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Background: Lung cancer is the most common primary malignant tumor of the lung, and as one of the malignant tumors that pose the greatest threat to the health of the population, the incidence rate has remained high in recent years. Previous studies have shown that KLRB1 is transcriptionally repressed in lung adenocarcinoma and correlates with lung adenocarcinoma prognosis. The objective of this study is to investigate the intrinsic mechanisms by which KLRB1 affects the malignant phenotypes of lung adenocarcinoma such as immune infiltration, proliferation, growth and metastasis. Methods: We assessed the expression levels of KLRB1 in publicly available databases and investigated its associations with clinical and pathological variables. Enrichment analysis was subsequently conducted to investigate possible signaling pathways and their associated biological functions. Statistical analysis, including Spearman correlation and the application of multigene prediction models, was utilized to assess the relationship between the expression of KLRB1 and the infiltration of immune cells. The diagnostic and prognostic value of KLRB1 was evaluated using Kaplan-Meier survival curves, diagnostic receptor operating characteristic (ROC) curves, histogram models, and Cox regression analysis. Specimens from lung adenocarcinoma (LUAD) patients were collected, the expression level of KLRB1 was detected by protein blotting analysis, and the expression level of KLRB1 was detected at the mRNA level by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Small interfering RNA (siRNA) was used to silence gene expression, and Transwell, Cell Counting Kit-8 (CCK-8) and colony formation assays were subsequently performed to analyze the effects of KLRB1 on LUAD cell migration, invasion and proliferation. Results: KLRB1 expression was lower in lung cancer tissue than in surrounding healthy tissue. Genes differentially expressed in the low and high KLRB1 expression groups were found to be significantly enriched in pathways related to immunity. KLRB1 exerted an impact on the MAPK/ERK signaling pathway, thereby modulating the growth and proliferation of LUAD cells. KLRB1 expression is linked to prognosis, immune infiltration, and cell migration and proliferation in LUAD. Conclusions: The evidence revealed a correlation between KLRB1 and both prognosis and immune infiltration in LUAD patients.
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Undifferentiated pleomorphic sarcoma (UPS) is a highly aggressive malignant soft tissue tumor with a poor prognosis; however, the identity and heterogeneity of tumor populations remain elusive. Here, eight major cell clusters were identified through the RNA sequencing of 79,569 individual cells of UPS. UPS originates from mesenchymal stem cells (MSCs) and features undifferentiated subclusters. UPS subclusters were predicted to exist in two bulk RNA datasets, and had a prognostic value in The Cancer Genome Atlas (TCGA) dataset. The functional heterogeneity of malignant UPS cells and the immune microenvironment were characterized. Additionally, the fused cells were innovatively detected by expressing both monocyte/macrophage markers and other subcluster-associated genes. Based on the ligand-receptor interaction analysis, cellular interactions with epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) were abundant. Furthermore, 73% of patients with UPS (48/66) showed positive EGFR expression, which was associated with a poor prognosis. EGFR blockade with cetuximab inhibited tumor growth in a patient-derived xenograft model. Our transcriptomic studies delineate the landscape of UPS intratumor heterogeneity and serve as a foundational resource for further discovery and therapeutic exploration.
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Receptores ErbB , Sarcoma , Análise de Célula Única , Humanos , Animais , Receptores ErbB/genética , Receptores ErbB/metabolismo , Camundongos , Sarcoma/patologia , Sarcoma/genética , Prognóstico , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão GênicaRESUMO
Lung cancer is one of the most common malignant tumors and has a poor prognosis and a low survival rate. Traditional treatments, such as radiotherapy and chemotherapy, still face some challenges because of high drug resistance and toxicity. Therefore, it is necessary to discover a new kind of targeted drug with low toxicity and high efficiency. CDK12 is a cell cycle-dependent kinase whose main function is to activate RNA polymerase II (RNAPII) and promote the transcriptional extension of RNA. However, the role and molecular mechanism of CDK12 in lung cancer are still unclear. In this study, the mutation and RNA-Seq data of CDK12 in lung adenocarcinoma and squamous cell carcinoma were downloaded from The Cancer Genome Atlas (TCGA) database and analyzed with the custom scripts. Cell proliferation was evaluated by Cell Counting Kit-8 (CCK-8) and cell colony formation assays. A subcutaneous tumor experiment in nude mice was used to examine the effects of CDK12 knockdown on the in vivo tumor growth of NSCLC cells. The cell cycle distribution and the apoptosis rate of lung cancer cells were assessed by flow cytometry. Regulation of TANK-binding kinase 1 (TBK1) by CDK12 was evaluated by quantitative PCR, immunoprecipitation and Western blot analysis. In this study we have analyzed the mutation and expression data of The Cancer Genome Atlas (TCGA) database and found that CDK12 is highly expressed in lung cancer tissues. Clinical correlation analysis showed that high expression of CDK12 in NSCLC reduces patient survival, but its high expression is only related to early tumor progression and has no significant correlation with late tumor progression and metastasis. Furthermore, we present evidence that CDK12 depletion in lung cancer cell lines not only leads to the inhibition of cell growth and induces apoptosis but also inhibits tumor growth of NSCLC cells in vivo. CDK12 positively regulates the expression of the oncogene TBK1 in lung cancer cells. These results revealed that CDK12 affects the progression of non-small cell lung cancer through positive regulation of TBK1 expression, suggesting that CDK12 might be a potential molecular target for the treatment of non-small cell lung cancer.
