NLRC4 methylation and its response to intravenous immunoglobulin therapy in Kawasaki disease: a case control study.

BACKGROUND: Kawasaki disease (KD) is a systemic vasculitis accompanied by many systemic physiological and biochemical changes. Elucidating its molecular mechanisms is crucial for diagnosing and developing effective treatments. NLR Family CARD Domain Containing 4 (NLRC4) encodes the key components of inflammasomes that function as pattern recognition receptors. The purpose of this study was to investigate the potential of NLRC4 methylation as a biomarker for KD. METHODS: In this study, pyrosequencing was utilized to analyze NLRC4 promoter methylation in blood samples from 44 children with initial complete KD and 51 matched healthy controls. Methylation at five CpG sites within the NLRC4 promoter region was evaluated. RESULTS: Compared to controls, NLRC4 methylation significantly decreased in KD patients (CpG1: p = 2.93E-06; CpG2: p = 2.35E-05; CpG3: p = 6.46E-06; CpG4: p = 2.47E-06; CpG5: p = 1.26E-05; average methylation: p = 5.42E-06). These changes were significantly reversed after intravenous immunoglobulin (IVIG) treatment. ROC curve analysis demonstrated remarkable diagnostic capability of mean NLRC4 gene methylation for KD (areas under ROC curve = 0.844, sensitivity = 0.75, p = 9.61E-06, 95% confidence intervals were 0.762-0.926 for mean NLRC4 methylation). In addition, NLRC4 promoter methylation was shown to be significantly negatively correlated with the levels of central granulocyte percentage, age, mean haemoglobin quantity and mean erythrocyte volume. Besides, NLRC4 promoter methylation was positively correlated with lymphocyte percentage, lymphocyte absolute value. CONCLUSIONS: Our work revealed the role of peripheral NLRC4 hypomethylation in KD pathogenesis and IVIG treatment response, could potentially serve as a treatment monitoring biomarker, although its precise functions remain to be elucidated.

Circulating plasma and exosome levels of the miR-320 family as a non-invasive biomarker for methamphetamine use disorder.

The neurobiological mechanism underlying methamphetamine (MA) use disorder was still unclear, and no specific biomarker exists for clinical diagnosis of this disorder. Recent studies have demonstrated that microRNAs (miRNAs) are involved in the pathological process of MA addiction. The purpose of this study was to identify novel miRNAs for the diagnosis biomarkers of MA user disorder. First, members of the miR-320 family, including miR-320a-3p, miR-320b, and miR-320c, were screened and analyzed in the circulating plasma and exosomes by microarray and sequencing. Secondly, plasma miR-320 was quantified by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in eighty-two MA patients and fifty age-gender-matched healthy controls. Meanwhile, we also analyzed exosomal miR-320 expression in thirty-nine MA patients and twenty-one age-matched healthy controls. Furthermore, the diagnostic power was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve. The expression of miR-320 significantly increased in plasma and exosomes of MA patients compared with healthy controls. The AUC of the ROC curves of miR-320 in plasma and exosomes of MA patients were 0.751 and 0.962, respectively. And the sensitivities of miR-320 were 0.900 and 0.846, respectively, whereas the specificities of miR-320 were 0.537 and 0.952, respectively, in plasma and exosomes in MA patients. And the increased plasma miR-320 was positively correlated with cigarette smoking, age of onset, and daily use of MA in MA patients. Finally, cardiovascular disease, synaptic plasticity, and neuroinflammation were predicted to be the target pathways related to miR-320. Taken together, our findings indicated that plasma and exosomal miR-320 might be used as a potential blood-based biomarker for diagnosing MA use disorder.

Differential expression profiling of tRNA-Derived small RNAs and their potential roles in methamphetamine self-administered rats.

