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The regulatory mechanisms underlying bone metastasis in lung adenocarcinoma (LUAD) are not yet fully understood despite the frequent occurrence of bone involvement. This study aimed to examine the involvement and mechanism of integrin subunit beta 3 (ITGB3) in the process of LUAD bone metastasis. Our findings indicate that ITGB3 facilitates the migration and invasion of LUAD cells in vitro and metastasis to the bone in vivo. Furthermore, ITGB3 stimulates osteoclast production and activation, thereby expediting osteolytic lesion progression. Extracellular vesicles (EVs) isolated from the conditioned medium (CM) of LUAD cells overexpressing ITGB3 determined that ITGB3 facilitates osteoclastogenesis and enhances osteoclast activity by utilizing EVs-mediated transport to RAW264.7 cells. Our in vivo findings demonstrated that ITGB3-EVs augmented the population of osteoclasts, thereby establishing an osteoclastic pre-metastatic niche (PMN) conducive to the colonization and subsequent growth of LUAD cells in the bone. ITGB3 is enriched in serum EVs of patients diagnosed with LUAD bone metastasis, potentially facilitating osteoclast differentiation and activation in vitro. Our research illustrates that ITGB3-EVs derived from LUAD cells facilitate osteoclast differentiation and activation by modulating the phosphorylation level of p38 MAPK. This process ultimately leads to the generation of osteolytic PMN and accelerates the progression of bone metastasis.
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BACKGROUND: Bone is a common site for metastasis in lung adenocarcinoma, but the mechanism behind lung adenocarcinoma bone metastasis is still unclear. And currently, there is a lack of easily traceable and stable lung adenocarcinoma bone metastasis cell models, which limits the research on the mechanism of lung adenocarcinoma bone metastasis. The establishment of human lung adenocarcinoma cell line that are highly metastatic to bone, labeled with green fluorescent proteins (GFP) and fireflies luciferase (LUC), along with transcriptomic characterization, would be beneficial for research on lung adenocarcinoma bone metastasis and provide new experimental methods. METHODS: The human lung adenocarcinoma cell line A549-GFP-LUC was injected into nude mice via the left ventricle to construct a bone metastasis model, and was domesticated in vivo for three consecutive times to obtain the human high bone metastasis lung adenocarcinoma cell line A549-GFP-LUC-BM3; cell counting kit-8 (CCK-8), colony formation assay, scratch wound assays, Transwell assay and Western blot were used to compare the proliferation and invasion abilities of A549-GFP-LUC-BM3 with the parental cells. A549-GFP-LUC-BM3 cells and parental cells were further analyzed by transcriptomic sequencing. RESULTS: Human high-bone metastatic lung adenocarcinoma cells A549-GFP-LUC-BM3 was successfully established. Compared to parental cells, this cells exhibited a significantly higher incidence of bone metastasis and enhanced in vitro proliferation, migration, and invasion abilities. Transcriptomic sequencing results revealed that the A549-GFP-LUC-BM3 cell line had 2954 differentially expressed genes compared to the parental cells, with 1021 genes up-regulated and 1933 genes down-regulated. Gene Ontology (GO) functional enrichment analysis indicated that the differentially expressed genes were primarily localized in cellular components such as the cell periphery. The molecular functions identified as significantly enriched included signaling receptor activity, calcium ion binding, and extracellular matrix structural constituent. Additionally, the biological processes found to be enriched were cell adhesion and biological adhesion. The enrichment analysis conducted using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that the differentially expressed genes were primarily involved in the metabolism of xenobiotics by cytochrome P450, retinol metabolism, drug metabolism-cytochrome P450, cell adhesion molecules, steroid hormone biosynthesis, and the nuclear factor kappa B (NF-κB) signaling pathway. CONCLUSIONS: The highly bone-metastatic human lung adenocarcinoma cell line with GFP and luciferase double labeling was successfully established. The biological behavior and transcriptome sequencing of the cell line suggest that it has a high bone-metastatic potential.
