WO2020103794A1 - 一种用于检测浸润性膀胱癌的标志物及其应用 - Google Patents

一种用于检测浸润性膀胱癌的标志物及其应用

Info

Publication number
WO2020103794A1
WO2020103794A1 PCT/CN2019/119190 CN2019119190W WO2020103794A1 WO 2020103794 A1 WO2020103794 A1 WO 2020103794A1 CN 2019119190 W CN2019119190 W CN 2019119190W WO 2020103794 A1 WO2020103794 A1 WO 2020103794A1
Authority
WO
WIPO (PCT)
Prior art keywords
imprinted gene
expression
copy number
bladder cancer
diras3
Prior art date
Application number
PCT/CN2019/119190
Other languages
English (en)
French (fr)
Inventor
成彤
周宁
Original Assignee
立森印迹诊断技术有限公司
李星
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 立森印迹诊断技术有限公司, 李星 filed Critical 立森印迹诊断技术有限公司
Priority to US17/295,629 priority Critical patent/US20220002817A1/en
Publication of WO2020103794A1 publication Critical patent/WO2020103794A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present application relates to the field of biotechnology, to the field of genetic diagnosis, to a marker for detecting tumors, and in particular to a marker for detecting invasive bladder cancer and its application.
  • Bladder cancer is the most common malignant tumor of the urinary system, with the highest incidence of urinary system malignant tumors. According to statistics from the World Health Organization (WHO), in 2012, there were 429,793 new cases worldwide, 165,084 deaths, 55,486 new diagnoses in China, and 26,820 deaths (World Cancer Report 2014). 85-90% of newly diagnosed bladder cancers are non-muscle invasive bladder cancer. Although the recurrence rate after the first surgical resection is as high as 75%, muscular invasion is rare and metastasis rarely occurs. -15% is muscular invasive bladder cancer, which will eventually metastasize to a distant place, which is life-threatening.
  • WHO World Health Organization
  • Genomic imprinting is a way of gene regulation in epigenetics. It is characterized by the methylation of alleles from a specific parent, so that only one allele of a gene is expressed, while the other is in a gene silence state. Genes of this kind are called imprinted genes. Imprint deletion is an epigenetic change in which demethylation of an imprinted gene causes the silent allele to be activated and start gene expression. A large number of studies have shown that this phenomenon (deletion of imprinting) is common in various types of cancer and occurs earlier than changes in cell and tissue morphology. At the same time, in healthy cells, the percentage of blotting loss is extremely low, in stark contrast to cancer cells. Therefore, the methylation status of imprinted genes can be used as a pathological marker to analyze the abnormal state of cells through specific molecular detection techniques.
  • imprinted genes cover cell signaling, cell cycle regulation, intracellular and extracellular transport, and formation of extracellular matrix, the role of imprinted genes in different cancers is different, and the amount of expression is also very different. Because of this, different sensitivities and specificities are formed, which play a huge role in the invasion, metastasis and prognosis of tumor development.
  • the current invasive or non-invasive bladder cancer does not have a clearly distinguishable diagnostic marker, and the molecular marker changes existing at the cell level of the invasive or non-invasive bladder cancer are analyzed to provide more accuracy The pre-diagnosis and diagnosis information.
  • this application provides a marker for detecting invasive bladder cancer and its application. By using imprinted gene detection, it can distinguish between invasive and non-invasive bladder cancer. Provide a basis for subsequent treatment.
  • the present application provides a marker for invasive bladder cancer, the marker being the imprinting gene Grb10 and / or the imprinting gene Diras3.
  • the ratio of the imprinted gene Grb10 and the imprinted gene Diras3 copy number abnormal expression is used as Bladder Infiltration Factor (BIF, Bladder Infiltration Factor), to judge the infiltration of bladder cancer, the higher the infiltration factor, the greater the infiltration of bladder cancer.
  • BIF Bladder Infiltration Factor
  • the formulas for the total expression level, normal expression level, missing expression level and copy number abnormal expression level of the imprinted gene are as follows:
  • Missing expression c / (b + c + d) ⁇ 100%;
  • a is the number of nuclei in which the cells are not stained after hematoxylin staining
  • b is the red / brown mark in which the imprinted genes are present after the cells are hematoxylin stained
  • the number of nuclei present c is the number of nuclei that have red / brown marks after the cells are hematoxylin-stained
  • the d is the number of nuclei where the cells are imprinted with the gene deleted
  • d is two
  • the above red / brown mark indicates the number of nuclei with abnormal gene copy number imprinting.
  • the infiltration factor is the ratio of the abnormal expression of the imprinted gene Grb10 copy number to the abnormal expression of the imprinted gene Diras3 copy number .
  • the bladder cancer infiltrating property does not obviously mean that it is not able to distinguish clearly whether it is invasive bladder cancer, and it needs to be grown and tested again.
  • the imprinted gene Diras3 when the total expression of the imprinted gene Diras3 is not less than 8% and the abnormal expression of the copy number of the imprinted gene Diras3 is not more than 1%, if the total expression of the imprinted gene Grb10 is not less than 8%, the imprinted gene Grb10 Abnormal expression of copy number is not more than 1.5% and infiltration factor is greater than 1.5, it is judged as mixed partial invasive bladder cancer; wherein, the infiltration factor is the abnormal expression of the imprinted gene Grb10 copy number and the abnormal expression of the imprinted gene Diras3 copy number ratio.