Transfer RNA-derived small RNAs (tsRNAs) are a novel class of short, non-coding RNAs that are closely associated with the pathogenesis of various diseases. Accumulating evidence has demonstrated their critical functional roles as regulatory factors in gene expression regulation, protein translation regulation, regulation of various cellular activities, immune mediation, and response to stress. However, the underlying mechanisms by which tRFs & tiRNAs affect methamphetamine-induced pathophysiological processes are largely unknown. In this study, we used a combination of small RNA sequencing, quantitative reverse transcription-polymerase chain reaction (qRT‒PCR), bioinformatics, and luciferase reporter assays to screen the expression profiles and identify the functional roles of tRFs and tiRNAs in the nucleus accumbens (NAc) of methamphetamine self-administration rat models. A total of 461 tRFs & tiRNAs were identified in the NAc of rats after 14 days of methamphetamine self-administration training. Of those, 132 tRFs & tiRNAs were significantly differentially expressed: 59 were significantly upregulated, whereas 73 were significantly downregulated in the rats with methamphetamine self-administration. Decreased expression levels of tiRNA-1-34-Lys-CTT-1 and tRF-1-32-Gly-GCC-2-M2, as well as increased expression levels of tRF-1-16-Ala-TGC-4 in the METH group compared with the saline control were validated by using RT‒PCR. Then, bioinformatic analysis was performed to analyse the possible biological functions of tRFs & tiRNAs in methamphetamine-induced pathogenesis. Furthermore, tRF-1-32-Gly-GCC-2-M2 was identified to target BDNF using the luciferase reporter assay. An altered tsRNA expression pattern was proven, and tRF-1-32-Gly-GCC-2-M2 was shown to be involved in methamphetamine-induced pathophysiologic processes by targeting BDNF. The current study provides new insights for future investigations to explore the mechanisms and therapeutic methods for methamphetamine addiction.

Association of GABA receptor delta subunit gene variations with increased risk of methamphetamine dependence.

OBJECTIVE: Evidence reveals that γ-aminobutyric acid (GABA) receptors are involved in the development of methamphetamine (METH) dependence. The GABA receptor delta subunit gene (GABRD) might be a good candidate gene for METH dependence. In a case-control study, we investigated the association between the single nucleotide polymorphisms (SNPs) in GABRD and METH dependence in a Chinese Han population. METHODS: A total of 300 METH dependent patients and 300 age and sex matched normal control subjects were recruited. Four SNPs (rs13303344, rs4481796, rs2376805, and rs2229110) in GABRD were determined with the TaqMan genotyping assay. The association of the SNPs with METH dependence was assessed. RESULTS: Only the allele frequency of rs2376805 significantly differed between the patients and controls (P = 0.030). The G allele frequency of rs2376805 was higher in the METH dependent group than in the controls (odds ratio = 1.332, 95 % CI: 1.028-1.724). This association was found in females but not in males. In females, the frequencies of genotype and allele at rs2376805 significantly differed between the patients and controls (P = 0.025, 0.022, respectively); the rs2376805 G allele may also be a risk factor for METH dependence (odds ratio = 1.548, 95 % CI: 1.063-2.257). The haplotype ACGT frequency significantly differed between the patients and controls in total subjects (P = 0.008, odds ratio = 1.815, 95 % CI: 1.183-2.782), as well as in females (P = 0.005, odds ratio = 2.702, 95 % CI: 1.313-5.562). In females only, the METH craving score was significantly lower in patients harboring the G allele at rs2376805 than in those harboring the homozygous AA genotype (P = 0.044). CONCLUSION: The preliminary results indicate that GABRD rs2376805 is associated with METH dependence, especially in females.

Association between rs1799971 in the mu opioid receptor gene and methadone maintenance treatment response.

