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BACKGROUND: Combining PARP inhibitors (PARPis) with immune checkpoint inhibitors may improve clinical outcomes in selected cancers. We evaluated rucaparib and atezolizumab in advanced gynaecological or triple-negative breast cancer (TNBC). METHODS: After identifying the recommended dose, patients with PARPi-naive BRCA-mutated or homologous recombination-deficient/loss-of-heterozygosity-high platinum-sensitive ovarian cancer or TNBC received rucaparib plus atezolizumab. Tumour biopsies were collected pre-treatment, during single-agent rucaparib run-in, and after starting combination therapy. RESULTS: The most common adverse events with rucaparib 600 mg twice daily and atezolizumab 1200 mg on Day 1 every 3 weeks were gastrointestinal effects, fatigue, liver enzyme elevations, and anaemia. Responding patients typically had BRCA-mutated tumours and higher pre-treatment tumour levels of PD-L1 and CD8 + T cells. Markers of DNA damage repair decreased during rucaparib run-in and combination treatment in responders, but typically increased in non-responders. Apoptosis signature expression showed the reverse. CD8 + T-cell activity and STING pathway activation increased during rucaparib run-in, increasing further with atezolizumab. CONCLUSIONS: In this small study, rucaparib plus atezolizumab demonstrated acceptable safety and activity in BRCA-mutated tumours. Increasing anti-tumour immunity and inflammation might be a key mechanism of action for clinical benefit from the combination, potentially guiding more targeted development of such regimens. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov (NCT03101280).
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Purpose: Exosomes are able to exchange their bioactive RNA cargo to recipient cells. In COPD, exosomes can be controlled and engineered for its use as targeted diagnostic and therapeutic tool. Our study explored novel lncRNAs and mRNAs in plasma exosomes that could be involved in the pathogenesis of COPD. Methods: High-throughput sequencing was conducted to detect the alterations in the expression of exosomal lncRNAs and mRNAs. Gene ontology (GO) functional analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to determine the significant functions and pathways associated with differentially expressed (DE) lncRNAs. The mRNA expression profile dataset, GSE76925, and microRNA expression profile dataset, GSE70080, were obtained from the GEO database. Venn diagrams were used to find common DE mRNAs between my mRNAs dataset and GSE76925. These common DEGs were subjected to PPI analyses to identify Hub genes. Subsequently, Venn diagrams were used to identify common genes between the target genes of DE-miRNAs and Hub genes as well as DE-miRNAs and my lncRNAs dataset. Finally, a lncRNA-miRNA-mRNA co-expression network was constructed by prediction using proprietary software. The lncRNA and mRNA expressions were then validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Results: We identified 1578 differentially regulated lncRNAs and 3071 differentially regulated mRNAs. GO and KEGG pathway analyses suggested that the DE lncRNAs are involved in the pathogenesis of COPD. A lncRNA-miRNA-mRNA meshwork was established to predict the potential interactions among these RNAs. RP3-329A5.8 and MRPS11 expression was then subjected to qRT-PCR for validation. Correlations between MRPS11 and clinic-pathological features were explored. Conclusion: Our study provided a set of lncRNAs and mRNAs that may be involved in the pathogenesis of COPD, thereby highlighting the need for further research on both diagnostic biomarkers and molecular mechanisms.