  • the mixed partial invasive bladder cancer refers to that bladder cancer includes invasive bladder cancer and non-invasive bladder cancer, and the growth of invasive bladder cancer is faster than that of non-invasive bladder cancer.
  • the imprinted gene Grb10 when the total expression of the imprinted gene Diras3 is not less than 8% and the copy number of the imprinted gene Diras3 is abnormally expressed by more than 1%, if the total expression of the imprinted gene Grb10 is not less than 8%, the imprinted gene Grb10 Abnormal expression of copy number is greater than 1.5% and infiltration factor is not greater than 1.5 or the total expression of imprinted gene Grb10 is not less than 8%, abnormal expression of imprinted gene Grb10 is not greater than 1.5% and infiltration factor is not less than 1.5, then judge It is a mixed bladder cancer; wherein the infiltration factor is the ratio of the abnormal expression of the imprinted gene Grb10 copy number to the abnormal expression of the imprinted gene Diras3 copy number.
  • the mixed bladder cancer refers to that bladder cancer includes invasive bladder cancer and non-invasive bladder cancer. With the development of bladder cancer, it needs to be tested again.
  • the total expression of the imprinted gene Diras3 is less than 8% or the abnormal expression of the copy number of the imprinted gene Diras3 is not more than 1%
  • the total expression of the imprinted gene Grb10 is not less than 8% and the imprinted gene Grb10 Abnormal expression of copy number is greater than 1.5%, it is judged as invasive bladder cancer.
  • the infiltration factor is the imprinted gene Grb10 copy number abnormal expression Ratio of imprinted gene Diras3 copy number abnormal expression.
  • the infiltration factor is the ratio of the abnormal expression amount of the imprinted gene Grb10 copy number to the abnormal expression amount of the imprinted gene Diras3 copy number.
  • Z15 is the imprinting gene Diras3
  • Z11 is the imprinting gene Grb10.
  • the imprinting gene Grb10 is expressed in infiltrating bladder cancer cell lines, and the imprinting gene Diras3 is not expressed in infiltrating bladder cancer cell lines.
  • the infiltrating bladder cancer cell line is any one of T24 cell line, J82 cell line or UMUC3 cell line or a combination of at least two.
  • the imprinting gene Grb10 is not expressed in non-invasive bladder cancer cell lines, and the imprinting gene Diras3 is expressed in non-invasive bladder cancer cell lines.
  • the non-invasive bladder cancer cell line is 5637 cell line.
  • the detection methods of this application are different from immunohistochemistry methods, DNA sequencing methods, DNA methylation analysis, and FISH methods, which reduce false positives and other negative effects.
  • the present application provides the marker as described in the first aspect for preparing a medicine or reagent for diagnosing bladder cancer.
  • the markers of this application can accurately detect the invasiveness of bladder cancer, guide the choice of bladder cancer surgery, and can reduce the recurrence and metastasis after surgery;
  • the detection method of this application is different from the immunohistochemistry method, which reduces false positives and other negative effects. Not only that, the deletion site of the imprinting gene related to the infiltration of bladder cancer caused the gene to be silenced, deleted, and rearranged. Targeted drugs or technical methods can be used to guide later treatment and medication.
  • Figure 1 shows the results of bladder infiltration factor detection for different bladder cancer cell lines
  • Figure 2 shows the results of bladder infiltration factor detection in 17 bladder cancer samples.
  • the method for detecting imprinted genes includes the following steps:
  • Design probe design specific primers based on the imprinted gene sequence
  • the designed probes are designed according to the imprinting genes Grb10 and Diras3. Specifically, a sequence is selected as the probe in the inner spinner of each gene.
  • the specific probe is designed by Advanced Cell Diagnostics.
  • Loss of expression (LOI) c / (b + c + d) ⁇ 100%;
  • a is the number of nuclei in which the cells are not stained after hematoxylin staining
  • b is the red / brown mark in which the imprinted genes are present after the cells are hematoxylin stained
  • the number of nuclei present c is the number of nuclei that have red / brown marks after the cells are hematoxylin-stained
  • the d is the number of nuclei where the cells are imprinted with the gene deleted
  • d is two
  • the above red / brown mark indicates the number of nuclei with abnormal gene copy number imprinting.
  • the bladder infiltration factor is less than 1.5, and in the infiltrating bladder cancer cell lines UMUC3, T24, and J82, the bladder infiltration factor is greater than 1.5.
  • the tissues of 17 bladder cancer patients including cystoscopy biopsy samples were obtained, and the samples were processed as follows:
  • the bladder infiltration factor for most non-invasive bladder cancer samples is less than 1.5, and the bladder infiltration factor for most invasive bladder cancer samples is greater than 1.5%.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biomedical Technology (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

