Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Unveiling DNA methylation in Alzheimer’s disease:a review of array-based human brain studies

The intricacies of Alzheimer’s disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms,particularly DNA methylation.This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer’s disease neuropathology.The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer’s disease progression.The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus.Notably,ANK1 hypermethylation,a protein implicated in neurofibrillary tangle formation,was recurrently identified in the entorhinal cortex.Further,the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3,RHBDF2,and MCF2L,potentially influencing neuroinflammatory processes.The complex role of BIN1 in late-onset Alzheimer’s disease is underscored by its association with altered methylation patterns.Despite the disparities across studies,these findings highlight the intricate interplay between epigenetic modifications and Alzheimer’s disease pathology.Future research efforts should address methodological variations,incorporate diverse cohorts,and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer’s disease progression.

Comparative DNA methylation reveals epigenetic adaptation to high altitude in snub-nosed monkeys

DNA methylation plays a crucial role in environmental adaptations.Here,using whole-genome bisulfite sequencing,we generated comprehensive genome-wide DNA methylation profiles for the high-altitude Yunnan snub-nosed monkey(Rhinopithecus bieti)and the closely related golden snub-nosed monkey(R.roxellana).Our findings indicated a slight increase in overall DNA methylation levels in golden snub-nosed monkeys compared to Yunnan snub-nosed monkeys,suggesting a higher prevalence of hypermethylated genomic regions in the former.Comparative genomic methylation analysis demonstrated that genes associated with differentially methylated regions were involved in membrane fusion,vesicular formation and trafficking,hemoglobin function,cell cycle regulation,and neuronal differentiation.These results suggest that the high-altitude-related epigenetic modifications are extensive,involving a complete adaptation process from the inhibition of single Ca^(2+)channel proteins to multiple proteins collaboratively enhancing vesicular function or inhibiting cell differentiation and proliferation.Functional assays demonstrated that overexpression or down-regulation of candidate genes,such as SNX10,TIMELESS,and CACYBP,influenced cell viability under stress conditions.Overall,this research suggests that comparing DNA methylation across closely related species can identify novel candidate genomic regions and genes associated with local adaptations,thereby deepening our understanding of the mechanisms underlying environmental adaptations.

Effectiveness of fecal DNA syndecan-2 methylation testing for detection of colorectal cancer in a high-risk Chinese population

BACKGROUND Colorectal cancer(CRC)is among the most prevalent and life-threatening malignancies worldwide.Syndecan-2 methylation(mSDC2)testing has emerged as a widely used biomarker for early detection of CRC in stool and serum samples.Cancer(CRC)is among the most prevalent and life-threatening malignancies worldwide.mSDC2 testing has emerged as a widely used biomarker for early detection of CRC in stool and serum samples.AIM To validate the effectiveness of fecal DNA mSDC2 testing in the detection of CRC among a high-risk Chinese population to provide evidence-based data for the development of diagnostic and/or screening guidelines for CRC in China.METHODS A high-risk Chinese cohort consisting of 1130 individuals aged 40-79 years was selected for evaluation via fecal mSDC2 testing.Sensitivity and specificity for CRC,advanced adenoma(AA)and advanced colorectal neoplasia(ACN)were determined.High-risk factors for the incidence of colorectal lesions were determined and a logistic regression model was constructed to reflect the efficacy of the test.RESULTS A total of 1035 high-risk individuals were included in this study according to established criteria.Among them,16 suffered from CRC(1.55%),65 from AA(6.28%)and 189 from non-AAs(18.26%);150 patients were diagnosed with polyps(14.49%).Diagnoses were established based upon colonoscopic and pathological examinations.Sensitivities of the mSDC2 test for CRC and AA were 87.50%and 40.00%,respectively;specificities were 95.61%for other groups.Positive predictive values of the mSDC2 test for CRC,AA and ACN were 16.09%,29.89%and 45.98%,respectively;the negative predictive value for CRC was 99.79%.After adjusting for other high-risk covariates,mSDC2 test positivity was found to be a significant risk factor for the occurrence of ACN(P<0.001).CONCLUSION Our findings confirmed that offering fecal mSDC2 testing and colonoscopy in combination for CRC screening is effective for earlier detection of malignant colorectal lesions in a high-risk Chinese population.

Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state

Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

DNA methylation in poultry:a review

As an important epigenetic modification,DNA methylation is involved in many biological processes such as animal cell differentiation,embryonic development,genomic imprinting and sex chromosome inactivation.As DNA methylation sequencing becomes more sophisticated,it becomes possible to use it to solve more zoological problems.This paper reviews the characteristics of DNA methylation,with emphasis on the research and application of DNA methylation in poultry.

Rice melatonin deficiency causes premature leaf senescence via DNA methylation regulation

In a study of DNA methylation changes in melatonin-deficient rice mutants,mutant plants showed premature leaf senescence during grain-filling and reduced grain yield.Melatonin deficiency led to transcriptional reprogramming,especially of genes involved in chlorophyll and carbon metabolism,redox regulation,and transcriptional regulation,during dark-induced leaf senescence.Hypomethylation of mCG and mCHG in the melatonin-deficient rice mutants was associated with the expression change of both protein-coding genes and transposable element-related genes.Changes in gene expression and DNA methylation in the melatonin-deficient mutants were compensated by exogenous application of melatonin.A decreased S-adenosyl-L-methionine level may have contributed to the DNA methylation variations in rice mutants of melatonin deficiency under dark conditions.

Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

Hypomethylation of glycine dehydrogenase promoter in peripheral blood mononuclear cells is a new diagnostic marker of hepatitis B virus-associated hepatocellular carcinoma

Background: Glycine dehydrogenase(GLDC) plays an important role in the initiation and proliferation of several human cancers. In this study, we aimed to detect the methylation status of GLDC promoter and its diagnostic value for hepatitis B virus-associated hepatocellular carcinoma(HBV-HCC). Methods: We enrolled 197 patients, 111 with HBV-HCC, 51 with chronic hepatitis B(CHB), and 35 healthy controls(HCs). The methylation status of GLDC promoter in peripheral mononuclear cells(PBMCs) was identified by methylation specific polymerase chain reaction(MSP). The mRNA expression was examined using real-time quantitative polymerase chain reaction(q PCR). Results: The methylation frequency of the GLDC promoter was significantly lower in HBV-HCC patients(27.0%) compared to that in CHB patients(68.6%) and HCs(74.3%)( P < 0.001). The methylated group had lower alanine aminotransferase level( P = 0.035) and lower rates of tumor node metastasis(TNM) Ⅲ/Ⅳ( P = 0.043) and T3/T4( P = 0.026). TNM stage was identified to be an independent factor for GLDC promoter methylation. GLDC mRNA levels in CHB patients and HCs were significantly lower than those in HBV-HCC patients( P = 0.022 and P < 0.001, respectively). GLDC mRNA levels were significantly higher in HBV-HCC patients with unmethylated GLDC promoters than those with methylated GLDC promoters( P = 0.003). The diagnostic accuracy of alpha-fetoprotein(AFP) combined with GLDC promoter methylation for HBV-HCC was improved compared with that of AFP alone(AUC: 0.782 vs. 0.630, P < 0.001). In addition, GLDC promoter methylation was an independent predictor for overall survival of HBV-HCC patients( P = 0.038). Conclusions: The methylation frequency of GLDC promoter was lower in PBMCs from HBV-HCC patients than that from patients with CHB and HCs. The combination of AFP and GLDC promoter hypomethylation significantly improved the diagnostic accuracy of HBV-HCC.

