CN112481273B - 结直肠癌抑癌基因及其启动子区高dna甲基化的验证方法 - Google Patents
结直肠癌抑癌基因及其启动子区高dna甲基化的验证方法 Download PDFInfo
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Abstract
本发明提供一种结直肠癌抑癌基因,为CPEB1基因。本发明还提供一种结直肠癌抑癌基因的启动子区高DNA甲基化的验证方法,通过DNA甲基化靶向测序技术检测甲基化程度、荧光定量PCR技术检测基因mRNA表达、Western印迹技术检测蛋白水平、流式细胞术检测细胞凋亡情况、CCK‑8实验和细胞克隆形成实验评估细胞增殖克隆能力、损伤修复实验评估细胞迁移能力、Transwell小室实验评估细胞侵袭能力、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力等实验,证明CPEB1基因是一新的结直肠癌抑癌基因,且其启动子区存在高DNA甲基化位点,基因表达呈低表达模式。
Description
技术领域
本发明属于医药卫生领域,具体涉及一种结直肠癌抑癌基因及其启动子区高DNA甲基化的验证方法。
背景技术
结直肠癌(CRC)是常见的消化系统恶性肿瘤,美国等发达国家CRC死亡人数约占癌症死亡人数的10%,仅次于肺癌而位居第2位。2017年中国肿瘤登记年报显示,我国CRC发病率和死亡率近年来呈明显上升趋势,每年新增CRC病例约40万,每年因CRC死亡病例约19.5万,年增速约为4.2%,超过国际平均水平2%,CRC防治形势依旧严峻。
DNA甲基化是发生在DNA序列上的甲基化修饰,密切而广泛的参与基因表达的转录调控和维持基因组稳定性,是表观遗传变异中最普遍而关键的修饰方式。病理条件下,异常的DNA甲基化可致相关基因表达的开放和关闭紊乱,从而导致相关疾病发生和/或疾病进展。研究发现,CRC表观遗传的不稳定性主要体现在,基因启动子区和5’端调控区的异常DNA甲基化以及全基因组DNA去甲基化。大量文献证实,DNA甲基化可作为CRC的第三种发病机制,特别是其在微卫星不稳定型CRC发病中发挥重要作用。尤其游离DNA(ctDNA)甲基化可作为CRC的早期诊断标志物。
发明内容
本发明要解决的技术问题是提供一种结直肠癌抑癌基因及其启动子区高DNA甲基化的验证方法,证明CPEB1基因在结直肠癌组织及癌旁组织的呈现低表达模式,在结直肠癌体外细胞模型SW480、HCT116细胞中CPEB1基因呈现高甲基化状态和低表达模式,采用去甲基化试剂DAC(5-Aza-2'-deoxycytidine)甲基化降低,表达量升高。
为解决上述技术问题,本发明的实施例提供一种结直肠癌抑癌基因,为CPEB1基因。
本发明还提供一种结直肠癌抑癌基因的启动子区高DNA甲基化的验证方法,所述结直肠癌抑癌基因为CPEB1基因,所述验证方法包括如下步骤:
S1、DNA甲基化靶向测序技术检测甲基化程度:以二代测序高通量测序平台为基础,结合Bisulfite处理和生物信息数据分析进行DNA甲基化水平图谱绘制;
S2、荧光定量PCR技术检测基因mRNA表达:收集总RNA,使用第一链cDNA合成试剂盒将其转化为cDNA;采用SYBR Green Master Mix在ABI 7500 PCR仪上进行实时荧光定量qPCR;
Transwell小室实验评估细胞侵袭能力:使用Transwell小室进行细胞侵袭实验;将转染pcDNA3.1-CPEB1、未转染对照和pcDNA3.1的细胞悬液加入到Transwell小室同时加入200μL不含血清的培养基,再将500μL含10%胎牛血清的培养基加入到下室作为迁移刺激;Transwell小室置于细胞培养箱中孵育24 h,对膜下表面的侵袭性细胞进行结晶紫染色10min,在显微镜40倍高倍视野下拍照;