本申请提供一种用于检测浸润性膀胱癌的标志物及其应用，所述标志物为印记基因Grb10和/或印记基因Diras3。本申请标志物可以准确检测膀胱癌的浸润性，对膀胱癌的手术方式选择具有指导作用，可以减少手术后的复发和转移；本申请检测方法区别于免疫组化方法，减少了假阳性和其他负面作用，不仅如此，通过发现的膀胱癌浸润性相关印记基因缺失位点的致该基因沉默、剔除、重排的靶向药物或技术方法，可用于指导后期的治疗和用药。

Description

一种用于检测浸润性膀胱癌的标志物及其应用 技术领域
本申请涉及生物技术领域，涉及基因诊断领域，涉及一种检测肿瘤的标志物，具体涉及一种用于检测浸润性膀胱癌的标志物及其应用。
背景技术
膀胱癌是泌尿***最常见的恶性肿瘤，发病率居泌尿***恶性肿瘤的首位。据世界卫生组织(WHO)统计，2012年全球新增病例429,793例，死亡165,084例，中国新诊断55,486例，死亡26,820例(World Cancer Report 2014)。临床上新诊断的膀胱癌85-90％都是非肌层浸润性膀胱癌，虽然第一次手术切除后复发率高达75％，但较少发生肌层浸润，也很少发生转移，而另外10-15％为肌层浸润性膀胱癌，最终会发生远处转移，危及生命。对于非肌层浸润性膀胱癌，只要按时进行膀胱镜检查，发现复发后及时在膀胱镜下切除，生存率一般较高。对于肌层浸润性膀胱癌，需要进行较大范围的膀胱手术切除甚至全膀胱切除，并结合放化疗，才能得到较好的疗效。因此，如果能够在早期检测出膀胱癌是否会发生肌层浸润，对治疗方法的选择具有重要指导意义，能够挽救病人的生命。
基因组印记是表观遗传学中基因调控的一种方式。其特点是，通过甲基化来自特定亲代的等位基因，使某个基因只有一个等位基因表达，而另一个则陷入基因沉默状态。该种类的基因，被称为印迹(记)基因。印迹缺失是印迹基因去甲基化导致沉默状态的等位基因被激活并且开始基因表达的一种表观遗传改变。大量研究表明，该现象(印迹缺失)普遍存在于各类癌症并且发生时间早于细胞和组织形态改变。与此同时，在健康细胞中，印迹缺失比例极低，与 癌细胞成鲜明对比。所以，印迹基因的甲基化状态可以作为病理标记，通过特定分子检测技术，对细胞异常状态进行分析。
由于印迹基因的功能涵盖细胞信号传递、细胞周期调控、细胞内外物质运输、细胞外基质形成等多个方面，所以在不同的癌症中印迹基因所表现的作用有所不同，表达的量也相差很大，故而形成了不同的敏感性和特异性，对肿瘤发生发展中的浸润和转移及预后起了巨大的作用。
基于上述原因，目前的膀胱癌的浸润性或非浸润性并没有明显区分的诊断标志物，解析膀胱癌的浸润性或非浸润性在细胞层面上存在的分子标记物变化，以此提供更精确的预诊和诊断信息。
发明内容
针对现有技术的不足及实际的需求，本申请提供了一种用于检测浸润性膀胱癌的标志物及其应用，通过采用印记基因的检测，能够区分膀胱癌的浸润和非浸润性，为后续的治疗提供依据。
为达到上述目的，本申请采用如下技术方案：
第一方面，本申请提供一种浸润性膀胱癌的标志物，所述标志物为印记基因Grb10和/或印记基因Diras3。
本申请中，通过对印记基因Grb10和印记基因Diras3进行总表达量、印记基因缺失表达量和印记基因拷贝数异常表达量的计算，以印记基因Grb10和印记基因Diras3的拷贝数异常表达量比值作为膀胱浸润因子(BIF,Bladder Infiltration Factor)，从而判断膀胱癌的浸润性，浸润因子越高，表明膀胱癌的浸润性越大。
根据本申请，所述印记基因的总表达量、正常表达量、缺失表达量和拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常表达量＝b/(b+c+d)×100％；
缺失表达量＝c/(b+c+d)×100％；
