUAD and LUSC, and discussed the relevant biological pathways of KMT5A involved in the pathogenesis of LUAD and LUSC. KMT5A was highly expressed in LUAD and LUSC cancer tissues. The overall survival analysis revealed that the prognosis of high expression group was poor for LUAD, but was opposite in LUSC. In LUAD, the expression of KMT5A was significantly correlated with age (P = 0.029), gender (P = 0.001) and m (P = 0.042). Logistic regression showed that gender was significantly correlated with poor prognosis of LUAD (P = 0.00175). Multivariate analysis of Cox proportional hazards model exhibited that KMT5A risk ratio (HR) was 0.97, 95% confidence interval (CI), 0.94-1.0, P = 0.026. Age (P = 0.044), t (P = 0.031), m (P = 0.047) were the independent prognostic factors of LUSC patients, while the stage was the independent prognostic factor of LUAD (P < 0.001). Genome enrichment analysis presented that LUSC was differentially enriched with antigen processing and presentation, cell adhesion molecules, cytokine receptor interaction, ECM receptor interaction, etc. LUAD was differentially enriched with apoptosis, cancer pathway, vascular endothelial growth factor signaling pathway and wnt signaling pathway. Overall, this study presented the clinical value of KMT5A in LUSC and LUAD and suggested the possible pathways involved.

The Combined Detection of Immune Genes for Predicting the Prognosis of Patients With Non-Small Cell Lung Cancer.

Lung cancer is one of the leading causes of cancer-related death. In recent years, there has been an increasing interest in the fields of tumor and immunity. This study focused on the possible prognostic value of immune genes in non-small cell lung cancer patients. We used The Cancer Genome Atlas (TCGA) to download gene expression data and clinical information of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). The immune gene list was downloaded from the Immport database. We then constructed immune gene prognostic models on the basis of Cox regression analysis. We further evaluated the clinical significance of the models via survival analysis, receiver operating characteristic (ROC) curves, and independent prognostic factor analysis. Moreover, we analyzed the associations of prognostic models with both mutation burdens and neoantigens. Using the Gene Expression Omnibus (GEO) and Kaplan-Meier plotter databases, we evaluated the validity of the prognostic models. The prognostic model of LUAD included 13 immune genes, and the prognostic model of LUSC contained 10 immune genes. High-risk patients based on prognostic models had a lower 5-year survival rate than did low-risk patients. The ROC curve analysis demonstrated the prediction accuracy of the prognostic models, as the area under the curve (AUC) was 0.742, 0.707, and 0.711 for LUAD, and 0.668, 0.703, and 0.668 for LUSC, when the predicted survival times were 1, 3, and 5 years, respectively. The mutation burden analysis showed that mutation level was associated with the risk score in patients with LUAD. The analysis based on GEO and Kaplan-Meier plotter demonstrated the prognostic validity of the models. Therefore, immune gene-related models of LUAD and LUSC can predict prognosis. Further study of these genes may enable us to better distinguish between LUAD and LUSC and lead to improvement in immunotherapy for lung cancer.

Integrative Bioinformatics Analysis Identifies NEK2 as a Potential Biomarker in Head and Neck Squamous Cell Carcinoma.

The purpose of this study is to explore the function and regulatory networks of differentially expressed genes (DEGs) in head and neck squamous cell carcinoma (HNSC) by bioinformatics analysis using bioinformatics methods. The gene expression profile associated with HNSC was upregulated from the Cancer RNA-Seq Nexus (CRN) database and Oncomine. Functional analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and gene ontology was performed on DEGs. The protein-protein interaction (PPI) network of DEGs was constructed using STRING and Cytoscape databases. A PPI network contained 101 genes. According to the comprehensive analysis of Degree and MCODE score, there are 12 genes. Based on the screened protein network and differential genes, further exploration was conducted according to The Cancer Genome Atlas (TCGA) normal and the genotype-tissue expression (GTEx) data. This investigation involved observing the expression of the first eight hub genes in tissues and the expression of NIMA-related kinase 2 (NEK2) in different stages and then survival analysis of HNSC. Results showed that NEK2 has important value in HNSC, which provides a reference for the molecular mechanism of HNSC.

[Generation of a novel HBeAg transgenic mice using CRISPR/Cas9 technique].