S4、损伤修复实验评估细胞迁移能力:细胞生长24h,处于70-80%融合密度时,接种到6孔细胞培养板中;用200μL的移液管尖端划过细胞层,划痕后,用培养基冲洗2次,去除脱落的细胞,并在0h测量间隙距离;为确定创面愈合程度,在划痕后24h和48h测量剩余间隙距离;
迁移距离的计算公式为:迁移距离=0h时的间隙距离- t时的间隙距离,其中,t =24h或48h;
S5、CCK-8实验和细胞克隆形成实验评估细胞增殖克隆能力:2x105细胞培养于24孔培养板中,转染pcDNA3.1空质粒、CPEB1过表达质粒,分别于0、24、48、72h收集细胞,采用CCK-8法计算细胞增殖能力;细胞克隆形成实验是将以每孔100个细胞的密度接种于24孔细胞培养板中,分别转染pcDNA3.1- CPEB1和pcDNA3.1-EGFP质粒,细胞培养14天;再用磷酸盐缓冲液洗3次后,用 500μL的4%多聚甲醛固定15分钟后,最后用Giemsa染料染色10分钟;统计紫色的细胞球体的数量,每细胞球体包括至少10细胞;细胞克隆形成率(%)=实验组细胞克隆数/对照组细胞克隆数×100%;
S6、Western印迹技术检测蛋白水平:使用放射免疫沉淀裂解缓冲液提取总蛋白,用BCA蛋白测定定量;蛋白样品分别与anti-CPEB1、anti-E-cadherin和anti-MMP-9孵育;内部参照采用GAPDH蛋白;采用免疫组织化学法检测Ki67蛋白,观察细胞原位增殖情况;
S7、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力:用构建好的CPEB1过表达及对照病毒感染HCT116细胞,分别用空细胞组、空载体组(空载病毒包装对照)做对照细胞系成瘤。取5只BALB/c-nu 裸鼠,细胞2x106/只/次,注射裸鼠腋下成瘤,连续2天,注射2次,细胞中加基质胶,提高成瘤率;最后一次注射细胞1周后,看是否有肿瘤凸起,如果没有,再注射一次以最终成瘤,实验期间记录小鼠的生活和精神状况,食欲,体重记录(每3天一次),待control组肿瘤长到1cm3左右,将实验组和对照组排在一起,拍照观察肿瘤大小和个数情况,做好数据统计;同时,人性化处死裸鼠,排成一排测对应肿瘤的大小,空细胞对照组在左边,过表达空载体组在中间,过表达实验组右边,在下方比对拍照;取移植瘤,免疫组化检测肿瘤增殖(Ki-67)指标,WB检测迁移(E-cadhein、MMP9)指标。
其中,步骤S1的理论依据为:将DNA样本经Bisulfite处理后,甲基化的胞嘧啶C保持不变,但非甲基化的胞嘧啶发生脱氨基被转化成脲嘧啶U;在利用该处理产物作为模板进行PCR的产物中,甲基化的胞嘧啶还是胞嘧啶C,但非甲基化胞嘧啶变成了胸腺嘧啶T,通过测序判断出目的片断哪些胞嘧啶是甲基化的。
其中,步骤S1中,DNA甲基化靶向测序实验用引物如下:
其中,步骤S2中,荧光定量PCR实验用引物如下:
本发明的上述技术方案的有益效果如下:本发明通过DNA甲基化靶向测序技术检测甲基化程度、荧光定量PCR技术检测基因mRNA表达、Western印迹技术检测蛋白水平、流式细胞术检测细胞凋亡情况、CCK-8实验和细胞克隆形成实验评估细胞增殖克隆能力、损伤修复实验评估细胞迁移能力、Transwell小室实验评估细胞侵袭能力、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力等实验,证明CPEB1基因是一新的结直肠癌抑癌基因,且其启动子区存在高DNA甲基化位点,基因表达呈低表达模式。
