CN114990217A - 检测17号染色体开放阅读框42表达水平的试剂在皮肤黑色素瘤中的应用 - Google Patents
检测17号染色体开放阅读框42表达水平的试剂在皮肤黑色素瘤中的应用 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及检测C17ORF42表达水平的试剂的应用和试剂盒。更具体地,涉及检测C17ORF42表达水平的试剂在制备用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的制剂中的应用以及一种用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的试剂盒。临床样本检测结果显示,皮肤黑色素瘤中C17ORF42表达较瘤旁组织显著升高;且C17ORF42高表达不利于皮肤黑色素瘤患者总体生存。检测该基因表达变化的试剂可用于皮肤黑色素瘤预后或者诊断、治疗。
Description
技术领域
本发明属于生物技术领域,更具体地,涉及检测C17ORF42表达水平的试剂在制备用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的制剂中的应用,以及一种用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的试剂盒。
背景技术
黑色素瘤,通常是指恶性黑色素瘤,是黑色素细胞来源的一种高度恶性的肿瘤,简称恶黑,多发生于皮肤。皮肤黑色素瘤占皮肤恶性肿瘤的第三位(约占6.8%~20%),好发于成人,皮肤白皙的白种人发病率高,而深色皮肤的亚洲人和非洲人发病率较低,极少见于儿童。近年来,恶性黑色素瘤的发生率和死亡率逐年升高,与其他实体瘤相比,其致死年龄更低。恶性黑色素瘤除早期手术切除外,缺乏特效治疗,预后差。因此,恶性黑色素瘤的早期诊断和治疗极其重要。
17号染色体开放阅读框42,又名C17ORF42,其编码蛋白质定位于细胞质和细胞核。目前,有关C17ORF42基因在皮肤黑色素瘤发生发展中的作用未见报道。本发明的发明人通过癌症和肿瘤基因图谱(The Cancer Genome Atlas,TCGA)高通量数据挖掘发现C17ORF42高表达不利于皮肤黑色素瘤患者预后,并通过收集临床皮肤黑色素瘤患者样本和随访信息,进一步验证C17ORF42表达差异对皮肤黑色素瘤患者的预后作用。
基于以上论述,迫切需要开发更有针对性的皮肤黑色素瘤预后标志物以满足临床需求。随着多组学高通量数据的快速增加,一些分子生物学标志物被发现与皮肤黑色素瘤发生和预后有关,这使得更准确、有效地诊疗皮肤黑色素瘤成为可能。
发明内容
本发明的目的就是发掘一种新的皮肤黑色素瘤预后标志物C17ORF42,由此进一步提供检测C17ORF42表达水平的试剂在制备用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的制剂中的应用以及一种用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的试剂盒。
本发明第一方面是面提供检测C17ORF42表达水平的试剂在制备用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的制剂中的应用。
具体地,所述C17ORF42表达水平包括检测C17ORF42的基因表达水平和/或检测C17ORF42的蛋白表达水平。
更具体地,所述检测C17ORF42表达水平的方法包括:RT-qPCR方法检测皮肤黑色素瘤和瘤旁组织中C17ORF42的表达量;通过分子探针技术检测皮肤黑色素瘤和瘤旁组织中C17ORF42 mRNA表达量;通过免疫组化或Western-Blot检测皮肤黑色素瘤组织中C17ORF42蛋白表达量变化。
更具体地,所述检测C17ORF42表达水平的试剂为靶向C17ORF42编码DNA序列的寡核酸探针、PCR引物,或者为靶向C17ORF42的抗体。
更具体地,所述检测C17ORF42表达水平的试剂为具有SEQ ID NO:1和SEQID NO:2所示核苷酸序列的实时荧光定量PCR特异性引物。
SEQ ID NO:1为5'-GTATATGCTCGAGAGAAGATGG-3'。
SEQ ID NO:2为5'-CTTGAGAGCACTCTAGATGG-3'。
本发明第二方面提供一种用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的试剂盒,该试剂盒包括:检测C17ORF42表达水平的试剂。
具体地,所述检测C17ORF42表达水平的试剂为靶向C17ORF42编码DNA序列的寡核酸探针、PCR引物,或者为靶向C17ORF42的抗体。
更具体地,所述检测C17ORF42表达水平的试剂为具有SEQ ID NO:1和SEQ ID NO:2所示核苷酸序列的实时荧光定量PCR特异性引物。
更具体地,所述试剂盒还可以含有其他常规的用于实时荧光定量的试剂。