Subject(s)
MicroRNAs , Pulmonary Disease, Chronic Obstructive , RNA, Long Noncoding , Humans , RNA, Long Noncoding/genetics , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/genetics , MicroRNAs/genetics , RNA, Messenger/genetics , Computational BiologyABSTRACT
Purpose: Child-Turcotte-Pugh class A (CTP-A) in unresectable hepatocellular carcinoma (HCC) is the standard criterion for active therapy and clinical trial enrollment. We hypothesized that insulin-like growth factor-1 (IGF-1) derived scores may provide improved survival prediction over CTP classification. This study aimed to evaluate the potential prognostic and predictive effects of IGF-1 derived scores in the phase III IMbrave150 study. Patients and Methods: Baseline and on-treatment serum IGF-1 levels from 371 patients were subjected to central analysis. Patients' IGF-1 score (1/2/3) and IGF-CTP score (A/B/C) were determined based on pre-specified cutoffs. Outcomes were analyzed by baseline and by on-treatment changes of the IGF-1 and IGF-CTP scores within and between the two treatment arms. The interaction between these scores and outcomes was assessed using univariate and multivariate analyses. Results: Baseline IGF-CTP score (A vs B/C) showed prognostic significance for OS in both the atezolizumab-bevacizumab (hazard ratio [HR], 0.33; 95% confidence interval [CI], 0.20-0.56; P<0.001) and sorafenib (HR, 0.32; 95% CI, 0.16-0.65; P=0.002) arms. Baseline IGF-1 score (1 vs 2/3) also showed prognostic significance for OS in both the atezolizumab-bevacizumab (HR, 0.33; 95% CI, 0.20-0.55; P<0.001) and sorafenib (HR, 0.48; 95% CI, 0.26-0.89; P=0.02) arms. HRs for PFS were consistent with those for OS. No significant predictive effects were observed for either score between the two arms. Kinetic analysis revealed that patients with increased IGF-1 score (1-> 2/3) at 3 weeks post treatment had shorter OS than patients with stable IGF-1 score of 1 in both the atezolizumab-bevacizumab (HR, 3.70; 95% CI, 1.56-8.77; P=0.003) and sorafenib (HR, 5.83; 95% CI, 1.88-18.12; P=0.0023) arms. Conclusion: Baseline and kinetic change of IGF-CTP and IGF-1 scores are independent prognostic factors for patients with unresectable HCC treated with atezolizumab-bevacizumab or sorafenib. These novel scores may provide improved patient stratification in future HCC clinical trials. IMbrave150 ClincialTrials.gov number, NCT03434379.
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Atezolizumab (anti-programmed death-ligand 1 (PD-L1)) and bevacizumab (anti-vascular endothelial growth factor (VEGF)) combination therapy has become the new standard of care in patients with unresectable hepatocellular carcinoma. However, potential predictive biomarkers and mechanisms of response and resistance remain less well understood. We report integrated molecular analyses of tumor samples from 358 patients with hepatocellular carcinoma (HCC) enrolled in the GO30140 phase 1b or IMbrave150 phase 3 trial and treated with atezolizumab combined with bevacizumab, atezolizumab alone or sorafenib (multikinase inhibitor). Pre-existing immunity (high expression of CD274, T-effector signature and intratumoral CD8+ T cell density) was associated with better clinical outcomes with the combination. Reduced clinical benefit was associated with high regulatory T cell (Treg) to effector T cell (Teff) ratio and expression of oncofetal genes (GPC3, AFP). Improved outcomes from the combination versus atezolizumab alone were associated with high expression of VEGF Receptor 2 (KDR), Tregs and myeloid inflammation signatures. These findings were further validated by analyses of paired pre- and post-treatment biopsies, in situ analyses and in vivo mouse models. Our study identified key molecular correlates of the combination therapy and highlighted that anti-VEGF might synergize with anti-PD-L1 by targeting angiogenesis, Treg proliferation and myeloid cell inflammation.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Antibodies, Monoclonal, Humanized , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bevacizumab/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Carcinoma, Hepatocellular/genetics , Clinical Trials, Phase I as Topic , Clinical Trials, Phase III as Topic , Humans , Inflammation/drug therapy , Liver Neoplasms/drug therapy , Liver Neoplasms/genetics , Liver Neoplasms/pathologyABSTRACT
Soil carbon (SC) is a key component of the carbon cycle and plays an important role in climate change; however, quantitatively assessing SC dynamics at the regional scale remains challenging. Earth system model (ESM) that considers multiple environmental factors and spatial heterogeneity has become a powerful tool to explore carbon cycle-climate feedbacks, although the performance of the ESM is diverse and highly uncertain. Thus, identifying reliable ESMs is a prerequisite for better understanding the response of SC dynamics to human activity and climate change. The 16 ESMs that participated in the fifth phase of the Coupled Model Intercomparison Project (CMIP5) were employed to evaluate the skill performance of SC density simulation by comparison with reference data from the International Geosphere-Biosphere Programme Data and Information System (IGBP-DIS). Although ESMs generally reflect spatial patterns with lower SC in northwest China and higher SC in southeast China, 11 of 16 ESMs underestimated the SC in China, and 5 of 16 ESMs overestimated the SC density as most ESMs had large discrepancies in capturing the SC density in the northern high latitudes of China and the Qinghai-Tibet Plateau. According to a series of model performance statistics, SC simulated by Institute Pierre Simon Laplace (IPSL) Coupled Model had a close spatial pattern with IGBP-DIS and showed higher skills for SC predictions in China relative to other CMIP5 ESMs. The multimodel ensemble average obtained by IPSL family ESMs showed that SC density exhibited increasing trends under both the RCP4.5 scenario and RCP8.5 scenario. The SC density increased slowly under RCP8.5 compared with that under RCP4.5 and even displayed a decreasing trend in the late 21st century. The findings of this study can provide a reference for identifying the shortcomings of SC predictions in China and guide SC parameterization improvement in ESMs.
