CN108277277A - 一种评估家族性乳腺癌风险的标记物及其应用 - Google Patents
一种评估家族性乳腺癌风险的标记物及其应用 Download PDFInfo
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Abstract
本发明公开了一种评估家族性乳腺癌风险的标记物,所述标记物为CNDP1基因或CNDP1蛋白,并进一步证实CNDP1在乳腺癌样本中表达上调。利用该分子标记物制备家族性乳腺癌早期诊断试剂或试剂盒,能够更早期、更便捷地检测乳腺癌高危人群中乳腺癌变的发生、发展趋势。同时利用该分子标记物检测乳腺癌还为基因治疗、药物治疗等临床应用提供了治疗靶点和重要依据。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种评估家族性乳腺癌风险的标记物及其应用。
背景技术
乳腺癌是女性最常见的恶性肿瘤,其发病率、死亡率逐年递增,远高于全国平均增长水平,且发病年龄呈现年轻化趋势(高发年龄段比世界平均发病年龄提前了10岁),为威胁女性健康的头号杀手。然而,由于大部分乳腺癌缺乏针对性治疗靶点,目前临床上多采用传统手段进行干预,不能有效地降低其复发、转移及死亡风险,给家庭社会带来沉重负担。
遗传因素在乳腺癌的发生发展中发挥重要作用。研究表明,约10%的乳腺癌与遗传相关。在早发性乳腺癌案例(<35岁)中,30%以上由遗传因素所致。BRCA1和BRCA2是最早被鉴定,也是最常见的家族遗传性乳腺癌特征基因。随着相关新技术的应用,已有大量基因陆续被报道与乳腺癌相关。这些基因大致可以分为“高风险”与“低至中度风险”乳腺癌易感基因,其中“高风险”易感基因包括:BRCA1和BRCA2(在年轻人中发病率高得多);而PTEN,TP53,CHEK2,TGFβ1,PALB2和ATM属于“中度风险”乳腺癌易感基因。研究表明,40岁以前被诊断为乳腺癌患者的女性中,约1%被发现携带p53基因的突变;30岁以前被诊断为乳腺癌患者的女性中,多达4%携带p53基因的突变。携带PTEN同源缺失突变的女性,其患乳腺癌的风险为25-50%,这在年轻女性中更为常见。PALB2基因突变的女性,患乳腺癌和胰腺癌的风险增高。另外还有90个常见“低度风险”突变。上述这些变异仅仅解释了37%的家族性乳腺癌的风险。尚有接近三分之二的家族性乳腺癌发病风险找不到对应的致病基因。
家族性乳腺癌是指一个家族中有两个或以上具有血缘关系的成员患乳腺癌,占乳腺癌总体的5-7%。与散发性乳腺癌相比,家族性乳腺癌的发病率更高,且年轻(<40岁)早发性患者,预后更差,存活率更低。大规模的流行病学研究表明,有一个一级亲属患病的个体,其乳腺癌发病的概率为5.5%;有两个一级亲属患病的个体,其发病率为13.3%。Evans等人进一步的回顾分析表明,与正常对照人群相比,有一级亲属患乳腺癌的家族个体,其发病率增加2-4倍。家族遗传性在年轻早发乳腺癌群体中较为常见。可见,探索寻找族性乳腺癌相关基因,将为家族性乳腺癌的早期诊断、治疗和预后提供新的理论依据和临床指导。
CNDP1(Carnosine dipeptidase 1,肌肽二肽酶1)位于人染色体18q22.3,是肌肽(Carnosine)水解为β-丙氨酸和组氨酸的限速酶。研究表明,其与糖尿病性肾病密切相关,在前列腺癌和胶质母细胞瘤中表达下调;一项大规模的临床研究表明,低水平表达的CNDP1与前列腺癌的淋巴结转移密切相关。ArnerP团队的研究发现,在胃肠道肿瘤中,低水平表达的CNDP1与患者体内的分解代谢和不良预后密切相关,综合前列腺癌和胶质瘤中外周血CNDP1的低表达,提示CNDP1可构成侵袭性肿瘤及癌症恶病质的标志物。但目前尚无该基因与家族性乳腺癌发生发展相关的报道。
发明内容