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis:Perspective of Different Water Iodine Exposure

Objective This study aimed to identify differentially methylated genes(DMGs) associated with natural killer cells in patients with autoimmune thyroiditis(AIT), focusing on the influence of varying water iodine exposure levels.Methods Participants were divided into categories based on median water iodine(MWI)concentrations: iodine-fortified areas(IFA, MWI < 10 μg/L), iodine-adequate areas(IAA, 40 ≤ MWI ≤ 100μg/L), and iodine-excessive areas(IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K Bead Chip analysis for 10/10 paired samples. Validation of DNA methylation and m RNA expression levels of the DMGs was conducted using Methyl Target^(TM) and QRT-PCR for 176/176paired samples.Results KLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA.Conclusion The DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

miR-214-5p通过DNMT1介导的AXIN2基因DNA甲基化修饰在皮肤基底细胞癌中的作用机制

目的探讨皮肤基底细胞癌中轴抑制蛋白2(Axis inhibition protein 2,AXIN2)基因启动子甲基化对基因转录的影响及miR-214-5p通过靶向DNA甲基转移酶1(DNA methyltransferase1,DNMT1)对AXIN2甲基化率的调控机制。方法收集2022年1月-2023年6月在广州市皮肤病防治所就诊治疗的102例皮肤基底细胞癌(Cutaneous basal cell carcinoma,BCC)患者作为研究对象,提取癌组织和癌旁正常组织标本及基线资料。焦磷酸测序法检测AXIN2基因启动子区甲基化率。实时荧光定量PCR检测AXIN2、DNMT1基因mRNA和miR-214-5p的表达水平。将miR-214-5p模拟物(mimic)、抑制物(inhibitor)及其阴性对照(mimic NC和inhibitor NC)分别对基底细胞癌A431细胞进行转染,48 h后检测DNMT1基因mRNA表达水平和AXIN2基因甲基化率。结果BCC癌组织的AXIN2基因甲基化率显著高于癌旁正常组织(t=5.128,P<0.001),AXIN2基因mRNA相对表达水平显著低于癌旁正常组织(t=7.826,P<0.001),DNMT1基因mRNA表达水平显著高于癌旁正常组织(t=4.838,P<0.001),miR-214-5p表达水平显著低于癌旁正常组织(t=5.426,P<0.001)。BCC癌组织的AXIN2基因甲基化率与其mRNA表达水平呈负相关(r=-0.793,P<0.001),DNMT1基因mRNA水平与AXIN2基因甲基化率呈正相关(r=0.814,P<0.001),miR-214-5p表达水平与DNMT1基因mRNA水平呈负相关(r=-0.747,P<0.001)。双荧光素酶报告基因实验结果证实,DNMT1是miR-214-5p的靶基因。细胞转染后,与mimic NC、inhibitor和inhibitor NC比较,mimic的DNMT1基因mRNA水平、AXIN2基因甲基化率显著降低(P<0.001);而inhibitor的DNMT1基因mRNA水平和AXIN2基因甲基化率相较于其他三组明显上升(P<0.001)。结论miR-214-5p可通过调控下游靶蛋白DNMT1表达,影响AXIN2基因的DNA甲基化率,调控AXIN2基因的表达水平,参与皮肤基底细胞癌的发生机制。

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

A Metaheuristic Technique for Cluster-Based Feature Selection of DNA Methylation Data for Cancer

