CN109234401B - 一种用于胃腺癌诊断的分子标志物 - Google Patents
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Abstract
本发明涉及一种用于胃腺癌诊断的分子标志物circ_PSMC3,其核苷酸序列如SEQ ID NO.1所示。通过qRT‑PCR对患者血液样本中circ_PSMC3的表达量进行检测,可实现对胃腺癌的早期诊断及预后评估,用于检测上述分子标志物的特异性引物,包括SEQ ID NO.2所示的上游引物和SEQ ID NO.3所示的下游引物。前期芯片筛选结合临床大样本验证发现,该分子标志物在胃腺癌患者中的表达水平显著低于正常人。临床检测仅需采集微量外周血,创伤性小,易被受检者接受,可成为胃腺癌患者的早诊、病情进展的判断以及预后评估的有效手段,该指标用于胃腺癌诊断的特异性强,敏感性高,结果稳定。
Description
技术领域
本发明涉及一种用于胃腺癌诊断的分子标志物,属于生物技术领域。
背景技术
胃癌是严重威胁人类健康的最常见恶性肿瘤之一,主要有两种病理分型:腺癌和其他类型。大多数胃癌是腺癌,其分布具有明显的地域差异。我国胃癌发病率在全世界处于高水平,预后较差,5年总体生存率较低。2016年临床医师癌症杂志报道,胃癌已经成为中国发病率最高的五种肿瘤之一,其发病率在男性中排第二位,女性中排第三位。
目前,胃腺癌的诊断率非常低,临床常用的诊断技术如胃镜、上消化道造影、CT等,由于较高的检查费用以及要承担一定的痛苦和风险很难普及,常规的血清学检查中肿瘤标记物如CEA等不论是敏感性特异性都表现的不尽人意,因此,寻找新型肿瘤标志物一直是肿瘤研究领域的重要课题。
环状RNA(circRNAs)是一种新的内源性非编码RNA,在20世纪90年代早期被确定为乱序外显子的转录本,其结构、功能和机制被相继报道,成为后来20年的研究热点。不同于以5'端和3'端为末端的传统线性RNA,circRNAs是缺少5'端帽和3'端poly(A)尾的特殊闭环结构,且与哺乳动物细胞中微小RNA(miRNAs)和长链非编码RNA(lncRNAs)相比,具有更高的稳定性和序列保守性,是因为它们具有核酸酶抗性。近期大量研究报道,在不同物种中circRNAs均有表达。基于生物信息学分析和高通量测序技术的迅速发展,研究人员发现了数以万计的circRNAs,发现它们参与血管病变、神经***疾病、肿瘤等疾病发生发展。CircRNAs芯片分析技术已应用于许多肿瘤,包括消化***肿瘤,神经***肿瘤,泌尿***肿瘤,头颈部肿瘤等,如下咽鳞状细胞癌、喉鳞状细胞癌、基底细胞癌、胰腺导管腺癌、食管癌、肝细胞癌、甲状腺***状癌等。然而,在胃癌中,血浆circRNAs鲜有报道。
发明内容
本发明的目的在于解决现有技术的不足,提供一种用于胃腺癌诊断的分子标志物,通过检测分子标志物的表达水平来判断患者是否患有胃腺癌或者患胃腺癌的风险,从而指导临床医生进行早期干预和治疗,提高患者的生存率和生存质量。
技术方案
一种用于胃腺癌诊断的分子标志物,所述分子标志物为circ_PSMC3,其核苷酸序列如SEQ ID NO.1所示。
环状RNA具有机构稳定、丰度度和样本特异性表达等特征,本发明人通过检测circ_PSMC3在胃腺癌患者和正常人血液样本中表达量,发现circ_PSMC3在胃腺癌患者和正常人血液样本中的表达存在差异,并且与分期相关。
用于检测上述分子标志物的特异性引物,包括SEQ ID NO.2所示的上游引物和SEQID NO.3所示的下游引物。
上游引物序列为:5′-GTTTAGGGTCCCTGCCCTTTG-3′;
下游引物序列为:5′-GTGTTGGGCTGGAAGCCATC-3′。通过对该引物的PCR产物测序,验证了引物扩增的特异性。
上述特异性引物在制备或筛选胃腺癌诊断药物中的应用。
一种胃腺癌的诊断试剂盒,包括上述特异性引物。
有益效果:本发明提供了一种用于胃腺癌诊断的分子标志物circ_PSMC3,通过qRT-PCR对患者血液样本中circ_PSMC3的表达量进行检测,可实现对胃腺癌的早期诊断及预后评估,前期芯片筛选结合临床大样本验证发现,该circ_PSMC3分子标志物在胃腺癌患者中的表达水平显著低于正常人,且其表达水平与临床分期及病人生存期高度相关。临床检测仅需采集微量外周血(5ml),创伤性小,更易被受检者接受,更可成为胃腺癌患者的早诊、病情进展的判断以及预后评估的有效手段,通过ROC曲线分析,AUC值为0.9326,P<0.0001,反映了该指标用于胃腺癌诊断的特异性强,敏感性高。结果稳定,具有广泛的临床应用前景。