OBJECTIVE: Genetic variations can affect individual response to methadone maintenance treatment (MMT) for heroin addiction. The A118G variant (rs1799971) in the mu opioid receptor gene (OPRM1) is a potential candidate single nucleotide polymorphism (SNP) for personalized MMT. This study determined whether rs1799971 is related to MMT response or dose. METHODS: We recruited 286 MMT patients from a Han Chinese population. The rs1799971 genotype was determined via TaqMan genotyping assay. The genetic effect of this SNP on MMT response or dose was evaluated using logistic regression. A meta-analysis was performed to merge all available data to evaluate the role of rs1799971 in MMT using RevMan 5.3 software. RESULTS: No statistical significance was observed in the association between the OPRM1 rs1799971 and MMT response or dose in our Chinese cohort. Meta-analysis indicated that the OPRM1 A118G variation was not significantly associated with MMT response or dose requirement. CONCLUSION: The results suggest that rs1799971 in OPRM1 might not play a critical role alone in influencing MMT response or dose.

Association study of genetic polymorphisms in GABRD with treatment response and dose in methadone maintenance treatment.

Aim: This study determined if gene variants in the GABA receptor delta subunit (GABRD) are associated with treatment response and dose in methadone maintenance treatment (MMT) for heroin addiction. Materials & methods: A total of 286 MMT patients were recruited and divided into response and nonresponse groups based on retention time in therapy. A total of 177 responders were classified into low dose and high dose subgroups according to the stabilized methadone dose. Four (single nucleotide polymorphisms) SNPs (rs13303344, rs4481796, rs2376805 and rs2229110) in GABRD were genotyped using the TaqMan SNP assay. Logistic regression was used to assess the genetic effects of the SNPs in MMT. Results: No significant associations were observed between the SNPs and treatment response or dose, except the frequency of haplotype ACGC at the four SNPs significantly differed between responders and nonresponders. Conclusion: The results indicated that GABRD variants may play a small role in modulating methadone treatment response.

Lay abstract This study determined if gene variants in the GABA receptor delta subunit (GABRD) are associated with treatment response and dose in methadone maintenance treatment (MMT) for heroin addiction. A total of 286 MMT patients were recruited and divided into response and nonresponse groups. A total of 177 responders were classified into low and high dose subgroups. Four single nucleotide polymorphisms (SNPs) (rs13303344, rs4481796, rs2376805 and rs2229110) in GABRD were genotyped and assessed the genetic effects of the SNPs in MMT. No significant associations were observed between the SNPs and treatment response or dose, except the frequency of haplotype ACGC significantly differed between responders and nonresponders. The results indicated that GABRD variants may play a small role in MMT, which may help provide a foundation for personalized solutions for MMT.

Role of nucleus accumbens microRNA-181a and MeCP2 in incubation of heroin craving in male rats.

RATIONALE: Epigenetic regulation has been implicated in the incubation of drug craving (the time-dependent increase in drug seeking after prolonged withdrawal from drug self-administration). There is little information available on the role of microRNAs in incubation of heroin craving. OBJECTIVE: This study aimed to investigate the roles and mechanisms of miR-181a and methyl CpG binding protein 2 (MeCP2) in the nucleus accumbens (NAc) in incubation of heroin seeking. METHODS: MiRNA sequencing was used to predict potential miRNAs, and miRNA profiles were performed in the NAc after 1 day or 14 days after withdrawal from heroin self-administration. Following 14 days of heroin self-administration, rats were injected of lentiviral vectors into the NAc and evaluated for the effects of overexpression of miR-181a or knockdown of MeCP2 on non-reinforced heroin seeking after 14 withdrawal days. RESULTS: Lever presses during the heroin-seeking tests were higher after 14 withdrawal days than after 1 day (incubation of heroin craving). miR-181a expression in NAc was lower after 14 withdrawal days than after 1 day, and meCP2 expression in NAc was higher after 14 days than after 1 day. Luciferase activity assay showed that the 3'UTR of MeCP2 is directly regulated by miR-181a. Overexpression of miR-181a in NAc decreased heroin seeking after 14 withdrawal days and decreased MeCP2 mRNA and protein expression. Knockdown of MeCP2 expression in NAc by LV-siRNA-MeCP2 also decreased heroin seeking after 14 withdrawal days. CONCLUSIONS: Results indicate that incubation of heroin craving is mediated in part by time-dependent decreases in NAc miR181a expression that leads to time-dependent increases in MeCP2 expression. Our data suggest that NAc miR-181a and MeCP2 contribute to incubation of heroin craving.