Subject(s)
Carbon , Soil , Carbon Cycle , China , Climate Change , HumansABSTRACT
We reported a patient with unresectable hepatocellular carcinoma (HCC) who initially received 15 cycles of atezolizumab plus bevacizumab combination and had best tumor response of partial response, but later experienced disease progression. After subsequent surgical resection, the patient enjoyed long-term disease-free status at the last follow-up 19 months after surgery. By investigating paired tumor tissues (pretreatment and post-progression samples) with immunohistochemistry, multiplex immunofluorescence, RNA sequencing, and DNA sequencing, we explored the dynamic changes in the tumor microenvironment (TME) and potential mechanisms underlying acquired resistance to the combination. In the post-progression HCC tissue compared with the baseline tissue, the expression of PD-L1 in tumor-infiltrating immune cells and the abundance of CD8+ T cells in the tumor area had decreased, and an immune-excluded TME had emerged. Transcriptomic analysis revealed a gene expression signature representing progenitor/hepatoblast features in the post-progression tumor tissue, with an increased expression of imprinted genes and decreased expression of cytochrome P450 family genes. Finally, tumor mutational burden and MHC class I expression in tumor cells were both increased in the post-progression tissue, suggesting that neoantigen depletion or loss-of-antigen presentation were unlikely causes of acquired resistance in this patient. Atezolizumab plus bevacizumab combination therapy enabled our patient to receive hepatectomy and achieve long-term remission. A comparison of paired tumor tissues suggested that immune-excluded TME and tumor dedifferentiation may have contributed to acquired resistance to the combination.
ABSTRACT
Distinct T cell infiltration patterns, i.e., immune infiltrated, excluded, and desert, result in different responses to cancer immunotherapies. However, the key determinants and biology underpinning these tumor immune phenotypes remain elusive. Here, we provide a high-resolution dissection of the entire tumor ecosystem through single-cell RNA-sequencing analysis of 15 ovarian tumors. Immune-desert tumors are characterized by unique tumor cell-intrinsic features, including metabolic pathways and low antigen presentation, and an enrichment of monocytes and immature macrophages. Immune-infiltrated and -excluded tumors differ markedly in their T cell composition and fibroblast subsets. Furthermore, our study reveals chemokine receptor-ligand interactions within and across compartments as potential mechanisms mediating immune cell infiltration, exemplified by the tumor cell-T cell cross talk via CXCL16-CXCR6 and stromal-immune cell cross talk via CXCL12/14-CXCR4. Our data highlight potential molecular mechanisms that shape the tumor immune phenotypes and may inform therapeutic strategies to improve clinical benefit from cancer immunotherapies.
Subject(s)
Biomarkers, Tumor/genetics , Fibroblasts/immunology , Ovarian Neoplasms/immunology , Single-Cell Analysis/methods , Stromal Cells/immunology , T-Lymphocytes/immunology , Tumor Microenvironment , Biomarkers, Tumor/immunology , Chemokine CXCL12/genetics , Chemokine CXCL12/immunology , Chemokine CXCL16/genetics , Chemokine CXCL16/immunology , Chemokines, CXC/genetics , Chemokines, CXC/immunology , Female , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , RNA-Seq , Receptors, CXCR4/genetics , Receptors, CXCR4/immunology , Receptors, CXCR6/genetics , Receptors, CXCR6/immunology , Stromal Cells/metabolism , Stromal Cells/pathology , T-Lymphocytes/metabolism , T-Lymphocytes/pathologyABSTRACT
PURPOSE: Exosomes contain abundant circRNAs and are determined to be involved in the pathogenesis of lung adenocarcinoma (LUAD). Thus, our study aimed to explore new circRNAs in plasma exosomes that could be involved in such pathogenesis. PATIENTS AND METHODS: High-throughput sequencing was used in identifying the alterations in exosomal circRNA expression. Gene ontology functional analysis (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to determine the significant functions and pathways associated with differentially expressed circRNAs. TargetScan and miRanda were used to predict circRNA-targeted microRNAs and mRNAs. CircRNA expression profiles were then validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Wound healing and Transwell assays were performed to determine the roles of has_circ_0102537 in LUAD progression. RESULTS: We identified six significantly upregulated and 214 significantly downregulated circRNAs. GO and KEGG pathway analysis suggested that the differentially expressed circRNAs are involved in the occurrence and development of LUAD. A circRNA-miRNA-mRNA meshwork was established to predict the potential interactions among these RNAs. The circRNA expression profile was then subjected to qRT-PCR for validation. We identified hsa_circ_0102537 to be downregulated in both LUAD plasma exosomes and tissues. GO, KEGG pathway analysis, circRNA-miRNA-mRNA meshwork, and further experiments suggest that hsa_circ_0102537 could be involved in LUAD progression. CONCLUSION: Our study explored a large number of circRNAs that may be involved in the LUAD pathogenesis, thereby supporting the need for further research on both diagnosis biomarkers and the potential intervention therapeutic targets.