为了实现乳腺癌的早期诊断、早期治疗,本发明的目的在于提供一种评估家族性乳腺癌风险的标记物及其在诊断试剂、试剂盒中的应用。
本发明的目的是通过下列技术方案实现的:
首先,本发明提供一种评估家族性乳腺癌风险的标记物,所述标记物为CNDP1基因或CNDP1蛋白。
优选的,所述CNDP1基因或CNDP1蛋白在乳腺癌样本中表达上调。
优选的,所述检测表达上调的方法包括基因水平检测和蛋白水平的检测;优选的,所述检测CNDP1基因表达上调的方法包括qRT-PCR、全外显子组测序、基因芯片;所述检测CNDP1蛋白表达上调的方法包括免疫组化、Westernblot、iTRAQ技术。
优选的,所述CNDP1基因在乳腺癌患者中发生突变,所述突变位点为NM_032649:exon6:c.G724A:p.G242R。
优选的,所述CNDP1基因突变为错义突变。
进一步地,本发明提供一种筛选评估家族性乳腺癌风险的标志物CNDP1的方法,所述方法如下:
(1)通过iTRAQ联合质谱方法筛选家族性乳腺癌外周血样本中差异表达的蛋白;
(2)利用全外显子组测序技术筛选家族性乳腺癌外周血样本中差异表达的基因;
(3)通过联合iTRAQ蛋白组学数据和全外显子组测序数据,结合生物学分析,筛选到在家族性乳腺癌外周血样本中既在蛋白水平又在基因水平表达差异的CNDP1;
(4)通过RT-PCR和Westernblot进一步验证CNDP1基因或CNDP1蛋白的表达情况。
本发明通过联合iTRAQ和全外显子组测序技术,筛选到在家族性乳腺癌外周血样本中既在蛋白水平又在基因水平表达差异的CNDP1作为评估家族性乳腺癌风险的标记物;并通过Western blot技术验证了家族性乳腺癌患者外周血中CNDP1蛋白表达上调;同时验证人乳腺癌细胞中CNDP1基因或蛋白的表达上调。
进一步,本发明提供了CNDP1基因或蛋白在制备评估、诊断或预后乳腺癌产品中的应用。
优选的,所述产品包括试剂、试剂盒或芯片。
进一步,本发明提供了一种用于测定CNDP1基因表达水平的试剂,所述试剂包括特异性扩增CNDP1的引物,所述引物序列如下:
正向引物如SEQ ID NO.1所示;
反向引物如SEQ ID NO.2所示。
优选的,所述试剂还包括RNA提取试剂、反转录试剂、定量PCR试剂、正常对照样本RNA。
具体为:
(1)RNA提取试剂:Trizol、氯仿、异丙醇和75%乙醇等。
(2)反转录试剂:逆转录缓冲液、逆转录酶、和T重复寡核苷酸Oligo dT或Random6mers。
(3)定量PCR试剂:PCR缓冲液、SYBR Green荧光染料、dNTPs组成的SYBR Green聚合酶链式反应体系和RNase Free H2O。
本发明所述的乳腺癌样本包括乳腺癌患者外周血、组织等。
优选的,所述乳腺癌样本为乳腺癌患者外周血。
进一步,本发明提供了用于测定样本中CNDP1基因表达水平的试剂在制备诊断或预示乳腺癌的试剂盒或试剂中的应用。
本发明有益效果:
1、本发明通过联合分析家族性乳腺癌家系样本蛋白质组和全外显子测序的数据,筛选到了同时在蛋白质和基因水平差异表达的新致病基因CNDP1;
2、进一步,在乳腺癌患者外周血中和体外细胞学实验均证实CNDP1表达显著升高;上述实验表明CNDP1基因与乳腺癌发生发展密切相关;
3、在患者外周血中检测到CNDP1基因突变,表明CNDP1基因表达差异是可以遗传的;
本发明公开了一种与乳腺癌相关的标记物CNDP1,并进一步证实CNDP1在乳腺癌样本中表达上调。利用该分子标记物制备乳腺癌早期诊断试剂或试剂盒,能够更早期、更便捷地检测乳腺癌高危人群中乳腺癌变的发生、发展趋势。同时利用该分子标记物检测乳腺癌还为基因治疗、药物治疗等临床应用提供了治疗靶点和重要依据。
附图说明
图1为家系1图谱;
图2RT-PCR检测CNDP1基因在人乳腺癌细胞中的表达水平;