附图说明
图1为本发明中CPEB1基因在结直肠癌组织中呈低表达模式的示意图;
图2为本发明中结直肠癌体外细胞模型SW480、HCT116细胞中CPEB1基因呈现高甲基化状态和低表达模式的示意图;
图3为本发明中体外过表达CPEB1对结直肠癌细胞系表型的影响的示意图;
图4为本发明中动物实验结果图。
具体实施方式
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。
本发明提供了一种结直肠癌抑癌基因,为CPEB1基因。
本发明还提供一种如权利要求1所述的结直肠癌抑癌基因的启动子区高DNA甲基化的验证方法,所述结直肠癌抑癌基因为CPEB1基因,所述验证方法包括如下步骤:
S1、DNA甲基化靶向测序技术检测甲基化程度:MethylTarget以二代测序高通量测序平台为基础,结合Bisulfite处理和生物信息数据分析进行低成本、高效率、高准确度的DNA甲基化水平图谱绘制;
其中,步骤S1的理论依据为:将DNA样本经Bisulfite处理后,甲基化的胞嘧啶C保持不变,但非甲基化的胞嘧啶发生脱氨基被转化成脲嘧啶U;在利用该处理产物作为模板进行PCR的产物中,甲基化的胞嘧啶还是胞嘧啶C,但非甲基化胞嘧啶变成了胸腺嘧啶T,通过测序判断出目的片断哪些胞嘧啶是甲基化的。
步骤S1中,DNA甲基化靶向测序实验用引物如下:
S2、荧光定量PCR技术检测基因mRNA表达:使用TRIzol (Invitrogen公司,美国)收集总RNA,使用第一链cDNA合成试剂盒(Vazyme Biotech公司,中国)按照说明书将其转化为cDNA;采用SYBR Green Master Mix (Vazyme Biotech)在ABI 7500 PCR仪(ABI,美国)上进行实时荧光定量qPCR;GAPDH基因作为本研究的内参。
步骤S2中,荧光定量PCR实验用引物如下:
S3、Transwell小室实验评估细胞侵袭能力:使用Transwell小室进行细胞侵袭实验;将转染pcDNA3.1-CPEB1、未转染对照和pcDNA3.1的细胞悬液加入到Transwell小室同时加入200μL不含血清的培养基,再将500μL含10%胎牛血清的培养基加入到下室作为迁移刺激;Transwell小室置于细胞培养箱中孵育24 h,对膜下表面的侵袭性细胞进行结晶紫染色10 min,在显微镜40倍高倍视野下拍照;
S4、损伤修复实验评估细胞迁移能力:细胞生长24h,处于70-80%融合密度时,接种到6孔细胞培养板中;用200μL的移液管尖端划过细胞层,划痕后,用培养基冲洗2次,去除脱落的细胞,并在0h测量间隙距离;为确定创面愈合程度,在划痕后24h和48h测量剩余间隙距离;
迁移距离的计算公式为:迁移距离=0h时的间隙距离- t时的间隙距离,其中,t =24h或48h;
S5、CCK-8实验和细胞克隆形成实验评估细胞增殖克隆能力:2x105细胞培养于24孔培养板中,转染pcDNA3.1空质粒、CPEB1过表达质粒,分别于0、24、48、72h收集细胞,采用CCK-8法计算细胞增殖能力;细胞克隆形成实验是将以每孔100个细胞的密度接种于24孔细胞培养板中,分别转染pcDNA3.1- CPEB1和pcDNA3.1-EGFP质粒,细胞培养14天。再用磷酸盐缓冲液洗3次后,用 500μL的4%多聚甲醛固定15分钟后,最后用Giemsa染料染色10分钟。统计紫色的细胞球体的数量 (每细胞球体包括至少10细胞);细胞克隆形成率(%)=实验组细胞克隆数/对照组细胞克隆数×100%;
