OBJECTIVE: To construct a luciferase reporter gene vector carrying human nuclear factor of activated T cells 2 (NFATc2) gene promoter and examine the effects of metformin and lipopolysaccharide (LPS) on the transcriptional activity of NFATc2 gene. METHODS: The promoter sequence of human NFATc2 gene was acquired from UCSC website for PCR amplification. NFATc2 promoter fragment was inserted into pGL3-basic plasmid double cleaved with Kpn Ⅰ and Hind Ⅲ. The resultant recombinant plasmid pGL3-NFATC2-promoter was co-transfected with the internal reference plasmid pRL-TK in 293F cells, and luciferase activity in the cells was detected. Reporter gene vectors of human NFATc2 gene promoter with different fragment lengths were also constructed and assayed for luciferase activity. The changes in transcription activity of NFATc2 gene were assessed after treatment with different concentrations of metformin and LPS for 24 h. We also examined the effect of mutation in RUNX2-binding site in NFATC2 gene promoter on the regulatory effects of metformin and LPS on NFATc2 transcription. RESULTS: We successfully constructed pGL3-NFATc2-promoter plasmids carrying different lengths (2170 bp, 2077 bp, 1802 bp, 1651 bp, 1083 bp, 323 bp) of NFATc2 promoter sequences as verified by enzymatic digestion and sequencing. Transfection of 293F cells with the plasmid carrying a 1651 bp NFATc2 promoter (pGL3-1651 bp) resulted in the highest transcriptional activity of NFATc2 gene, and the luciferase activity was approximately 3.3 times that of pGL3-2170 bp (1.843 ± 0.146 vs 0.547 ± 0.085). Moderate (5 mmol/L) and high (10 mmol/L) concentrations of metformin significantly upregulated the transcriptional activity of pGL3-1651 bp by up to 2.5 and 3 folds, respectively. LPS at different doses also upregulated the transcriptional activity of pGL3-1651 bp by at least 1.6 folds. The mutation in the RUNX2 binding site on pGL3-1651 bp obviously reduced metformin- and LPS-induced enhancement of pGL3-1651bp transcription by 1.7 and 2 folds, respectively. CONCLUSION pGL3-NFATc2-promoter can be transcribed and activated in 293F cells, and LPS and metformin can activate the transcription of pGL3- NFATc2-promoter in a RUNX2-dependent manner.
Core Binding Factor Alpha 1 Subunit/genetics* ; Humans ; Lipopolysaccharides/pharmacology* ; Luciferases/genetics* ; Metformin/pharmacology* ; NFATC Transcription Factors/genetics* ; Promoter Regions, Genetic ; T-Lymphocytes ; Transcription, Genetic/drug effects* ; Transfection

OBJECTIVETo investigate the effect of Lang-chuang-ding Decoction (, LCD) on the expression of DNA methylation of CD70 gene promoter in peripheral blood mononuclear cells (PBMCs) of females with systemic lupus erythematosus (SLE).

METHODSPBMCs isolated from female patients with SLE or healthy donors were cultured and treated with LCD medicated serum or normal serum for 24 or 48 h. The mRNA expressions of CD70 gene in PBMCs were detected by reverse transcription polymerase chain reaction (PCR); the DNA methylation of the CD70 gene promoter region was detected by methylation-specific PCR.

RESULTSAfter treated with medicated serum for 48 h, the mRNA expression levels of CD70 in PBMCs of SLE patients were signifificantly higher than those of healthy donors (P<0.05); the DNA methylation levels of CD70 promoter region in PBMCs of SLE patients treated with medicated serum for 48 h were signifificantly higher than those treated with fetal bovine serum (P<0.01).

CONCLUSIONLCD could inhibit CD70 gene expression in PBMCs of SLE patients by promoting the DNA methylation of CD70 gene promoter.