Epigenetics is the study of phenotypic variations that do not alter DNA sequences.Cancer epigenetics has grown rapidly over the past few years as epigenetic alterations exist in all human cancers.One of these alterations is DNA methylation;an epigenetic process that regulates gene expression and often occurs at tumor suppressor gene loci in cancer.Therefore,studying this methylation process may shed light on different gene functions that cannot otherwise be interpreted using the changes that occur in DNA sequences.Currently,microarray technologies;such as Illumina Infinium BeadChip assays;are used to study DNA methylation at an extremely large number of varying loci.At each DNA methylation site,a beta value(β)is used to reflect the methylation intensity.Therefore,clustering this data from various types of cancers may lead to the discovery of large partitions that can help objectively classify different types of cancers aswell as identify the relevant loci without user bias.This study proposed a Nested Big Data Clustering Genetic Algorithm(NBDC-GA);a novel evolutionary metaheuristic technique that can perform cluster-based feature selection based on the DNA methylation sites.The efficacy of the NBDC-GA was tested using real-world data sets retrieved from The Cancer Genome Atlas(TCGA);a cancer genomics program created by the NationalCancer Institute(NCI)and the NationalHuman Genome Research Institute.The performance of the NBDC-GA was then compared with that of a recently developed metaheuristic Immuno-Genetic Algorithm(IGA)that was tested using the same data sets.The NBDC-GA outperformed the IGA in terms of convergence performance.Furthermore,the NBDC-GA produced a more robust clustering configuration while simultaneously decreasing the dimensionality of features to a maximumof 67%and of 94.5%for individual cancer type and collective cancer,respectively.The proposed NBDC-GA was also able to identify two chromosomes with highly contrastingDNAmethylations activities that were previously linked to cancer.

Comparative detection of syndecan-2 methylation in preoperative and postoperative stool DNA in patients with colorectal cancer

BACKGROUND Early detection of colorectal cancer(CRC)is essential to reduce cancer-related morbidity and mortality.Stool DNA(sDNA)testing is an emerging method for early CRC detection.Syndecan-2(SDC2)methylation is a potential biomarker for the sDNA testing.Aberrant DNA methylation is an early epigenetic event during tumorigenesis and can occur in the normal colonic mucosa during aging,which can compromise the sDNA test results.METHODS In this prospective study,we enrolled 151 patients with CRC who underwent curative surgical resection between September 2016 and May 2020.Preoperative stool samples were collected from 123 patients and postoperative samples were collected from 122 patients.A total of 104 samples were collected from both preoperative and postoperative patients.Aberrant promoter methylation of SDC2 in sDNA was assessed using linear target enrichment quantitative methylation-specific real-time polymerase chain reaction.Clinicopathological parameters were analyzed using the results of SDC2 methylation.RESULTS Detection rates of SDC2 methylation in the preoperative and postoperative stool samples were 88.6%and 19.7%,respectively.Large tumor size(3 cm,P=0.019)and advanced T stage(T3–T4,P=0.033)were positively associated with the detection rate of SDC2 methylation before surgery.Female sex was associated with false positives after surgery(P=0.030).Cycle threshold(CT)values were significantly decreased postoperatively compared with preoperative values(P<0.001).The postoperative negative conversion rate for preoperatively methylated SDC2 was 79.3%(73/92).CONCLUSION Our results suggested that the SDC2 methylation test for sDNA has acceptable sensitivity and specificity.However,small size and early T stage tumors are associated with a low detection rate of SDC2 methylation.As the cycle threshold values significantly decreased after surgery,SDC2 methylation test for sDNA might have a diagnostic value for CRC.