Association between GABA receptor delta subunit gene polymorphisms and heroin addiction.

Evidence suggests that γ-aminobutyric acid (GABA) receptors are involved in the development of drug dependence. Considering its exclusively extrasynaptic localization, GABA receptor delta subunit (GABRD) is likely involved in heroin addiction. The purpose of this study was to explore the association between the single nucleotide polymorphisms (SNPs) of GABRD and heroin addiction. Genotyping of five SNPs (rs13303344, rs4481796, rs2376805, rs2229110, and rs41307846) in GABRD gene was performed by using TaqMan SNP assay. The association between heroin addiction and these SNPs was assessed in 446 heroin dependent patients and 400 normal control subjects of male Han Chinese origin. Only the genotype and allele frequencies at rs13303344 differed significantly between the cases and controls (nominal P values were 0.028 and 0.019, respectively). The C allele of rs13303344 was associated with an increased risk of heroin addiction (OR = 1.281, 95 % CI: 1.042-1.575). After Bonferroni correction, the association lost significance. The frequencies of the haplotype C-C-A and A-C-A at GARBD (rs13303344-rs4481796- rs2376805) differed significantly between the cases and controls. The heroin craving score was significantly higher in patients with CC/AC genotypes at rs13303344 than in those with the AA genotype (nominal P = 0.017). The results suggest that GABRD rs13303344 may contribute to the susceptibility to heroin addiction and is associated with the drug cravings of heroin dependent patients.

Akt and its phosphorylation in nucleus accumbens mediate heroin-seeking behavior induced by cues in rats.

Akt is initially identified as one of the downstream targets of phosphatidylinositol-3 kinase (PI3K) and is involved in morphine reward and tolerance. However, whether phospholyration of Akt (p-Akt) mediates heroin relapse remains unclear. Here, we aimed to explore the role of p-Akt in the nucleus accumbens (NAc) in cue-induced heroin-seeking behaviors after withdrawal. First, rats were trained to self-administer heroin for 14 days, after which we assessed heroin-seeking behaviors induced by a context cue (CC) or by discrete conditioned cues (CS) after 1 day or 14 days of withdrawal. We found that the active responses induced by CC or CS after 14 days of withdrawal were higher than those after 1 day of withdrawal. Meanwhile, the expression of p-Akt in the NAc was also greatest when rats were exposed to the CS after 14 days of withdrawal. Additionally, a microinjection of LY294002, an inhibitor of PI3K, into the NAc inhibited the CS-induced heroin-seeking behaviors after 14 days of withdrawal, paralleling the decreased levels of p-Akt in the NAc. Finally, Akt1 or ß-arrestin 2 was downregulated via a lentiviral injection to assess the effect on heroin seeking after 14 days of withdrawal. CS-induced heroin-seeking behavior was inhibited by downregulation of Akt1, but not ß-arrestin 2, in the NAc. These data demonstrate that Akt phosphorylation in the NAc may play an important role in the incubation of heroin-seeking behavior, suggesting that the PI3K/Akt pathways may be involved in the process of heroin relapse and addiction.

Somatically acquired mutations in primary myelofibrosis: A case report and meta-analysis.

Familial myeloproliferative disease (MPD) cases account for 7.6% of the global MPD cases. The present study reported 2 cases of primary myelofibrosis (PMF). The patients were two sisters; the older sister succumbed to the disease at the age of 37, whereas the younger sister maintained a stable disease status and gave birth to a son through in vitro fertilization. Genetic analysis of bone marrow DNA samples showed that both sisters carried a Janus kinase 2 (JAK2) V617F mutation, and the older sister also had a trisomy 8 chromosomal abnormality (47, XX, +8). A systematic literature search was also performed using PubMed, CNKI and Wanfang databases, to determine the association between JAK2 and PMF. Following comprehensive screening of the published literature, 19 studies were found to be eligible for the current meta-analysis. The results showed that JAK2 V617F was a risk factor of PMF, and no sex dimorphism was observed in JAK2 V617F mutation prevalence amongst all PMF cases. In addition, there was a lack of association between the JAK2 V617F mutation and PMF-related mortality.