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Adenocarcinoma de Pulmão , Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Animais , Camundongos , Adenocarcinoma de Pulmão/genética , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Pulmonares/patologia , Camundongos Nus , HumanosRESUMO
It is urgent to identify new biomarkers and therapeutic targets to ameliorate the clinical prognosis of patients with lung cancer. The functional significance and molecular mechanism of dynein cytoplasmic 1 heavy chain 1 (DYNC1H1) in nonsmall cell lung cancer (NSCLC) progression is still elusive. In our current study, publicly available data and Western blotting experiments confirmed that DYNC1H1 expression was upregulated in lung cancer samples compared with noncancerous samples. Quantitative real-time PCR (qPCR) results indicated that high DYNC1H1 expression in lung cancer tissues was significantly associated with clinical tumor stage and distal metastasis; moreover, its high expression was negatively correlated with prognosis. Functional experiments demonstrated that DYNC1H1 loss of function caused a significant decrease in cell viability and cell proliferative ability, inhibition of the cell cycle, and promotion of both migration potential and invasion potential in vitro. Animal experiments by tail vein injection of lung cancer cells showed that DYNC1H1 knockdown significantly decreased lung cancer metastasis. Mechanistically, the results from a human protein array showed changes in the IFN-γ-JAK-STAT signaling pathway, and analysis of The Cancer Genome Atlas (TCGA) immune data demonstrated that disturbance of the immune microenvironment might be involved in the impaired growth and metastatic ability mediated by DYNC1H1 loss in NSCLC. DYNC1H1 might serve as a promising biological marker of prognosis and a potential clinical therapeutic target for patients with NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/genética , Proliferação de Células/genética , Dineínas do Citoplasma/genética , Imunidade/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Transdução de Sinais/genética , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Interferon gama/genética , Janus Quinase 1/genética , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Fatores de Transcrição STAT/genética , Microambiente Tumoral , Regulação para Cima/genéticaRESUMO
The existence of cancer stem cells (CSCs), marked by CD133, is the primary cause of death in hepatocellular carcinoma (HCC). Here, we generated a new risk model comprising the signatures of four genes highly correlated with CD133 (CD133(hi)) that help improve survival in HCC. Three datasets were used to identify the differential CD133(hi) genes by comparing sorted CD133+ liver CSCs and CD133- differentiated counterparts. Univariate analysis was used to screen significantly differential CD133(hi) genes associated with overall survival in the training dataset, which were used for risk model construction. High-risk patients were strongly associated with poor survival by Kaplan-Meier survival analysis in both the training and validation datasets. Clinical stratification analyses further demonstrated that the risk factors acted as independent factors and that high-risk patients were characterized by more aggressive cancer features. Functional enrichment analyses performed by gene set enrichment analysis (GSEA) and the Database for Annotation, Visualization and Integrated Discovery (DAVID) revealed that high-risk patients showed the disturbance of immune hepatic homeostasis involving aberrant immune cells, including macrophages and T and B cells, and an abnormal inflammatory response including the IL6/Jak/STAT3 pathway and TNF signaling pathway. In conclusion, our constructed CD133(hi) gene risk model provides a resource for understanding the role of CD133+ CSCs in the progression of HCC in terms of tumor-immune interactions.