附图说明
图1为circ_PSMC3的结构示意图;
图2为circ_PSMC3对胃腺癌患者与健康人群的ROC曲线;
图3为qRT-PCR检测circ_PSMC3在胃腺癌患者血浆和健康人群血浆中的表达水平;
图4为circ_PSMC3在胃腺癌患者癌组织和癌旁组织中的表达水平;
图5为生存曲线分析circ_PSMC3表达水平与患者生存率的相关性。
具体实施方式
下面结合附图和具体实施例对本发明作进一步说明。
实施例1
1.病例选择
研究患者组织及血液来源于2013年至2016年8月期间在南京医科大学附属南京医院接受根治或姑息性手术切除的胃腺癌患者。正常对照血液样本均来源于南京市第一医院健康体检人群,健康对照人群排除恶性肿瘤病史以及胃肠道相关疾病病史。本实验所有标本均获得患者或其委托人签署知情同意,所有涉及人体标本的研究均获得南京市第一医院伦理委员会批准。
研究患者纳入标准如下:
a.经术后病理学证实为胃腺癌(按照第7版(2009年)胃腺癌TNM分期标准进行临床分期);
b.住院期间接受根治或姑息性切除的患者,同时排除术前放化疗者;
c.现病史、家族史、个人史以及各种检查资料均完善。
本研究中GC癌症患者血样106例,正常人血样21例。
临床资料的收集主要分为胃腺癌患者和正常人两部分,均需包含一般资料:包括患者的姓名、性别、年龄、职业、既往病史、家族史、女性月经史以及婚育史等。胃腺癌患者主要由胃腺癌患者的住院资料整理而来。主要包括上述一般资料外,还应包括临床特征,包括肿瘤的组织学类型、部位、大小、浸润程度、***有无转移,肿瘤细胞分化程度,肿瘤TNM分期,手术时间,术后有无放化疗以及常规辅助检查等资料。同时获得所有人的随访同意,检查结果资料完善保存。
2.标本收集
是血液的收集,每位胃腺癌患者术前采血一次(5ml/管)用于后续实验研究。所有血液样本应避免发生溶血。其中血浆样本收集采血管为乙二胺四乙酸二钠(Ethylenediaminetetraacetic acid,EDTA)抗凝管,全血样本应在0.5h内完成血浆的离心收集冻存;血清样本收集采血管为常规生化促凝管,样本应在2h内完成血清的析出离心冻存。全血样本通过两步离心法以去除细胞成分影响,首先在2000g,4℃,离心10min,随后小心转移上清至新的EP管中,然后12000g,4℃,离心10min,最后完成血浆及血清样本分装(400μl/管),放入-80℃冰箱长期保存。
3.总RNA提取
血浆、血清总RNA提取使用mirVana PARIS Kit(Ambion1566,USA)试剂盒,具体步骤如下:
1)所有试剂均需恢复到室温下使用,取出血浆/血清样本冰面上融化备用,洗脱液95℃预热备用;
2)400μl血浆/血清样本加入已经添加等体积的2X Denaturing Solution的EP管中,充分震荡混匀;
3)加入等体积的Acid-Phenol:Chloroform,震荡混匀1min,随后室温下10000g离心5min;
4)小心转移上层水相至新的EP管,并记录转移水相体积,随后添加1.25倍体积100%乙醇至EP管中,充分混匀后转移裂解液/乙醇混合物通过滤器(10000g离心30s);
5)加700μl mi RNA Wash Solution 1清洗滤器1次(10000g离心15s),随后加入500μl Wash Solution 2/3清洗滤器2次(10000g离心15s);
6)50μl 95℃预热的无核糖核酸酶水洗脱滤器上的RNA(10000g离心30s),-80℃长期保存;
7)紫外分光光度计测定样本浓度和纯度。
4.RNA反转录和qPCR扩增
(1)逆转录体系的配置
①从-20℃冰箱中取出逆转录试剂盒,将2种酶***冰盒冰中,其它试剂冰上融化,RNase Free H2O常温融化备用。
②根据所测RNA浓度,计算逆转录每管所需RNA体积,并同时计算出DEPC水体积。RNA与DEPC水总体积不能超过7ul。注意:RNA反复冻融极易降解,一旦确定样本提取的RNA质量较好,应按照癌与癌旁配对标准尽可能的将RNA一次性全部逆转录成cDNA。如配对样本的RNA量大,超过10份逆转录的量,考虑到逆转录的成本,可仅按10份的量做逆转录。