拷贝数异常表达量＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，其中不存在标记，即印记基因没有表达的细胞核的数量；所述b为将细胞进行苏木素染色后，其中存在一个红色/棕色标记，即印记基因存在的细胞核的数量；所述c为将细胞进行苏木素染色后，其中存在两个红色/棕色标记，即印记基因缺失的细胞核的数量；所述d为将细胞进行苏木素染色后，其中存在两个以上红色/棕色标记，即印记基因拷贝数异常的细胞核的数量。
根据本申请，当所述印记基因Diras3的总表达量小于8％或印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量小于8％或印记基因Grb10拷贝数异常表达量不大于1.5％且浸润因子不大于1.5，则判断为膀胱癌浸润属性不明显；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
本申请中，所述膀胱癌浸润属性不明显指的是并不能够显著区分是否为浸润性膀胱癌，还需要让其生长并再次进行检测。
根据本申请，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10拷贝数异常表达量不大于1.5％且浸润因子大于1.5，则判断为混合偏浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
本申请中，所述混合偏浸润性膀胱癌指的是膀胱癌中包括了浸润性膀胱癌 和非浸润性膀胱癌，其中浸润性膀胱癌的生长相比于非浸润性膀胱癌快一些。
根据本申请，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量大于1.5％且浸润因子不大于1.5或印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量不大于1.5％且浸润因子不小于于1.5，则判断为混合膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
本申请中，所述混合膀胱癌指的是膀胱癌中包括了浸润性膀胱癌和非浸润性膀胱癌，随着膀胱癌的发展，还需要再次进行检测。
根据本申请，当所述印记基因Diras3的总表达量小于8％或印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量不小于8％且印记基因Grb10的拷贝数异常表达量大于1.5％，则判断为浸润性膀胱癌。
根据本申请，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量大于1％时，若印记基因Grb10的总表达量小于8％或印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量不大于1.5％且浸润因子小于1.5，则判断为非浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
根据本申请，当所述印记基因Diras3的总表达量不小于8％且拷贝数异常大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量大于1.5％且浸润因子大于1.5，则判断为浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
本申请中，上述各种膀胱癌浸润性的判断具体如下：
Figure PCTCN2019119190-appb-000001
其中，Z15为印记基因Diras3，Z11为印记基因Grb10。
本申请中，作为本领域技术人员应当知晓，随着样本量的增多，其标准也会随着浮动，所述标志物的总表达量和的拷贝数异常表达量与浸润性膀胱癌的关系会存在上下15％的浮动，其也是在本申请的范围之内的。
根据本申请，所述印记基因Grb10在浸润性膀胱癌细胞株中表达，所述印记基因Diras3在浸润性膀胱癌细胞株中不表达。
根据本申请，所述浸润性膀胱癌细胞株为T24细胞株、J82细胞株或UMUC3细胞株中的任意一种或至少两种的组合。
根据本申请，所述印记基因Grb10在非浸润性膀胱癌细胞株中不表达，所述印记基因Diras3在非浸润性膀胱癌细胞株中表达。
