CN116334223B - 可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用 - Google Patents
可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用 Download PDFInfo
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Abstract
本发明涉及可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用,本发明从分子生物学及基因诊断水平上,提供了筛选结直肠癌高危人群的技术方法。基于我们前期通过结直肠癌sQTLs遗传图谱显示,rs61746794位点与中国人群的结直肠癌易感性相关。通过针对rs61746794位点进行引物和探针设计,能够依靠荧光定量PCR,即可对正常人群进行rs61746794危险位点的检测,从而鉴别结直肠癌的高危人群,辅助结直肠癌早期筛查及诊断,该技术方法设计巧妙、简易可行、结果准确可靠,有助于临床上对该类人群进行结直肠癌筛查和早期干预。
Description
技术领域
本发明涉及于基因工程及肿瘤医学领域,具体涉及一种可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用。
背景技术
结直肠癌是一种常见的消化道恶性肿瘤,严重威胁我国人民健康,其发病率位列我国恶性肿瘤第三位,死亡率排名第二位。且随着国家经济水平的发展以及居民生活方式和饮食结构的改变,我国结直肠癌的发病率逐年上升,使其成为严重影响国民健康的重大公共卫生问题。尽管科技和医疗水平不断提高,癌症防治方法不断取得进步,但其造成的疾病负担依然严重。目前,针对结直肠癌最有效的筛查方法是粪便免疫化学试验(FIT)结合结肠镜检查。但是,这些方法存在检出率不高和患者接受度不高等弊端。如能采用灵敏度高、简便、更为精准的针对结直肠癌的筛查和早期诊断方法,则可以有效提高患者肿瘤的治疗率,且为国家节省大量的医疗支出。
近年来,以分子遗传学为基础的个体化筛查方案,成为结直肠癌高危人群筛查的重点。单核苷酸多态性(Single Nucleotide Polymorphism,SNP)是最常见的遗传变异,是一类已被证实与肿瘤发生风险相关的分子标志物,其反映了个体的遗传背景差异。随着高通量基因分型技术的发展,全基因组关联研究(Genome-Wide Association Study,GWAS)已经鉴别出了一系列与中国人群结直肠癌易感性密切相关的遗传变异位点。然而,目前发现的易感位点只能解释结直肠癌约15%的遗传度,仍有相当一部分易感位点有待发现,以填补丢失的遗传度;另一方面,这些统计学关联位点并非真正的功能性位点,缺乏对其生物学功能的具体解读,很难直接并精准的应用于肿瘤高风险人群的识别和筛查当中。可变剪接是指前体mRNA去除内含子保留外显子产生不同的基因转录本和蛋白异构体的过程,是调节基因表达和产生蛋白质多样性的重要机制。异常可变剪接已被发现与包括肿瘤在内的多种疾病关系密切,而位于剪接位点或剪接调控元件上的基因遗传变异均可能导致基因异常剪接,进而影响疾病的发生发展。因此,在全基因组范围内系统鉴定影响可变剪接的遗传变异(splicing quantitative trait locus,sQTL),是解析易感位点生物学功能的一个重要的切入点,也是对肿瘤遗传易感性的一个非常有效的补充。
发明内容
本发明的目的在于克服现有技术的不足,本发明的目的是提供一种可变剪接功能性位点的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用。
具体为可变剪接功能性位点rs61746794的检测试剂在制备结直肠癌辅助诊断试剂盒中的应用。
本发明的有益效果为:本发明从分子生物学及基因诊断水平上,提供了筛选结直肠癌高危人群的技术方法。基于我们前期通过结直肠癌sQTLs遗传图谱显示,rs61746794位点与中国人群的结直肠癌易感性相关。通过针对rs61746794位点进行巧妙的引物和探针设计,能够依靠荧光定量PCR,即可对正常人群进行rs61746794危险位点的检测,从而鉴别结直肠癌的高危人群,辅助结直肠癌早期筛查及诊断,该技术方法设计巧妙、简易可行、结果准确可靠,可在各级医院进行推广,对于评估结直肠癌的患病风险提供了帮助,有助于临床上对该类人群进行结直肠癌筛查和早期干预。