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Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/mortalidade , Neoplasias Hepáticas/mortalidade , Células-Tronco Neoplásicas/metabolismo , Microambiente Tumoral/imunologia , Antígeno AC133/genética , Antígeno AC133/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Conjuntos de Dados como Assunto , Feminino , Humanos , Estimativa de Kaplan-Meier , Fígado/imunologia , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Masculino , Modelos Genéticos , Células-Tronco Neoplásicas/imunologia , Prognóstico , RNA-Seq , Medição de Risco/métodos , Fatores de Risco , Taxa de Sobrevida , Transcriptoma/imunologia , Microambiente Tumoral/genéticaRESUMO
Objective: Bone metastasis from patients with advanced lung adenocarcinoma (LAC) is a very serious complication. To better understand the molecular mechanism, our current study sheds light on identification of hub genes mediating bone metastatic spread by combining bioinformatic analysis with functional verification. Methods: First, we downloaded a lung adenocarcinoma dataset (GSE76194) from Gene Expression Omnibus, analyzed differentially expressed genes (DEGs) through Limma package in R software and constructed a protein-protein interaction network. Based on that preliminary data, we further performed modular and topological analysis using Cystoscope to obtain biological connected genes. Through literature searching and performing mRNA expression analysis on the other independent public dataset (GSE10799), we finally focused on TBX2. Functional effects of TBX2 were performed in tumorigenicity assays including migration and invasion assays, cell proliferation assay, and cell cycle assay. In addition, mechanically, we found enriched pathways related to bone metastasis using Gene Set Enrichment Analysis (GSEA) and validated our results by western blot. Result: A total of 1132 significant genes were sorted initially. We selected common significant genes (log FC>2; p<0.01) from both the biological network data and microarray data. In total, 44 such genes were identified. we found TBX2, along with 10 other genes, to be reported with relevance to bone metastasis in other cancer types. Moreover, TBX2 showed significantly higher expression levels in patients that were found positive for metastasis to bone marrow compared to patients that did not exhibit this type of metastasis in the other separated cohort (GSE10799). Thus, we finally focused on TBX2. We found that TBX2 had detectable expression in LAC cell lines and silencing endogenous TBX2 expression in A549 and H1299 cell lines markedly suppressed migration and invasion, cell proliferation and arrested cell-cycle. Pathway enrichment analyses suggested that TBX2 drove LAC oncogenesis and metastasis through various pathways with epithelial mesenchymal transition (EMT) figuring prominently in the bone metastatic group, which was evidenced by western blot. Conclusion: Collectively, TBX2 plays as a potential predictor of bone metastasis from LAC, yielding a better promise view towards "driver" gene responsible for bone metastasis.
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BACKGROUND: Long noncoding RNAs (lncRNAs) play a key role in the development and progression of many cancer types, including lung cancer. The objective of this study is to examine the function and molecular mechanism of lncRNAs involved in non-small cell lung cancer (NSCLC). METHODS: First, 7 lung cancer-related differentially expressed LncRNAs were screened from 2 genomic profiling datasets. Of these lncRNAs, FOXF1 adjacent noncoding developmental regulatory RNA (FENDRR) was found to be the only one that was both significantly down-regulated in the patients with advanced pathology and negatively correlated with prognosis. Thus, lncRNA FENDRR was further studied in this project. Clinical correlation analysis was further conducted in the GSE30219 dataset and 73 paired lung cancer and noncancerous tissues stored in our lab; Subsequently, we evaluated FENDRR coding potential with the Phylogenetic Codon Substitution Frequencies (PhyloCSF), Coding-Potential Assessment Tool (CPAT), and Coding Potential Calculator (CPC) online analytical tool. The cell growth ability was measured by CCK8 assay and clonogenicity assay, the metastatic capacities were evaluated using Transwell migration and invasion assays. Mechanistically, we analyzed the correlation of FENDRR function in NSCLC with immune response by utilizing The Cancer Genome Atlas (TCGA) data. RESULTS: Results indicated a negative clinical correlation of FENDRR. Coding potential analysis showed FENDRR as a noncoding RNA. Elevated expression of FENDRR led to cell growth arrest, inhibition of proliferative ability, declined migration and invasion potential of NSCLC cells in vitro. Mechanistically, we discovered that FENDRR expression might be involved in aberrant immune response regulation. CONCLUSIONS: Taken together, our results provide a greater understanding of lncRNA FENDRR as a tumor suppressor with respect to tumor-immune interactions in NSCLC.