Subject(s)
Adenocarcinoma of Lung , Bone Neoplasms , Lung Neoplasms , Mice, Nude , Humans , Adenocarcinoma of Lung/genetics , Adenocarcinoma of Lung/pathology , Adenocarcinoma of Lung/metabolism , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Lung Neoplasms/metabolism , Bone Neoplasms/secondary , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Mice , Animals , A549 Cells , Gene Expression Profiling , Transcriptome , Cell Line, Tumor , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Adenocarcinoma/metabolism , Mice, Inbred BALB C , Cell ProliferationABSTRACT
Background: Bone metastasis significantly impact the prognosis of non-small cell lung cancer (NSCLC) patients, reducing their quality of life and shortening their survival. Currently, there are no effective tools for the diagnosis and risk assessment of early bone metastasis in NSCLC patients. This study employed machine learning to analyze serum indicators that are closely associated with bone metastasis, aiming to construct a model for the timely detection and prognostic evaluation of bone metastasis in NSCLC patients. Methods: The derivation cohort consisted of 664 individuals with stage IV NSCLC, diagnosed between 2015 and 2018. The variables considered in this study included age, sex, and 18 specific serum indicators that have been linked to the occurrence of bone metastasis in NSCLC. Variable selection used multivariate logistic regression analysis and Lasso regression analysis. Six machine learning methods were utilized to develop a bone metastasis diagnostic model, assessed with Area Under the Curve (AUC), Decision Curve Analysis (DCA), sensitivity, specificity, and validation cohorts. External validation used 113 NSCLC patients from the Medical Alliance (2019-2020). Furthermore, a prospective validation study was conducted on a cohort of 316 patients (2019-2020) who were devoid of bone metastasis, and followed-up for at least two years to assess the predictive capabilities of this model. The model's prognostic value was evaluated using Kaplan-Meier survival curves. Findings: Through variable selection, 11 serum indictors were identified as independent predictive factors for NSCLC bone metastasis. Six machine learning models were developed using age, sex, and these serum indicators. A random forest (RF) model demonstrated strong performance during the training and internal validation cohorts, achieving an AUC of 0.98 (95% CI 0.95-0.99) for internal validation. External validation further confirmed the RF model's effectiveness, yielding an AUC of 0.97 (95% CI 0.94-0.99). The calibration curves demonstrated a high level of concordance between the anticipated risk and the observed risk of the RF model. Prospective validation revealed that the RF model could predict the occurrence of bone metastasis approximately 10.27 ± 3.58 months in advance, according to the results of the SPECT. An online computing platform (https://bonemetastasis.shinyapps.io/shiny_cls_1model/) for this RF model is publicly available and free-to-use by doctors and patients. Interpretation: This study innovatively employs age, gender, and 11 serological markers closely related to the mechanism of bone metastasis to construct an RF model, providing a reliable tool for the early screening and prognostic assessment of bone metastasis in NSCLC patients. However, as an exploratory study, the findings require further validation through large-scale, multicenter prospective studies. Funding: This work is supported by the National Natural Science Foundation of China (NO.81974315); Shanghai Municipal Science and Technology Commission Medical Innovation Research Project (NO.20Y11903300); Shanghai Municipal Health Commission Health Industry Clinical Research Youth Program (NO.20204Y034).
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BACKGROUND AND AIMS: Clear and effective indicators for early detection of severe coronavirus disease 2019 (COVID-19) are insufficient. We investigated the clinical value of the plasma SARS-CoV-2 N antigen (plasma N antigen) for severe COVID-19 early identification and disease progression monitoring. MATERIALS AND METHODS: A cross-sectional study compared the diagnostic value of plasma N antigen levels detected within two days after hospital admission in 957 patients with COVID-19 during the BA2.2 outbreak in Shanghai (April 6-June 15, 2022). A follow-up study analyzed the plasma N antigen prognostic value in 274 non-severe patients, and a longitudinal study evaluated its continuous monitoring value in 16 patients with COVID-19 grade changes. RESULTS: Plasma N antigen concentrations were significantly higher in severely ill than in non-severely ill patients. The plasma N antigen was superior to nasopharyngeal nucleic acid CT values and established COVID-19 blood biomarkers in identifying severe COVID-19. Patients with high plasma N-antigen concentrations at initial admission were more prone to developing severe COVID-19. The changes in plasma N antigen concentrations were consistent with disease progression. Two logistic regression models, including and excluding plasma N antigen, were established, with model 1 (including plasma N antigen) (AUC = 0.971, 0.958-0.980) yielding a better diagnostic value for severe COVID-19 than Model 2 (plasma N antigen excluded). CONCLUSION: The plasma N antigen is superior to nasopharyngeal nucleic acids and established COVID-19 blood biomarkers for severe COVID-19 early recognition and progression monitoring, enabling the most accurate patient triaging and efficient utilization of medical resources.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2 , Follow-Up Studies , Longitudinal Studies , Cross-Sectional Studies , China , Biomarkers , Disease ProgressionABSTRACT
Due to its key roles in malignant tumor progression and reprograming of the tumor microenvironment, integrin ß3 has attracted great attention as a new target for tumor therapy. However, the structure-function relationship of integrins ß3 remains incompletely understood, leading to the shortage of specific and effective targeting probes. This work uses a purified extracellular domain of integrin ß3 and integrin ß3-positive cells to screen aptamers, specifically targeting integrin ß3 in the native conformation on live cells through the SELEX approach. Following meticulous truncation and characterization of the initial aptamer candidates, the optimized aptamer S10yh2 was produced, exhibiting a low equilibrium dissociation constant (Kd) in the nanomolar range. S10yh2 displays specific recognition of cancer cells with varying levels of integrin ß3 expression and demonstrates favorable stability in serum. Subsequent analysis of docking sites revealed that S10yh2 binds to the seven amino acid residues located in the core region of integrin ß3. The S10yh2 aptamer can downregulate the level of integrin heterodimer αvß3 on integrin ß3 overexpressed cancer cells and partially inhibit cell migration behavior. In summary, S10yh2 is a promising probe with a small size, simple synthesis, good stability, high binding affinity, and selectivity. It therefore holds great potential for investigating the structure-function relationship of integrins.
