ABSTRACT
OBJECTIVES: Human cell-free circulating DNA (cf-DNA) derived mainly from cell apoptosis and necrosis can be measured by a variety of laboratory techniques, but almost all of these methods require sample preparation. We have developed a branched DNA (bDNA)-based Alu assay for quantifying cf-DNA in myocardial infarction (MI) patients. DESIGN AND METHODS: A total of 82 individuals were included in the study; 22 MI and 60 normal controls. cf-DNA was quantified using a bDNA-based Alu assay. RESULTS: cf-DNA was higher in serum compared to plasma and there was a difference between genders. cf-DNA was significantly higher in MI patients compared to the controls. There was no correlation between cf-DNA and creatine kinase-MB (CK-MB), troponin I (cTnI) or myoglobin (MYO). In serial specimens, cf-DNA was sensitive and peaked earlier than cTnI. CONCLUSIONS: The bDNA-based Alu assay is a novel method for quantifying human cf-DNA. Increased cf-DNA in MI patients might complement cTnI, CK-MB and MYO in a multiple marker format.
Subject(s)
DNA/blood , Mass Screening/methods , Myocardial Infarction/diagnosis , Biomarkers/blood , DNA/analysis , Female , Humans , Male , Mass Screening/standards , Methods , Myocardial Infarction/blood , Sensitivity and Specificity , Sex FactorsABSTRACT
OBJECTIVE: To study the effect of pGCsi-H1-APRIL on the growth of human colorectal cancer cells in transplated tumor in nude mice and to improve the effect of APRIL on proliferation and apoptosis of colorectal cancer (CRC). METHODS: Human CRC model was established in nude mice, and the nude mice were treated with APRIL siRNA twice per week for 2 weeks. APRIL mRNA expression was surveyed by PCR and APRIL protein expression was detected by immunohistochemistry. The expression of PCNA protein was detected by ELISA. The expression of bcl-2 and bcl-xl was assessed by immunohistochemical staining, and TUNEL staining was used to detect apoptosis. RESULTS: The expression of APRIL mRNA in the APRIL siRNA group was (0.13 ± 0.05) × 10(-3), significantly lower than that in the vector group (0.95 ± 0.04) × 10(-3) and the PBS group (0.96 ± 0.05) × 10(-3). The expression of APRIL protein in the APRIL siRNA group was (87.5 ± 5.0)% lower than that in the vector and PBS groups (P < 0.05). APRIL siRNA significantly suppressed the growth of SW480 tumor: the IR (inhibitory rate) of APRIL siRNA group was (60.7 ± 1.5)% (P < 0.05). The expression of PCNA in APRIL siRNA group was (176.8 ± 18.1) ng/ml, was (56.5 ± 2.0)% lower than that of PBS group (328.4 ± 22.8) ng/ml. Furthermore, the expressions of anti-apoptosis proteins bcl-2 and bcl-xl of APRIL siRNA group were (82.6 ± 4.5)% and (79.2 ± 3.5)% lower than those of the PBS group. The apoptotic rate of the APRIL siRNA group was 40.1% ± 2.5%, significantly higher than that in the vector group (2.5 ± 0.1)% and PBS group (2.5 ± 0.2)% (P < 0.05). CONCLUSION: APRIL siRNA may significantly suppress the growth and promote apoptosis in transplanted tumor of human colorectal cancer in nude mice. APRIL may become a candidate gene of gene therapy of human colorectal cancer.
Subject(s)
Apoptosis , Cell Proliferation , Colorectal Neoplasms/pathology , RNA, Small Interfering/genetics , Tumor Necrosis Factor Ligand Superfamily Member 13/biosynthesis , Animals , Cell Line, Tumor , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Female , Humans , Ligands , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Transplantation , Proliferating Cell Nuclear Antigen/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/metabolism , Random Allocation , Tumor Necrosis Factor Ligand Superfamily Member 13/genetics , bcl-X Protein/metabolismABSTRACT
The receptor for advanced glycation end products (RAGE) interacts with several ligands and is involved in various human diseases. Several splicing forms of the RAGE gene have been characterized, and two general mechanisms are usually responsible for the generation of soluble receptors. However, variants distribution and respective roles in different tumors are not clear. We analyzed RAGE and hRAGEsec mRNA expression in esophageal and lung cancer by RT-polymerase chain reaction. The Agilent clipper 1000 Bioanalyzer using lab-on-a-chip technology was applied to size and quantify the polymerase chain reaction products. Western blotting was performed to measure total soluble RAGE protein levels. The results showed that RAGE and its splice variants increased in esophageal cancers and decreased in lung cancers. We conclude that RAGE presents as a major isoform; soluble RAGE may also play certain roles in esophageal cancer and lung cancer.
Subject(s)
Esophageal Neoplasms/metabolism , Gene Expression Regulation, Neoplastic , Lung Neoplasms/metabolism , Receptors, Immunologic/metabolism , Aged , Alternative Splicing , Blotting, Western , Computational Biology , Down-Regulation , Electrophoresis , Esophageal Neoplasms/genetics , Esophageal Neoplasms/pathology , Female , Gene Expression Profiling , Humans , Ligands , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Male , Microfluidic Analytical Techniques , Middle Aged , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor for Advanced Glycation End Products , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Solubility , Tissue Distribution , Up-RegulationABSTRACT
OBJECTIVE: To establish a real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) method for quantifying a proliferation-inducing ligand (APRIL) mRNA in sputum samples from patients with non-small cell lung cancer (NSCLC), and to evaluate its role in the diagnosis of NSCLC. METHODS: Seventy-one cases of NSCLC and 62 cases of benign pulmonary disease were enrolled in this study from August 2007 to May 2008 in Affiliated Hospital of Nantong University, Jiangsu. Sixty-five healthy volunteers served as the control. The fluorescence of the PCR products was detected continuously during the amplification by RFQ-PCR. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples was determined using software. The results were presented as the ratios of target genes to beta(2)-microglobulin (beta(2)-M) mRNA, and compared with those obtained by conventional cytological method. RESULTS: The detection range of the assay was from 38 copies/microl to 3.8 x 10(6) copies/microl. The coefficients of variation values of both intra-experimental and inter-experimental reproducibility were 8.5% and 13.6%, respectively. The expression of APRIL mRNA in tumor sputum was higher than that in benign pulmonary disease and healthy volunteers (t = 10.50, 11.32, P < 0.01). The positive rate for APRIL mRNA expression was 81.7% (58 of 71) in sputum samples of NSCLC, 3.2% (2/62) in benign pulmonary disease and 1.5% (1/65) in healthy volunteers when cut-off values for positivity were set at the x(-) +/- 2 s of mRNA expression in health volunteers. The level of APRIL mRNA of NSCLC was not related to sex, age, smoking status, TNM stage and lymph node metastasis (P > 0.05, respectively), but was related to pathology subtype and the location of tumors (P < 0.05, respectively). The APRIL mRNA assay (82%) produced a higher detection rate than conventional cytological method (14%) (chi(2) = 67.68, P < 0.01). CONCLUSION: Measurement of the expression of APRIL mRNA in sputum by RFQ-PCR showed high sensitivity and specificity, which maybe useful in diagnosing NSCLC.