S6、Western印迹技术检测蛋白水平:使用放射免疫沉淀裂解缓冲液提取总蛋白,用BCA蛋白测定定量;蛋白样品分别与anti-CPEB1、anti-E-cadherin和anti-MMP-9孵育;内部参照采用GAPDH蛋白;采用免疫组织化学法检测Ki67蛋白,观察细胞原位增殖情况;
S7、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力:用构建好的CPEB1过表达及对照病毒感染HCT116细胞,分别用空细胞组、空载体组(空载病毒包装对照)做对照细胞系成瘤。取5只BALB/c-nu 裸鼠,细胞2x106/只/次,注射裸鼠腋下成瘤,连续2天,注射2次,细胞中加基质胶,提高成瘤率;最后一次注射细胞1周后,看是否有肿瘤凸起,如果没有,再注射一次以最终成瘤,实验期间记录小鼠的生活和精神状况,食欲,体重记录(每3天一次),待control组肿瘤长到1cm3,将实验组和对照组排在一起,拍照观察肿瘤大小和个数情况,做好数据统计;同时,人性化处死裸鼠,排成一排测对应肿瘤的大小,空细胞对照组在左边,过表达空载体组在中间,过表达实验组右边,在下方比对拍照;取移植瘤,免疫组化检测肿瘤增殖(Ki-67)指标,WB检测迁移(E-cadhein、MMP9)指标。
下面结合具体实验进一步阐述本发明的技术方案。
一、通过TCGA数据库中387例结直肠癌组织和45例癌旁组织的Illumina
HumanMethylation 450K甲基化芯片数据,发现了一个新的结直肠癌抑癌基因CPEB1,其在
结直肠癌组织中呈低表达模式,且其9个甲基化位点呈高甲基化状态。如图1所示,其中,图
1a为TCGA数据库发现结直肠癌组织中CPEB1基因有9个高甲基化位点;图1b为TCGA数据库中
387例结直肠癌组织和45例癌旁组织的甲基化值(β value = ,M表示甲基化,
U表示未甲基化);图1c为本发明收集的104例结直肠癌组织和对应癌旁组织验证CPEB1基因
甲基化状态,发现有20个高甲基化位点;图1d为本发明收集的104例结直肠癌组织和对应癌
旁组织的甲基化值(β value);图1e为CPEB1基因受试者工作曲线显示曲线下面积(AUC)=
0.88,敏感度(Sens)=0.78,特异度(Spec)=0.95,显示可做为结直肠癌的生物标志物;图1f
为本发明收集的结直肠癌数据显示的Septin 9基因(市场已成熟的甲基化产品)的甲基化
结果,可做为本发明的阳性对照。
二、CPEB1基因在结直肠癌组织及癌旁组织的呈现低表达模式,在结直肠癌体外细胞模型SW480、HCT116细胞中CPEB1基因呈现高甲基化状态和低表达模式,采用去甲基化试剂DAC(5-Aza-2'-deoxycytidine)甲基化降低,表达量升高。如图2所示,其中,图2a为TCGA数据库发现结直肠癌组织中CPEB1基因呈现低表达模式;图2b为TCGA数据库发现结直肠癌组织中CPEB1基因表达量与甲基化状态呈负相关,P<0.0001;图2c为本发明收集的49例结直肠癌组织和对应癌旁组织中CPEB1基因表达量,发现癌中呈低表达模式;图2d为本发明收集的49例结直肠癌组织和对应癌旁组织中CPEB1基因表达量与甲基化状态呈负相关,P<0.0001;图2e为结直肠癌细胞系SW480中CPEB1基因呈现高甲基化状态,采用去甲基化试剂DAC处理后其甲基化降低;图2f为结直肠癌细胞系HCT116细胞中CPEB1基因呈现高甲基化状态,采用去甲基化试剂DAC处理后其甲基化降低;图2g为定量PCR检测发现,结直肠癌细胞系SW480和HCT116中CPEB1基因低表达模式,采用去甲基化试剂DAC处理后其表达量升高;图2h为通过WB印迹技术进一步验证,结直肠癌细胞系SW480和HCT116中CPEB1基因低表达模式,采用去甲基化试剂DAC处理后其表达量升高。