血清CYFRA21-1、CA125联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征研究

目的分析血清细胞角蛋白19片段(CYFRA21-1)、糖类抗原125(CA125)联合HPV DNA检测在宫颈癌早期筛查中的价值及病理特征。方法选取2019年8月至2023年6月沧州市中心医院收治的宫颈癌患者152例为观察组,另选取同期在本院行体检且各项正常女性80名为对照组;对比两组血清CYFRA21-1、CA125水平以及HPV DNA检测结果;对比观察组不同手术病理结果及血清CYFRA21-1、CA125表达水平以及HPV DNA检测结果;以病理学检查为金标准,分析血清CYFRA21-1、CA125水平以及HPV DNA检测单独以及联合诊断宫颈癌的一致性;绘制ROC曲线,分析血清CYFRA21-1、CA125联合HPV DNA单独检测及联合检测宫颈癌的效能。结果152例患者中,鳞状细胞癌104例,腺癌患者48例;其中轻度不典型增生66例、中度不典型增生57例、重度不典型增生29例。观察组血清CYFRA21-1、CA125水平以及HPV DNA阳性率均高于对照组,差异有统计学意义(P<0.05);血清CYFRA21-1、CA125水平:鳞状细胞癌<腺癌,轻度不典型增生<中度不典型增生<重度不典型增生,差异均有统计学意义(P<0.05)。观察组不同分期、不同肿瘤增生类型的HPV DNA阳性率比较,差异无统计学意义(P>0.05);血清CYFRA21-1、CA125水平、HPV DNA检测单独以及联合诊断宫颈癌与病理学检查结果的一致性Kappa值分别为0.677、0.731、0.756、0.902;CA125+CYFRA21-1+HPV DNA联合检测的AUC为0.894,高于CA125、CYFRA21-1、HPV DNA单独检测(P>0.05)。结论血清CYFRA21-1、CA125联合HPV DNA检测的诊断效能高于单一检测,提示三指标联合检测可显著提高宫颈癌早期筛查的诊断价值,可为制定临床治疗方案提供参考资料。

华裔科学家陈志坚发现感知DNA免疫监控的cGAS-STING信号通路——2024年拉斯克奖基础医学研究奖

北京时间2024年9月19日,生物医学领域重要奖项拉斯克奖(The Lasker Award)揭晓,华人学者陈志坚博士(德克萨斯大学西南医学中心)因发现感知自身及外源DNA的环鸟嘌呤-腺嘌呤核苷酸合成酶[cyclic guanosine monophosphate(GMP)-adenosine monophosphate(AMP)synthase,cGAS],阐明了DNA如何刺激免疫和炎症反应的分子机制荣获基础医学研究奖。

精子DNA碎片对体外受精-胚胎移植妊娠结局及胚胎发育的影响

目的:分析精子DNA碎片指数(DFI)对体外受精-新鲜胚胎移植(IVF-ET)助孕及活产结局的影响。方法:选取2015年6月至2020年12月因女方因素首次行IVF并进行新鲜胚胎移植的患者共947例,根据DFI分为低、中、高3组:DFI<15%、15≤DFI≤30%和30%<DFI,比较3组双方基础数据,女方包括年龄、体质量指数(BMI)、基础生殖激素、AMH等;男方包括年龄、BMI、精液参数;超促排卵参数;处理前和处理后IVF精液参数;促排卵结局、胚胎质量及妊娠结局。结果:不同DFI水平间相比,双方年龄在3组间均有显著性差异,15≤DFI≤30%和30%<DFI组双方年龄显著大于DFI<15%组;基础状态精液参数中前向运动精子百分率、非前向运动精子百分率、不活动精子百分率,以及IVF处理前精子总活率、前向运动精子百分率在3组间也均有显著差异(P<0.01);中和高DFI组中基础精子正常形态率、IVF处理前精子浓度、精子总数和处理后精子浓度均显著低于低DFI组;30%<DFI组无论是精液处理前还是精液处理后精子总数均显著低于其他两组(P<0.01)。此外,中和高DFI组受精率显著低于低DFI组,其他临床数据包括促排卵参数、胚胎质量、妊娠结局组间均无显著差异。结论:高DFI水平对精子活率产生显著影响,IVF精液处理后DFI水平与精子浓度、精子总数、受精率均呈显著负相关,对胚胎质量及妊娠结局无影响。