附图说明
图1为sQTLs图谱鉴定结直肠癌易感位点rs61746794的流程图;
图2为sQTL rs61746794人群筛选和潜在调控的目标基因;
图3为基于SNP位点rs61746794的结直肠癌风险预测模型AUC曲线;
图4为rs61746794minigene实验结果;
图5为RNA结合蛋白PRPF8等位基因特异性结合于rs61746794上下游;
图6为rs61746794对靶基因PRMT7蛋白酶活性的影响;
图7为靶基因PRMT7不同转录本过表达对结直肠癌细胞系增殖能力的影响。
具体实施方式
以下对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
我们系统识别和鉴定了中国人群结直肠癌功能性sQTLs,用于结直肠癌高风险人群的识别。首先整合基因组、转录组和临床数据,在全基因组范围内系统识别和鉴定结直肠癌sQTLs,进一步通过两阶段、多中心病例-对照研究识别影响结直肠癌风险的sQTLs,通过一系列分子生物学实验,发现sQTL rs61746794具有剪接调控功能,相比于rs61746794[C]个体,携带[T]等位基因的个体结直肠癌患病风险显著增加。机制上,rs61746794[T]促进靶基因16号外显子剪接,进而影响蛋白结构,增强蛋白精氨酸甲基化酶活性,激活下游多条癌症相关通路,比如,YAP,AKT和KRAS通路,使结直肠癌发病风险增加。此位点筛选流程具体见图1。因此,剪接相关变异位点rs61746794与结直肠癌患病风险有着密切的关系,有望在临床上应用,辅助结直肠癌的早期诊断。
具体来说本发明研究的实验方法主要包括以下内容:
发现阶段结直肠癌病例对照研究样本招募于北京(中国医学科学院)及武汉多家医院(同济医院、武汉大学人民医院和武汉大学中南医院),共收集到包含4,293名结直肠癌患者和7,176名健康对照的血液样本。基本人口学特征如表1所示。研究对象包括7,397名男性,4,072名女性,病例组的男/女比例与对照组没有统计学差异。病例组的年龄大于对照组,没有统计学差异。我们采用Logistic回归,校正年龄、性别、吸烟饮酒混杂因素后,在加性模型下计算sQTLs与结直肠癌易感性的关联,并由此绘制曼哈顿图,结果如图2中a图所示。我们以P值<0.05作为显著性阈值,共鉴定出1,217个与结直肠癌风险相关的可变剪接SNP,其中16q22区域人群最显著。在该区域我们发现,rs61746794可调控靶基因PRMT7的16号外显子剪接,结果如图2中b图所示。病例对照研究结果表明,携带rs61746794风险基因型的个体,在中国人群样本中,结直肠癌的患病风险分别较正常人增加22%(OR=1.22,95%CI:1.11-1.35,5.32×10-5)。
表1.发现阶段中国人群病例对照研究样本的基本资料
采用双侧独立t检验计算;*采用双侧χ2检验计算
表2.中国人群样本rs61746794结直肠癌风险关联分析结果
然后,我们分两阶段从北京武汉两地区共募集到的具有完整病历资料及分型明确的6,024例结直肠癌患者和10,022例无肿瘤病史的正常对照的中国人群,然后对这些人进行了基因分型。结直肠癌患者和正常对照均为中国汉族。患者经组织病理学确诊,无年龄限制;正常对照无肿瘤病史,经体检无肿瘤体征。收集了研究对象的性别、年龄等信息。每个研究对象均知情同意参加本研究并捐献2ml外周静脉血,用于分离制备淋巴细胞基因组DNA。基本人口学特征如表3所示。
表3.验证阶段中国人群病例对照研究样本的基本资料
*采用双侧χ2检验比较
表4.中国人群样本rs61746794结直肠癌风险关联分析结果
我们运用非条件logistic回归加性模型(additive model)对两个阶段分别计算SNP位点rs61746794与结直肠癌易感性的关联,并校正性别、年龄、吸烟和饮酒情况。中国人群两阶段病例对照分析结果,在校正了混杂因素后,携带rs61746794[T]风险基因型的个体,结直肠癌的患病风险增加。为加强病例对照研究的检验效能,我们将两阶段的样本合并,检验rs61746794与结直肠癌易感性的关联。结果显示中国人群病例对照研究结果与发现阶段样本的结果一致,结直肠癌的患病风险分别较正常人增加25%(OR=1.25,95%CI:1.15-1.37,1.62×10-7),详细结果见表4。