三、结直肠癌细胞系中CPEB1基因过表达可抑制结直肠细胞的生长、增殖、迁移、和侵袭能力,并使肿瘤细胞凋亡增加,证明CPEB1基因是一新的结直肠癌抑癌基因。如图3所示,其中,图3a为 RT-PCR检测SW480和HCT116细胞CPEB1 mRNA表达。图3b和图3c为吸光度测定(450 nm)显示CPEB1上调可抑制SW480和HCT116细胞的增殖;图3d和图3e中创面愈合实验显示,SW480和HCT116细胞中过表达CPEB1明显抑制了细胞的迁移能力。图3f为细胞克隆形成实验,发现CPEB1在SW480和HCT116细胞中过表达明显抑制了细胞的增殖和生长能力;图3g为通过Transwell实验显示,在SW480和HCT116细胞中过表达CPEB1降低了细胞的迁移能力;图3h为流式细胞术检测CPEB1在SW480和HCT116细胞中过表达可显著增加细胞凋亡率。其中,图3d、图3e、图3f、图3g和图3h中,(i)、实验的代表性图片;(ii)、相应实验的统计直方图。数据以三次独立实验的平均值±标准差表示。对照、未转染的CRC细胞系;空、转染pcDNA3.1空载体到CRC细胞系;CPEB1,转染pcDNA3.1-CPEB1重组载体到CRC细胞系;*P <0.05,**P < 0.01,***P < 0.001。
四、动物实验证实,抑制异种移植瘤动物模型中CPEB1基因表达可增加结直肠癌的侵袭和转移,进一步证明CPEB1基因是一新的结直肠癌抑癌基因。如图4所示,体内小鼠模型显示CPEB1表达上调抑制了人结直肠癌的生长和转移。其中,图4a为通过pcDNA3.1组(空质粒对照)、pcDNA3.1-CPEB1组(CPEB1过表达)和未转染质粒组(WT)显示小鼠移植瘤大体;图4b和图4c为过表达CPEB1可显著降低小鼠模型的肿瘤大小(图4b)和肿瘤体积(图4c);图4d为CPEB1过表达组、对照组和野生型组小鼠肿瘤组织的HE染色;图4e为小鼠细胞系注射过表达CPEB1质粒,WB检测E-cadherin表达增加,MMP9表达减少;图4f为通过免疫组织化学(IMH)染色实验,CPEB1过表达显著减弱肿瘤细胞的增殖能力;图4g为在整个移植实验过程中记录小鼠的体重。其中,图4e、图4f和图4g 中,(i)、WB实验、IMH实验和小鼠体重实验各组的代表性图像;(ii)、三种实验的统计直方图。图中,WT,未转染载体CRC小鼠模型;空、转染pcDNA3.1空载体到CRC小鼠模型;CPEB1,转染pcDNA3.1-CPEB1重组质粒载体到CRC小鼠模型;*P < 0.05,**P < 0.01,***P < 0.001。
本发明通过DNA甲基化靶向测序技术检测甲基化程度、荧光定量PCR技术检测基因mRNA表达、Western印迹技术检测蛋白水平、流式细胞术检测细胞凋亡情况、CCK-8实验和细胞克隆形成实验评估细胞增殖克隆能力、损伤修复实验评估细胞迁移能力、Transwell小室实验评估细胞侵袭能力、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力等实验,证明CPEB1基因是一新的结直肠癌抑癌基因,且其启动子区存在高DNA甲基化位点,基因表达呈低表达模式。
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 南通大学附属医院
<120> 结直肠癌抑癌基因及其启动子区高DNA甲基化的验证方法
<141> 2020-12-29
<160> 8
<170> SIPOSequenceListing 1.0
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Claims (1)