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Close proximity between cytotoxic T lymphocytes and tumour cells is required for effective immunotherapy. However, what controls the spatial distribution of T cells in the tumour microenvironment is not well understood. Here we couple digital pathology and transcriptome analysis on a large ovarian tumour cohort and develop a machine learning approach to molecularly classify and characterize tumour-immune phenotypes. Our study identifies two important hallmarks characterizing T cell excluded tumours: 1) loss of antigen presentation on tumour cells and 2) upregulation of TGFß and activated stroma. Furthermore, we identify TGFß as an important mediator of T cell exclusion. TGFß reduces MHC-I expression in ovarian cancer cells in vitro. TGFß also activates fibroblasts and induces extracellular matrix production as a potential physical barrier to hinder T cell infiltration. Our findings indicate that targeting TGFß might be a promising strategy to overcome T cell exclusion and improve clinical benefits of cancer immunotherapy.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Carcinoma, Ovarian Epithelial/immunology , Gene Expression Regulation, Neoplastic/immunology , Ovarian Neoplasms/immunology , Transforming Growth Factor beta/metabolism , Tumor Microenvironment/immunology , Antigen Presentation/immunology , Carcinoma, Ovarian Epithelial/genetics , Carcinoma, Ovarian Epithelial/metabolism , Carcinoma, Ovarian Epithelial/pathology , Cell Line, Tumor , Cohort Studies , DNA Methylation , Endopeptidases , Female , Gelatinases/metabolism , Gene Expression Profiling , Histocompatibility Antigens Class I/metabolism , Humans , Machine Learning , Membrane Proteins/metabolism , Multigene Family , Ovarian Neoplasms/genetics , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Prognosis , RNA-Seq , Serine Endopeptidases/metabolism , Stromal Cells/metabolismABSTRACT
Lung cancer is one of the leading causes of cancer-related mortality worldwide. Non-small cell lung cancer (NSCLC) is the most common pathological type of lung cancer. Long non-coding RNAs (lncRNAs) are promising novel diagnostic and prognostic biomarkers, as well as potential therapeutic targets for lung cancer. Long non-coding RNAs (lncRNAs) have been demonstrated to modulate tumor cells proliferation, cell cycle progression, invasion, and metastasis by regulating gene expression at transcriptional, post-transcriptional, and epigenetic levels. The oncogenic aberrant Wnt/ß-catenin signaling is prominent in lung cancer, playing a vital role in tumorigenesis, prognosis, and resistance to therapy. Interestingly, compelling studies have demonstrated that lncRNAs exert either oncogenic or tumor suppressor roles by regulating Wnt/ß-catenin signaling. In this review, we aim to present the current accumulated knowledge regarding the roles of Wnt/ß-catenin signaling-regulated lncRNAs in the pathogenesis of non-small cell lung cancer (NSCLC). Better understanding of the effects of lncRNAs on Wnt/ß-catenin signaling might contribute to the improved understanding of the molecular tumor pathogenesis and to the uncovering of novel therapeutic targets in NSCLC.