Increased expression of plasma hsa-miR-181a in male patients with heroin addiction use disorder.

BACKGROUND: Drug addiction is an uncontrolled, chronic, and recurrent encephalopathy that presently lacks specific and characteristic biomarkers for diagnosis and treatment. As regulators of gene expression, microRNAs (miRNAs) are increasingly used for diagnostic and prognostic purposes in various disease states. Previous studies indicated that miRNAs play important roles in the development and progression of drug addictions, including addiction to methamphetamine, cocaine, alcohol, and heroin. METHODS: We identified significant miRNAs using the microarray method and then validated the hsa-miR-181a expression levels in 53 heroin addiction patients and 49 normal controls using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Finally, the potential associations between transcriptional levels in heroin addiction patients and their clinicopathological features were analyzed. RESULTS: A total of 2006 miRNAs were differentially expressed between heroin addiction patients and normal controls. The top 10 up-regulated miRNAs in patients were hsa-miR-21a, hsa-miR-181a, hsa-miR-4459, hsa-miR-4430, hsa-miR-4306, hsa-miR-22-3P, hsa-miR-486-5P, hsa-miR-371b-5P, hsa-miR-92a-3P, and hsa-miR-5001-5P. The top 10 down-regulated miRNAs in patients were hsa-miR-3195, hsa-miR-4767, hsa-miR-3135b, hsa-miR-6087, hsa-miR-1181, hsa-miR-4785, hsa-miR-718, hsa-miR-3141, hsa-miR-652-5P, and hsa-miR-6126. The expression level of hsa-miR-181a in heroin addiction patients was significantly increased compared with that in normal controls (P < .001). The area under the receiver operating characteristic curve of hsa-miR-181a was 0.783, the sensitivity was 0.867, and the specificity was 0.551. CONCLUSIONS: The increased expression of hsa-miR-181a in the plasma of heroin patients may be a consequence of the pathological process of heroin abuse. This study highlights the potential of hsa-miR-181a as a novel biomarker for the diagnosis of heroin addiction.

Histone 3 lysine 9 acetylation of BRG1 in the medial prefrontal cortex is associated with heroin self­administration in rats.

Heroin addiction is a chronic relapsing brain disorder with negative social consequences. Histone acetylation serves a role in drug­induced behavior and neuroplasticity impairment. Brahma/SWI2­related gene­1 (BRG1) participates in cerebellar development, embryogenesis and transcriptional regulation of neuronal genes concurrent with histone modifications. However, little is known about the relationship between histone H3 lysine 9 acetylation (H3K9ac) and BRG1 in response to heroin. The present study aimed to assess the contribution of histone 3 lysine 9 acetylation of BRG1 to heroin self­administration. The present study established a Sprague­Dawley rat model of heroin self­administration under a fixed­ratio­1 paradigm. Chromatin immunoprecipitation followed by reverse transcription­quantitative PCR (RT­qPCR) was used to detect the accumulation of H3K9ac on BRG1 in the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc) following heroin self­administration. The relative expression levels of BRG1 were analyzed by RT­qPCR. H3K9ac at the promoter region of BRG1 was significantly elevated (P=0.002), and the expression of BRG1 in the mPFC increased 1.47­fold in the heroin self­administration group compared with the control group. No significant difference in H3K9ac at the BRG1 locus was observed in the NAc (P=0.323), with the expression of BRG1 decreasing 1.38­fold in the heroin self­administering rats compared with the control group. H3K9ac is associated with transcriptional activation, and the increased BRG1 expression suggested an essential and novel role for BRG1 and its H3K9ac­mediated regulation in the mPFC after heroin self­administration; and this may function through epigenetically modulating the activation of neuroplasticity­associated genes. This association may provide a novel therapeutic target for the treatment of heroin addiction.