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BACKGROUND: KH-type splicing regulatory protein (KHSRP) plays an important role in cancer invasion, but the relevant mechanism is not well known. In the present study, we investigated the function and potential molecular mechanism of KHSRP in non-small cell lung cancer (NSCLC) metastasis and elucidated its clinical significance. METHODS: Isobaric tags for relative and absolute quantitation and the SWATH™ approach were combined with nanoliquid chromatography-tandem mass spectrometry analysis to identify metastasis-associated nucleoproteins in NSCLC. Real-time PCR and Western blot were used to screen for metastasis-associated candidate molecules. Gene knockdown and overexpression were used to investigate their functions and molecular mechanisms in lung cancer cells. Coimmunoprecipitation (Co-IP) experiments were performed to identify the interactions between candidate molecules and their interacting proteins. Gene expression and its association with multiple clinicopathologic characteristics were analyzed by immunohistochemistry (IHC) and Western blot in human lung cancer specimens. RESULTS: KHSRP was identified as a metastasis-associated candidate molecule. In NSCLC cell lines, knockdown of KHSRP significantly reduced lung cancer cell proliferation, migration, and invasion in vitro and in vivo, whereas overexpression of KHSRP did the opposite. Mechanistically, the protein heterogeneous nuclear ribonucleoprotein C (C1/C2) (HNRNPC) was identified to interact with KHSRP using Co-IP experiments. In NSCLC cell lines, overexpression of HNRNPC significantly promoted lung cancer cell proliferation, migration, and invasion in vitro and in vivo. KHSRP and HNRNPC may induce human lung cancer cell invasion and metastasis by activating the IFN-α-JAK-STAT1 signaling pathway. Drastically higher expression levels of KHSRP and HNRNPC were observed in lung cancer tissues compared to those in adjacent noncancerous tissues. Increased KHSRP and HNRNPC expression was significantly associated with advanced tumor stages and metastasis (both lymph node and distant). Kaplan-Meier survival analysis showed that patients with high KHSRP and HNRNPC expression levels were predicted to have the shortest survival times and to have a poor prognosis. CONCLUSIONS: KHSRP plays an important role in NSCLC metastasis and may serve as a potential prognostic marker and novel therapeutic target for lung cancer metastasis treatment.
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Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Transativadores/metabolismo , Células A549 , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo C/metabolismo , Xenoenxertos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Metástase Neoplásica , Proteínas de Ligação a RNA/genética , Transativadores/genética , TransfecçãoRESUMO
Increasing evidence has confirmed that deubiquitinating enzymes play an important role in lung cancer progression. In the current study, we investigated the expression profile of deubiquitinating enzymes in non-small cell lung cancer (NSCLC) tissues and identified OTUB2 as an upregulated deubiquitinating enzyme. The role of OTUB2 in NSCLC is unknown. Methods: Quantitative, real-time PCR and Western blot were used to detect OTUB2 and U2AF2 expression in NSCLC tissues. The correlations between OTUB2 and U2AF2 expression and clinicopathologic features were then analyzed. We used In vitro Cell Counting Kit-8 (CCK-8) , colony formation , and trans-well invasion assays to investigate the function of OTUB2 and U2AF2 in tumorigenesis. The regulation of glycolysis by OTUB2 and U2AF2 was assessed by determining the extracellular acid ratio, glucose consumption, and lactate production. The mechanism of OTUB2 was explored through co-immunoprecipitation and mass spectrometry analyses. A xenograft model was also used to study the tumorigenesis role of OTUB2 In vivo. Results: OTUB2 expression was significantly upregulated in primary NSCLC tissues and greatly associated with metastasis, advanced tumor stages, poor survival, and recurrence. In NSCLC cell lines, OTUB2 promoted cell growth, colony formation, migration, and invasive activities. Mechanistic investigations showed that OTUB2 stimulated the Warburg effect and induced the activation of the serine/threonine kinase/mechanistic target of rapamycin kinase (AKT/mTOR) pathway in different NSCLC cells. More importantly, OTUB2 promoted NSCLC progression, which was largely dependent on the direct binding to and deubiquitination of U2AF2, at least in NSCLC cells. U2AF2 expression was also significantly upregulated in primary NSCLC tissues and dramatically associated with metastasis, advanced tumor stages, poor survival, and recurrence. Importantly, a positive correlation between the protein expression of OTUB2 and U2AF2 in NSCLC tissues was found. In vivo experiments indicated that OTUB2 promoted xenograft tumor growth of NSCLC cell. In addition, our results suggest that high expression of OTUB2, U2AF2 and PGK1 is significantly associated with worse prognosis in NSCLC patients. Conclusion: Taken together, the present study provides the first evidence that OTUB2 acts as a pivotal driver in NSCLC tumorigenesis by stabilizing U2AF2 and activating the AKT/mTOR pathway and the Warburg effect. It may serve as a new potential prognostic indicator and therapeutic target in NSCLC.