组蛋白去乙酰化酶抑制剂CUDC-101对前列腺癌DU145细胞DNA损伤、迁移和上皮-间质转化的影响

目的:探讨泛素化组蛋白(H2AX)在前列腺癌(PCa)和癌旁良性前列腺组织中的表达及与其PCa患者临床病理参数之间的关系,阐明新型组蛋白去乙酰化酶抑制剂(HDACi)CUDC-101对PCa的DNA损伤、迁移及上皮-间质转化(EMT)的影响。方法:肿瘤基因组谱(TCGA)数据库和UALCAN数据库检索各种癌症组织中H2AX mRNA的表达情况及其在PCa与正常前列腺组织中的表达差异,分析其表达与PCa患者临床预后的关系;采用免疫组织化学染色法检测在PCa组织和癌旁良性前列腺组织中H2AX蛋白的表达情况,分析H2AX蛋白表达与PCa患者临床病理参数的关系;体外培养DU145细胞,分为对照组、5%FBS组、5%FBS+100μmol·L^(-1)CUDC-101组和5%FBS+200μmol·L^(-1)CUDC-101组,采用细胞划痕实验和Transwell小室实验检测CUDC-101处理前后PCa DU145细胞的细胞划痕面积及迁移细胞数;免疫荧光染色法检测CUDC-101处理后PCa DU145细胞中上皮细胞标志物E钙黏蛋白(E-cadherin)和磷酸化组蛋白γ-H2AX表达情况;Western blotting法检测CUDC-101处理后EMT相关蛋白、γ-H2AX和磷酸化蛋白激酶B(p-AKT)表达情况。结果:TCGA数据库和UALCAN数据库分析,H2AX mRNA在PCa组织中高表达,并且H2AX低表达组患者的无病生存期(DFS)明显高于H2AX mRNA高表达组(P<0.001);免疫组织化学染色,在PCa组织中H2AX蛋白强阳性表达率高于在癌旁良性前列腺组织(64.34%vs 14.29%),其过表达与PCa的T分期(P=0.001)和世界卫生组织/国际泌尿科病理学会(WHO/ISUP)预后分级分组(P=0.004)有关,但与PCa患者的年龄、Gleason评分、淋巴结转移、神经和脉管浸润无关(P>0.05);免疫荧光染色法检测,与对照组和EMT诱导组比较,CUDC-101处理组E-cadherin和γ-H2AX蛋白的荧光表达增强;细胞划痕实验和Transwell小室实验检测,与对照组比较,CUDC-101处理组DU145细胞愈合面积和迁移细胞数明显下调;Western blotting法检测,与EMT诱导组比较,CUDC-101处理组E-cadherin和γ-H2AX蛋白表达水平升高(P<0.05或P<0.01),波形蛋白(Vimentin)和p-AKT蛋白表达水平降低(P<0.05或P<0.01)。结论:H2AX蛋白过表达与PCa患者的不良预后密切关联。新型HDACi抑制剂CUDC-101可调控H2AX和AKT的磷酸化,抑制PCa细胞EMT过程。

