5-Methylcytosine(5mC)is the most important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5mC with the genera...5-Methylcytosine(5mC)is the most important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC).It has been known that 5mC,5hmC and 5fC play critical roles in modulating gene expression.However,unlike the 5mC,5hmC,and 5fC,the functions of 5caC are still underexplored.Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA.In the current study,we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR(qPCR)for the site-specific quantification of 5caC in DNA.This method depends on the selective conversion of 5caC to form dihydrouracil(DHU)by pyridine borane treatment.DHU behaves like thymine and pairs with adenine(DHU-A).Thus,the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A,which is utilized to achieve the site-specific detection and quantification of 5caC in DNA.As a proof-of-concept,the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples.The method is rapid,straightforward and cost-effective,and shows promising in promoting the investigation of the functional roles of 5caC in future study.展开更多
文摘目的探讨二氢嘧啶脱氢酶(DPYD)在抑郁状态小鼠脑胶质瘤中的表达及其在脑胶质瘤恶性进展中的作用。方法采用慢性社会挫败应激实验(由10只14周龄CD-1小鼠轮流攻击C57BL/6J小鼠10 d)构建C57BL/6J小鼠(6周龄)抑郁模型(抑郁组,n=5),采用社会交互实验、糖水偏好实验和悬尾实验评价抑郁组与对照组(n=5)C57BL/6J小鼠,判断抑郁模型是否构建成功。采用立体定向技术将GL-261鼠源性胶质瘤细胞注射于两组小鼠尾状核头(各组n=5),构建脑胶质瘤原位模型,并通过7.0 T MRI观察肿瘤原位生长情况。通过RNA测序检测两组小鼠肿瘤的差异表达基因,将表达上调的差异基因匹配至中国脑胶质瘤基因组图谱计划(CGGA)(325例)和癌症基因组图谱(TCGA)(699例)数据库中,通过比较其在高级别胶质母细胞瘤与低级别脑胶质瘤中的表达差异确定目标基因。采用免疫组织化学染色法验证目标基因在小鼠脑胶质瘤组织中的表达情况。通过转染siRNA分别敲低LN229和HG9细胞株中目标基因的表达;根据转染的siRNA定义为敲低#1、#2、#3组及其对照组,并采用蛋白质免疫印迹实验检测各组目标基因的表达量。在LN229和HG9细胞株中应用CCK-8增殖实验、Transwell侵袭实验和划痕修复实验分别验证敲低目标基因对胶质瘤细胞增殖、侵袭和迁移能力的影响(n=3)。结果行为学实验结果显示,与无CD-1小鼠时比较,有CD-1小鼠时抑郁组小鼠在角落区域的停留时间延长,抑郁组较对照组糖水摄入比率下降、悬尾体位时静止时间延长(均P<0.05),提示抑郁模型构建成功。种瘤后第20天的头颅MRI显示抑郁组脑胶质瘤体积[(292.41±8.88)cm^(3)]较对照组[(223.22±41.44)cm^(3)]明显增大(t=2.79,P=0.026)。两组鼠脑组织转录组测序结果显示,1528个差异表达的基因中DPYD(差异倍数为14.51,P=0.012)等670个基因表达上调;经生物信息学分析,将DPYD作为目标基因。免疫组织化学染色结果显示,DPYD蛋白在抑郁组小鼠肿瘤中的阳性表达率高于对照组(t=4.44,P=0.016)。在LN229和HG9细胞株中,与对照组比较,DPYD敲低#1、#2、#3组的DPYD表达量均下降(均P<0.001),其中敲低#1组最为显著。在LN229和HG9细胞株中,与对照组相比,敲低#1组的划痕修复率均下降、60 h时的吸光度值均较低、穿膜细胞数量均减少(均P<0.05)。结论脑胶质瘤进展可能与抑郁情绪相关,抑郁状态下DPYD的表达增高,敲低DPYD可显著降低胶质瘤细胞的侵袭、增殖和迁移能力。
基金supported by the National Natural Science Foundation of China(Nos.22074110,21635006,21721005)the Fundamental Research Funds for the Central Universities(2042021 kf0212).
文摘5-Methylcytosine(5mC)is the most important epigenetic modification in mammals.The active DNA demethylation could be achieved through the ten-eleven translocation(TET)protein-mediated oxidization of 5mC with the generation of 5-hydroxymethylcytosine(5hmC),5-formylcytosine(5fC)and 5-carboxylcytosine(5caC).It has been known that 5mC,5hmC and 5fC play critical roles in modulating gene expression.However,unlike the 5mC,5hmC,and 5fC,the functions of 5caC are still underexplored.Investigation of the functions of 5caC relies on the accurate quantification and localization analysis of 5caC in DNA.In the current study,we developed a method by chemical conversion in conjugation with ligation-based real-time quantitative PCR(qPCR)for the site-specific quantification of 5caC in DNA.This method depends on the selective conversion of 5caC to form dihydrouracil(DHU)by pyridine borane treatment.DHU behaves like thymine and pairs with adenine(DHU-A).Thus,the chemical conversion by pyridine borane leads to the transformation of base paring from 5caC-G to DHU-A,which is utilized to achieve the site-specific detection and quantification of 5caC in DNA.As a proof-of-concept,the developed method was successfully applied in the site-specific quantification of 5caC in synthesized DNA spiked in complex biological samples.The method is rapid,straightforward and cost-effective,and shows promising in promoting the investigation of the functional roles of 5caC in future study.