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Carcinogênese , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Fator de Processamento U2AF/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Tioléster Hidrolases/metabolismo , Aerobiose , Animais , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Glicólise , Humanos , Ácido Láctico/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Análise de SobrevidaRESUMO
E3 ubiquitin ligases, which are key enzymes in the ubiquitin proteasome system, catalyze the ubiquitination of proteins to target them for proteasomal degradation. Emerging evidence suggests that E3 ubiquitin ligases play important roles in the development and progression of lung cancer. In our study, we characterized the gene expression landscape of lung cancer using data obtained from TCGA to explore the changes in E3 ubiquitin ligase containing the regulators of E3 ubiquitin ligase activity. Overall, most gene expression changes occurred in NSCLC tissues compared with adjacent normal ones. In total, 48 E3 ubiquitin ligases containing the regulators were up-regulated in NSCLC tissues compared with their levels in normal tissues. We analyzed the expression of up-regulated E3 ubiquitin ligases containing the regulators in two publicly available transcriptome data sets (GSE13213 and GSE30219). We found that four E3 ubiquitin ligases (UHRF1, BRCA1, TRAIP and HLTF) and one regulator of ubiquitin E3 activity DCUN1D1 that were dramatically up-regulated in cancer were significantly associated with tumor metastasis and patient's poor prognosis both in two transcriptome data sets. Next, clinical analysis indicated that the expression levels of DCUN1D1 correlated with clinical stage and lymph node metastasis in NSCLC patients as determined by quantitative reverse transcription-PCR. Furthermore, functional assays showed that DCUN1D1 promoted NSCLC cell invasion and migration as determined by transwell assay in vitro. Mechanistically, we found that the C-terminal Cullin binding domain leads to oncogenic activity and the UBA domain acts as a negative regulator of DCUN1D1 function in NSCLC. Moreover, DCUN1D1 activated the FAK oncogenic signaling pathway and up-regulated PD-L1. Taken together, our results demonstrate that DCUN1D1 is a metastasis regulator and suggest a new therapeutic option for NSCLC metastasis.
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Antígeno B7-H1/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Quinase 1 de Adesão Focal/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Metástase Neoplásica/genética , Transdução de Sinais/genética , Células A549 , Carcinogênese/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Complexo de Endopeptidases do Proteassoma/genética , Ligação Proteica/genética , Ativação Transcricional/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação/genética , Regulação para Cima/genéticaRESUMO
Approximately 90% of patients diagnosed with non-small cell lung cancer (NSCLC) die due to distant metastases. However, the complicated molecular and cellular mechanisms involved in lung cancer metastasis remain poorly understood. Copine III (CPNE3), a member of a Ca2+-dependent phospholipid-binding protein family, was identified as a novel metastasis-associated protein in NSCLC in our previous study, however, its function in metastasis remains unclear. Here, we found that CPNE3 was expressed at high levels in NSCLC tissues and advanced TNM stages and was significantly associated with poor prognosis. In addition, CPNE3 interacted with phosphorylated erb-b2 receptor tyrosine kinase 2 (pErbB2) and receptor of activated protein C kinase 1 (RACK1) and activated the focal adhesion (FA) signaling pathway in NSCLC cells. Moreover, knockdown of RACK1 inhibited cell motility in the CPNE3-overexpressed NSCLC cells. These findings offer mechanistic insights into the oncogenic roles of CPNE3 and the pivotal effects of CPNE3 as a biomarker and therapeutic target for NSCLC metastasis.
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MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression and protein synthesis. Our previous study demonstrated that miR-148a suppressed the metastasis of non-small cell lung cancer (NSCLC) in vitro and in vivo. However, the modulatory mechanism of this effect remains unclear. In the present study, quantitative proteomic technology was used to study the protein expression profile of SPC-A-1 cells subsequent to the downregulation of miR-148a expression, in order to elucidate the molecular mechanism of the suppression of NSCLC metastasis by miR-148a. The differentially expressed proteins identified were analyzed using bioinformatics tools, including the Database for Annotation, Visualization and Integrated Discovery and the Search Tool for the Retrieval of Interacting Genes/proteins. In two experiments, 4,048 and 4,083 proteins were identified, and 4,014 and 4,039 proteins were quantified, respectively. In total, 44 proteins were upregulated and 40 proteins were downregulated. This was verified at the protein and mRNA levels by western blotting and reverse transcription-quantitative polymerase chain reaction, respectively. Bioinformatics analysis was used to identify potential interactions and signaling networks for the differentially expressed proteins. This may have provided an appropriate perspective for the comprehensive analysis of the modulatory mechanism underlying the metastasis-suppressive effects of miR-148a in NSCLC. In conclusion, quantitative proteomic technology revealed that miR-148a may regulate a panel of tumor-associated proteins to suppress metastasis in NSCLC.