Subject(s)
Aptamers, Nucleotide , Neoplasms , Humans , Integrin beta3/chemistry , Integrin beta3/metabolism , Aptamers, Nucleotide/pharmacology , Integrin alphaVbeta3/metabolism , Cell Movement , Tumor MicroenvironmentABSTRACT
BACKGROUND: Lung adenocarcinoma (LUAD) is the most common non-small-cell lung cancer, with a high incidence and a poor prognosis. AIM: To construct effective predictive models to evaluate the prognosis of LUAD patients. METHODS: In this study, we thoroughly mined LUAD genomic data from the Gene Expression Omnibus (GEO) (GSE43458, GSE32863, and GSE27262) and the Cancer Genome Atlas (TCGA) datasets, including 698 LUAD and 172 healthy (or adjacent normal) lung tissue samples. Univariate regression and LASSO regression analyses were used to screen differentially expressed genes (DEGs) related to patient prognosis, and multivariate Cox regression analysis was applied to establish the risk score equation and construct the survival prognosis model. Receiver operating characteristic curve and Kaplan-Meier survival analyses with clinically independent prognostic parameters were performed to verify the predictive power of the model and further establish a prognostic nomogram. RESULTS: A total of 380 DEGs were identified in LUAD tissues through GEO and TCGA datasets, and 5 DEGs (TCN1, CENPF, MAOB, CRTAC1 and PLEK2) were screened out by multivariate Cox regression analysis, indicating that the prognostic risk model could be used as an independent prognostic factor (Hazard ratio = 1.520, P < 0.001). Internal and external validation of the model confirmed that the prediction model had good sensitivity and specificity (Area under the curve = 0.754, 0.737). Combining genetic models and clinical prognostic factors, nomograms can also predict overall survival more effectively. CONCLUSION: A 5-mRNA-based model was constructed to predict the prognosis of lung adenocarcinoma, which may provide clinicians with reliable prognostic assessment tools and help clinical treatment decisions.
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BACKGROUND: Early stratification of disease progression remains one of the major challenges towards the post-coronavirus disease 2019 (COVID-19) era. The clinical relevance of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid load is debated due to the heterogeneity in patients' underlying health conditions. We determined the prognostic value of nasopharyngeal viral load dynamic conversion for COVID-19. METHODS: The cycling threshold (Ct) values of 28,937 nasopharyngeal SARS-CoV-2 RT-PCRs were retrospectively collected from 3,364 COVID-19 patients during hospitalization and coordinated to the onset of disease progression. The ROC curve was utilized to determine the predictive performance of the rate of Ct value alteration between two consecutive RT-PCR runs within 48 h (ΔCt%) for disease transformation across patients with different COVID-19 severity and immune backgrounds, and further validated with 1,860 SARS-CoV-2 RT-PCR results from an independent validation cohort of 262 patients. For the 67 patients with severe COVID-19, Kaplan-Meier analysis was performed to evaluate the difference in survival between patients stratified by the magnitude of Ct value alteration between the late and early stages of hospitalization. RESULTS: The kinetics of viral nucleic acid conversion diversified across COVID-19 patients with different clinical characteristics and disease severities. The ΔCt% is a clinical characteristic- and host immune status-independent indicator for COVID-19 progression prediction (AUC = 0.79, 95 % CI = 0.76 to 0.81), which outperformed the canonical blood test markers, including c-reactive protein (AUC = 0.57, 95 % CI = 0.53 to 0.61), serum amyloid A (AUC = 0.61, 95 % CI = 0.54 to 0.68), lactate dehydrogenase (AUC = 0.61, 95 % CI = 0.56 to 0.67), d-dimer (AUC = 0.56, 95 % CI = 0.46 to 0.66), and lymphocyte count (AUC = 0.62, 95 % CI = 0.58 to 0.66). Patients with persistent high SARS-CoV-2 viral load (an increase of mean Ct value < 50 %) during the first 3 days of hospitalization demonstrated a significantly unfavorable survival (HR = 0.16, 95 % CI = 0.04 to 0.65, P = 2.41 × 10-3). CONCLUSIONS: Viral nucleic acid dynamics of SARS-CoV-2 eliminates the inter-patient variance of basic health conditions and therefore, can serve as a prognostic marker for COVID-19.
