CN110358831B - 检测跨膜蛋白41a表达水平的试剂的应用和试剂盒 - Google Patents

检测跨膜蛋白41a表达水平的试剂的应用和试剂盒 Download PDF

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CN110358831B
CN110358831B CN201910624164.4A CN201910624164A CN110358831B CN 110358831 B CN110358831 B CN 110358831B CN 201910624164 A CN201910624164 A CN 201910624164A CN 110358831 B CN110358831 B CN 110358831B
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杜欣娜
李春明
戚家峰
胡明
凌存宝
张虎
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Abstract

本发明属于生物技术领域，涉及检测跨膜蛋白41A表达水平的试剂的应用和试剂盒。更具体地，涉及检测TMEM41A表达水平的试剂在制备用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的制剂中的应用以及一种用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的试剂盒。临床样本检测结果显示，乳腺癌中TMEM41A表达较癌旁组织显著升高；且TMEM41A高表达不利于乳腺癌患者总体生存。因此，检测该基因表达变化的试剂可用于乳腺癌预后或者诊断、治疗。

Description

检测跨膜蛋白41A表达水平的试剂的应用和试剂盒
技术领域
本发明属于生物技术领域，更具体地，涉及检测跨膜蛋白41A(TMEM41A)表达水平的试剂在制备用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的制剂中的应用以及一种用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的试剂盒。
背景技术
乳腺癌是最为常见的妇科恶性肿瘤。全球肿瘤流行病统计数据显示，2012年世界范围内乳腺癌新发病例总数1677000例，是仅次于肺癌的第二高发肿瘤，也是女性发病率和死亡率最高的恶性肿瘤。我国乳腺癌发病率高达25.89/10万，约占所有女性恶性肿瘤发病率的16.83％。而且，我国乳腺癌患病数和新诊断患者数在全世界的比例正在逐年增加，且呈现出发病年轻化趋势，严重影响女性的身心健康、甚至危及生命。因此，乳腺癌发病机制和预后的研究对乳腺癌患者意义深远。乳腺癌在细胞起源、组织学形态、疾病分级、临床表现、治疗反应以及转移潜能等方面都表现出极大的复杂性与异质性，限制了乳腺癌预后标志物应用的广泛性。因此，迫切需要开发更有针对性的乳腺癌预后标志物，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乳腺癌发生和预后有关，这使得更准确、有效地诊疗乳腺癌成为可能。
发明内容
本发明的目的是提供一种新的乳腺癌预后标志物跨膜蛋白41A(TMEM41A)，由此进一步提供检测TMEM41A表达水平的试剂在制备用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的制剂中的应用以及一种用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的试剂盒。
TMEM41A，是一种蛋白编码基因，定位于3q27.2，包含8个外显子，Gene ID:90407，其编码蛋白质主要定位于细胞膜。目前，有关TMEM41A基因在乳腺癌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(Cancer Genome Atlas，TCGA)高通量数据挖掘发现TMEM41A高表达不利于乳腺癌患者预后，并通过收集临床乳腺癌患者样本和随访信息，进一步验证TMEM41A表达差异对乳腺癌患者的预后作用。
为了实现上述目的，本发明的第一方面提供检测跨膜蛋白41A(TMEM41A)表达水平的试剂在制备用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的制剂中的应用。
进一步地，所述检测TMEM41A表达水平包括检测TMEM41A的基因表达水平和/或检测TMEM41A的蛋白表达水平。
更具体地，所述检测TMEM41A表达水平的方法包括：通过RT-qPCR方法检测乳腺癌和癌旁组织中TMEM41A的表达量；通过分子探针技术检测乳腺癌和癌旁组织中TMEM41AmRNA表达量；通过免疫组化或Western-Blot检测乳腺癌和癌旁组织中TMEM41A蛋白表达量。
更具体地，所述检测TMEM41A表达水平的试剂为靶向TMEM41A编码DNA序列的寡核酸探针、PCR引物，或者为靶向TMEM41A的抗体。
根据本发明，优选地，所述检测TMEM41A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。
5’-GCTGTGCTGTGTGTTGACCT-3’，SEQ ID NO：1；
5’-GCAGGGCCACTTTATCAGGAA-3’，SEQ ID NO：2。
本发明的第二方面提供一种用于乳腺癌辅助诊断和/或乳腺癌患者预后判断的试剂盒，该试剂盒包括：检测TMEM41A表达水平的试剂。
进一步地，所述检测TMEM41A表达水平的试剂为靶向TMEM41A编码DNA序列的寡核酸探针、PCR引物，或者为靶向TMEM41A的抗体。
具体地，所述检测TMEM41A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。
根据本发明，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，优选地，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。
5’-GGCACCCAGCACAATGAAGA-3’，SEQ ID NO：3；
5’-ACTCCTGCTTGCTGATCCAC-3’，SEQ ID NO：4。