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Long noncoding RNAs (lncRNA) participate in carcinogenesis and tumor progression in lung cancer. Here, we report the identification of a lncRNA signature associated with metastasis of non-small cell lung cancer (NSCLC). In particular, elevated expression of LINC00963 (MetaLnc9) in human NSCLC specimens correlated with poor prognosis, promoted migration and invasion of NSCLC cells in vitro, and enhanced lung metastasis formation in vivo Mechanistic investigations showed that MetaLnc9 interacted with the glycolytic kinase PGK1 and prevented its ubiquitination in NSCLC cells, leading to activation of the oncogenic AKT/mTOR signaling pathway. MetaLnc9 also interacted with P54nrb/NonO (NONO) to help mediate the activity of CRTC, a coactivator for the transcription factor CREB, reinforcing a positive feedback loop for metastasis. Taken together, our results establish MetaLnc9 as a driver of metastasis and a candidate therapeutic target for treating advanced NSCLC. Cancer Res; 77(21); 5782-94. ©2017 AACR.
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Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , Fosfoglicerato Quinase/genética , Proteínas Proto-Oncogênicas c-akt/genética , RNA Longo não Codificante/genética , Serina-Treonina Quinases TOR/genética , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Ativação Enzimática , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Fosfoglicerato Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Transplante HeterólogoRESUMO
Lung cancer is the most prevalent cancer in humans and has the lowest survival outcomes due to its high metastatic potential. The aim of the present study was to screen for metastasisrelated genes (MRGs) by investigating the differential expression genes (DEGs) identified by the mRNA expression profiles in SPCA1sci (highly metastatic) and SPC-A-1 (parental) cells. DEGs were screened using Genespring software. Gene Ontology and pathway enrichment analyses of these DEGs were performed. Interaction networks between the proteins encoded by the DEGs were identified using the database BioGRID and were visualized by Cytoscape. Modular analysis of the proteinprotein interaction network was performed in CFinder. Among these DEGs, the expression levels of 18 genes were examined in SPCA1sci and SPCA1 cell lines with reverse transcriptionquantitative polymerase chain reaction, and 10 of the 18 genes were assessed by western blotting to validate the results of the microarray. Furthermore, the role of metallothionein 1X (MT1X) in nonsmall cell lung cancer was explored in functional assays and 72 pairs of clinical samples in vitro. Finally, 4,838 DEGs were screened, including 798 upregulated and 4,040 downregulated genes. The significantly enriched functions included gene expression, cytosol and poly(A) RNA binding, and the most enriched pathway was biosynthesis of antibiotics. Furthermore, MT1X was revealed to promote the migration and invasion ability in SPCA1sci and PC9 lung cancer cell lines. Therefore, MT1X was identified as a candidate MRG through systematic analysis in the present microarray, which was demonstrated to offer potential reference value in screening MRGs.
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Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Perfilação da Expressão Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Transcriptoma , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Linhagem Celular Tumoral , Movimento Celular/genética , Biologia Computacional/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/mortalidade , Metástase Neoplásica , Estadiamento de Neoplasias , Prognóstico , Mapeamento de Interação de Proteínas , Mapas de Interação de ProteínasRESUMO
BACKGROUND/AIMS: microRNAs (miRNAs) are noncoding RNAs that regulate multiple targets through either the degradation of mRNAs or the inhibition of protein translation, thereby altering several functions simultaneously. Growing evidence indicates that miRNAs are involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). METHODS: In this study, the mRNA expression levels of miR-148a were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The functions of miR-148a in migration/invasion and lung metastasis formation were determined by using transwell and tail vein injection assays, respectively. RESULTS: We demonstrated that miR-148a was down-regulated in NSCLC metastatic samples, and its expression was suppressed in NSCLC compared with the corresponding nonmalignant lung tissues. Clinical analysis indicated that miR-148a expression was lower in NSCLC patients compared with nonmalignant lung tissues . Decreased miR-148a was significantly associated with tumor node metastasis stage and lymph node metastasis. Furthermore, functional assays showed that miR-148a expression suppressed NSCLC cell invasive and migratory abilities in vitro and suppressed cancer metastasis in vivo, while inhibition of miR-148a enhanced NSCLC cell invasion and lung metastasis formation in a mouse model. CONCLUSIONS: Evidence from this study demonstrated that miR-148a exerts tumor-suppressive effects in NSCLC and suggests a new therapeutic option for NSCLC.