根据本申请，所述非浸润性膀胱癌细胞株为5637细胞株。
本申请中，本申请检测方法区别于免疫组化方法、DNA测序方法、DNA甲 基化分析和FISH等方法，减少了假阳性和其他负面作用。
另一方面，本申请提供如第一方面所述的标志物用于制备诊断膀胱癌的药物或试剂。
与现有技术相比，本申请具有如下有益效果：
(1)本申请标志物可以准确检测膀胱癌的浸润性，对膀胱癌的手术方式选择具有指导作用，可以减少手术后的复发和转移；
(2)本申请取材简单，可以使用膀胱镜活检细胞或尿液脱落细胞，在早期区分浸润性和非浸润性膀胱癌，用在早期普查和癌症术后随访，尤其是对于疑似复发病人的跟踪随访，可以争取时间，为挽救病人生命做出重大贡献；
(3)本申请检测方法区别于免疫组化方法，减少了假阳性和其他负面作用，不仅如此，通过发现的膀胱癌浸润性相关印记基因缺失位点的致该基因沉默、剔除、重排的靶向药物或技术方法，可用于指导后期的治疗和用药。
附图说明
图1为不同膀胱癌细胞株的膀胱浸润因子检测结果；
图2为17例膀胱癌样本的膀胱浸润因子检测结果。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1膀胱癌细胞株浸润性检测
所述的印记基因的检测方法，包括如下步骤：
(1)将培养的膀胱癌细胞株5637、UMUC3、T24和J82放入10％中性***溶液中进行固定，以防RNA降解，固定时间为24小时，粘附到玻片上， 所述玻片需要用正电荷脱载玻片，在37℃烤箱烘烤3h以上；
(2)按照RNASCope的样品处理方法封闭样本中内源性过氧化物酶活性，增强通透性并暴露出RNA分子；
(3)设计探针：根据印记基因序列设计特异性引物；
所述设计探针是根据印记基因Grb10和Diras3进行设计的，具体在每个基因的内旋子内选择一段序列作为探针，具体的探针由Advanced Cell Diagnostics公司设计。
(4)将步骤(3)的探针与待测样本通过试剂盒进行RNA SCope原位杂交；
(5)信号扩增和苏木精染色，用显微镜成像分析印记基因的表达情况；
所述模型中的计算印记基因的总表达量、正常表达量、缺失表达量和拷贝数异常表达量的公式如下：
总表达量＝(b+c+d)/(a+b+c+d)×100％；
正常表达量＝b/(b+c+d)×100％；
缺失表达量(LOI)＝c/(b+c+d)×100％；
拷贝数异常表达量(CNV)＝d/(b+c+d)×100％；
其中，所述a为将细胞进行苏木素染色后，其中不存在标记，即印记基因没有表达的细胞核的数量；所述b为将细胞进行苏木素染色后，其中存在一个红色/棕色标记，即印记基因存在的细胞核的数量；所述c为将细胞进行苏木素染色后，其中存在两个红色/棕色标记，即印记基因缺失的细胞核的数量；所述d为将细胞进行苏木素染色后，其中存在两个以上红色/棕色标记，即印记基因拷贝数异常的细胞核的数量。
计算膀胱浸润因子，所述膀胱浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
从图1可以看出，非浸润膀胱癌细胞株5637中，膀胱浸润因子小于1.5，浸润性膀胱癌细胞株UMUC3、T24和J82中，膀胱浸润因子都大于1.5。
实施例2 17例膀胱癌样本浸润性检测
获取17例膀胱癌病人的组织包括膀胱镜活检样本，按如下步骤处理样本：
(1)获取恶性淋巴瘤的组织细胞切片(10微米)，放入10％中性***溶液中进行固定，以防RNA降解，固定时间为24小时，石蜡包埋(FFPE)，所述玻片需要用正电荷脱载玻片，所述切片在40℃烤箱烘烤3h以上；
(2)按照RNASCope的样品处理方法进行脱蜡处理，封闭样本中内源性过氧化物酶活性，增强通透性并暴露出RNA分子；
其他检测方法同实施例1。
从图2可以看出，绝大部分非浸润性膀胱癌样本的膀胱浸润因子都小于1.5，绝大部分浸润性膀胱癌样本的膀胱浸润因子都大于1.5％。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (13)