前期人群结果显示该位点的确是结直肠癌风险位点,因此我们进一步用这个SNP位点建立了结直肠癌的风险预测模型。中国人群来自发现阶段GWAS研究(4,293例病例和7,176例对照),欧洲人群来自GECCO项目(17,789例病例和19,951例对照)我们构建了一个公式,综合考虑SNP的三种基因型和性别、年龄、吸烟及饮酒情况。其中,对于SNP基因分型,野生纯合型=“1”,杂合型=“2”,突变纯合型=“3”;对于性别,男性为“1”,女性为“0”;对于年龄,大于或等于60岁为“1”,小于60岁为“0”。分析时以多因素logistic回归系数β为权重,得到基于rs61746794分型危险评分的公式如下:
中国人群危险评分=(0.1791×性别的评分)+(-0.0285×年龄的评分)+(0.8553×吸烟评分)+(-0.4464×饮酒评分)+(0.2141×rs61746794分型的评分)。
欧洲人群危险评分=(0.1977×性别的评分)+(0.4799×年龄的评分)+(-0.1620×rs61746794分型的评分)
通过绘制AUC曲线,可得该模型曲线下面积在欧洲人群和中国人群分别为0.573和0.585,具体见图3。
为在功能上解析rs61746794功能位点标记的结直肠癌风险关联,我们运用sQTL分析发现该致病位点不同等位基因型与靶基因PRMT7的16号外显子剪接显著相关,随后利用minigene实验在两种结直肠癌细胞系中证实了rs61746794对PRMT7的16号外显子的剪接调控,具体结果见图4。机制上,我们发现rs61746794[T]风险等位基因与RNA结合蛋白PRPF8的结合能力更强,使得的PRMT7的16号外显子剪接增加,短转录本PRMT7-V2的表达增加,具体见图5。短转录本PRMT7-V2的蛋白酶活性更高,使得组蛋白H4R3与H3R2精氨酸甲基化水平增加,激活下游肿瘤相关通路,比如YAP,AKT和KRAS等通路,具体见图6。
为深入探究PRMT7-V1或PRMT7-V2对结直肠癌细胞增殖的影响,我们将不同等位基因型对应不同转录本的质粒在HCT116和SW480结直肠癌细胞系中过表达,采用CCK-8和克隆形成实验检测细胞增殖能力,结果见图7。结果表明,靶基因PRMT7在结直肠癌中发挥致癌作用,且相较于PRMT7-V1,过表达PRMT7-V2使得结直肠癌细胞增值活力更强。
结合以上大规模人群数据和生物学功能实验结果表明,rs61746794[C>T]位点通过促进靶基因PRMT7的16号外显子剪接而增加经典短转录本相对含量,蛋白酶活性增加,促进肿瘤细胞增殖,最终导致个体结直肠癌患病风险增加。因此,对人群进行rs61746794变异的检测,从而鉴别结直肠癌的高危人群,有助于辅助结直肠癌患者的诊断。
实验方法:
1.外周血DNA提取:
我们用常规酚-氯仿法提取DNA,具体步骤如下:
1)取约3ml抗凝血,室温下5,000×g离心15min,弃去上层,留约0.3ml血细胞。加入0.5ml新鲜配制的终浓度为20μg/ml RNA酶的抽提缓冲液,混匀后37℃孵育1h。
2)加入终浓度为100μg/ml的蛋白酶K,混匀后37℃孵育过夜。
3)每管加入Tris缓冲液平衡的酚(pH=7.0)0.7ml,充分混匀,室温下8,000×g离心15min。
4)将上层液体转移到另一1.5ml离心管中,加入等体积酚–氯仿(1:1)0.7ml,充分混匀15min;室温8,000×g离心15min。
5)将上层液体转移到另一干净1.5ml离心管中,加入10%体积的10M乙酸铵溶液,加入2倍体积预冷的无水乙醇,–20℃静置2h以沉淀DNA。
6)沉淀出的DNA用75%乙醇洗涤,12,000×g离心15min后弃上层液体;再用75%乙醇洗涤,12,000×g离心15min后弃上层液体。
7)将管倒立在吸水纸上,待乙醇挥发干净后,每管加适当TE缓冲液,4℃放置一周后保存于–20℃备用。
2.基因分型
采用的分型平台为TaqMan基因分型技术(ABI 7900HT Real Time PCR system,Applied Biosystems),5μl PCR反应体系如表5所示:
表5
反应条件为:95℃预变性10min,然后95℃15sec和60℃1min共45个循环,降温到4℃。
反应所用的引物和探针如下:
rs61746794引物:
正向引物:TGTGAGAGGCCTGCGATACA
反向引物:AGGCTTCGACGTGCACATC
rs61746794探针:
正向探针:FAM-CTACCTTAATCATGTCGTC-MGB
反向探针:VIC-CTACCTTAATCATGTCATC-MGB