图3Westernblot检测CNDP1蛋白在人乳腺癌细胞中的表达水平;
图4Westernblot检测CNDP1蛋白在家系外周血中的表达水平。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例中所用的技术手段为本领域技术人员所熟知的常规手段,所用的试剂可以商业购买得到。
实施例中未注明具体条件的实验方法,通常为本领域常规方法。
本发明通过联合分析家族性乳腺癌家系样本蛋白质组和全外显子测序的数据,筛选到了同时在蛋白质和基因水平差异表达的,且在乳腺癌患者外周血中均是高表达的新致病基因CNDP1;家系外周血样本进一步验证结果表明,与正常人相比,家系中乳腺癌患者外周血CNDP1蛋白表达水平明显升高,家系中其他女性成员CNDP1蛋白表达水平亦升高,同时患者一级亲属外周血CNDP1表达水平高于其二级亲属;细胞学实验也证明,与正常细胞相比,乳腺癌细胞中CNDP1的表达显著升高。CNDP1基因或蛋白同时在乳腺癌家系外周血样本和乳腺癌细胞株中差异表达,表明该基因与乳腺癌发生发展密切相关。而在患者外周血中检测到该基因突变,表明CNDP1基因表达差异是可以遗传的。
本发明涉及的细胞如下:
MCF-10A(非致瘤的上皮细胞株),人乳腺癌细胞MCF-7、MDA-MB-231、MDA-MB-453、MDA-MB-468均购自国家细胞资源共享平台;
MCF10A:乳腺上皮细胞生长培养基MEGM试剂盒(Lonza,货号CC-3150),加入100ng/ml霍乱毒素,37℃5%CO2;
MCF-7:DMEM培养基(HyClone SH30243.01B),加入0.01mg/ml人重组胰岛素;胎牛血清至终浓度为10%;37℃5%CO2;
MDA-MB-231:Leibovitz's L-15Medium(GIBCO 11415064),胎牛血清至终浓度为10%;37℃100%空气,无CO2;
MDA-MB-453:Leibovitz's L-15Medium(GIBCO 11415064),胎牛血清至终浓度为10%;37℃100%空气,无CO2;
MDA-MB-468:Leibovitz's L-15Medium(GIBCO 11415064),胎牛血清至终浓度为10%;37℃100%空气,无CO2;
本发明主要的仪器和试剂如下:
涡旋振荡器(海门市其林贝尔仪器制造有限公司,型号:QL-901);
离心机(Thermo,型号:PICO17);
超声波细胞破碎仪(南京先欧仪器制造有限公司,型号:XO);
酶标仪(Thermo,型号:MμLtiskanMK3);
恒温孵浴器(上海浦东荣丰科学仪器有限公司,型号:HH.S4);
真空冷冻干燥机(Thermo,型号:SPD2010-230);
高pH条件下的反相色谱分离:RIGOL L-3000高效液相色谱系统(北京普源精电科技有限公司),流动相A:98%ddH2O,2%乙腈(pH 10);流动相B:98%乙腈,2%ddH2O(pH 10);
色谱柱:Durashell-C18,4.6mm×250mm,5μm,(Agela,货号:DC952505-0);
纳升级反相色谱-TripleTOFTM 5600进行蛋白质分析:液相色谱:(AB Sciex,型号:EksigentNano LCμLtra 2D系统),流动相A:98%超纯水,2%乙腈,0.1%甲酸;流动相B:98%乙腈,2%超纯水,0.1%甲酸;
质谱系统(AB Sciex,型号:TripleTOFTM5600);
C18预柱:(cHiPLC,200μmX 500μm,ChromeXP C18-CL3μm );
分析柱:(cHiPLC,75μm X 15cm,ChromeXP C18-CL 3μm );
进样瓶(Agilent,5183-2072);