临床样本检测结果显示，乳腺癌中TMEM41A表达较癌旁组织显著升高(P<0.001)；且TMEM41A高表达不利于乳腺癌患者总体生存(P<0.001)。因此，检测该基因表达变化的试剂可用于乳腺癌预后或者诊断、治疗。
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示出了TCGA高通量数据分析乳腺癌中TMEM41A的表达。乳腺癌组织中TMEM41A表达显著高于正常组织(P<0.001)。
图2示出了TCGA高通量数据分析TMEM41A高表达对乳腺癌患者生存的影响。TMEM41A高表达不利于乳腺癌患者总体生存(P＝0.00338)。
图3示出了乳腺癌组织中TMEM41A的表达。乳腺癌组织中TMEM41A表达显著高于癌旁组织(P<0.001)。
图4示出了乳腺癌组织中TMEM41A高表达对乳腺癌患者生存的影响。TMEM41A高表达不利于乳腺癌患者总体生存(P<0.001)。
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高通量数据分析乳腺癌中TMEM41A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“TMEM41A”，选择TCGAdataset“Breast invasivecarcinoma”，检索，点击“Expression”，记录结果。利用GraphPad 12.0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：乳腺癌组织中TMEM41A的表达较正常组织显著升高(P<0.001)，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TMEM41A高表达与乳腺癌预后关系。
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“Breast Invasive Carcinoma(TCGAProvisional)”，组学数据选择“mRNAExpression z-Scores(RNASeq V2RSEM)”，基因输入“TMEM41A：EXP>＝2”，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：TMEM41A高表达不利于乳腺癌患者总体生存(P＝0.00338)(图2)。
实施例3
本实施例用于说明制备检测TMEM41A表达量的试剂用于制备乳腺癌患者预后的试剂盒(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/μl M-MLV逆转录酶50.0μl；
11.ABI 2×PCR Mix 2.0ml；
12.10.0μM TMEM41A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
Figure BDA0002126510090000061
实施例4
本实施例用于说明临床乳腺癌组织样本TMEM41A的检测。
1.本项研究在患者知情同意下进行。57例乳腺癌患者临床信息来自患者就诊记录。乳腺癌标本分为两部分：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。
RNA(mg/ml)＝40×OD260×稀释倍数(n)/1000
3.反转录：每25μl反转录体系包括100pmol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，ddH2O 8.2μl。反应条件为：94℃2min，94℃15s，60℃40s，40个循环。
5.2-ΔΔCT法计算TMEM41A相对表达量：本实验检测57例乳腺癌组织和18例癌旁组织中TMEM41A的相对表达量变化。ACTB作为内参基因，将qPCR测得的靶基因TMEM41A 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(取癌旁样本ΔCT的平均值为ΔCT对照)，利用Excell表格中Power函数计算每组TMEM41A相对表达量。利用软件GraphPad Prism 6.0绘图，T检验分析乳腺癌和癌旁TMEM41A表达差异，P<0.05为差异具有统计学意义。
6.TMEM41A高表达与乳腺癌患者预后：患者随访时间为1-32个月，成功接受随访患者数为57例。TMEM41A相对表达量高于癌旁组织相对表达量均数2倍的为高表达，共7例，其他为TMEM41A低表达，共50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，乳腺癌中TMEM41A表达较癌旁组织显著升高(P<0.001)，如图3所示；且TMEM41A高表达不利于乳腺癌患者总体生存(P<0.001)，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> 检测跨膜蛋白41A表达水平的试剂的应用和试剂盒
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Claims (5)

1.检测TMEM41A表达水平的试剂在制备用于乳腺癌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TMEM41A表达水平包括检测TMEM41A的基因表达水平和/或检测TMEM41A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TMEM41A表达水平的方法包括：通过RT-qPCR方法检测乳腺癌和癌旁组织中TMEM41A的表达量；通过分子探针技术检测乳腺癌和癌旁组织中TMEM41A mRNA表达量；通过免疫组化或Western-Blot检测乳腺癌和癌旁组织中TMEM41A蛋白表达量。
4.根据权利要求1所述的应用，其中，所述检测TMEM41A表达水平的试剂为靶向TMEM41A编码DNA序列的寡核酸探针、PCR引物，或者为靶向TMEM41A的抗体。
5.根据权利要求4所述的应用，其中，所述检测TMEM41A表达水平的试剂为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。
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