1.一种结直肠癌抑癌基因CPEB1作为结直肠癌生物标志物的应用,其特征在于,验证CPEB1启动子区高DNA甲基化,验证方法包含以下步骤:
S1、DNA甲基化靶向测序技术检测甲基化程度:以二代测序高通量测序平台为基础,结合Bisulfite处理和生物信息数据分析进行DNA甲基化水平图谱绘制;
DNA甲基化靶向测序实验用引物如下:
S2、荧光定量PCR技术检测基因mRNA表达:收集总RNA,使用第一链cDNA合成试剂盒将其转化为cDNA;采用SYBR Green MasterMix在ABI 7500PCR仪上进行实时荧光定量qPCR;
荧光定量PCR实验用引物如下:
S3、Transwell小室实验评估细胞侵袭能力:使用Transwell小室进行细胞侵袭实验;将转染pcDNA3.1-CPEB1、未转染对照和pcDNA3.1的细胞悬液加入到Transwell小室同时加入200μL不含血清的培养基,再将500μL含10%胎牛血清的培养基加入到下室作为迁移刺激;Transwell小室置于细胞培养箱中孵育24h,对膜下表面的侵袭性细胞进行结晶紫染色10min,在显微镜40倍高倍视野下拍照;
S4、损伤修复实验评估细胞迁移能力:细胞生长24h,处于70-80%融合密度时,接种到6孔细胞培养板中;用200μL的移液管尖端划过细胞层,划痕后,用培养基冲洗2次,去除脱落的细胞,并在0h测量间隙距离;为确定创面愈合程度,在划痕后24h和48h测量剩余间隙距离;
迁移距离的计算公式为:迁移距离=0h时的间隙距离-t时的间隙距离,其中,t=24h或48h;
S5、CCK-8实验和细胞克隆形成实验评估细胞增殖克隆能力:2x105细胞培养于24孔培养板中,转染pcDNA3.1空质粒、CPEB1过表达质粒,分别于0、24、48、72h收集细胞,采用CCK-8法计算细胞增殖能力;细胞克隆形成实验是将以每孔100个细胞的密度接种于24孔细胞培养板中,分别转染pcDNA3.1-CPEB1和pcDNA3.1-EGFP质粒,细胞培养14天;再用磷酸盐缓冲液洗3次后,用500μL的4%多聚甲醛固定15分钟后,最后用Giemsa染料染色10分钟;统计紫色的细胞球体的数量,每细胞球体包括至少10细胞;细胞克隆形成率%=实验组细胞克隆数/对照组细胞克隆数×100%;
S6、Western印迹技术检测蛋白水平:使用放射免疫沉淀裂解缓冲液提取总蛋白,用BCA蛋白测定定量;蛋白样品分别与anti-CPEB1、anti-E-cadherin和anti-MMP-9孵育;内部参照采用GAPDH蛋白;采用免疫组织化学法检测Ki67蛋白,观察细胞原位增殖情况;
S7、裸鼠成瘤实验模拟CPEB1基因在体内的致瘤能力:用构建好的CPEB1过表达及对照病毒感染HCT116细胞,分别用空细胞组、空载病毒包装对照做对照细胞系成瘤,取5只BALB/c-nu裸鼠,细胞2x106/只/次,注射裸鼠腋下成瘤,连续2天,注射2次,细胞中加基质胶,提高成瘤率;最后一次注射细胞1周后,看是否有肿瘤凸起,如果没有,再注射一次以最终成瘤,实验期间记录小鼠的生活和精神状况,食欲,体重记录,每3天一次,待control组肿瘤长到1cm3,将实验组和对照组排在一起,拍照观察肿瘤大小和个数情况,做好数据统计;同时,人性化处死裸鼠,排成一排测对应肿瘤的大小,空细胞对照组在左边,过表达空载体组在中间,过表达实验组右边,在下方比对拍照;取移植瘤,免疫组化检测肿瘤增殖Ki-67指标,WB检测迁移E-cadherin、MMP9指标。
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