瓶盖(Agilent,5185-5829);
内称管(Agilent,5182-0549);
喷针(New Objective,PN:FS360-20-10-N-20-C12);
尿素(Bio-Rad,货号:161-0731,美国);
硫尿(Sigma-Aldrich,货号:T7875,美国);
CHAPS(Bio-Rad,货号:161-0460,美国);
Protease Inhibitor Cocktail(Roche,货号:04693116001,美国);
蛋白定量染液(Thermo Scientific,货号:23238,美国);
牛血清白蛋白(Bovine SerumAlbumin,BSA)(Sigma-Aldrich,货号:A2058,美国);
DTT(Bio-Rad,货号:161-0611,美国);
碘乙酰胺(Bio-Rad,货号:163-2109,美国);
试剂盒中的Dissolution Buffer(AB Sciex,PN:4381664);
胰酶(Promega,货号:V5111,美国);
10K超滤管(milipore,PN:UFC5010BK);
8标试剂盒(AB Sciex,PN:4390812,PN:4381664);
Ziptip(Millipore,PN:ZTC18M096(2μg));
乙腈(Merck,货号:100030,德国);
氨水(Sigma-Aldrich,货号:17837,美国);
ddH2O用氨水调pH值到10;
FastQuant cDNA第一链合成试剂盒(TIANGEN);
Talent qPCRPreMix(SYBR Green)(TIANGEN);
本发明涉及的iTRAQ(isobaric tags for relative and absolutequantitation,同位素标记相对和绝对定量)技术是由AB SCIEX公司研发的一种体外同种同位素标记的相对与绝对定量技术。该技术利用多种同位素试剂标记蛋白多肽N末端或赖氨酸侧链基团,经高精度质谱仪串联分析,可同时比较多达8种样品之间的蛋白表达量,是近年来定量蛋白质组学常用的高通量筛选技术。iTRAQ定量蛋白质组学是蛋白酶切后形成多肽,用iTRAQ同位素试剂标记多肽N末端或赖氨酸侧链基团。标记后的肽段经过液相分离,进行一级质谱和二级质谱分析,在二级质谱前,被标记的不同样本中的同一肽段表现为相同的质荷比和其他理化性质。而在二级质谱中,信号离子表现为不同质荷比(114~121)的峰,根据波峰的高度及面积,可以鉴定出蛋白质和分析出同一蛋白质不同处理的定量信息。
iTRAQ试剂包括三部分:报告部分、肽反应部分、平衡部分。(1)报告部分有八种:113-121(无120),因此iTRAQ试剂可同时标记8组样品。(2)肽反应部分:能与肽段N端及赖氨酸侧链氨基发生共价连接而标记上肽段。(3)平衡部分:保证被标记的同一肽段的质荷比相同。与传统的双向电泳定量分析相比,iTRAQ具有以下技术服务优势:(1)灵敏度高,检测限低,可检测出低丰度蛋白;(2)分离能力强,分析范围广,iTRAQ可以对任何类型的蛋白质进行分离鉴定,包括高分子量蛋白质、酸性蛋白和碱性蛋白,膜蛋白和不溶性蛋白;(3)高通量:同时对8个样本进行分析,提高了实验通量,可同时对多个时间点或不同处理的蛋白质进行分析;(4)结果可靠:定性与定量分析结果更加可靠;(5)自动化程度高:液相与质谱连用,自动化操作,分析速度快,分离效果好。
实施例1不同家族遗传性乳腺癌样本收集和临床资料采集
1、乳腺癌患者家系纳入标准
1)一个家族中除先证者外,在一级亲属中有1例或更多的乳腺癌患者,且至少有1例满足下列条件之一:发病时<40岁;同时或先后出现双侧乳腺癌;同时或先后出现非乳腺恶性肿瘤;