ABSTRACT
Vascular smooth muscle cell (VSMC) proliferation and migration are vital to atherosclerosis (AS) development and plaque rupture. MicroRNA-377-3p (miR-377-3p) has been reported to inhibit AS in apolipoprotein E knockout (ApoE-/-) mice. Herein, the mechanism underlying the effect of miR-377-3p on alleviating AS is explored. In vivo experiments, ApoE-/- mice were fed with high-fat diet (HFD) to induce AS and treated with miR-377-3p agomir or negative control agomir (agomir-NC) on week 0, 2, 4, 6, 8, 10 after HFD feeding. MiR-377-3p was found to restore HFD-induced AS lesions and expressions of matrix metalloproteinase (MMP)-2, MMP-9, α-smooth muscle actin (α-actin) and calponin. In in vitro experiments, human VSMCs were tranfected with miR-377-3p agomir or agomir-NC, followed by treatment with oxidized low-density lipoprotein (ox-LDL). MiR-377-3p was observed to significantly inhibit ox-LDL-induced VSMC proliferation characterized by inhibited cell viability, expressions of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E and cell cycle transition from G1 to S phase accompanied with less 5-Ethynyl-2'-deoxyuridine (EdU)-positive cells. Furthermore, MiR-377-3p significantly inhibited ox-LDL-induced VSMC migration characterized by inhibited wound closure and decreased relative VSMC migration. Besides, neuropilin2 (NRP2) was verified as a target of miR-377-3p. MiR-377-3p was observed to inhibit NRP2 expressions in vivo and in vitro. Moreover, miR-377-3p significantly inhibited MMP-2 and MMP-9 expressions in human VSMCs. Additionally, miR-377-3p-induced inhibition of VSMC proliferation and migration could be attenuated by NRP2 overexpression. These results indicated that miR-377-3p inhibited VSMC proliferation and migration via targeting NRP2. The present study provides an underlying mechanism for miR-377-3p-based AS therapy.
Subject(s)
Atherosclerosis/metabolism , Cell Movement , Cell Proliferation , MicroRNAs/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Neuropilin-2/metabolism , Animals , Atherosclerosis/genetics , Atherosclerosis/pathology , Cell Cycle Proteins/metabolism , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Diet, High-Fat , Disease Models, Animal , Humans , Lipoproteins, LDL/toxicity , Male , Mice, Knockout, ApoE , MicroRNAs/genetics , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Neuropilin-2/genetics , Signal TransductionABSTRACT
BACKGROUND: The aim of this study was to gain further investigation of non-small cell lung cancer (NSCLC) tumorigenesis and identify biomarkers for clinical management of patients through comprehensive bioinformatics analysis. METHODS: miRNA and mRNA microarray datasets were downloaded from GEO (Gene Expression Omnibus) database under the accession number GSE102286 and GSE101929, respectively. Genes and miRNAs with differential expression were identified in NSCLC samples compared with controls, respectively. The interaction between differentially expressed genes (DEGs) and differentially expressed miRNAs (DEmiRs) was predicted, followed by functional enrichment analysis, and construction of miRNA-gene regulatory network, protein-protein interaction (PPI) network, and competing endogenous RNA (ceRNA) network. Through comprehensive bioinformatics analysis, we anticipate to find novel therapeutic targets and biomarkers for NSCLC. RESULTS: A total of 123 DEmiRs (5 up- and 118 down-regulated miRNAs) and 924 DEGs (309 up- and 615 down-regulated genes) were identified. These genes and miRNAs were significantly involved in different pathways including adherens junction, relaxin signaling pathway, and axon guidance. Furthermore, hsa-miR-9-5p, has-miR-196a-5p and hsa-miR-31-5p, as well as hsa-miR-1, hsa-miR-218-5p and hsa-miR-135a-5p were shown to have higher degree in the miRNA-gene regulatory network and ceRNA network, respectively. Furthermore, BIRC5 and FGF2, as well as RTKN2 and SLIT3 were hubs in the PPI network and ceRNA network, respectively. CONCLUSION: Several pathways (adherens junction, relaxin signaling pathway, and axon guidance) miRNAs (hsa-miR-9-5p, has-miR-196a-5p, hsa-miR-31-5p, hsa-miR-1, hsa-miR-218-5p and hsa-miR-135a-5p) and genes (BIRC5, FGF2, RTKN2 and SLIT3) may play important roles in the pathogenesis of NSCLC.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Computational Biology/methods , Gene Expression Profiling/methods , Gene Regulatory Networks , Lung Neoplasms/pathology , MicroRNAs/genetics , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , Prognosis , Protein Interaction Maps , Signal TransductionABSTRACT