Adult ; CD27 Ligand ; genetics ; metabolism ; DNA Methylation ; drug effects ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; drug effects ; metabolism ; Lupus Erythematosus, Systemic ; drug therapy ; genetics ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; metabolism

BACKGROUNDDuring the past decades, the incidence of invasive aspergillosis (IA) caused by Aspergillus fumigatus has increased dramatically. The aims of this study were to investigate the susceptibility of clinical isolates of A. fumigatus to triazole and the underlying cyp51A mutations in triazole-resistant A. fumigatus.

METHODSA total of 126 A. fumigatus clinical isolates from 126 patients with proven or probable IA were obtained from four large tertiary hospitals in Nanjing, China, between August 2012 and July 2015. The determination of minimal inhibitory concentrations (MICs) for itraconazole, voriconazole, and posaconazole was performed by broth microdilution according to the European Committee on Antimicrobial Susceptibility Testing reference method.

RESULTSA total of 4 A. fumigatus isolates (3.17%) were confirmed to be itraconazole resistant, with MICs of ≥8 mg/L, and one isolate (0.8%) was confirmed to be voriconazole resistant and posaconazole resistant, with MICs of 4 mg/L and 0.5 mg/L, respectively. We found that two of the 4 isolates of triazole-resistant A. fumigatus had the L98H amino acid substitution in combination with a 34-base pair tandem repeat in the promoter region, one isolate had an M220I mutation, and another itraconazole-resistant isolate did not have a substitution in the cyp51A gene.

CONCLUSIONSThis study shows that triazole-resistant A. fumigatus clinical isolates are present in Nanjing, China, which is a new challenge to the clinical management of IA.

Antifungal Agents ; pharmacology ; Aspergillus fumigatus ; drug effects ; genetics ; China ; Drug Resistance, Fungal ; Itraconazole ; pharmacology ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; Tandem Repeat Sequences ; genetics ; Triazoles ; pharmacology ; Voriconazole ; pharmacology

BACKGROUNDClaudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported.

METHODSIn this study, HCMECs were stimulated with CoCl2to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs.

RESULTSWe found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription.

CONCLUSIONSThe results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs.

Cell Hypoxia ; Cells, Cultured ; Claudins ; analysis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Histones ; metabolism ; Humans ; Myocardium ; cytology ; metabolism ; Promoter Regions, Genetic

Although methyltransferase has been recognized as a major element that governs the epigenetic regulation of the genome during temozolomide (TMZ) chemotherapy in glioblastoma multiforme (GBM) patients, its regulatory effect on glioblastoma chemoresistance has not been well defined. This study investigated whether DNA methyltransferase (DNMT) expression was associated with TMZ sensitivity in glioma cells and elucidated the underlying mechanism. DNMT expression was analyzed by western blotting. miR-20a promoter methylation was evaluated by methylation-specific PCR. Cell viability and apoptosis were assessed using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and TdT-mediated dUTP-biotin nick end labeling assays, respectively. The results showed that compared with parental U251 cells, DNMT1 expression was downregulated, miR-20a promoter methylation was attenuated and miR-20a levels were elevated in TMZ-resistant U251 cells. Methyltransferase inhibition by 5-aza-2\'-deoxycytidine treatment reduced TMZ sensitivity in U251 cells. In U251/TM cells, DNMT1 expression was negatively correlated with miR-20a expression and positively correlated with TMZ sensitivity and leucine-rich repeats and immunoglobulin-like domains 1 expression; these effects were reversed by changes in miR-20a expression. DNMT1 overexpression induced an increase in U251/TM cell apoptosis that was inhibited by the miR-20a mimic, whereas DNMT1 silencing attenuated U251/TM cell apoptosis in a manner that was abrogated by miR-20a inhibitor treatment. Tumor growth of the U251/TM xenograft was inhibited by pcDNA-DNMT1 pretreatment and boosted by DNMT1-small hairpin RNA pretreatment. In summary, DNMT1 mediated chemosensitivity by reducing methylation of the microRNA-20a promoter in glioma cells.
Animals ; Antineoplastic Agents, Alkylating/*pharmacology/therapeutic use ; Apoptosis/drug effects ; Brain/drug effects/metabolism/pathology ; Brain Neoplasms/drug therapy/*genetics/pathology ; DNA (Cytosine-5-)-Methyltransferase/antagonists & inhibitors/*genetics/metabolism ; DNA Methylation ; Dacarbazine/*analogs & derivatives/pharmacology/therapeutic use ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation, Neoplastic ; Glioma/drug therapy/*genetics/pathology ; Humans ; Mice, Inbred C57BL ; MicroRNAs/*genetics ; Promoter Regions, Genetic