Subject(s)
COVID-19 , Humans , COVID-19/diagnosis , SARS-CoV-2 , Retrospective Studies , Prognosis , Time Factors , Viral Load , Disease ProgressionABSTRACT
BACKGROUND: The prognosis is very poor for lung cancer patients with bone metastasis. Unfortunately, a suitable method has yet to become available for the early diagnosis of bone metastasis in lung cancer patients. The present work describes an attempt to develop a novel model for the early identification of lung cancer patients with bone metastasis risk. METHODS: As the test group, 205 primary lung cancer patients were recruited, of which 127 patients had bone metastasis; the other 78 patients without bone metastasis were set as the negative control. Additionally, 106 healthy volunteers were enrolled as the normal control. Serum levels of several cytokines in the bone microenvironment (CaN, OPG, PTHrP, and IL-6) and bone turnover markers (tP1NP, ß-CTx) were detected in all samples by ECLIA or ELISA assay. Receiver operating characteristic (ROC) curves and multivariate analyses were performed to evaluate diagnostic abilities and to assess the attributable risk of bone metastasis for each of these indicators; the diagnostic model was established via logistic regression analysis. The prospective validation group consisted of 44 patients with stage IV primary lung cancer on whom a follow-up of at least 2 years was conducted, during which serum bone biochemical marker concentrations were monitored. RESULTS: The serological molecular model for the diagnosis of bone metastasis was logit (p). ROC analysis showed that when logit (p) > 0.452, the area under curve of the model was 0.939 (sensitivity: 85.8%, specificity: 89.7%). Model validation demonstrated accuracy with a high degree of consistency (specificity: 85.7%, specificity: 87.5%, Kappa: 0.770). The average predictive time for bone metastasis occurrence of the model was 9.46 months earlier than that of the bone scan diagnosis. Serum OPG, PTHrP, tP1NP, ß-CTx, and the diagnostic model logit (p) were all positively correlated with bone metastasis progression (P < 0.05). CONCLUSIONS: This diagnostic model has the potential to be a simple, non-invasive, and sensitive tool for diagnosing the occurrence and monitoring the progression of bone metastasis in patients with lung cancer.
Subject(s)
Biomarkers, Tumor/blood , Bone Neoplasms/diagnosis , Early Detection of Cancer/methods , Lung Neoplasms/pathology , Models, Biological , Aged , Bone Neoplasms/blood , Bone Neoplasms/secondary , Bone and Bones/diagnostic imaging , Bone and Bones/pathology , Disease Progression , Female , Follow-Up Studies , Humans , Lung Neoplasms/blood , Male , Middle Aged , Predictive Value of Tests , Prognosis , Prospective Studies , ROC Curve , Radionuclide Imaging , Risk Assessment/methodsABSTRACT
Early and precise cancer diagnosis substantially improves patient survival. Recent work has revealed that the levels of multiple microRNAs in serum are informative as biomarkers for the diagnosis of cancers. Here, we designed a DNA molecular computation platform for the analysis of miRNA profiles in clinical serum samples. A computational classifier is first trained in silico using miRNA profiles from The Cancer Genome Atlas. This is followed by a computationally powerful but simple molecular implementation scheme using DNA, as well as an effective in situ amplification and transformation method for miRNA enrichment in serum without perturbing the original variety and quantity information. We successfully achieved rapid and accurate cancer diagnosis using clinical serum samples from 22 healthy people (8) and people with lung cancer (14) with an accuracy of 86.4%. We envision that this DNA computational platform will inspire more clinical applications towards inexpensive, non-invasive and rapid disease screening, classification and progress monitoring.