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Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/metabolismo , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Regulação para Baixo , Feminino , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Metástase Linfática , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Pessoa de Meia-Idade , Oligonucleotídeos Antissenso/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
MicroRNAs (miRNAs) are involved in human cancer including non-small cell lung cancer (NSCLC). In this study, we compared miRNA expression microarray of SPC-A-1sci (high metastatic) and SPC-A-1 (weakly metastatic) cells. We found that miRNA-10a was up-regulated in NSCLC compared with corresponding normal tissues. High expression of miR-10a was associated with tumor node metastasis and lymph node metastasis. Furthermore, overexpression of miR-10a promoted NSCLC cell proliferation, migration and invasion in vitro. We found that PTEN was a direct target of miR-10a in NSCLC. Also miR-10a activated the PTEN/AKT/ERK pathway. We suggest that miR-10a contributes to NSCLC by targeting PTEN.
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Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Divisão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fase G2 , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Transdução de Sinais , Transfecção , Regulação para CimaRESUMO
Lung cancer is the leading cause of malignancy-related death worldwide, and metastasis always results in a poor prognosis. However, therapeutic progress is hampered by a deficiency of appropriate pre-clinical metastatic models. To bridge this experimental gap, we developed an in vivo metastatic model via subcutaneous (s.c.) injection. The original cell line (XL-2) adopted in this model was newly isolated from the ascites of a patient with extensive metastases of lung adenocarcinoma, thereby avoiding any alteration of its initial molecular biology features from artificial serial cultivation. After comprehensive phenotypical and histological analysis, it was identified as a lung adenocarcinoma cell line. Additionally, the drug test showed that XL-2 cell line was sensitive to docetaxel, and resistant to doxorubicin, indicating it might serve as a cell line model of drug resistance for identifying mechanisms of tumors resistant to doxorubicin. Through this s.c. model, we further obtained a highly metastatic cell line (designated XL-2sci). The metastatic rate of mice in XL-2 group was 3/10, in contrast to the rate of 9/10 in XL-2sci group. Optical imaging, micro-computed tomography (micro-CT) scanning and Transwell assays were further applied to identify the enhanced metastatic capacity of Xl-2sci cells both in vivo and in vitro. Compared with XL-2 cells, ITRAQ labeled proteomics profiling study showed that some tumor metastasis-associated proteins were upregulated in XL-2sci cells, which also indicated the reliability of our model. Proliferation ability of XL-2 and XL-2sci were also evaluated. Results showed that highly metastatic XL-2sci possessed a decreased proliferation capacity versus XL-2, which demonstrated that its increased metastatic activity was not facilitated by a faster growth rate. In conclusion, we successfully developed an in vivo metastatic model using a newly established lung adenocarcinoma cell line, which will be beneficial to further investigations of lung cancer metastasis and to the development of anti-metastasis drugs.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Adenocarcinoma/metabolismo , Adenocarcinoma de Pulmão , Animais , Proliferação de Células , Docetaxel , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/secundário , Proteômica , Taxoides/farmacologia , Células Tumorais CultivadasRESUMO
BACKGROUND: microRNAs can repress the expression of target genes by destabilizing their mRNAs or by inhibiting their translation. Our previous findings suggested that miR-193a-3p inhibited the progression of NSCLC both in vitro and in vivo. However, the biological processes and molecular pathways through which this miRNA exerts its positive effects are unknown. METHODS: To explore the molecular mechanisms by which miR-193a-3p inhibited NSCLC metastasis, we investigated the changes in the protein profile of SPC-A-1sci (highly metastatic) cells in response to the up-regulation of miR-193a-3p expression using a proteomics approach (iTRAQ combined with NanoLC-MS/MS). Changes in the profiles of the expressed proteins were verified using western blotting and were analyzed using the DAVID and STRING programs. RESULTS: In the two replicated experiments, 4962/4946 proteins were identified, and the levels of expression of 4923/4902 proteins were quantified. In total, 112 of these proteins were differentially expressed. Among them, the up-regulated levels of expression of two of the 62 proteins with up-regulated expression (PPP2R2A and GSN) and the down-regulated levels of expression four of the 50 proteins with down-regulated expression (LMNB2, UHRF1, G3BP1, and HNRNPU) were verified using western blotting. The bioinformatics analysis revealed the interactions and signaling networks of these differentially expressed proteins. CONCLUSION: miR-193a-3p inhibited the metastasis of lung cancer cells by deregulating the expression of tumor-related proteins. These findings may improve the understanding of the molecular mechanisms underlying the metastatic-inhibitory effect of miR-193a-3p on lung cancer cells.