2)术前未接受过任何相关治疗,如放疗、化疗、内分泌治疗以及靶向治疗;
3)符合国际临床分期I-IV期而无禁忌者,行相关手术治疗者(包括乳腺肿物切除术备乳腺癌手术,乳腺癌根治术、改良根治术以及保乳术,结合或不结合腋窝淋巴结清扫术)。
2、乳腺癌患者家系排除标准
1)术前行辅助放/化疗治疗;
2)结合临床症状、体征、辅助检查,或者经B超引导下乳腺肿块穿刺活检病理学诊断为良性病变,无明确手术指针者;
3)存在其他手术禁忌症而不能实行手术取材的患者;
4)手术切除后,癌肿过小(最大直径<1cm),存在病理诊断难度的;
5)合并有类风湿性关节炎、风湿热等其他风湿免疫疾病的患者;
6)患有其他严重系统性疾病的患者;
7)流行病学资料、临床资料和影像资料不完整者;
8)未获得知情同意者。
实施例2采用iTRAQ技术进行血液蛋白质组学研究鉴定分析
1、检测样本
家系1(图1为家系1图谱)中包括成员14人,其中1号病故,2号为患者,家庭成员4号、5号患病,其他10位成员检测未患病;4人(1、2、4、5号)罹患恶性肿瘤,包括乳腺癌、胰腺癌、宫颈癌、甲状腺癌和肝癌等为病例;1人(4号)同时患三种恶性肿瘤(乳腺癌、甲状腺癌及肝癌)。其中病例组为3例(2号、4号、5号),对照组为10例(其他成员),采集该家族所有成员的血液,4℃静置1h,3000g离心10min,收集上清,冰上分装后存至-80℃备用。
2、iTRAQ定量实验
2.1样品蛋白质提取
1)将血清去除高丰度蛋白;
2)将上清取出,待样本处理;
3)将粉末按照1:10(W/V)加入lysis buffer(7M尿素,2M硫脲,0.1%CHAPS,片/50ml Protease Inhibitor Cocktail),涡旋混匀;15,000g 10℃离心1hr,小心取出上清,分装后冻存于-80℃。
2.2蛋白定量(Bradford法)
采用Bradford法[Marion M.Bradford.Analytical Biochemistry,1976,72:248-254]测定样本提取的蛋白浓度。先将样本用lysis buffer(7M尿素、2M硫脲、0.1%CHAPS)进行一定倍数稀释使其终浓度落在标曲范围内,稀释好的样本和标准品(将BSA用lysisbuffer溶解成系列浓度的标准蛋白)各取10μL分别和300μL蛋白定量染料避光反应20min,用酶标仪同时测定标准品和样本在595nm下的吸光值,根据标准品每管吸光值和浓度的关系绘制标准曲线
根据曲线公式计算各样品蛋白浓度。
2.3蛋白酶解(FilterAided Sample Preparation,FASP)
1)蛋白定量后取200μg蛋白溶液置于离心管中;
2)加入终浓度25mM DTT,60度反应1小时;
3)加入终浓度50mM碘乙酰胺,室温10分钟;
4)将还原烷基化后的蛋白溶液加入10K的超滤管中,12,000转离心20分钟,弃掉收集管底部溶液;
5)加入iTRAQ试剂盒中的DissolutionBuffer 100μL,12,000转离心20分钟,弃掉收集管底部溶液,重复3次(为节省试剂,这步可以将Dissolution Buffer用水稀释5倍后使用);
6)更换新的收集管,在超滤管中加入胰蛋白酶,总量4μg(与蛋白质量比1:50),体积50μL,37摄氏度反应过夜;
7)次日,12,000转离心20分钟,酶解消化后的肽段溶液离心于收集管底部;
8)在超滤管中加入50μL Dissolution Buffer,12,000转再次离心20分钟,与上步合并,收集管底部共得到100μL酶解后的样品。
2.4iTRAQ标记
1)从冰箱中取出iTRAQ试剂,平衡到室温,将试剂离心至管底;
2)向每管试剂中加入150μL异丙醇,涡旋振荡,离心至管底;
3)取50μL样品(100μg酶解产物)转移到新的离心管中;