miR-576-5p通过结合CADM2调节胃腺癌DNA损伤和放化疗敏感性

目的 探究miR-576-5p通过调控细胞黏附分子(CADM)2基因对胃腺癌DNA损伤和放化疗敏感性的影响。方法 GEO2R分析胃腺癌数据集(GSE26595、GSE6174)中表达上调的基因,GSEA分析miR-576-5p与细胞增殖和DNA损伤的关系。按照转染物的不同,分为空白对照组(miR-NC)和miR-576-5p模拟物组(miR-576-5p),向miR-576-5p组的BGC-823、SGC-7901细胞中转染miR-576-5p模拟物组,miR-NC组细胞仅转染对应的对照片段。实时荧光定量PCR(qRT-PCR)检测BGC-823、SGC-7901细胞中miR-576-5p的表达水平,细胞计数试剂盒(CCK8)检测细胞活力。将miR-576-5p过表达的BGC-823细胞皮下注射至裸鼠体内,成瘤后检测小鼠瘤重和体积,qRT-PCR检测肿瘤组织中miR-576-5p表达水平。以不同剂量的γ射线照射BGC-823、SGC-7901细胞,Western印迹检测γH2AX的蛋白表达。CCK8检测细胞对多柔比星化疗的敏感性。双荧光素酶活性实验验证miR-576-5p和CADM2的关系,qRT-PCR和Western印迹检测转染miR-576-5p模拟物的BGC-823、SGC-7901细胞中CADM2表达。向miR-576-5p高表达的BGC-823、SGC-7901细胞中转染CADM2过表达质粒,设为miR-576-5p+CADM2组,Western印迹检测CADM2的表达,CCK8检测细胞活力。结果 与miR-NC组相比,miR-576-5p组细胞活力显著增强(P<0.05)。体内实验结果表明,miR-576-5p过表达显著诱导了胃腺癌BGC-823细胞的肿瘤体内增殖,肿瘤体积和质量显著增加(P<0.05),miR-576-5p在miR-576-5p过表达的肿瘤组织中显著上调(P<0.05)。与miR-NC组相比,miR-576-5p组γH2AX蛋白表达显著下调,且呈照射剂量依赖性。同时剂量依赖性抑制胃腺癌细胞对多柔比星的化疗敏感性,抑制细胞活力(P<0.05)。与miR-NC组相比,miR-576-5p组荧光素酶活性被显著抑制(P<0.05),miR-576-5p过表达显著抑制了CADM2 mRNA和蛋白表达(P<0.05)。与miR-NC组相比,miR-576-5p组CADM2的表达显著下调,而与miR-576-5p组相比,miR-576-5p+CADM2组CADM2表达上调。与miR-NC组相比,miR-576-5p组细胞活性显著增强,而与miR-576-5p组相比,miR-576-5p+CADM2组细胞活性显著被抑制(P<0.05)。结论 miR-576-5p在胃腺癌中高表达,通过靶向抑制CADM2的表达促进胃腺癌细胞体内外增殖,抑制DNA损伤和降低对放化疗的敏感性。

磷酸三苯酯和磷酸三(1,3-二氯-2-丙基)酯对小鼠精母细胞DNA损伤和细胞周期的影响

目的 探讨不同剂量的磷酸三苯酯(TPhP)和磷酸三(1,3-二氯-2-丙基)酯(TDCPP)对小鼠精母细胞DNA损伤和细胞周期的影响。方法 选取小鼠精母细胞(GC-2)为细胞模型,经TPhP和TDCPP(0、3、10、30和50μmol/L)染毒48 h后,利用CCK-8方法检测GC-2的细胞存活率,采用高内涵分析系统检测TPhP和TDCPP对细胞核碎片化程度、磷酸化组蛋白(pH2AX)、DNA同源重组修复蛋白(Rad51)及细胞周期等参数的影响。实时荧光定量PCR法分析精母细胞生长发育关键调控基因,包括环腺苷酸应答元件结合蛋白(CREB-1)、抑制素-α (inhibin-α)、粘连蛋白2(nectin-2)和增殖标记蛋白(Ki67)mRNA的表达水平。结果 与对照组相比,30和50μmol/L TPhP和TDCPP均显著降低GC-2细胞存活率(P<0.05),并引起细胞核碎片化程度加剧(P<0.01)。DNA损伤标志物pH2AX在10、30和50μmol/L TPhP组及TDCPP组均显著升高(P<0.05),Rad51蛋白在30和50μmol/L TPhP组和10、30和50μmol/L TDCPP组均显著升高(P<0.01),表明较高浓度的TPhP和TDCPP可引起DNA损伤。细胞周期分析显示,TPhP主要将细胞阻滞在G0/G1期,而TDCPP主要将细胞阻滞在G0/G1期和G2/M期。荧光定量PCR结果可见,与对照组相比,TPhP(30和50μmol/L)和TDCPP(10、30和50μmol/L)抑制精母细胞生长发育关键基因(CREB-1、inhibin-α、nectin-2和Ki67)的表达(P<0.01)。结论 TPhP和TDCPP均可引起GC-2细胞DNA损伤和细胞周期阻滞,并最终影响精母细胞的生长发育。