Xiaolei Feng

Changes in the expression of genes involved in cell cycle regulation and the relative telomere length in the process of canceration induced by omethoate

Organophosphorous pesticides (OPs), with high efficiency, broad-spectrum and low residue, are widely used in China. Omethoate is a broad category of organophosphorous pesticides and is more domestically utilized which has chronic toxic effect on human health caused by long-term, low-dose exposure to Ops, recently its potential genotoxicity has attracted wide attention which can cause chromosomal DNA damage. Thus, the aim of this study is screen susceptible biomarkers and explore the mechanism of canceration induced by omethoate. 180 long-term organophosphorus pesticide–exposed workers and 115 healthy controls were recruited. Quantitative polymerase chain reaction method was applied to determine the relative telomere length in peripheral lymphocyte DNA as well as p53 and p21 gene expression levels. Genetic polymorphisms were determined by the polymerase chain reaction–restriction fragment length polymorphism method. Multiple linear regression was conducted to explore the effects of exposure, expression levels, and polymorphisms in genes on the telomere length. The results showed the relative telomere lengths in the exposure group were significantly longer than that in the control group. The messenger RNA expression levels of p53 and p21 in exposure group were significantly lower than that in the control group; telomere lengths of the CA genotype individuals of p21 rs1801270 polymorphism locus were significantly longer than that of the CC genotype in the control group that were estimated using the Bonferroni method; and bivariate correlation analysis showed that the messenger RNA expression level of gene p53 was negatively correlated with telomere length, and the messenger RNA expression level of gene p21 was positively correlated with telomere length. Multivariate analysis found that p53 messenger RNA and p21 messenger RNA had an impact on telomere length. These results demonstrated that the messenger RNA expression levels of p53 and p21 may have a relationship with the changes in telomere length induced by omethoate and provided strong evidence for the mechanism of canceration induced by poison.

An Improved PCR-RFLP Assay for the Detection of a Polymorphism rs2289487 of PLIN1 Gene.

In recent research, it has been shown that rs2289487 within the PLIN1 gene has different variants that have been associated with obesity, type 2 diabetes, and other diseases. However, the isochizomers such as the BsmI enzyme required for detection of this polymorphism through polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method are expensive. In this study, we aimed to explore a novel PCR‐RFLP method for identifying the single‐nucleotide polymorphism (SNP) of rs2289487 of PLIN1 gene.

Detecting the polymorphism of TERF1 gene by an improved PCR-RFLP method

Polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) is a common and mature method of detecting the single nucleotide polymorphism (SNP). But, for the polymorphism site rs3863242 of telomeric repeat binding factor 1(TERF1) gene, there is no appropriate restriction enzyme to recognize it, which limits the research between the variants of rs3863242 and human diseases.

Establishment of two data mining models of lung cancer screening based on three gene promoter methylations combined with telomere damage

Objective To identify the significance of a support vector machine (SVM) model and a decision tree (DT) model for the diagnosis of lung cancer combined with the detection of fragile histidine triad (FHIT), RAS association domain family 1 (RASSF1A) and cyclin-dependent kinase inhibitor 2A (p16) promoter methylation levels and relative telomere length (RTL) of white blood cells from peripheral blood DNA. Methods The levels of p16, RASSF1A and FHIT promoter methylation and the RTL of white blood cells in peripheral blood DNA of 200 healthy individuals and 200 lung cancer patients were analyzed by SYBR Green-based quantitative methylation-specific PCR and quantitative PCR. Based on the 4 biomarkers, SVM and DT models were developed. Results The levels of FHIT, RASSF1A and p16 promoter methylation were 3.33 (1.86-6.40) and 2.85 (1.39-5.44) (p = 0.002); 27.62 (9.09-52.86) and 17.17 (3.86-50.87) (p = 0.038); and 0.59 (0.16-4.50) and 0.36 (0.06-4.00) (p = 0.008) in cases and controls, respectively. RTL was 0.93 ± 0.32 and 1.16 ± 0.57 (p<0.001). The areas under the receiver operating characteristic (ROC) curves of the Fisher discriminant analysis, SVM and DT models were 0.670 (0.569-0.761), 0.810 (0.719-0.882) and 0.810 (0.719-0.882), respectively. Conclusions The SVM and DT models for diagnosing lung cancer were successfully developed through the combined detection of p16, RASSF1A and FHIT promoter methylation and RTL, which provided useful tools for screening lung cancer.

Application of Created Restriction Site PCR-RFLP to Identify POT1 Gene Polymorphism.

Protection of telomeres protein 1 (POT1) plays pivotal roles in protection of chromosome ends and regulation of telomere length with other telomere binding proteins; its genetic polymorphisms are associated with many diseases. In this study, we explored a novel PCR-RFLP method for typing the single nucleotide polymorphism (SNP) rs1034794 of the human POT1 gene. A new restriction enzyme site was introduced into a POT1 gene amplification product by created restriction site PCR (CRS-PCR). One primer was designed based on changed sequence; after PCR amplification, a new restriction enzyme site for AluI was introduced into the PCR products. One hundred and seventy eight samples from Han Chinese individuals were tested to evaluate this new method. The 3′-end of the forward primer was next to the polymorphic site, and the third base from the 3′-end was the mismatched base A. The final PCR product contained the AGCT sequence (AluI recognition site) when the ancestral POT1 alleles were amplified. The data obtained with the new method perfectly matched those obtained with the sequencing method. Thus, CRS-PCR is a new low-cost and high-efficiency alternative for rs1034794 typing.