  1. 一种用于检测浸润性膀胱癌的标志物，其为印记基因Grb10和/或印记基因Diras3。
  2. 根据权利要求1所述的标志物，其中，所述印记基因的总表达量、正常表达量、缺失表达量和拷贝数异常表达量的公式如下：
    总表达量＝(b+c+d)/(a+b+c+d)×100％；
    正常表达量＝b/(b+c+d)×100％；
    缺失表达量＝c/(b+c+d)×100％；
    拷贝数异常表达量＝d/(b+c+d)×100％；
    其中，所述a为将细胞进行苏木素染色后，其中不存在标记，即印记基因没有表达的细胞核的数量；所述b为将细胞进行苏木素染色后，其中存在一个红色/棕色标记，即印记基因存在的细胞核的数量；所述c为将细胞进行苏木素染色后，其中存在两个红色/棕色标记，即印记基因缺失的细胞核的数量；所述d为将细胞进行苏木素染色后，其中存在两个以上红色/棕色标记，即印记基因拷贝数异常的细胞核的数量。
  3. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量小于8％或印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量小于8％或印记基因Grb10拷贝数异常表达量不大于1.5％且浸润因子不大于1.5，则判断为膀胱癌浸润属性不明显；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
  4. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10拷贝数异常表达量不大于 1.5％且浸润因子大于1.5，则判断为混合偏浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
  5. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量大于1.5％且浸润因子不大于1.5或印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量不大于1.5％且浸润因子不小于于1.5，则判断为混合膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
  6. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量小于8％或印记基因Diras3的拷贝数异常表达量不大于1％时，若印记基因Grb10的总表达量不小于8％且印记基因Grb10的拷贝数异常表达量大于1.5％，则判断为浸润性膀胱癌。
  7. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量不小于8％且印记基因Diras3的拷贝数异常表达量大于1％时，若印记基因Grb10的总表达量小于8％或印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量不大于1.5％且浸润因子小于1.5，则判断为非浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
  8. 根据权利要求1或2所述的标志物，其中，当所述印记基因Diras3的总表达量不小于8％且拷贝数异常大于1％时，若印记基因Grb10的总表达量不小于8％、印记基因Grb10的拷贝数异常表达量大于1.5％且浸润因子大于1.5，则 判断为浸润性膀胱癌；其中，所述浸润因子为印记基因Grb10拷贝数异常表达量与印记基因Diras3拷贝数异常表达量的比值。
  9. 根据权利要求1-8中任一项所述的标志物，其中，所述印记基因Grb10在浸润性膀胱癌细胞株中表达，所述印记基因Diras3在浸润性膀胱癌细胞株中不表达。
  10. 根据权利要求9所述的标志物，其中，所述浸润性膀胱癌细胞株为T24细胞株、J82细胞株或UMUC3细胞株中的任意一种或至少两种的组合。
  11. 根据权利要求1-10中任一项所述的标志物，其中，所述印记基因Grb10在非浸润性膀胱癌细胞株中不表达，所述印记基因Diras3在非浸润性膀胱癌细胞株中表达。
  12. 根据权利要求11所述的标志物，其中，所述非浸润性膀胱癌细胞株为5637细胞株。
  13. 根据权利要求1-12中任一项所述的标志物用于制备诊断膀胱癌的药物或试剂。
PCT/CN2019/119190 2018-11-21 2019-11-18 一种用于检测浸润性膀胱癌的标志物及其应用 WO2020103794A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/295,629 US20220002817A1 (en) 2018-11-21 2019-11-18 Marker used to detect invasive bladder cancer, and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811391567.0A CN111206093A (zh) 2018-11-21 2018-11-21 一种用于检测浸润性膀胱癌的标志物及其应用
CN201811391567.0 2018-11-21