OBJECTIVETo explore the correlation between UGT1A1 (*28, *60 and * 93) polymorphism and the adverse reactions in small cell lung cancer patients after irinotecan chemotherapy.

METHODSClinical data of 58 small cell lung cancer patients in extensive stage treated in our hospital were retrospectively analyzed. Polymerase chain reaction was used to amplify the UTG, and direct sequencing was performed to determine the UGT polymorphism. The adverse reactions ≥ grade 3 after irinotecan chemotherapy in patients with different UGT genotype were analyzed.

RESULTSAmongthe 58 patients with extensive stage small cell lung cancer, there were 45 (77.6%) cases of wild type UGT1A1*28, 40 (69.0%) cases of wild type UGT1A1* 93, 38 (65.5%) cases of wild type UGT1A1*60, 18 cases of mutation in UGT1A1* 93 and 20 cases of mutation in UGT1A1*60. In UGT1A1 promoter position 28, there were 8 (13.8%) cases of TA5 mutation and 5 (8.6%) cases of TA7 mutation. Among the patients with TA5 mutation, 5 cases had ≥ grade 3 diarrhea, 3 cases had ≥ grade 3 leucopenia and 3 cases had ≥ grade 3 neutropenia, while among the patients with UGT1A1 * 93 mutation, 7 cases had ≥ grade 3 diarrhea, 6 cases had ≥ grade 3 leucopenia and 4 cases had ≥ grade 3 neutropenia.

CONCLUSIONSTA5 and UGT1A1* 93 mutation increase the risk of diarrhea and ≥ grade 3 leukopenia and neutropenia, however, wild type UGT1A1 (*28, * 93, *60) and mutant UGT1A1*60 do not increase those risks. Further prospective study in a larger number of patients is needed to clarify the association between UGT1A1*28, UGT1A1* 93 and UGT1A1*60 polymorphism and adverse reactions of irinotecan, and to help clinicians in choosing a better therapeutic modality for personalized chemotherapy to improve curative effect and reduce adverse reactions.

Antineoplastic Agents, Phytogenic ; adverse effects ; Camptothecin ; adverse effects ; analogs & derivatives ; Diarrhea ; Genotype ; Glucuronosyltransferase ; genetics ; metabolism ; Humans ; Neutropenia ; Polymorphism, Genetic ; Promoter Regions, Genetic ; Prospective Studies ; Retrospective Studies ; Small Cell Lung Carcinoma ; drug therapy ; genetics

OBJECTIVETo investigate the promoter methylation of p16, FHIT and RASSF1A gene and telomere damage in the workers exposed to coal tar pitch, and to explore the effective biomarker of occupational exposure to coal tar pitch.

METHODS180 cases of workers exposed to coal tar pitch in a certain carbon plant named as exposure group, and 145 healthy cases with a medical examination in the first affiliated hospital of Zhengzhou University were selected as control group. Relative telomere length in peripheral blood DNA was detected using real-time quantitative PCR, and the promoter methylation rate of p16, RASSF1A and FHIT gene in peripheral blood DNA were determined by real-time quantitative methylation specific PCR. The relative telomere length and gene promoter methylation in two groups were compared, and influencing factors were analyzed.

