OBJECTIVE: To analyze the relationship between and gene methylation with aging in the general population. METHODS: We collected peripheral blood samples from 284 male and 246 female healthy subjects for detection of methylation levels of and genes using quantitative methylation-specific PCR (qMSP). The relationship between the methylation levels of and genes and aging was analyzed using Spearman or Pearson correlation test. RESULTS: We found a significant positive correlation between the methylation levels of the two genes in these subjects ( < 0.05). In the overall population as well in the female subjects, methylation was found to be inversely correlated with age ( < 0.05). The methylation levels of and genes were inversely correlated with TG, ApoE, Lp(a) and AST in the overall population ( < 0.05). In both the female and male subjects, the methylation levels of the two genes were inversely correlated with Lp(a) ( < 0.05). In the male subjects, methylation was inversely correlated with AST ( < 0.05), while methylation was inversely correlated with HDL and ApoE ( < 0.05). In the female subjects, methylation was positively correlated with LDL and inversely correlated with ApoE and AST ( < 0.05). CONCLUSIONS The methylation levels of and are closely related to age and the levels of multiple proteins in healthy subjects.
Aging ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; DNA Methylation ; Female ; Humans ; Male ; Real-Time Polymerase Chain Reaction

OBJECTIVE: The aim of this study is to establish whether cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) gene polymorphisms are associated with premature triple-vessel disease (PTVD). METHODS: Nine single-nucleotide polymorphisms (rs1063192, rs10757274, rs1333042, rs1333049, rs2285327, rs3217986, rs3217992, rs4977574, and rs9632884) were genotyped in 884 PTVD patients and 907 control subjects (males ⪕ 50 years old and females ⪕ 60 years old) using the improved multiplex ligase detection reaction method. RESULTS: The allele frequencies of rs10757274 G, rs1333049 C, rs4977574 G (all P < 0.001), and rs3217986 G (P = 0.040) were significantly higher in the PTVD group than in the control group, but those of rs1063192 A, rs1333042 G, and rs9632884 C (all P < 0.001) were significantly lower in the former than in the latter. Logistic regression analysis revealed that homozygote AA of rs1333042 is associated with decreased risk for PTVD (OR = 0.42, 95% CI: 0.22-0.82, P = 0.011). In addition, the allele frequencies observed differed between genders. The G allele of rs3217986 was associated with increased risk for PTVD in male patients only (OR = 2.94, 95% CI: 1.27-6.80, P = 0.012) in the dominant model, and no positively mutated allele was found in female patients. CONCLUSION Polymorphisms of the CDKN2B-AS1 gene are associated with the incidence of PTVD in the Chinese population. Furthermore, the frequencies of mutated alleles differed between genders.
Adult ; Asian Continental Ancestry Group ; genetics ; China ; Coronary Artery Disease ; genetics ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; RNA, Antisense ; genetics ; Sex Factors

Gastric cancer is the result of multiple risk factors, including environmental factors, genetic factors and the interaction between them. The environmental factors mainly include dietary, Helicobacter pylori infection and family history of gastric cancer. Genetic factors mainly refer to the susceptible genes that cause epigenetic alterations in oncogenes, tumor suppress genes, cell cycle regulators, DNA repair genes and signaling molecules. This paper summarizes the susceptible genes of gastric cancer and explores the genetic basis of it.
Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Genes, Tumor Suppressor ; Genes, p16 ; Humans ; Oncogenes ; Stomach Neoplasms ; etiology ; genetics

OBJECTIVETo detect the presence of p15 antisense RNA(p15AS) in acute myeloid leukemia(AML).

METHODSp15AS and p15 mRNA in two leukemia cell lines was detected with strand-specific primer RT-qPCR. To explore the connection between p15AS and AML, 43 patients with newly diagnosed AML and 21 patients with benign diseases (Iron deficiency anemia) as controls were enrolled. The expression level of p15AS in bone marrow cells derived from the patients and the controls were determined by strand-specific primer RT-qPCR, and its relationship with clinical features was analyzed.

RESULTSThe two AML lines displayed high p15AS and low p15 expression. Samples derived from the AML patients showed relatively increased expression of p15AS and down-regulated p15 expression in their bone cells. In contrast, the 21 controls showed high expression of p15 but relatively low expression of the p15AS. Compared with the normal controls, the expression levels of p15 protein were significantly lower among the AML patients (P<0.01). No relationships were detected between the level of p15AS and the patient's age, gender, FAB subtype, total white blood cell count, platelet count, proliferative degree of bone marrow cell and karyotype classification (P>0.05 for all comparisons).