Subject(s)
Computers, Molecular , Early Detection of Cancer/methods , Gene Expression Profiling/methods , Lung Neoplasms/diagnosis , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Computational Biology , Humans , Lung Neoplasms/genetics , Lung Neoplasms/metabolism , MicroRNAs/blood , MicroRNAs/geneticsABSTRACT
The study was conducted to ascertain whether the quantification of circulating cell-free DNA (cfDNA) in serum has value as a diagnostic or for monitoring the progression of non-small cell lung cancer (NSCLC). The serum/plasma cfDNA concentration was quantified by absolute qPCR of long interspersed nuclear element-1 (LINE1) in 60 NSCLC patients and 68 controls in good health. Receiver operating characteristic (ROC) curve analysis was performed to determine the diagnostic utility and cut-off levels of cfDNA, CEA, and CYFRA21-1 in NSCLC patients. Correlations between cfDNA and age, sex, tumour stage and progression-free survival (PFS) were analysed. A follow-up study was conducted on 4 NSCLC patients, and serum cfDNA, CEA, and CYFRA21-1 were quantified throughout disease progression. Serum cfDNA levels were significantly higher in NSCLC patients than those in normal controls. Elevated serum cfDNA concentration was also significantly associated with advanced tumour stage. Serum cfDNA had a ROC area under the curve comparable to that of CEA and CYFRA21-1 for the diagnosis of NSCLC, and the combined cfDNA/CEA/CYFRA21-1 indicator had the highest diagnostic efficiency. Moreover, increased serum cfDNA levels were strongly correlated with tumour progression and poor PFS. This study preliminarily confirmed that cfDNA can monitor disease progression in NSCLC patients, and the lead time was 1-7 months compared with clinical medical imaging. Serum cfDNA may be useful in monitoring NSCLC progression, suggesting that the non-invasive quantification of serum cfDNA by LINE1 qPCR is a viable option for predicting progression and disease severity when repeated invasive tissue biopsy is not possible.
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The involvement of HOXA4 in colorectal cancer and epithelial ovarian cancer has been reported. Although it has been reported that the Hoxa4 gene is involved in the patterning of the mouse lung during embryonic development, little is known about the biological functions of HOXA4 in lung cancer. In the current study, HOXA4 expression was down-regulated in lung cancer tissues when compared with non-cancerous tissues. HOXA4 expression was associated with tumor size, TNM stage, lymph node metastasis and prognosis. Bioinformatics analysis revealed that HOXA4 expression was negatively correlated with cell cycle, metastasis, and the Wnt signaling pathway. Moreover, HOXA4 overexpression in lung cancer cell lines suppressed cell proliferation, migration, and invasion. HOXA4 decreased the protein expression levels of ß-catenin, Cyclin D1, c-Myc and Survivin, indicating the inhibition of Wnt signaling. HOXA4 significantly increased the protein and mRNA levels of glycogen synthase kinase-3ß (GSK3ß) by promoting its transcription. Furthermore, inhibition of GSK3ß by LiCl abolished the suppression of cell growth, migration, and invasion mediated by HOXA4. Overexpression of HOXA4 in xenograft tumors also decreased tumor growth and Wnt signaling. Collectively, these data suggest that HOXA4 is a potential diagnostic and prognostic marker in lung cancer, and its overexpression could inhibit lung cancer progression in part by promoting GSK3ß transcription.
Subject(s)
Biomarkers, Tumor/biosynthesis , Cell Movement , Down-Regulation , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/biosynthesis , Lung Neoplasms/metabolism , Neoplasm Proteins/metabolism , A549 Cells , Animals , Female , Humans , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness , Transcription Factors , Wnt Signaling PathwayABSTRACT
Stanniocalcin 1 (STC1) is reported to functionally participate in the development of several cancers. However, the role of STC1 in the tumorigenesis and progression of lung adenocarcinoma remains to be fully elucidated. Here, we found that the average levels of serum STC1 were 5.47, 5.53, and 6.94 ng/mL (P = 0.0045) in the healthy subjects and patients with lung adenocarcinoma at tumor stages I-II and III-IV according to Union for International Cancer Control (UICC), respectively. Subsequently, the positive correlation between the STC1 expression level in lung adenocarcinoma tissues and tumor stages was confirmed by immunohistochemical staining assay. Additionally, studies in the STC1-overexpressing or STC1-silenced stable cell lines showed that STC1 increased cell proliferation by promoting G1/S transition in cell cycle progression via up-regulating cyclin B1 and cyclin E. Moreover, studies in the STC1-overexpressing or STC1-silenced stable cell lines also showed that STC1 inhibited cell apoptosis by up-regulating the expression of anti-apoptosis proteins Bcl-2 and Bcl-xl and down-regulating the expression of pro-apoptosis proteins Bax, Bak, and Bid via the activation of the ERK and JNK signaling pathway. In addition, neutralization of STC1 with monoclonal antibody significantly increased the apoptosis of A549 cells. Taken together, our findings strongly suggest that elevated expression of STC1 protein at the III-IV stage of lung adenocarcinoma promotes tumorigenesis of lung adenocarcinoma and positively associates with the cancer progression, which may be of potential value as tumor marker in clinical tracking lung adenocarcinoma progression.