RESULTSRelative telomere length in exposed group was lower than that in the control group, and the difference was statistically significant (Z = -5.395, P < 0.001). There was no significant difference in the promoter methylation rate of p16, FHIT and RASSF1A gene between the two groups (P > 0.05). Stratification analysis by gender, age, and smoking, we found that when the age was less than or equal to 40, the promoter methylation rate of p16 in exposed group was more than that in control group, and the difference was statistically significant (Z = -1.914, P = 0.011).

CONCLUSIONOccupational exposure to coal tar pitch may induce leukocyte DNA telomere length of human peripheral blood shortened, and may not change the promoter methylation rates of p16, FHIT and RASSF1A gene.

Acid Anhydride Hydrolases ; genetics ; Coal Tar ; adverse effects ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Humans ; Leukocytes ; drug effects ; Neoplasm Proteins ; genetics ; Occupational Exposure ; adverse effects ; Promoter Regions, Genetic ; Telomere ; drug effects ; ultrastructure ; Tumor Suppressor Proteins ; genetics

BACKGROUND/AIMS: The T-helper 1 (TH1) immune reaction is essential for the eradication of hepatitis C virus (HCV) during pegylated interferon alpha (PEG-IFN-alpha)- and ribavirin (RBV)-based therapy in chronic HCV patients. Secreted phosphoprotein 1 (SPP1) was shown to be a crucial cytokine for the initiation of a TH1 immune response. We aimed to investigate whether SPP1 single nucleotide polymorphisms (SNPs) may influence sustained virological response (SVR) rates. METHODS: Two SNPs in the promoter region of SPP1 at the -443 C>T and -1748 G>A loci were genotyped in 100 patients with chronic HCV genotype 4 infection using a TaqMan SNP genotyping assay. RESULTS: Sixty-seven patients achieved a SVR, and 33 patients showed no SVR. Patients carrying the T/T genotype at the -443 locus showed a significantly higher SVR rate than those carrying the C/T or C/C genotype (83.67% vs 50.98%, p<0.001). At the -1748 locus, the SVR rate was significantly higher in patients with the G/G genotype than in those with the A/A genotype (88.89% vs 52.63%, p=0.028) and in patients with the G/A genotype than in those with the A/A genotype (85.29% vs 52.63%, p=0.001). CONCLUSIONS: SPP1 SNPs at -443 C>T and -1748 G>A loci may be useful markers for predicting the response to PEG-IFN-alpha-2b plus RBV therapy in Egyptian patients with chronic HCV genotype 4 infection.
Adult ; Antiviral Agents/*therapeutic use ; Biomarkers/blood ; Drug Therapy, Combination ; Egypt ; Female ; Genotype ; Hepacivirus/drug effects/genetics ; Hepatitis C, Chronic/*drug therapy/virology ; Humans ; Interferon-alpha/*therapeutic use ; Male ; Middle Aged ; Osteopontin/*genetics ; Polyethylene Glycols/*therapeutic use ; Polymorphism, Single Nucleotide/genetics ; Predictive Value of Tests ; *Promoter Regions, Genetic ; Recombinant Proteins/therapeutic use ; Ribavirin/*therapeutic use ; Treatment Outcome

BACKGROUNDExtended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae (K. pneumoniae) is one of the most popular pathogens that cause refractory respiratory tract infection. The genetic environment, including insertion sequences and the types of promoter, plays a key role in exploration of the mechanism of prevalence and dismission of the ESBL-producing K. pneumoniae isolates. The aim of the investigation was to target analysis the genetic environment and promoter sequences of blaCTX-M, blaSHV and blaTEM, the most popular β-lactamase genes harbored by ESBL-producing K. pneumoniae isolates.