Most patients with lung adenocarcinoma (LUAD) present high recurrence rate and poor prognosis after therapy. Therefore, the purpose of this study was to identify prognostic factors involved in LUAD. Five microarray datasets (including GSE75037, GSE63459, GSE43458, GSE32863, and GSE10072) were downloaded. After data preprocessing and quality control, meta-analysis was performed to screen differentially expressed genes (DEGs) using the MetaDE.ES method in MetaDE package. Subsequently, network construction and module identification were conducted by the Weighted Gene Co-expression Network Analysis method. Moreover, survival-associated genes were identified using the univariate and multivariate Cox regression method in survival package. The risk score model was constructed by prognosis associated genes, followed by the Kaplan-Meier survival analysis. Oncomine expressions analysis of several prognosis associated genes was conducted. The expression levels of key genes were detected using quantitative real-time PCR experiments. A total of 1434 DEGs between LUAD and normal samples were identified. Nine disease-associated modules were identified, in which M8 module was most correlated with LAUD phenotype. A total of 89 indicators (including T stage, M stage, and ADIPOR2) were significantly associated with LAUD prognosis, while only T stage and 9 DEGs (e.g., ARHGEF3, GTSE1, RBM15 and CD52) were retained as the potential prognostic factors following multivariate COX regression analysis. The upregulated adiponectin receptor 2 (ADIPOR2), rho guanine nucleotide exchange factor 3 (ARHGEF3), and CD52 molecule (CD52), and downregulated GTSE1 were validated in LAUD samples of Oncomine database. Importantly, ADIPOR2 and ARHGEF3 were confirmed to be down-regulated in LUAD tissues. ADIPOR2, ARHGEF3, G2 and S-phase expressed 1 (GTSE1) and CD52 might be promising prognostic factors in LUAD.
Subject(s)
Adenocarcinoma of Lung/diagnosis , Adenocarcinoma of Lung/genetics , Computational Biology , Oligonucleotide Array Sequence Analysis , Gene Regulatory Networks , Humans , PrognosisABSTRACT
Circulating tumor cells (CTCs) have a great potential for noninvasive diagnosis and real-time monitoring of cancer. A comprehensive evaluation of four whole genome amplification (WGA)/next-generation sequencing workflows for genomic analysis of single CTCs, including PCR-based (GenomePlex and Ampli1), multiple displacement amplification (Repli-g), and hybrid PCR- and multiple displacement amplification-based [multiple annealing and loop-based amplification cycling (MALBAC)] is reported herein. To demonstrate clinical utilities, copy number variations (CNVs) in single CTCs isolated from four patients with squamous non-small-cell lung cancer were profiled. Results indicate that MALBAC and Repli-g WGA have significantly broader genomic coverage compared with GenomePlex and Ampli1. Furthermore, MALBAC coupled with low-pass whole genome sequencing has better coverage breadth, uniformity, and reproducibility and is superior to Repli-g for genome-wide CNV profiling and detecting focal oncogenic amplifications. For mutation analysis, none of the WGA methods were found to achieve sufficient sensitivity and specificity by whole exome sequencing. Finally, profiling of single CTCs from patients with non-small-cell lung cancer revealed potentially clinically relevant CNVs. In conclusion, MALBAC WGA coupled with low-pass whole genome sequencing is a robust workflow for genome-wide CNV profiling at single-cell level and has great potential to be applied in clinical investigations. Nevertheless, data suggest that none of the evaluated single-cell sequencing workflows can reach sufficient sensitivity or specificity for mutation detection required for clinical applications.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , DNA Copy Number Variations , Lung Neoplasms/genetics , Neoplastic Cells, Circulating/metabolism , Single-Cell Analysis/methods , Carcinoma, Non-Small-Cell Lung/blood , Carcinoma, Non-Small-Cell Lung/pathology , DNA Mutational Analysis/methods , Genome, Human , High-Throughput Nucleotide Sequencing/methods , Humans , Lung Neoplasms/blood , Lung Neoplasms/pathology , PC-3 Cells , Polymerase Chain Reaction/methods , Reproducibility of Results , Sensitivity and Specificity , Whole Genome Sequencing/methodsABSTRACT
Long non-coding RNA (lncRNA) colon cancer-associated transcript-1 (CCAT1) has been reported to play important roles in the development and progression of multiple human malignancies. However, the functional role and molecular mechanism of CCAT1 on gefitinib resistance in non-small cell lung cancer (NSCLC) are largely unclear. The aim of this study is to explore the roles of CCAT1 on gefitinib resistance in NSCLC and to explore the underlying mechanisms. The quantitative real-time PCR (qRT-PCR) analysis was to investigate the expression pattern of CCAT1 in gefitinib-resistant NSCLC patient tissues and cell lines, and then the effects of CCAT1 on gefitinib resistance of NSCLC in vitro and in vivo. Furthermore, bioinformatics online program predictions and luciferase reporter assay were used to validate the association of CCAT1 and miR-218 in NSCLC cells. In this study, CCAT1 was observed to be upregulated in gefitinib-resistant patient tissues and cell lines. In vitro and in vivo experiments demonstrated that CCAT1 knockdown impaired cell proliferation and promoted the gefitinib-induced cell apoptosis. Furthermore, we demonstrated that CCAT1 acts as a sponge for miR-218, and verified that HOXA1 is a novel target of miR-218. These results suggest that CCAT1 may serve as a promising therapeutic target for the treatment of epidermal growth factor receptor (EGFR) plus NSCLC patients.