Subject(s)
Adenocarcinoma/blood , Biomarkers, Tumor/blood , Glycoproteins/blood , Lung Neoplasms/blood , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Adult , Aged , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Disease Progression , Female , Humans , Lung Neoplasms/pathology , Male , Middle Aged , Neoplasm StagingABSTRACT
INTRODUCTION: Few serum markers are valid and useful for the diagnosis or therapeutic effect monitoring of osteosarcoma. This study aimed to investigate the role of ß-isomerized C-terminal telopeptides (ß-CTx) and total procollagen type 1 amino-terminal propeptide (tP1NP) as serological biomarkers for osteosarcoma patients. MATERIALS AND METHODS: A total of 48 patients with osteosarcoma and 55 healthy volunteers were investigated. Serum ß-CTx and tP1NP levels were measured by electrochemiluminescence immunoassay. Data were analyzed by t test with Walth's correction and receiver operating characteristic (ROC) curve analysis. RESULTS: The baseline levels of ß-CTx and tP1NP were found to be significantly higher in patients with osteosarcoma than the healthy volunteers. The mean areas under the ROC curves were 0.919 (range, 0.864-0.973) for ß-CTx and 0.866 (range, 0.792-0.939) for tP1NP. The levels of ß-CTx and tP1NP were lower in patients with stable disease after operation than those before operation. CONCLUSION: These findings support our hypothesis that ß-CTx and tP1NP are promising serum biomarkers for diagnosing or monitoring osteosarcoma.
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BACKGROUND AND OBJECTIVES: Previous studies have suggested that the aberrant expression of Stanniocalcin-1 (STC1) occurs in tumor cells. In this study, we explored whether the circulating STC1 mRNA is a promising biomarker in the peripheral blood in patients with non-small cell lung cancer (NSCLC). METHODS: The level of circulating STC1 mRNA was determined with a sensitive quantitative real-time reverse transcription PCR assay. and the data were analyzed by the statistical methods of one-way ANOVA, Mann-Whitney-Wilcoxon U-Test, and Receiver operating characteristic (ROC) curve analysis. RESULTS: The level of circulating STC1 mRNA in patients with NSCLC was significantly higher than in patients with benign pulmonary disease (P < 0.001) or healthy volunteers (P < 0.001). Higher levels of circulating STC1 mRNA were associated with more advanced tumor stages and histological subtypes. Using a cutoff of 0.055, the sensitivity and specificity of STC1 mRNA levels to differentiate between patients with NSCLC and patients with benign pulmonary diseases was 66.7 and 90.9%, and it was 63.7 and 99.8% for patients with NSCLC and healthy volunteers, respectively. CONCLUSIONS: These findings support our hypothesis that circulating STC1 mRNA is a promising biomarker in the peripheral blood.
Subject(s)
Biomarkers, Tumor/blood , Carcinoma, Non-Small-Cell Lung/diagnosis , Glycoproteins/blood , Lung Neoplasms/diagnosis , Neoplastic Cells, Circulating/metabolism , RNA, Messenger/analysis , Adult , Aged , Biomarkers, Tumor/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Case-Control Studies , Diagnosis, Differential , Female , Glycoproteins/genetics , Humans , Lung Diseases/diagnosis , Lung Diseases/genetics , Lung Diseases/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Middle Aged , RNA, Messenger/blood , ROC Curve , Real-Time Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
The aim of this study was to investigate the prevalences of plasmid-mediated AmpC beta-lactamases (PABLs) in isolates of Escherichia coli and Klebsiella spp. from a university hospital in China. A total of 1,935 consecutive nonrepeat clinical isolates of Escherichia coli, Klebsiella pneumoniae, and Klebsiella oxytoca were collected between January 2003 and July 2005. The isolates with cefoxitin zone diameters less than 18 mm (screen positive) were selected for PCR of the bla(AmpC) genes and sequencing. Fifty-four (2.79%) isolates harbored PABLs, as demonstrated by PCR and isoelectric focusing. Sequence analysis revealed the presence of bla(DHA-1) and bla(CMY-2) genes. The Southern blot hybridization studies confirmed that bla(CMY-2) and bla(DHA-1) were located on plasmids. Based on species, PABLs were detected in 4.29% (29 isolates of DHA-1 and 1 isolate of CMY-2) of K. pneumoniae, 1.91% (11 isolates of DHA-1 and 12 isolates of CMY-2) of E. coli, and 3.03% (1 isolate of DHA-1) of K. oxytoca isolates. In contrast to our anticipation, the occurrence rate of DHA-1-producing K. pneumonia significantly decreased (P < 0.01), from 7.54% in 2003 to 2.72% in 2004. The results of random amplified polymorphic DNA analysis indicate that the prevalences of DHA-1-producing K. pneumoniae and CMY-2-producing E. coli strains were not due to epidemic strains. In conclusion, DHA-1 was the most prevalent acquired AmpC beta-lactamase in this collection of isolates from a medical center in China, and DHA-1-producing K. pneumoniae was the most prevalent bacterium harboring a PABL. To the best of our knowledge, this is the first report of CMY-2-type AmpC beta-lactamases in the Chinese mainland.