4)将iTRAQ试剂填加到样品中,涡旋振荡,离心至管底,室温反应2小时;
5)加入100μL水终止反应;
6)为了检测标记效率及定量准确性,从各组样品中各取出1μL混合,用Ziptip脱盐后进行MALDI-TOF-TOF(AB SCIEX 4800Plus)鉴定,确认标记反应良好;
7)混合标记后的样品,涡旋振荡,离心至管底;
8)真空冷冻离心干燥;
9)抽干后的样品冷冻保存待用。
3、酶解肽段离线预分离及LC-MS/MS质谱分析
3.1高pH条件下的反相色谱分离
1)混合标记后的样品用100μL流动相A溶解,14000g离心20min,取上清待用;
2)使用400μg酶解好的BSA进行分离(柱温45℃,检测波长214nm),检测系统情况;
3)取100μL准备好的样本上样;
4)流速0.7ml/min,分离梯度如下表1所示:
表1分离梯度
3.2纳升级反相色谱-TripleTOFTM5600进行蛋白质分析
1)根据紫外监测情况,将RP分离得到的组份合并为10个,合并时采用30μL 2%ACN,0.1%FA,加入第一个离心管,涡旋振荡并离心后,转入第二个离心管,依次直至合并组份最后一管;
2)12,000转离心10分钟,吸取上清上样;
3)上样体积8μL,采取夹心法上样;
4)Loading Pump流速2μL/min,15分钟;
5)分离流速0.3μL/min,分离梯度如下:
表2分离梯度
4、蛋白质组数据筛选及生物信息学分析
数据库的选择是以所需物种、数据库注释完备性及序列可靠性为参考依据的。在本次实验中选择的数据库来自uniprot(www.uniprot.org/),数据库本版为:Homosapiens_SwissProt_2016_03database;iTRAQ的质谱分析是由AB scix 5600型质谱完成,产生的质谱原始文件*.wiff采用Mascot软件搜库处理,采用scaffold软件对搜库结果进行质控。
基于iTRAQ技术,在家系血清样本中共筛选到55个差异表达的蛋白,其中16个下调,39个上调。
为了更好分析差异蛋白的功能,对差异表达的蛋白数据进行了Gene Ontology和通路分析,并对差异蛋白进项功能注释和蛋白质互相作用网络分析。
实施例3全外显子组测序
北京诺禾致源科技股份有限公司采用Agilent的液相芯片捕获系统,对人的全外显子区域DNA进行高效富集,然后在IlluminaHiseq平台上进行高通量、高深度测序。3例患者和10例对照样本均来自家系1。
1)外周血提取基因组DNA;
2)随机打断成长度为180-280bp的片段,经末端修复和加A尾后在片段两端分别连接上接头制备DNA文库;
3)采用Agilent的液相芯片捕获试剂盒(Agilent SureSelect HumanAll ExonV5试剂盒)对人的全外显子区域DNA进行高效富集;
4)在带有特异index的文库pooling后与多达543,872个生物素标记的探针进行液相杂交;
5)使用带链霉素的磁珠将20,965个基因的334,378个外显子捕获下来;
6)经PCR线性扩增后进行文库质检,合格后即在Illumina平台上进行高通量、高深度测序。
外显子组测序后获得原始数据,经过去污染等数据过滤、与人类参考基因组进行比对分析,检测个体携带的变异,并完成相应的注释和统计分析工作;经过全外显子组测序分析,筛选出1945个发生突变的基因。
通过联合分析蛋白质组和全外显子组数据,我们筛选到家族性乳腺癌患者既在蛋白水平又在基因水平表达差异的CNDP1,并发现在乳腺癌患者中CNDP1基因发生突变CNDP1:NM_032649:exon6:c.G724A:p.G242R。
实施例4CNDP1在细胞中表达水平
检测MCF10A、MCF-7、MDA-MB-231、MDA-MB-453、MDA-MB-468细胞中CNDP1的表达水平;