OBJECTIVE: To generate a new strain of HBeAg transgenic mice using CRISPR/Cas9 technique. METHODS: Hepatitis B virus (HBV) HBeAg gene was cloned and inserted in the pliver-HBeAg expression frame at the site of Rosa26 gene using CRISPR/Cas9 and homologous recombination techniques to construct the pliver-HBeAg expression vector containing HBeAg gene. The linear DNA fragment containing HBeAg gene was obtained by enzyme digestion. Cas9 mRNA, gRNA and the donor vector were microinjected into fertilized eggs of C57BL/6J mice, which were then transplanted into the uterus of C57BL/6J female surrogate mice to obtain F0 generation mice. The F0 generation mice were identified by long fragment PCR to obtain F0 transgenic mice with HBeAg gene. The positive F0 generation mice were bred with wild-type C57BL/6J mice to produce the F1 mice, which were identified by PCR and sequencing. The positive F1 transgenic mice carrying HBeAg gene were backcrossed until the homozygous offspring transgenic mice were obtained. The genotypes of the offspring mice were identified. The expressions of HBeAg and HBeAb in the heterozygous and homozygous HBeAg transgenic mice were detected by automatic chemiluminescence immunoassay, immune colloidal gold technique and immunohistochemistry method. RESULTS: A total of 56 F0 mice were obtained, and 2 of them carried homologous recombined HBeAg gene. Six positive F1 mice were obtained, from which 22 homozygous and 29 heterozygous F2 generation HBeAg transgenic mice were obtained. High concentration of HBeAg protein was detected in the peripheral blood of all the positive HBeAg transgenic mice without HBeAb expression. HBeAg expression was detected in the hepatocytes of HBeAg transgenic mice. CONCLUSIONS: We obtained a new strain of HBeAg transgenic mice with stable expression of HBeAg in the hepatocytes and immune tolerance to HBeAg using CRISPR/Cas9 technique, which provide a new animal model for studying HBV.

Potential Role of Exosomes in Cancer Metastasis.

High cancer mortality is attributed to metastasis to a large extent. However, cancer metastasis remains devoid of dynamic monitoring and early prevention in terms of current advances in diagnostic means and therapeutic modalities. Meanwhile, studies have shown that reciprocal crosstalk among cells via exosomes plays a critical role in maintaining normal physiological state or triggering disease progression, including cancer metastasis. Therefore, in this review, we focus on the latest literature (primarily from 2018) to summarize action mechanisms and experimental studies of exosomes in cancer metastasis and put forward some problems as well as new outlooks of these studies.

Outcomes after sofosbuvir-containing regimens for hepatitis C virus in patients with decompensated cirrhosis: a real-world study.

BACKGROUND: Direct-acting antivirals have been used for decompensated cirrhotic patients with hepatitis C virus (HCV) infection. However, the benefits in Chinese patients with decompensated cirrhosis are unclear. METHODS: Thirty patients with HCV infection and decompensated cirrhosis were administered sofosbuvir-containing regimens at our hospital between April and December 2015. The efficacy and safety of the treatments was determined by sustained virological response at week 12 (SVR 12), change of liver function and adverse events. RESULTS: The cohort included 13 treatment-experienced and 17 treatment-naïve patients. A total of 27 patients (90%) achieved SVR 12. No baseline characteristics (sex, age, treatment-experience, genotype, viral load, liver function or splenectomy) was association with achievement of SVR 12. Patients achieved SVR 12 had significantly improved liver function by post-treatment week 12 (P < 0.05). Of the 30 patients, six developed anemia, one developed hepatic decompensation, two developed impaired renal function and one developed a severe upper respiratory tract infection during the treatment. There was no death or HCC development during 12 months of follow-up off-therapy. Two patients (7.4%) with SVR 12 experienced new decompensated episodes during the follow-up. CONCLUSION: Sofosbuvir-containing regimens are effective in Chinese HCV patients with decompensated cirrhosis, regardless of baseline characteristics, as demonstrated by a high rate of SVR 12, as well as improvement in liver function. Although antiviral therapy is generally well tolerated, a vigilant monitoring of anemia and renal function should be mandatory.

Membrane proteomic analysis comparing squamous cell lung cancer tissue and tumour-adjacent normal tissue.

Lung cancer is the leading cause of cancer-related deaths worldwide. Squamous cell carcinoma is one of the predominant histological subtypes of lung cancer. Detecting lung cancer at an early stage is essential for successful therapy and increasing survival. There are still no satisfactory biomarkers for the early detection of lung cancer. In this study, tumour tissue paired with tumour-adjacent normal bronchial epithelial tissue was obtained from patients with squamous cell lung carcinoma without metastasis. The proteins extracted from the cell membrane were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) and were analysed with the Image Master two-dimensional platinum software. Twenty-five significantly different protein spots were selected and identified with matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). A total of 19 proteins were successfully identified. Twelve proteins were up-regulated, and seven proteins were down-regulated in the cancerous tissue compared with the tumour-adjacent normal tissue. One up-regulated protein and one down-regulated protein in squamous cell lung carcinoma were verified by Western blot analysis and RT-PCR; the results were consistent with the 2-DE analysis. In conclusion, membrane proteomics identified a number of candidate biomarker proteins that were differentially expressed between squamous cell lung cancer tissue and adjacent normal tissue. These biomarker candidates have the potential to elucidate the underlying pathogenesis of squamous cell lung cancer.