Assuntos
MicroRNAs/metabolismo , Proteômica , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Análise por Conglomerados , Bases de Dados Factuais , Regulação para Baixo , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Metástase Neoplásica , Proteínas de Neoplasias/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Mapas de Interação de Proteínas , Espectrometria de Massas em Tandem , Regulação para CimaRESUMO
BACKGROUND: Growing evidence indicates that miR-200c is involved in carcinogenesis and tumor progression in non-small-cell lung cancer (NSCLC). However, its precise biological role remains largely elusive. METHODS: The functions of miR-200c and USP25 in migration/invasion and lung metastasis formation were determined by transwell and tail vein injection assays, respectively. The potential regulatory targets of miR-200c were determined by prediction tools, correlation with target protein expression, and luciferase reporter assay. The mRNA expression levels of miR-200c and USP25 were examined in NSCLC cell lines and patient specimens using quantitative reverse transcription-PCR. The protein expression levels of USP25 were examined in NSCLC cell lines and patient specimens using western blot and immunohistochemical staining. RESULTS: We demonstrated that over-expression of miR-200c inhibited NSCLC cells migration, invasion, epithelial-mesenchymal transition (EMT) in vitro and lung metastasis formation in vivo. Further studies revealed that USP25 was a downstream target of miR-200c in NSCLC cells as miR-200c bound directly to the 3'-untranslated region of USP25, thus reducing both the messenger RNA and protein levels of USP25. Silencing of the USP25 gene recapitulated the effects of miR-200c over-expression. Clinical analysis indicated that miR-200c was negatively correlated with clinical stage, lymph node metastasis in NSCLC patients. Moreover, USP25 protein and mRNA level expressions were higher in NSCLC patients, compared to healthy control, and correlated with clinical stage and lymphatic node metastasis. CONCLUSIONS: These findings indicate that miR-200c exerts tumor-suppressive effects for NSCLC through the suppression of USP25 expression and suggests a new therapeutic application of miR-200c in the treatment of NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Neoplasias Pulmonares/genética , MicroRNAs/genética , Ubiquitina Tiolesterase/genética , Idoso , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/patologia , Metástase Linfática , Masculino , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologiaRESUMO
To identify cancer-related proteins, we used isobaric tags in a relative and absolute quantitation (iTRAQ) proteomic approach and SWATH™ quantification approach to analyze the secretome of an isogenic pair of highly metastatic and low metastatic non-small-cell lung cancer (NSCLC) cell lines. In addition, we compared two groups of pooled serum samples (12 early-stage and 12 late-stage patients) to mine data for candidates screened by iTRAQ-labeled proteomic analysis. A total of 110 proteins and 71 proteins were observed to be significantly differentially expressed in the cell line secretome and NSCLC sera, respectively. Among these proteins, CD109 was found to be highly expressed in both the highly metastatic cell line secretome and the group of late-stage patients. A sandwich ELISA assay also demonstrated an elevation of serum CD109 levels in individual NSCLC patients (n=30) compared with healthy subjects (n=19). Furthermore, CD109 displayed higher expression in lung cancer tissues compared with their matched noncancerous lung tissues (n=72). In addition, the knockdown of CD109 influenced several NSCLC cell bio-functions, for instance, depressing cell growth, affecting cell cycle phases. These phenomena suggest that CD109 plays a critical role in NSCLC progression. BIOLOGICAL SIGNIFICANCE: We simultaneously applied two quantitative proteomic approaches-iTRAQ-labeling and SWATH™-to analyze the secretome of metastatic cell lines, in order to explore the cancer-associated proteins in conditioned media. In this study, our results indicate that CD109 plays a critical role in non-small-cell lung cancer (NSCLC) progression, and is overexpressed in advanced NSCLC.