Role of GABRD Gene Methylation in the Nucleus Accumbens in Heroin-Seeking Behavior in Rats.

Epigenetic modifications such as DNA methylation play important roles in regulating gene expression and may mediate neuroplasticity and lead to drug-induced aberrant behaviors. Although several brain regions and neurobiological mechanisms have been suggested to be involved in these processes, there is remarkably little known about the effects of DNA methylation on heroin-seeking behavior. Using a Sprague-Dawley rat model, we show that heroin self-administration resulted in gamma-aminobutyric acid type A receptor subunit delta (GABRD) gene hypomethylation, which was associated with transcriptional upregulation of GABRD in the nucleus accumbens (NAc). Systemic l-methionine (MET) administration significantly strengthened the reinstatement of heroin-seeking behavior induced by heroin priming, whereas intra-NAc injections of the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (5-Aza-dC) had the opposite effect on heroin-seeking. Meanwhile, 5-Aza-dC treatment decreased DNA methylation and upregulated the expression of GABRD in the NAc, whereas MET had the opposite effect. Our results also reveal that 5-Aza-dC might alter the methylation landscape of the GABRD gene by directly repressing DNMT1 and DNMT3A expression. Furthermore, reinstatement of heroin-seeking behavior was significantly inhibited by directly overexpressing GABRD and remarkably reinforced by GABRD gene silencing in the NAc. Collectively, these results suggest that targeting the GABRD gene and its methylation might represent a novel pharmacological strategy for treating heroin addiction and relapse.

Hypermethylation of protocadherin γ subfamily A12 and solute carrier family 19 A 1 promoters contributes to the occurrence and metastasis of colorectal cancer.

The development of colorectal cancer (CRC) involves genetic and epigenetic modifications, and aberrant DNA methylation within gene promoters is a primary mediator of epigenetic inheritance in CRC. The present study evaluated whether promoter methylation of four CRC candidate genes [protocadherin γ subfamily A12 (PCDH-γ-A12), solute carrier family 19 A 1 (SLC19A1), cAMP responsive element binding protein (CREB) and cylindromatosis (CYLD) contributed to the risk and metastasis of CRC by screening a total of 42 CRC and 42 adjacent normal tissue samples. DNA methylation was measured by methylation-specific polymerase chain reaction (MSP). Polymerase chain reaction (PCR) products were bisulfite converted and validated by sequencing. The χ2 test was employed to assess the association between promoter methylation and a series of clinicopathological characteristics. The promoters of PCDH-γ-A12 and SLC19A1 were observed to be more frequently methylated in CRC tissues than normal tissues. In addition, significantly higher methylation of the PCDH-γ-A12 and SLC19A1 promoters was also observed in CRC tissues with lymph metastasis compared with those without lymph metastasis. In addition, no association was observed between CREB and CYLD methylation and the occurrence and metastasis of CRC. These results suggest that the hypermethylation of the PCDH-γ-A12 and SLC19A1 promoters may contribute to the occurrence and metastasis of CRC in the Han Chinese population.

AGTR1 promoter hypermethylation in lung squamous cell carcinoma but not in lung adenocarcinoma.