Subject(s)
Bacterial Proteins/genetics , Escherichia coli/enzymology , Hospitals, University/statistics & numerical data , Klebsiella oxytoca/enzymology , Klebsiella pneumoniae/enzymology , beta-Lactam Resistance , beta-Lactamases/genetics , beta-Lactamases/metabolism , Anti-Bacterial Agents/pharmacology , Cephalosporinase/genetics , Cephalosporinase/metabolism , China/epidemiology , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/isolation & purification , Escherichia coli Infections/epidemiology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Isoelectric Focusing , Klebsiella Infections/epidemiology , Klebsiella Infections/microbiology , Klebsiella oxytoca/drug effects , Klebsiella oxytoca/genetics , Klebsiella oxytoca/isolation & purification , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Klebsiella pneumoniae/isolation & purification , Microbial Sensitivity Tests , Plasmids/genetics , Polymerase Chain Reaction/methods , Prevalence , Sequence Analysis, DNAABSTRACT
Lymphatic spread of colorectal cancer cells to regional lymph nodes is one of the early events in metastatic cancers and often is associated with distant metastatic spread and a poor prognosis. The expression levels of newly described lymphatic endothelial markers, LYVE-1 and podoplanin, were assessed in our study. Paired (tumor and corresponding normal tissue) samples were obtained. The expression level of each factor was determined by using RT-PCR and quantified by using a real-time quantitative PCR (RT-QPCR) technique. The expression of podoplanin was significantly greater in patients with lymph node metastasis than in those without metastasis, but no different expression level of LYVE-1 was detected in 2 groups of patients. These results indicate that quantitative analysis of lymphangiogenic marker podoplanin in colorectal cancer specimens may be useful in predicting metastasis of colorectal cancer to regional lymph nodes, but the role of LYVE-1 in predicting metastasis of colorectal cancer still needs further analysis.
Subject(s)
Biomarkers, Tumor/genetics , Colorectal Neoplasms/pathology , Lymphangiogenesis/genetics , Membrane Glycoproteins/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Vesicular Transport Proteins/genetics , Female , Humans , Lymphatic Metastasis , Male , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA, Neoplasm/analysis , RNA, Neoplasm/metabolismABSTRACT
This study was conducted to investigate the circadian rhythms and light responses of Clock and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the environmental conditions of a 12 h light (05:00-17:00 h): 12 h-dark (17:00-05:00 h) cycle (LD) and constant darkness (DD). The pineal gland of Sprague-Dawley rats housed under a LD regime (n=42) for four weeks and of a regime (n=42) for eight weeks were sampled at six different times, every 4 h (n=7 animals per time point), during a 24 h period. Total RNA was extracted from each sample, and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine temporal changes in mRNA levels of Clock and NAT genes during different circadian or zeitgeber times. The data and parameters were analyzed by the cosine function software, Clock Lab software, and the amplitude F test was used to reveal the circadian rhythm. In the DD or LD condition, both the Clock and NAT mRNA levels in the pineal gland showed robust circadian oscillation (p<0.05) with the peak at the subjective night or at nighttime. In comparison with the DD regime, the amplitudes and mRNA levels at the peaks of Clock and NAT expressions in LD in the pineal gland were significantly reduced (p<0.05). In the DD or LD condition, the circadian expressions of NAT were similar in pattern to those of Clock in the pineal gland (p>0.05). These findings indicate that the transcriptions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also respond to the ambient light signal in a reduced manner.