ABSTRACT
AIMS: MicroRNAs have been demonstrated to be involved in the development of atherosclerosis. The present study aimed to evaluate the effect of miR-99a-5p and its target gene Homeobox A1 (HOXA1) in atherosclerosis. MAIN METHODS: The biological functions of miR-99a-5p on human aortic smooth muscle cells (ASMCs) were assessed by MTT, wound healing and transwell assays. The target genes of microRNAs were predicted by TargetScan and miRDB. The binding of miR-99a-5p and HOXA1 was confirmed by luciferase reporter assay. In the in vivo study, high-fat diet-induced atherosclerosis model was established in Apolipoprotein E knockout mice. Hematoxylin-eosin (H&E), oil Red O and Masson trichrome staining were performed for determination of atherosclerotic lesion. The levels of miR-99a-5p and HOXA1 mRNA were detected by real-time PCR. HOXA1 and migration-associated protein levels were detected by western blot or immunohistochemistry analysis. KEY FINDINGS: MiR-99a-5p inhibited HOXA1 expression by targeting 3'UTR of HOXA1 mRNA. Enforced HOXA1 significantly promoted the proliferation, migration, and invasion of ASMCs. Furthermore, miR-99a-5p overexpression inhibited the proliferation, migration, and invasion of ASMCs stimulated by HOXA1, whereas miR-99a-5p inhibition reversed the effects of HOXA1 knockdown on these behaviours of ASMCs. In vivo, the specific overexpression of miR-99a-5p significantly abated atherosclerotic lesions formatted, accompanied with a significant down-regulation of HOXA1 mRNA and protein expression levels. SIGNIFICANCE: We demonstrate for first time that miR-99a-5p may serve as a potential inhibitor of the atherosclerosis, and miR-99a-5p plays its role partially through targeting HOXA1.
Subject(s)
Atherosclerosis/prevention & control , Gene Expression Regulation , Genes, Homeobox , Homeodomain Proteins/genetics , MicroRNAs/physiology , Transcription Factors/genetics , 3' Untranslated Regions , Animals , Atherosclerosis/genetics , Cell Movement/physiology , Cell Proliferation/physiology , Cells, Cultured , Homeodomain Proteins/physiology , Humans , Male , Mice , Mice, Knockout , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Transcription Factors/physiologyABSTRACT
BACKGROUND: Lotensin has been shown to have a protective function in the early stage of chronic renal failure. However, its role in the intermediate and late stages of chronic renal failure remains largely unknown. The present study aimed to investigate the role and underlying mechanism of lotensin in advanced chronic kidney disease. METHODS: Female Wistar rats were randomly divided into three groups (n = 10): sham group, 5/6 nephrectomy (5/6 Nx) group, and lotensin group (oral administration of lotensin for 9 weeks following 5/6 Nx). Rats were sacrificed and pathological parameters were measured. Western blot assay and immunohistochemical staining were performed to detect the expression of transforming growth factor-ß1 (TGF-ß1) and α-smooth muscle actin (α-SMA) in kidney tissues. RESULTS: Compared to the 5/6 Nx group, lotensin administration significantly decreased 5/6 Nx-induced elevation in blood urea nitrogen, serum creatinine and 24-h urinary protein excretion (UPE) rates, but markedly increased red blood cell count, plasma albumin and hemoglobin levels, along with improved renal morphology. Mechanistically, lotensin dramatically downregulated the renal expression of TGF-ß1 and α-SMA induced by 5/6 Nx. CONCLUSIONS: Lotensin protects against advanced chronic kidney disease in rats with 5/6 Nx through the downregulation of TGF-ß1 and α-SMA.