Aberrant DNA methylation is associated with non-small cell lung cancer (NSCLC), suggesting that gene promoter methylation may be a potential biomarker for the detection or risk prediction of NSCLC. The present study aimed to evaluate the potential usage of angiotensin II receptor type 1 (AGTR1) methylation in two major pathologic subtypes: Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Quantitative methylation-specific polymerase chain reaction was used to investigate the effect of AGTR1 promoter methylation in the tumor and the paired adjacent non-tumor tissue samples from 42 patients with LUSC, and 69 with LUAD. The percentage of methylated reference was calculated and presented as the median (interquartile range 25th-75th percentile). The results of the current study revealed that there was significantly increased AGTR1 promoter methylation in the tumor tissues compared with the paired adjacent non-tumor tissue [97.4 (57.22-130.5) vs. 85 (48.25-123); P=0.024]. Furthermore, higher AGTR1 promoter methylation was observed in patients with LUSC compared with LUAD (odds ratio=2.483; 95% confidence interval=1.125-5.480; P=0.023). Significant differences were identified in AGTR1 methylation between non-tumor and the tumor tissues in LUSC [113.5 (68.33-148.73) vs. 93.04 (45.94-140); P=0.008]. In addition, the Cancer Genome Atlas data of 378 patients with LUSC and 477 with LUAD revealed an inverse correlation between gene expression and the methylation status of AGTR1 promoter.. These data suggest that AGTR1 hypermethylation is a promising biomarker to assist in LUSC detection and diagnosis.

Chemotherapy-induced hypomethylation of N-myc downstream-regulated gene 4 in the bone marrow of patients with acute myeloid leukemia.

N-myc downstream-regulated gene 4 (NDRG4) has previously been investigated as a possible tumor suppressor. Hypermethylation of tumor suppressor genes contributes to the occurrence and development of certain types of cancer, including acute myeloid leukemia (AML). The current study aimed to assess the contribution of chemotherapy-induced NDRG4 changeable methylation to the development of AML. A total of 30 patients (13 males and 17 females) were involved in the present study. The DNA methylation levels of five C-phosphate-G sites of the NDRG4 gene were measured using bisulfite pyrosequencing techniques. The results indicated significantly reduced gene-body methylation levels of NDRG4 during chemotherapy (prior to chemotherapy: 9.35±4.22%; following chemotherapy: 7.54±3.11%; P=0.030). Further analysis of AML subtypes revealed the methylation reductions were principally contributed by patients with M2 subtype AML (prior to chemotherapy: 9.91±4.76%; following chemotherapy: 5.26±1.16%; P=0.038). A significant association was also observed between the patient age and the altered levels of NDRG4 gene-body methylation in patients with M2 subtype AML (r=0.761; P=0.047), suggesting that reductions in induced-methylation may be age-dependent in patients with M2 subtype AML during chemotherapy. Therefore, age may affect the induced methylation levels of NDRG4 gene-body in patients with AML (particularly patients with M2 subtype AML) during chemotherapy.

Prognostic value of MLH1 promoter methylation in male patients with esophageal squamous cell carcinoma.

The DNA mismatch repair (MMR) gene MutL homolog 1 (MLH1) is critical for the maintenance of genomic integrity. Methylation of the MLH1 gene promoter was identified as a prognostic marker for numerous types of cancer including glioblastoma, colorectal, ovarian and gastric cancer. The present study aimed to determine whether MLH1 promoter methylation was associated with survival in male patients with esophageal squamous cell carcinoma (ESCC). Formalin-fixed, paraffin-embedded ESCC tissues were collected from 87 male patients. MLH1 promoter methylation was assessed using the methylation-specific polymerase chain reaction approach. Kaplan-Meier survival curves and log-rank tests were used to evaluate the association between MLH1 promoter methylation and overall survival (OS) in patients with ESCC. Cox regression analysis was used to obtain crude and multivariate hazard ratios (HR), and 95% confidence intervals (CI). The present study revealed that MLH1 promoter methylation was observed in 53/87 (60.9%) of male patients with ESCC. Kaplan-Meier survival analysis demonstrated that MLH1 promoter hypermethylation was significantly associated with poorer prognosis in patients with ESCC (P=0.048). Multivariate survival analysis revealed that MLH1 promoter hypermethylation was an independent predictor of poor OS in male patients with ESCC (HR=1.716; 95% CI=1.008-2.921). Therefore, MLH1 promoter hypermethylation may be a predictor of prognosis in male patients with ESCC.

Elevated OPRD1 promoter methylation in Alzheimer's disease patients.