Interaction between polymorphisms in cell-cycle genes and environmental factors in regulating cholinesterase activity in people with exposure to omethoate

Cholinesterase activity (ChA), the effective biomarker for organophosphate pesticide exposure, is possibly affected by single nucleotide polymorphisms (SNPs) in cell-cycle-related genes. One hundred and eighty workers with long-term exposure to omethoate and 115 healthy controls were recruited to explore the gene–gene and gene–environment interactions. The acetylthiocholine and dithio-bis-(nitrobenzoic acid) method was used to detect the cholinesterase activities in whole blood, erythrocytes and plasma. Genetic polymorphisms were determined by the PCR-RFLP and direct PCR electrophoresis methods. Statistical results showed that the cholinesterase activities of whole blood, erythrocytes and plasma in the exposure group were significantly lower than those in the control group (p < 0.001), and erythrocyte cholinesterase activities were associated with gender, smoking and drinking in the exposure group (p < 0.05). Single-locus analyses showed that there is a statistically significant difference in the ChA among the genotypes CC, CA and AA of the p21 rs1801270 locus in the control group (p = 0.033), but not in the exposure group. A significant interaction between genes and environmental factors (i.e. p53, p21, mdm2, gender, smoking and drinking) affecting ChA was found through a generalized multifactor dimensionality reduction analysis. These obtained markers will be useful in further marker-assisted selection in workers with exposure to omethoate.

Association of genetic polymorphisms of telomere binding proteins with cholinesterase activity in omethoate-exposed workers

Omethoate, an organophosphorous pesticide, can cause a variety of health effects, especially the decrease of cholinesterase activity. The aim of this study is to explore the association of genetic polymorphisms of telomere binding proteins with cholinesterase activity in omethoate-exposed population. Cholinesterase activities in whole blood, red blood cell and plasma were detected using acetylthiocholine and dithio-bis-(nitrobenzoic acid) method; Genetic Genotyping of POT1 rs1034794, POT1 rs10250202, TERF1 rs3863242 and TERT rs2736098 were performed with PCR-RFLP. The cholinesterase activities of whole blood, red blood cells and plasma in exposure group are significantly lower than that of the control group (P < 0.001). Multivariate analysis indicates that exposure group (b = - 1.016, P < 0.001), agender (b = 0.365, P < 0.001), drinking (b = 0.271, P = 0.004) and TERF1rs3863242 (b = - 0.368, P = 0.016) had an impact on cholinesterase activities. The results suggest that individual carrying AG+GG genotypes in TERF1 gene rs3863242 polymorphism were susceptible to damage in cholinesterase induced by omethoate.

Cross-sectional associations between genetic polymorphisms in metabolic enzymes and longer leukocyte telomere length induced by omethoate

Purpose This study aimed to explore the effects of genetic polymorphisms in metabolic enzymes on relative telomere length changes and explore the mechanism of canceration induced by omethoate. Materials and Methods 180 long-term omethoate-exposed workers and 115 healthy controls were recruited. Real-time PCR method was applied to determine the relative telomere length in peripheral blood leukocytes DNA, and Six polymorphic loci of GSTT1(+/−), GSTM1(+/−), GSTP1 rs1695, CYP2E1 rs6413432, CYP2E1 rs3813867 and PON2 rs12026 were detected by polymerase chain reaction and restriction fragment length polymorphism method; Multiple linear regression was conducted to explore the effects of omethoate exposure and genetic polymorphisms on the telomere length. Results The relative telomere lengths in the control group (0.94 [0.76, 1.32]) were significantly shorter than that in the exposure group (1.50 [1.11, 2.57]) (Z = 7.910, P < 0.001). Univariate analysis showed that the relative telomere lengths of the GSTM1-deletion individuals were significantly longer than that of the non - deletion genotype in the control group (Z = 2.911, P = 0.004), and the relative telomere lengths of GSTP1 rs1695 polymorphism locus (GG+AG) genotype individuals were longer than that of AA genotype in the exposure group. The difference was statistically significant (Z = 2.262, P = 0.024). Multivariate analysis found that pesticide-exposure (b = 0.524, P < 0.001) and GSTM1 polymorphism (b = −0.136, P = 0.029) had an impact on telomere length. Conclusions The relative telomere lengths of omethoate-exposure workers were longer than that in the control population. Also GSTM1 genetic polymorphism may influence the changes of the telomere length induced by omethoate.

Application of CRS-PCR-RFLP to identify CYP1A1 gene polymorphism

Cytochrome P4501A1 (CYP1A1) is a member of the cytochrome P450 gene family and plays an important role in the metabolism of exogenous and endogenous material. In recent research, it has been shown that its genetic polymorphisms are associated with many diseases. But the isoschizomers such as the BsrDI enzyme required for the detection of this polymorphism are expensive.