3.Minigene实验
人工合成minigene目的片段,包含PRMT7 15号外显子至16号外显子之间的基因组序列,不同之处在于rs61746794等位基因多态性[C/T]。然后将其克隆至pcDNA3.1(+)表达载体,构建PRMT7等位基因特异性minigene质粒,测序验证序列合成准确性,质粒图谱如图4c所示。该质粒将用于体外检测遗传变异对于基因剪接调控影响的minigene实验。
1)RNA提取:细胞转染minigene质粒36h后,从培养箱取出六孔板,提取RNA。使用微量紫外分光光度计检测RNA的纯度和浓度。稀释RNA至100ng/μl左右,置于4℃冰箱,当天进行逆转录。
2)RNA逆转录:计算并配制逆转录反应体系,充分混匀,置于冰上备用。将反应管置于9700基因扩增仪,设置反应条件进行逆转录。
3)cDNA扩增:cDNA扩增采用HotStart DNA Polymerase试剂盒,计算并配制扩增反应体系,充分混匀,置于冰上备用。将EP管置于9700基因扩增仪,设置反应条件进行cDNA扩增。
4)琼脂糖凝胶电泳检测:在碱性环境中,核酸磷酸基团带负电,每个核苷酸携带一个负电荷,DNA的荷质比恒定。所以在凝胶电泳中DNA迁移率主要取决于分子大小。受到电场作用,DNA分子将从负极移向正极,较短的DNA片段迁移较快,从而实现基于DNA片段大小的分离。因此琼脂糖凝胶电泳可以用于检测等位基因特异性minigene产物的表达情况。
以下为详细操作过程:
(1)向三角锥瓶加入100ml TAE电泳缓冲液和2g琼脂糖粉。
(2)瓶口盖上牛皮纸,在微波炉中加热2min。期间戴上防热手套小心摇动锥瓶数次,直至琼脂糖完全溶解。
(3)当溶液冷却至50℃-60℃不烫手时,加入10μl GoldView II型核酸染色剂,轻轻摇匀,避免产生气泡。
(4)将溶液倒入制胶模,厚度为3-5mm,插上梳子。待胶完全凝固后放入装满1×TAE电泳缓冲液的电泳槽内,加样孔一侧位于负极。
(5)取9μl样品和1μl上样缓冲液(10×)混合均匀,用微量移液枪小心加入样品槽中。同时单独加入6μl DNA marker作为参照。
(6)180V恒压电泳约20min,待染料迁移至凝胶3/4处取出,使用凝胶成像系统显影。
4.细胞增殖实验
1)细胞计数
在进行CCK-8增殖实验和克隆形成实验前,需要进行细胞计数,保证不同组别孔板中接种数量合适的细胞。本实验使用血球计数板进行细胞计数,该计数板由两个大小相同的计数池组成,每个计数池又由九个1mm×1mm方格组成,每个方格可容纳0.1mm3液体体积。具体计数步骤如下:
①将已培养一段时间后的细胞孔板进行消化,获得细胞悬液,尽量充分混匀打散细胞。
②对计数板进行消毒后盖上盖玻片。
③吸取10μL细胞悬液,沿玻片边缘缓慢滴入计数板中,保证盖玻片和计数板中间无气泡产生)。
④放置1min,于显微镜下观察计数板,并按如下原则对四角的方格进行计数:a.对于压住方格边线的细胞,只对上边和左边的细胞进行计数,b.若细胞成团块状,则计为一个细胞。
⑤细胞悬液浓度=四角方格内细胞总数/4×104个/mL。
2)CCK-8细胞增殖实验
本研究采用CCK-8试剂盒(Dojindo,日本)检测结直肠癌细胞增殖能力。
①吸取细胞悬液100μL并按计数浓度稀释至细胞量为2,000左右,接种于96孔板中。
②在24h、48h、72h和96h四个时间点进行细胞活性检测。向每个孔内加入10μLCCK-8试剂,上下左右摇晃以轻柔混匀,放回细胞培养箱继续培养1.5h。
③使用酶标仪进行吸光度测定,波长设定为450nm,根据四个时间点的吸光度值绘制不同实验组别的细胞增殖曲线以比较其差异。
3)克隆形成实验
①在转染细胞36h后,进行细胞计数并稀释至合适浓度。在6孔板的每孔中加入2mL细胞悬液使得细胞量为1,000左右,继续培养2周左右。
②期间每2~3天观察细胞形态并更换新鲜培养基。若显微镜下观察到细胞克隆时,可进行固定染色。
③从培养箱中取出6孔板,吸去废液并缓慢加入PBS清洗2次。吸净每孔PBS后加入2mL甲醇溶液,于室温放置30min。
④吸去甲醇后加入0.25%结晶紫溶液,避光放置30min进行染色。
⑤染色完成后,吸去孔中废液,并用双蒸水清洗孔板直至视野清晰,进行拍照保存。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
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