更具体地,所述试剂盒还包括以下组分中的至少一种:Trizol、异丙醇、氯仿、无水乙醇、无RNA酶水、随机引物、5×M-MLV缓冲液、dNTPs、RNA酶抑制剂、M-MLV逆转录酶、具有SEQ ID NO:3和SEQ ID NO:4所示核苷酸序列的ACTB实时荧光定量PCR特异性引物。
SEQ ID NO:3为5'-GGCACCCAGCACAATGAAGA-3'。
SEQ ID NO:4为5'-ACTCCTGCTTGCTGATCCAC-3'。
临床样本检测结果显示,皮肤黑色素瘤中C17ORF42表达较瘤旁组织显著升高;且C17ORF42高表达不利于皮肤黑色素瘤患者总体生存。检测该基因表达变化的试剂可用于皮肤黑色素瘤预后或者诊断、治疗。
本发明的有益效果在于:发掘一种新的皮肤黑色素瘤预后标志物C17ORF42,由此进一步提供检测C17ORF42表达水平的试剂在制备用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的制剂中的应用以及一种用于皮肤黑色素瘤辅助诊断和/或皮肤黑色素瘤患者预后判断的试剂盒。
本发明的其它特征和优点将在随后具体实施方式部分予以详细说明。
附图说明
通过结合附图对本发明示例性实施方式进行更详细的描述,本发明的上述以及其它目的、特征和优势将变得更加明显。
图1为TCGA高通量数据分析皮肤黑色素瘤中C17ORF42的表达。皮肤黑色素瘤组织中C17ORF42表达显著高于正常组织。
图2为TCGA高通量数据分析C17ORF42高表达对皮肤黑色素瘤患者生存的影响。C17ORF42高表达不利于皮肤黑色素瘤患者总体生存。
图3为皮肤黑色素瘤组织中C17ORF42的表达。皮肤黑色素瘤组织中C17ORF42表达显著高于正常组织。
图4为皮肤黑色素瘤组织中C17ORF42高表达对皮肤黑色素瘤患者生存的影响。C17ORF42高表达不利于皮肤黑色素瘤患者总体生存。
具体实施方式
下面将更详细地描述本发明的优选实施方式。虽然以下描述了本发明的优选实施方式,然而应该理解,可以以各种形式实现本发明而不应被这里阐述的实施方式所限制。
实施例中未注明具体条件者,皆按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例1:本实施例用于说明TCGA高通量数据分析皮肤黑色素瘤中C17ORF42的表达变化。
1、TCGA高通量数据分析过程。
登录TCGA门户网站UALCAN(http://ualcan.path.uab.edu/index.html)首页,点击“Analysis”,输入基因名称“C17ORF42”,选择TCGA dataset下的“Skin CutaneousMelanoma”进行检索,点击“Expression”,记录结果。利用GraphPad 6.0软件作图,统计方法为T检验,P<0 .05为差异有统计学意义。
2、结果。
皮肤黑色素瘤组织中C17ORF42的表达较正常组织显著升高,如图1所示。
实施例2:本实施例用于说明TCGA高通量数据分析C17ORF42高表达与皮肤黑色素瘤预后关系。
1、TCGA高通量数据分析过程。
登录TCGA门户网站cBioPortal(http://www.cbioportal.org/),选择肿瘤数据集“Skin Cutaneous Melanomas”,组学数据选择“mRNA Expression z-Scores(RNA Seq V2RSEM)”,基因输入“C17ORF42:EXP>=2”,然后进行检索,弹出页面中点击“Survival”,记录结果。Kaplan-Meier法绘制生存曲线,log-rank检验生存曲线差异,P<0 .05为差异有统计学意义。
2、结果。
C17ORF42高表达不利于皮肤黑色素瘤患者总体生存,如图2所示。
实施例3:本实施例用于说明制备检测C17ORF42表达量的试剂,用于制备皮肤黑色素瘤患者预后的试剂盒(50次反应)。
1)Trizol 50.0mL;
2)异丙醇50.0mL;
3)氯仿50.0mL;
4)无水乙醇50.0mL;
5)无RNA酶水5.0mL
6)1.0μM随机引物(Random primers)50.0μL;
7)5×M-MLV缓冲液2.0mL;
8)10.0mM三磷酸碱基脱氧核苷酸(dNTPs)100.0μL;
9)40U/μL RNA酶抑制剂50.0μL;
10)200U/μLM-MLV逆转录酶50.0μL;
11)ABI 2×PCR Mix 2.0ml;
12)10.0μM C17ORF42实时荧光定量PCR特异性引物30.0μL,引物序列见表1;
13)10.0μM ACTB实时荧光定量PCR特异性引物30.0μL,引物序列见表1。
表1:荧光定量RT-PCR引物序列。
实施例4:本实施例用于说明临床上皮肤黑色素瘤组织样本中C17ORF42的检测。
1、标本制备。
本项研究在患者知情同意下进行。28例皮肤黑色素瘤患者临床信息来自患者就诊记录。皮肤黑色素瘤标本分为两部分:一部分在液氮中立即冻结,保存在-80℃直到进行RNA提取,另一部分则用于组织病理学评估。