CONCLUSIONHigh expression of p15 antisense RNA was frequently found among AML patients, and may play an important role in epigenetic silencing of p15.

Adult ; Aged ; Bone Marrow Cells ; metabolism ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Epigenesis, Genetic ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Male ; Middle Aged ; RNA, Antisense ; genetics ; metabolism ; Up-Regulation ; Young Adult

OBJECTIVETo investigate the role of Tal1 gene, which is aberrantly expressed in 40%-60% of patients with T lymphocytic leukemia (T-ALL), in the proliferation of T-ALL cells.

METHODSWe established stable Jurkat-siTal1 and Jurkat-T1 cell lines by trasnfecting T-ALL Jurkat cells with lentiviral vectors to knock-down or overexpress Tal1. Jurkat cells transfected with negative control siRNAs for Tal1 knock-down (Jurkat-mock1) and over-expression(Jurkat-mock2) served as the control cells. The proliferation of the cells lines was assessed using CCK-8 assay, and the cell cycle distribution was determined by flow cytometry. The mRNA and protein expressions of cyclin-dependent kinase inhibitor 2 (CDKN2A) and cyclin-dependent kinase inhibitor 1 (CDKN2B) were measured by real-time RT-PCR and Western blotting, respectively.

RESULTSJurkat-T1 cells showed more active proliferation in vitro than Jurkat-mock2 cells, while Jurkat-siTal1 cells showed slower growth than Jurkat-mock1 cells. In Jurkat-T1 cells, G0/G1 phase cells were decreased and S phase cells increased compared with Jurkat-mock2 cells, and Jurkat-siTal1 cells showed increased G0/G1 phase cells and decreased S phase cells compared with Jurkat-mock1 cells. Real-time RT-PCR and Western blotting showed that Tal1 inhibited the cellular expression of CDKN2A and CDKN2B at both mRNA and protein levels.

CONCLUSIONTal1 promotes the growth and the transition from G0/G1 phase to S phase in T-ALL cells Jurkat by inhibiting the expressions of G0/G1 and S phase negative regulatory proteins CDKN2A and CDKN2B.

Apoptosis ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; metabolism ; Humans ; Jurkat Cells ; Lentivirus ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Proto-Oncogene Proteins ; metabolism ; RNA, Small Interfering ; T-Cell Acute Lymphocytic Leukemia Protein 1

OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.

METHODSThe effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.

RESULTS① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.

CONCLUSIONSPhenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.

Acetylation ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Histones ; metabolism ; Humans ; Methylation ; Phenelzine ; pharmacology

OBJECTIVETo investigate the methylation rate of cyclin-dependent kinase inhibitor 2A (CDKN2A) and cyclin-dependent kinase inhibitor 2B (CDKN2B) in the 9P21 region in children with acute myeloid leukemia (AML) and the association of gene methylation with clinical features and outcomes.

METHODSThe clinical data of 58 children who were newly diagnosed with AML between January 2010 and December 2012 were retrospectively analyzed. Thirty-eight healthy children were recruited as the control group. Genomic DNA was extracted from bone marrow or peripheral blood of the 58 patients and 38 healthy children. The methylation status of CDKN2A and CDKN2B was analyzed by methylation-specific multiplex ligation-dependent probe amplification.

RESULTSGene methylation was not found in healthy children. Methylation probes of 44 patients were detected in 58 patients. The methylation of CDKN2A was detected with 136 bp and 237 bp methylation probes. The methylation of CDKN2B was detected with 130 bp, 210 bp, 220 bp, and 417 bp methylation probes. The methylation rate of CDKN2A was 5%, while the methylation rate of CDKN2B was 76%. The methylation detected by some probes was associated with sex, hemoglobin, and platelet count at the first visit.

CONCLUSIONSThe methylation of CDKN2B is a common event in children with AML, while the methylation of CDKN2A is relatively rare.

Adolescent ; Child ; Child, Preschool ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; DNA Methylation ; Female ; Humans ; Infant ; Leukemia, Myeloid, Acute ; genetics ; Male

OBJECTIVETo explore the proliferation and invasive effects of inhibitors of kinase 4(INK4)(P15(ink4b) and P16(ink4a)/CDKN2) gene protein activation on RKO human colorectal cell in vivo and in vitro.

METHODSRKO human colorectal cell line was exposed to the specific DNA methyltransferase inhibitor 5-Aza-CdR and INK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression was detected by Western blotting. Soft agar cloning experiment and Transwell chamber assay were used to detect the proliferative and invasive ability in vitro. Tumorigenicity in nude mice was analyzed in vivo.

RESULTSINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein expression of RKO human colorectal cells after exposure to 1×10(-7), 5×10(-7) and 1×10(-6) mol/L 5-Aza-CdR concentrations(A, B, C groups) were 1.13, 1.38, 1.92 folds and 1.11, 1.45, 2.14 folds compared to positive control group respectively. Soft agar cloning experiment showed the number of cell colony significantly decreased from 36.8±5.1(positive control group) to 32.4±7.2, 21.3±5.4 and 19.5±6.4 (3 experiment groups, all P<0.05) respectively. Transwell chamber assay showed that migrated cell number in positive control group(67.4±7.2) was significantly higher than those of 3 experimental groups(35.3±4.6, 29.5±7.3 and 25.3±6.2, respectively). The tumor volume of metastasis model in nude mice was inhibited in experimental groups, but not significantly lower compared to control group (P>0.05). There were significant differences of tumor weight and inhibition rate between control group and 3 experimental groups in nude mice respectively(all P<0.01).

CONCLUSIONINK4(P15(ink4b) and P16(ink4a)/CDKN2) protein activation can inhibit tumor proliferation, migration and suppress the tumor formation ability.

Animals ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p16 ; genetics ; metabolism ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Metastasis ; Neoplasm Transplantation ; Transcriptional Activation

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia.
Acetylation/drug effects ; Adolescent ; Adult ; Aged ; Apoptosis/*drug effects ; Cadherins/metabolism ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p15/metabolism ; DNA Methylation/drug effects ; Enzyme Inhibitors/therapeutic use/*toxicity ; Frizzled Receptors/metabolism ; Gene Expression Regulation/drug effects ; HL-60 Cells ; Histone Deacetylase Inhibitors/therapeutic use/*toxicity ; Histone-Lysine N-Methyltransferase/*antagonists & inhibitors/metabolism ; Histones/genetics/metabolism ; Humans ; Hydroxamic Acids/therapeutic use/*toxicity ; K562 Cells ; Leukemia/drug therapy/metabolism/pathology ; Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; Male ; Middle Aged ; Piperazines/therapeutic use/toxicity ; Promoter Regions, Genetic ; Young Adult

OBJECTIVETo detect p15(INK4B) methylation levels and the kinetics of the methylation status before and after decitabine to explore its influences on prognosis and response to decitabine in myelodysplastic syndromes (MDS) patients.

METHODSWe examined 261 MDS patients (143 male and 118 female) with the median age of 52 years (32-78). Of them, 172 cases were low-risk group (low-risk 104 cases, intermediate-1 68 cases), 89 cases high-risk group (intermediate-2 52 cases, high risk 37 cases). Collections of bone marrow mononuclear cells of MDS patients and extracted the genomic DNA, the methylation status of p15(INK4B) was detected by methylation-specific PCR (MSP) method. Survival analysis was conducted according to the level of p15(INK4B) methylation in the cohort of patients. The kinetics of the methylation levels of p15(INK4B) in 58 MDS patients before and after 2 courses of decitabine have been assessed with the method of MSP.

RESULTSThe methylation level of p15(INK4B) in low-risk group patients was significantly lower than that in high-risk group (117.22 vs 157.63, P<0.05 ). The expected 2-year survival rate of p15(INK4B) methylation positive patients was lower than that of negative ones (91.8% vs 69.8%, P<0.05); the expected 2-year survival rate of p15(INK4B) methylation positive patients was shorter than that of negative ones in low-risk group（78.2% vs 92.0%, P<0.05）, meanwhile there was no significant difference in terms of expected 2-year survival rate and median expected survival between p15(INK4B) methylation positive and negative patients in high-risk group [35.6% vs 38.5%, (17.0±9.3) month vs (18.0±5.7) month, P>0.05]. Multivariate analysis showed p15(INK4B) methylation degree was an independent prognostic factor for overall survival. No statistical difference of overall response (OR) rates were found between p15(INK4B) methylation positive patients and negative patients before decitabine（65.9% vs 76.5%, P>0.05）, and complete remission (CR) rates between these two groups also showed no statistical difference（22.0% vs 29.4%, P>0.05）. p15(INK4B) methylation levels had no obvious change before and after treatment in decitabine responders（P>0.05）.

CONCLUSIONThe survival of newly diagnosed MDS patients with positive p15(INK4B) methylation was comparatively shorter, but p15(INK4B) methylation had no influence on response to decitabine.

Adult ; Aged ; Azacitidine ; analogs & derivatives ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; drug therapy ; genetics ; Prognosis ; Survival Rate ; Treatment Outcome