Subject(s)
Arylalkylamine N-Acetyltransferase/genetics , Circadian Rhythm/genetics , Gene Expression Regulation/radiation effects , Light , Pineal Gland/radiation effects , Trans-Activators/genetics , Transcription, Genetic/radiation effects , Animals , Arylalkylamine N-Acetyltransferase/metabolism , CLOCK Proteins , Circadian Rhythm/radiation effects , Gene Expression Regulation, Enzymologic/radiation effects , Male , Pineal Gland/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Time Factors , Trans-Activators/metabolismABSTRACT
The aim of this study was to observe and compare the endogenous circadian rhythm and photoresponse of Clock gene transcription in the suprachiasmatic nucleus (SCN) and pineal gland (PG) of rats. With free access to food and water in special darkrooms, Sprague-Dawley rats were housed under the light regime of constant darkness (DD) for 8 weeks (n=36) or 12 hour-light: 12 hour-dark cycle (LD) for 4 weeks (n=36), respectively. Then, their SCN and PG were dissected out every 4 h in a circadian day, 6 rats at each time (n=6). All animal treatments and sampling during the dark phases were conducted under red dim light (<0.1 lux). The total RNA was extracted from each sample and the semi-quantitative RT-PCR was used to determine the temporal mRNA changes of Clock gene in the SCN and PG at different circadian times (CT) or zeitgeber times (ZT). The grayness ratio of Clock/H3.3 bands was served as the relative estimation of Clock gene expression. The experimental data were analyzed by the Cosine method and the Clock Lab software to fit original results measured at 6 time points and to simulate a circadian rhythmic curve which was then examined for statistical difference by the amplitude F test. The main results are as follows: (1) The mRNA levels of Clock gene in the SCN under DD regime displayed the circadian oscillation (P<0.05). The endogenous rhythmic profiles of Clock gene transcription in the PG were similar to those in the SCN (P>0.05) throughout the day with the peak at the subjective night (CT15 in the SCN or CT18 in the PG) and the trough during the subjective day (CT3 in the SCN or CT6 in the PG). (2) Clock gene transcription in the SCN under LD cycle also showed the circadian oscillation (P<0.05), and the rhythmic profile was anti-phasic to that under DD condition (P<0.05). The amplitude and the mRNA level at the peak of Clock gene transcription in the SCN under LD were significantly increased compared with that under DD (P<0.05), while the value of corresponding rhythmic parameters in the PG under LD were remarkably decreased (P<0.05). (3) Under LD cycle, the circadian profiles of Clock gene transcription induced by light in the PG were quite different from those in the SCN (P<0.05). Their Clock transcription rhythms were anti-phasic, i.e., showing peaks at the light phase ZT10 in the SCN or at the dark time ZT17 in the PG and troughs during the dark time ZT22 in the SCN or during the light phase ZT5 in the PG. The findings of the present study indicate a synchronous endogenous nature of the Clock gene circadian transcriptions in the SCN and PG, and different roles of light regime in modulating the circadian transcriptions of Clock gene in these two central nuclei.
Subject(s)
CLOCK Proteins/genetics , Circadian Rhythm/physiology , Photoreceptor Cells, Vertebrate/physiology , Pineal Gland/physiology , Suprachiasmatic Nucleus/physiology , Animals , Male , Rats , Rats, Sprague-Dawley , Transcription, GeneticABSTRACT
In this paper, the circadian pattern of Clock and genes mediated by the Clock was investigated in peripheral lymphocytes of rats. Circadian rhythms of Clock are found under the regimes of constant darkness (DD) and 12-h light-12-h dark (LD12:12h), with the peak phase at CT7 and ZT21, respectively. Ten differential cDNA fragments were identified to be mediated by the Clock, including three known genes (catalase, myelin proteolipid protein, and histone acetylase), four known expressed sequence tags (ESTs), and three novel ESTs. Experiment of the RNA interference revealed that these ESTs were down-regulated by the Clock gene and three of them were identified as clock-controlled genes. Understanding of clock-mediated genes may lead to a new direction in drug design for control of circadian rhythms.
Subject(s)
Circadian Rhythm/physiology , Lymphocytes/physiology , Trans-Activators/biosynthesis , Animals , Biological Clocks/physiology , CLOCK Proteins , Catalase/metabolism , Expressed Sequence Tags , Gene Expression Regulation , In Vitro Techniques , Light , Male , Myelin Proteolipid Protein/biosynthesis , Myelin Proteolipid Protein/genetics , RNA, Small Interfering/genetics , Rats , Rats, Sprague-Dawley , Trans-Activators/geneticsABSTRACT
This study was to investigate the circadian rhythms and light responses of Clock gene and arylalkylamine N-acetyltransferase (NAT) gene expressions in the rat pineal gland under the 12 h-light : 12 h-dark cycle condition (LD) and constant darkness (DD). Sprague-Dawley rats housed under the light regime of LD (n=36) for 4 weeks and of DD (n=36) for 8 weeks were sampled for the pineal gland once a group (n=6) every 4 h in a circadian day. The total RNA was extracted from each sample and the semiquantitative reverse transcription polymerase chain reaction (RT-PCR) was used to determine the temporal changes in mRNA levels of Clock and NAT genes during different circadian times or zeitgeber times. The data were analysed by the cosine function software, Clock Lab software and the amplitude F test was used to reveal the circadian rhythm. The main results obtained are as follows. (1) In DD or LD condition, both of Clock and NAT genes mRNA levels in the pineal gland showed robust circadian oscillation (P< 0.05) with the peak at the subjective night or at night-time. (2) In comparison with DD regime, the amplitudes and the mRNA levels at peaks of Clock and NAT genes expressions in LD in the pineal gland were significantly reduced (P< 0.05). (3) In DD or LD condition, the circadian expressions of NAT gene were similar in pattern to those of Clock gene in the pineal gland (P> 0.05). These findings suggest that the expressions of Clock and NAT genes in the pineal gland not only show remarkably synchronous endogenous circadian rhythmic changes, but also response to the ambient light signal in a reduced manner.