METHODSFrom February 2010 to July 2011, 158 of 416 K. pneumoniae isolates producing ESBL from patients with lower respiratory tract infection were collected from seven tertiary hospitals from Beijing, Anhui, Fujian, Liaoning, Hebei and Inner Mongolia Autonomous Region in China. The genetic environment including promoters of 10 types of blaCTX-M, 18 types of blaSHV and 2 types of blaTEM were analyzed by amplification and direct sequencing with various sets of PCR primers.

RESULTSISEcp1 was located upstream of the 5' end of the blaCTX-M gene in 130 (97.0%) out of 134 K. pneumoniae isolates harboring blaCTX-M and provided a conserved promoter to blaCTX-M. A non-coding sequence preceded by kdpC and recF was identified in all of the blaSHV genes except blaSHV-12 and blaSHV-2a. IS26 was also found upstream of 1 blaCTX-M-15, 10 blaSHV-1 strains, 4 blaTEM-1 and all of the blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12. Eighty-seven of 91 strains harboring blaTEM-1 carried a copy of Tn2 and IS26-blaTEM-1 fragments were also detected in 4 strains. With respect to K. pneumoniae, the genetic environment of blaCTX-M-38, blaSHV-142 and blaTEM-135 were firstly elaborated, and four kinds of novel genetic environment of blaCTX-M-3, blaCTX-M-15 and blaTEM-1 have been detected as well.

CONCLUSIONSPerfective implementation of the genetic environment information of bgr;-lactamase gene needs to be further explored and supplemented. ISEcp1 and IS26 elements are widespread upstream of the blaCTX-M, blaSHV and blaTEM genes and contribute to horizontal transmission and genetic expression.

Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; metabolism ; China ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; genetics ; Microbial Sensitivity Tests ; Promoter Regions, Genetic ; genetics ; beta-Lactamases ; genetics ; metabolism

BACKGROUNDHerpes simplex virus thymidine kinase phosphorylates ganciclovir to ganciclovir monophosphate, which is then converted to ganciclovir triphosphate by endogenous cellular nucleoside kinases. The ganciclovir triphosphate acts as a DNA chain terminator due to the lack of a functional 3'-OH group and terminates the process of DNA replication, hence leading to cell apoptosis. At present, HSVtk gene usually acts as suicide gene to kill tumor cells. The aim of this study was to investigate the selective cytotoxicity of the herpes simplex virus thymidine kinase/ganciclovir (HSVtK/GCV) suicide gene system controlled by the a-fetoprotein (AFP) promoter on hepatocellular carcinoma (HCC) cells in vitro.

METHODSpAFP-HSVtk-IRES2-EGFP recombinant plasmid vectors driven by the AFP promoter were constructed. HL-7702 liver cells, HUH-7 HCC, and HepG2 HCC were transfected with the recombinant plasmids. HSVtK gene expression was detected using Western blotting analysis. HepG2 cells line stably expressing HSVtk gene was selected by G418 reagent. The cytotoxicity of HSVtK/GCV suicide gene system on hepatoma cells was measured by CCK-8 reagents when different doses of ganciclovir were added.

RESULTSPlasmid pAFP-TK-IRES2-EGFP-expressed HSVtk gene was constructed successfully. HSVtk gene expression level was significantly higher in AFP-positive hepatoma cells than in AFP-negative liver cells. After G418 selection, a HepG2 cells line stably expressing HSVtk gene was acquired. With the increase of the dose of ganciclovir the optical density at 450 nm of HepG2 cells stably expressing HSVtk gene gradually decreased (P < 0.05).

CONCLUSIONThe HSVtK gene-specific expression in hepatoma cells as well as the cytotoxicity of the suicide gene system in HepG2 cells provided the basis for the targeted gene therapy of HCC.

Apoptosis ; drug effects ; genetics ; Carcinoma, Hepatocellular ; genetics ; Cell Line, Tumor ; Fetal Proteins ; genetics ; Ganciclovir ; analogs & derivatives ; pharmacology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; genetics ; Promoter Regions, Genetic ; genetics ; Transfection