1、RT-PCR
1.1RNA提取
使用总RNA极速抽提试剂盒(飞捷生物)提取外周血或细胞的RNA:
1)悬浮培养细胞离心收集,留下细胞团块和适量上清,使细胞浓度<2×107/ml,充分震荡直至没有细胞团块,取100μL细胞悬液放入1.5ml eppendorf中;
2)处理好的样本管中,加入RA2液500μL,充分颠倒混匀5-10次,静置1min;
3)将样本裂解物全部吸入或倒入内套管,12000rpm离心1min;
4)取出内套管,吸去外套管中液体后放回内套管,加入500μL洗液,12000rpm离心1min;
5)重复步骤四再洗一次;
6)取出内套管,吸去外套管中液体后放回内套管,不加洗液,12000pm离心1min;
7)内套管移入新的1.5ml eppendorf管,在膜中央加入1‰DEPC水25μL;
8)室温静置1min后,离心1min,获得总RNA;
9)取2μLRNA用Nanodrop测浓度;
10)取2μL RNA,1.2%琼脂糖凝胶电泳质检。
1.2反转录
反转录体系如下表3所示:
表3反应体系
1.3Real-time PCR
所用引物由上海生工设计合成,如表4所示:
表4引物序列
SYBR GreenⅠPCR体系如下表5所示:
表5反应体系
PCR程序如下表6所示:
表6反应程序
2、结果分析
从定量软件中导出原始数据,通过计算ΔCT,ΔΔCT,2-ΔΔCT值,比较CNDP1在人乳腺癌细胞系和对照细胞系(MCF-10A)中的表达水平,结果显示,CNDP1在人乳腺癌细胞系(MDA-MB-453/468)中表达显著高于对照细胞系,如图2所示。
qPCR的结果与外显子组测序结果一致。
2、Westernblot检测
检测MCF10A和乳腺癌细胞系MCF-7、MDA-MB-231、MDA-MB-453、MDA-MB-468中CNDP1蛋白的表达量。
收集80%汇合度时细胞,离心后弃上清,PBS润洗两次,弃上清。加入RIPA裂解液,冰上裂解20min。12000g离心10min收集上清。加入1xSDS上样缓冲液,吹打混匀后煮沸变性5min。10%SDS-PAGE凝胶分离总蛋白,然后转移到PVDF膜。5%BSA室温封闭2h,与CNDP1抗体(abcam)4℃孵育过夜,TBST洗涤3次。二抗室温孵育1h,TBST洗涤3次。ECL超敏化学发光液显影,经Tannon成像系统成像。以GAPDH作为内参比较不同细胞中CNDP1蛋白的表达水平。
结果如图3所示,与对照细胞相比,乳腺癌细胞系CNDP1蛋白表达明显升高。
实施例5 CNDP1蛋白在外周血中表达水平
家系2中成员6人,其中2名患有乳腺癌(姐妹关系),其他女性均未发现患病(一级亲属2名(孩子),二级亲属2名(外甥女)),采集该家系成员的外周血和4名正常健康人的对照(Con)的外周血。
血液4℃静置1h,3000g离心10min,收集上清,冰上分装后加入1xSDS上样缓冲液,吹打混匀后煮沸变性5min。10%SDS-PAGE凝胶分离总蛋白,然后转移到PVDF膜。5%BSA室温封闭2h,与CNDP1抗体(abcam)4℃孵育过夜,TBST洗涤3次。二抗室温孵育1h,TBST洗涤3次。ECL超敏化学发光液显影,经Tannon成像系统成像。以Transferrin作为内参比较不同细胞中CNDP1蛋白的表达水平。
结果显示,与家系中正常人相比,家系中乳腺癌患者外周血CNDP1蛋白表达水平明显升高;与正常对照相比,家系中其他女性成员CNDP1蛋白表达水平亦升高,同时患者一级亲属(Firstdegree relatives)外周血CNDP1表达水平高于其二级亲属(Second degreerelatives)(图4)。
细胞学实验也证明,与正常细胞(MCF-10A)相比,乳腺癌细胞中CNDP1的基因和蛋白表达水平也是显著升高的。