③取PCR管架置于冰上,按需要放入无菌无酶200ul PCR管并按编号标记。移液器吸取适量RNA后,将枪头垂直***PCR管底,匀速轻柔打出液体,可减少甚至不产生气泡,完后确认枪头无明显残留。
④移液器吸取适量DEPC水,枪头贴着后壁打入PCR管内。因为之前的样本RNA是垂直加入管底,那么管四周侧壁均可视为未污染区,可以自己习惯的任何角度,贴着八联管前/后/左/右侧壁缓慢匀速打出液体,可减少甚至不产生气泡。
⑤逆转录Step1:每管加入gDNAEraser 1ul及5x gDNA Eraser Buffer 2ul。按步骤4操作,分别贴不同方向侧壁加入。
⑥置于八联管离心机上,轻轻弹掉管底或管壁气泡,短时离心。
⑦RT-PCR仪上42℃2分钟,反应后产物置于冰上备用。
⑧逆转录Step2:取1.5ml EP管,按照以下配方,配置每管反应
5x PrimerScript Buffer2 4ul
Rnase Free dH2O 4ul
PrimerScript RT Enzyme Mix I 1ul
RTPrimer Mix 1ul
设定反应条件:37℃15min,85℃5sec,4℃维持。
⑨扣紧管盖,将其从管架上取下来,置于离心管架上,有气泡者可轻弹管底,短时离心,确认每管的液平一致且管内无气泡后,复置于冰上。
⑩上机操作,设置反应条件,15-20min后将cDNA置于-20℃保存。
(2)QPCR扩增
用于检测分子标志物circ_PSMC3的特异性引物,包括上游引物和下游引物,上游引物序列为:5′-GTTTAGGGTCCCTGCCCTTTG-3′(SEQ ID NO.2),下游引物序列为:5′-GTGTTGGGCTGGAAGCCATC-3′(SEQ ID NO.3)。
以甘油醛3-磷酸脱氢酶(GAPDH)用作内部对照,GAPDH_F:5′-TGCACCACCAACTGCTTAGC-3′,GAPDH_R:5′-GGCATGGACTGTGGTCATGAG-3′。每个样本设置3个平行管,所有扩增反应均重复三次以上以保证结果的可靠性。
①准备好冰盒,从-20℃冰箱中取出待测样本的cDNA、引物、PCR试剂(SYBR PremixEx TaqTM II、ROC Reference Dye II),冰上解冻。
②备好八联管及管盖、八联管架、镊子、无菌1.5ml EP管、EP管架、适宜量程的移液器(2ul、20ul为必用量程)、无菌薄膜手套。
③将解冻好的cDNA、引物、PCR试剂,先依次放在振荡器上短时振荡,再放到离心机上短暂离心,冰上静置备用。
④取1.5ml EP管,按以下每孔配方配制八联管PCR Mix(按照19ul总体积配):
SYBR Premix Ex TaqTM II 10ul
Forward Primer
1ul*3为简化操作可配制成混合引物
Reverse Primer
ROC Reference Dye/II 0.4ul*4
cDNA 1ul
dH2O 7.6ul
反应条件:
ABI 7300/7500 Real-Time PCR System,StepOnePlusTM
Stage1:95℃30sec Reps 1
Stage2:95℃5sec,60℃30-34sec,Reps 40
Stage3:95℃15sec,60℃1min,95℃5sec,Reps 1
注意:Stage2中60℃时间设置:7500 Fast Real-Time PCR System/StepOnePlusTM:30 sec,7300:31 sec,7500:34 sec。
⑤按预设好每个样本及复孔在八联管的位置,将2ul移液器量程调节至1ul,依次加入样本的cDNA。
⑥关掉日光灯,屏蔽所有明亮光源,将ROC Reference Dye/II取相应的量加入到之前静置的承装PCR Mix的EP管中,后震荡混匀,再短时离心,以确保分装的Mix是混匀的。
⑦将20ul移液器量程调节至19ul,吸取Mix再依次贴着八联管后壁打出液体,每次打完后确认枪头无明显残留。
⑧用镊子夹取八联管盖一侧边缘,轻轻置于管口。戴新的无菌薄膜手套,轻轻将八联管连同八联管架一起从冰盒中取出置于桌面,轻压一侧管盖固定,再迅速用力扣紧管盖。记号笔于管盖两端做好标记,再由下至上轻扣八联管两端底部,将其从管架上取下来,置于八联离心架上,有气泡者可轻弹管底,短时离心,确认每管的液平一致且管内无气泡后,置于冰上。