Subject(s)
Actins/metabolism , Benzazepines/pharmacology , Kidney Failure, Chronic/drug therapy , Transforming Growth Factor beta1/metabolism , Animals , Blood Urea Nitrogen , Creatinine/blood , Female , Immunohistochemistry , Kidney/drug effects , Kidney/pathology , Kidney Failure, Chronic/metabolism , Random Allocation , Rats , Rats, WistarABSTRACT
The present study aimed to identify key pathways and genes in the pathogenesis of lung cancer. The GSE10072 dataset was downloaded from the Gene Expression Omnibus database. Protein-protein interaction data were collected from Human Protein Reference Database, and 201 pathways were downloaded from the Kyoto Encyclopedia of Genes and Genomes database. Signaling network impact analysis was performed to identify enriched pathways, followed by the construction of a pathway-pathway crosstalk network. Benzopyrene was used to treat normal human lung cells at concentrations of 0.01, 0.1, 1 and 10 µM, and cell viability was measured. Furthermore, growth arrest and DNA damage inducible ß (GADD45B), p53, cyclin B, Akt and nuclear factor (NF)-κB protein levels were also measured via western blotting. Impact analysis identified 11 enriched lung cancer-associated KEGG pathways, including 'complement and coagulation cascades', 'ECM-receptor interaction', 'P53 signaling pathway', 'cell adhesion molecules' and 'focal adhesion'. In addition, cell cycle, 'drug metabolism-cytochrome P450', 'metabolic pathways', 'pathways in cancer', 'focal adhesion' and 'antigen processing and presentation' were central in the pathway-pathway cross-talk network. Furthermore, the upregulated gene GADD45B was associated with three of the pathways, including an activated pathway ('MAPK signaling pathway') and two repressed pathways ('cell cycle' and 'P53 pathway'). Western blotting demonstrated that the expression of NF-κB, Akt and GADD45B increased over time in lung cells treated with benzopyrene, whereas the expression levels of cyclin B and P53 decreased. In conclusion, GADD45B may contribute to lung carcinogenesis via affecting the MAPK, P53 signaling and cell cycle pathways.
ABSTRACT
We evaluated the key genes related with recurrent respiratory tract infections (RRTIs), and then elucidated the possible molecular mechanisms of RRTIs. Neutrophil was isolated from peripheral bloods of the recurrent lower respiratory tract infection patients and healthy volunteers, respectively. The next generation sequencing information was obtained after RNA extraction, purification, library construction and sequencing. The sequencing information was preprocessed. Bioinformatics analysis including analysis of differentially expre-ssed genes (DEGs), Gene Ontology (GO) and pathway enrichment analysis, protein-protein interaction (PPI) analysis and transcription factors analysis were performed. The key genes were verified by real-time PCR. In total, 17 significant DEGs were obtained in case group compared with the control group by bioinformatics analysis. Then, 6 of 17 genes were detected by real-time PCR. There was statistical significance between case and control groups for peroxisome proliferator-activated receptor-γ (PPARG), prostaglandin-endoperoxide synthase 2 (PTGS2), transferrin (TF) and interleukin-10 (IL-10) (P<0.05), and there was no statistical significance between case and control groups for TIMP metallopeptidase inhibitor 1 (TIMP1) and matrix metallopeptidase 1 (MMP1). PPARG, PTGS2, TF and IL-10 are key genes associated with the progression of RRTIs. We speculate that TIMP1 and MMP1 may also be involved in the progression of RRTIs, but further studies with large number of samples are needed for verification.
Subject(s)
Computational Biology/methods , Gene Expression Regulation , Respiratory Tract Infections/genetics , Adult , Aged , Demography , Female , Gene Expression Profiling , Gene Ontology , Gene Regulatory Networks , Humans , Male , Middle Aged , Protein Interaction Maps/genetics , Reproducibility of Results , Transcription Factors/metabolismABSTRACT
Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.