Aberrant DNA methylation has been observed in the patients with Alzheimer's disease (AD), a common neurodegenerative disorder in the elderly. OPRD1 encodes the delta opioid receptor, a member of the opioid family of G-protein-coupled receptors. In the current study, we compare the DNA methylation levels of OPRD1 promoter CpG sites (CpG1, CpG2, and CpG3) between 51 AD cases and 63 controls using the bisulfite pyrosequencing technology. Our results show that significantly higher CpG3 methylation is found in AD cases than controls. Significant associations are found between several biochemical parameters (including HDL-C and ALP) and CpG3 methylation. Subsequent luciferase reporter gene assay shows that DNA fragment containing the three OPRD1 promoter CpGs is able to regulate gene expression. In summary, our results suggest that OPRD1 promoter hypermethylation is associated with the risk of AD.

APC2 and CYP1B1 methylation changes in the bone marrow of acute myeloid leukemia patients during chemotherapy.

Aberrant promoter DNA methylation is a major mechanism of leukemogenesis in hematologic malignancies, including acute myeloid leukemia (AML). However, the association between promoter methylation with chemotherapeutic outcomes remains unknown. In the present study, bone marrow samples were collected prior to and following chemotherapy in 30 AML patients. Methylation-specific polymerase chain reaction technology was used to examine the promoter methylation status of adenomatous polyposis col 2 (APC2) and cytochrome P450 family 1 subfamily B polypeptide 1 (CYP1B1). The results revealed no change in the methylation status of the APC2 promoter in patients following various chemotherapy regimens. However, the methylation status of the CYP1B1 promoter changed in response to 6 different chemotherapy regimens. AML patients of the M3 subtype displayed an induction of the CYP1B1 promoter methylation levels more frequently (57.1%) than patients affected by the other subtypes (M1: 33.3%; M2: 12.5%; M4: 16.7%; M5: 0% and M6: 0%). In addition, a higher frequency of male patients (4/13) exhibited modulation of the CYP1B1 promoter methylation status compared with female patients (3/17). Furthermore, of five AML patients with a poor prognosis, two exhibited changes leading to CYP1B1 hypomethylation and two leading to CYP1B1 hypermethylation. By contrast, three other patients exhibited hypermethylation changes along with remission. This may be explained by the different chemotherapy regimens used to treat these patients or by other unknown factors. The present study revealed that CYP1B1 promoter methylation was induced during chemotherapy, whereas the APC2 promoter remained hemimethylated. Furthermore, the changes in CYP1B1 methylation were dependent on the AML subtypes and the gender of the patients.

H4K5 histone acetylation of BRG1 is associated with heroin administration rather than addiction.

Diacetylmorphine hydrochloride (heroin) addiction is a chronic relapsing brain disorder that is a heavy public health burden worldwide. Brm/SWI2-related gene-1 (BRG1) is a tumor suppressor gene that can influence embryogenesis and the development of the cerebellum. The current study aimed to investigate the effect of histone H4 lysine 5 (H4K5) modifications on the BRG1 gene in brain tissue of the ventral tegmental area (VTA) of heroin-addicted rats. A total of 21 male Sprague Dawley rats were raised in a standard manner and underwent heroin self-administration training. Rats were randomly divided into three equal groups: Group A, self-administered delivery of heroin; group B, yoked delivery of heroin; and group C, yoked delivery of saline. The VTA was harvested and subjected to chromatin immunoprecipitation (ChIP) analysis. Gene expression was evaluated by quantitative polymerase chain reaction. We calculated the recovery rate, which indicated the percentage of the total input BRG1 recovered by ChIP. Our results showed that BRG1 was less associated with H4K5 histone modification in the group of rats that underwent heroin self-administration than in the other two groups (A vs. B, P=0.031; A vs. C, P=0.067). The recovery fold changes of the self-administration group and the passive-administration group were significantly different from those of the group with yoked saline (A vs. C, P=0.013; B vs. C, P=0.009; A vs. B, P=0.731). The results of the current study demonstrated that H4K5 histone acetylation of BRG1 in the VTA may be associated with heroin administration, but not addiction.