以上结果说明新型致病基因CNDP1在家族性乳腺癌的发生发展中发挥重要作用。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
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<120> 一种评估家族性乳腺癌风险的标记物及其应用
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Claims (10)
1.一种评估家族性乳腺癌风险的标记物,其特征在于,所述标记物为CNDP1基因或CNDP1蛋白。
2.如权利要求1所述的标记物,其特征在于,所述CNDP1基因或CNDP1蛋白在乳腺癌样本中表达上调。
3.如权利要求1所述的标记物,其特征在于,所述CNDP1基因在乳腺癌患者中发生突变,所述突变位点为NM_032649:exon6:c.G724A:p.G242R。
4.如权利要求3所述的标记物,其特征在于,所述CNDP1基因突变为错义突变。
5.一种筛选评估家族性乳腺癌风险的标志物CNDP1的方法,其特征在于,所述方法如下:
(1)通过iTRAQ联合质谱方法筛选家族性乳腺癌外周血样本中差异表达的蛋白;
(2)利用全外显子组测序技术筛选家族性乳腺癌外周血样本中差异表达的基因;
(3)通过联合iTRAQ蛋白组学数据和全外显子组测序数据,结合生物学分析,筛选到在家族性乳腺癌外周血样本中既在蛋白水平又在基因水平表达差异的CNDP1;
(4)通过RT-PCR和Westernblot进一步验证CNDP1基因或CNDP1蛋白的表达情况。
6.CNDP1基因或蛋白在制备评估、诊断或预后乳腺癌产品中的应用。
7.如权利要求6所述的应用,其特征在于,所述产品包括试剂、试剂盒或芯片。
8.一种用于测定CNDP1基因表达水平的试剂,其特征在于,所述试剂包括特异性扩增CNDP1的引物,所述引物序列如下:
正向引物如SEQ ID NO.1所示;
反向引物如SEQ ID NO.2所示。
9.如权利要求8所述的试剂,其特征在于,还包括RNA提取试剂、反转录试剂、定量PCR试剂、正常对照样本RNA。
10.用于测定样本中CNDP1基因表达水平的试剂在制备诊断或预示乳腺癌的试剂盒或试剂中的应用。
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CN109371130A (zh) * | 2018-11-19 | 2019-02-22 | 北京大学深圳医院(北京大学深圳临床医学院) | Ripor3在制备用于乳腺癌检测和治疗的生物制品中的应用 |
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Cited By (3)
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CN109182523A (zh) * | 2018-09-28 | 2019-01-11 | 北京致成生物医学科技有限公司 | 一种评估家族性乳腺癌风险的标记物及其应用 |
CN109371130A (zh) * | 2018-11-19 | 2019-02-22 | 北京大学深圳医院(北京大学深圳临床医学院) | Ripor3在制备用于乳腺癌检测和治疗的生物制品中的应用 |
CN109371130B (zh) * | 2018-11-19 | 2021-08-13 | 北京大学深圳医院(北京大学深圳临床医学院) | Ripor3在制备用于乳腺癌检测和治疗的生物制品中的应用 |
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