⑨上机操作,设置反应条件,加跑溶解曲线,通过融解曲线分析和电泳确定目的条带,ΔΔCT法进行相对定量。
5.数据分析及结果
采用t检验和卡方检验及生存分析的方法circ_PSMC3与GC患者临床分期资料及预后的相关性。进行受试者工作特征(ROC)曲线来评估其诊断价值。所有统计分析均使用SPSSfor Windows v.17.0进行。对于所有结果,P<0.05被认为有统计学意义。
图1为circ_PSMC3的结构示意图,可以看出,circ_PSMC3来自PSMC基因,长度为502bp。
图2为circ_PSMC3对胃腺癌患者与健康人群的ROC曲线,可以看出,AUC为0.9326(95%CI=0.8862–0.9791,特异性为95.24%,敏感性为85.85%,P<0.0001),这说明circ_PSMC3应用于胃腺癌的诊断具有较高的准确性,其可作为胃腺癌患者诊断的分子标志物。
图3为qRT-PCR检测circ_PSMC3在胃腺癌患者血浆和健康人群血浆中的表达水平(106例胃腺癌患者血浆和21例健康人群血浆),可以看出,Circ_PSMC3在胃癌患者血浆中表达水平低于健康人群,淋巴转移胃癌患者表达低于未转移胃癌患者。
图4为circ_PSMC3在胃腺癌患者癌组织和癌旁组织中的表达水平(106例胃腺癌患者癌组织和癌旁组织),可以看出,circ_PSMC3在胃癌患者癌组织表达显著低于对应癌旁组织。
对106例胃腺癌患者预后生存曲线进行分析,图5为生存曲线分析circ_PSMC3表达水平与患者生存率的相关性,可以看出,在胃腺癌患者中,Circ_PSMC3表达水平和预后生存率呈负相关,说明circ_PSMC3可以作为胃腺癌患者诊断及预后评估的标志物。
序列表
<110> 曹红勇
<120> 一种用于胃腺癌诊断的分子标志物
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 502
<212> DNA
<213> 环状RNa(circ_PSMC3)
<400> 1
ctttgacagt gagaaggctg gggaccggga ggtgcagagg acaatgctgg agcttctgaa 60
ccagctggat ggcttccagc ccaacaccca agttaaggta attgcagcca caaacagggt 120
ggacatcctg gaccccgccc tcctccgctc gggccgcctt gaccgcaaga tagagttccc 180
gatgcccaat gaggaggccc gggccagaat catgcagatc cactcccgaa agatgaatgt 240
cagtcctgac gtgaactacg aggagctggc ccgctgcaca gatgacttca atggggccca 300
gtgcaaggct gtgtgtgtgg aggcgggcat gatcgcactg cgcaggggtg ccacggagct 360
cacccacgag gactacatgg aaggcatcct ggaggtgcag gccaagaaga aagccaacct 420
acaatactac gcctagggca cacaggccag ccccagtctc acggctgaag tgcgcaataa 480
aagatggttt agggtccctg cc 502
<210> 2
<211> 21
<212> DNA
<213> 环状RNa(circ_PSMC3)
<400> 2
gtttagggtc cctgcccttt g 21
<210> 3
<211> 20
<212> DNA
<213> 环状RNa(circ_PSMC3)
<400> 3
gtgttgggct ggaagccatc 20
Claims (2)
1.分子标志物circ_PSMc3在制备胃腺癌诊断试剂盒中的应用,所述circ_PSMc3的核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所述的应用,其特征在于,所述胃腺癌诊断试剂盒包含用于检测分子标志物circ_PSMc3的特异性引物,所述特异性引物为如SEQ ID NO.2所示的上游引物和SEQID NO.3所示的下游引物。
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