2、组织中总RNA提取。
本实验在冰浴中进行。取30~50mg组织(新鲜或-70℃及液氮中保存的组织均可)置1.5mL离心管中,加入1mL Trizol充分匀浆,室温静置5min;每管加入200μL氯仿,剧烈混匀30sec,静置15min,4℃12000rpm离心15min;轻轻吸取上层液体400μL至另一新离心管中,加入等体积异丙醇,轻轻颠倒混匀,4℃12000rpm离心10min;弃上清,加入1mL 75%酒精洗涤沉淀物,4℃12000rpm离心10min;尽可能弃掉上清,室温下晾干10min,每管加入10μL无RNA酶水,溶解(65℃促溶10~15min)。测定OD260,计算RNA浓度。
3、反转录。
每25μL反转录体系包括100p moL随机引物,2μg总RNA,M-MLV反转录酶1μL,RNase抑制剂0.625μL,dNTPs(10mM)1.25μL,5×M-MLV缓冲液5μL,无RNA酶水补齐至25μL。反应条件为:37℃1h,95℃5min。
4、定量PCR。
每20μL反应体系包含2×PCR Mix 10μL,上、下游引物各0.4μL,cDNA 1μL,ddH2O8.2μL。反应条件为:94℃2min,94℃15s,60℃40s,40个循环。
5、2-ΔΔCT法计算C17ORF42相对表达量。
本实验检测28例皮肤黑色素瘤组织和6例瘤旁组织中C17ORF42的相对表达量变化。ACTB作为内参基因,将qPCR测得的靶基因C17ORF42 CT值与同组织来源的内参基因ACTB的CT值相减得到ΔCT,再将ΔCT与对照组ΔCT相减得到ΔΔCT(取瘤旁样本ΔCT的平均值为ΔCT对照),利用Excell表格中Power函数计算每组C17ORF42相对表达量。利用软件GraphPad Prism 6.0绘图,T检验分析皮肤黑色素瘤和瘤旁C17ORF42表达差异,P<0 .05为差异具有统计学意义。
6、C17ORF42高表达与皮肤黑色素瘤患者预后。
患者随访时间为1~40个月,成功接受随访患者数为28例。C17ORF42相对表达量高于瘤旁组织相对表达量均数2倍的为高表达,共12例,其他为C17ORF42低表达,共16例。Kaplan-Meier法绘制生存曲线,log-rank检验生存曲线差异,P<0 .05为差异有统计学意义。
7、结果。
临床样本检测结果显示,皮肤黑色素瘤中C17ORF42表达较瘤旁组织显著升高,如图3所示;且C17ORF42高表达不利于皮肤黑色素瘤患者总体生存,如图4所示。
以上已经描述了本发明的各实施例,上述说明是示例性的,并非穷尽性的,并且也不限于所披露的各实施例。在不偏离所说明的各实施例的范围和精神的情况下,对于本技术领域的普通技术人员来说许多修改和变更都是显而易见的。
序列表
<110> 江苏医药职业学院
<120> 检测17号染色体开放阅读框42表达水平的试剂在皮肤黑色素瘤中的应用
<141> 2022-06-02
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 1
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<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2
cttgagagca ctctagatgg 20
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 3
ggcacccagc acaatgaaga 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 4
actcctgctt gctgatccac 20
Claims (5)
1.检测17号染色体开放阅读框42(C17ORF42)表达水平的试剂在制备用于皮肤黑色素瘤患者预后判断的制剂中的应用。
2.根据权利要求1所述的应用,其中,所述检测C17ORF42表达水平包括检测C17ORF42的基因表达水平和/或检测C17ORF42的蛋白表达水平。
3.根据权利要求1所述的应用,其中,所述检测C17ORF42表达水平的方法包括:RT-qPCR方法检测皮肤黑色素瘤和瘤旁组织中C17ORF42的表达量;通过分子探针技术检测皮肤黑色素瘤和瘤旁组织中C17ORF42 mRNA表达量;通过免疫组化或Western-Blot检测皮肤黑色素瘤组织中C17ORF42蛋白表达量变化。
4.根据权利要求1所述的应用,其中,所述检测C17ORF42表达水平的试剂为靶向C17ORF42编码DNA序列的寡核酸探针、PCR引物,或者为靶向C17ORF42的抗体。
5.根据权利要求4所述的应用,其中,所述检测C17ORF42表达水平的试剂为具有SEQIDNO:1和SEQ ID NO:2所示核苷酸序列的实时荧光定量PCR特异性引物。
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