Publications (1)

Publication Number Publication Date
WO2020103794A1 true WO2020103794A1 (zh) 2020-05-28

Family

ID=70773666

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/119190 WO2020103794A1 (zh) 2018-11-21 2019-11-18 一种用于检测浸润性膀胱癌的标志物及其应用

Country Status (3)

Country Link
US (1) US20220002817A1 (zh)
CN (1) CN111206093A (zh)
WO (1) WO2020103794A1 (zh)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400804A (zh) * 2006-01-11 2009-04-01 基因组健康股份有限公司 用于结肠直肠癌预后的基因表达标志物
CN107422125A (zh) * 2016-05-23 2017-12-01 中国医学科学院肿瘤医院 与肌层浸润性膀胱癌相关的尿液蛋白质标志物
CN107532217A (zh) * 2015-05-29 2018-01-02 豪夫迈·罗氏有限公司 用于癌症的治疗和诊断方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6087164A (en) * 1997-10-03 2000-07-11 Yissum Research Development Company Of The Hebrew University Of Jerusalem Methods and compositions for inducing tumor-specific cytotoxicity
US7998695B2 (en) * 2005-02-10 2011-08-16 Oncotherapy Science, Inc. Method of diagnosing bladder cancer
US20110028333A1 (en) * 2009-05-01 2011-02-03 Brown University Diagnosing, prognosing, and early detection of cancers by dna methylation profiling
CN102409085B (zh) * 2010-09-26 2013-03-20 中国医学科学院肿瘤研究所 一种辅助诊断非浸润性膀胱癌和浸润性膀胱癌的试剂
GB201511152D0 (en) * 2015-06-24 2015-08-05 Ucl Business Plc Method of diagnosing bladder cancer
CN108245679B (zh) * 2018-02-12 2020-04-21 中南大学湘雅三医院 Spag5作为靶位点在制备治疗膀胱癌药物中的应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101400804A (zh) * 2006-01-11 2009-04-01 基因组健康股份有限公司 用于结肠直肠癌预后的基因表达标志物
CN107532217A (zh) * 2015-05-29 2018-01-02 豪夫迈·罗氏有限公司 用于癌症的治疗和诊断方法
CN107422125A (zh) * 2016-05-23 2017-12-01 中国医学科学院肿瘤医院 与肌层浸润性膀胱癌相关的尿液蛋白质标志物

Also Published As

Publication number Publication date
CN111206093A (zh) 2020-05-29
US20220002817A1 (en) 2022-01-06

Similar Documents

Publication Publication Date Title
CN105463070A (zh) Uca1基因在制备诊断结直肠癌产品中的应用
JP2022542987A (ja) 腫瘍マーカーおよびその応用
EP3726221B1 (en) Hierarchical model for detecting benign and malignant degrees of colorectal tumors and application thereof
CN111105842B (zh) 一种用于检测淋巴瘤、淋巴转移癌良恶性程度的分级模型及其应用
WO2018218737A1 (zh) 一种印记基因在***中的分级模型及其组成的***
WO2020103794A1 (zh) 一种用于检测浸润性膀胱癌的标志物及其应用
CN111100930B (zh) 一种用于检测胰腺肿瘤良恶性程度的分级模型及其应用
WO2019129144A1 (zh) 一种用于检测食道肿瘤和/或胃肿瘤良恶性程度的分级模型及其应用
WO2018219342A1 (zh) 一种印记基因分级模型和诊断方法及其应用
CN109971848B (zh) 一种用于检测食道肿瘤和/或胃肿瘤良恶性程度的分级模型及其应用
CN110890128B (zh) 一种用于检测皮肤肿瘤良恶性程度的分级模型及其应用
WO2019105381A1 (zh) 一种用于检测乳腺肿瘤良恶性程度的分级模型及其应用
WO2018214249A1 (zh) 一种印记基因分级模型及其组成的***和应用
CN110791563B (zh) 一种用于检测甲状腺肿瘤良恶性程度的分级模型及其应用
CN109837342A (zh) 一种用于检测乳腺肿瘤良恶性程度的分级模型及其应用
CN110714075B (zh) 一种用于检测肺肿瘤良恶性程度的分级模型及其应用
WO2023083335A1 (zh) 一种用于检测肿瘤良恶性程度的分级模型及其应用
WO2020098604A1 (zh) 用于检测***肿瘤良恶性程度的分级模型及其应用
CN111261219B (zh) 一种用于检测肝肿瘤良恶性程度的分级模型及其应用
WO2019218338A1 (zh) 通过活检细胞样本诊断癌症的方法
Visvanathan et al. Evaluating DNA Methylation in Random Fine Needle Aspirates from the Breast to Inform Cancer Risk

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19887124

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 19887124

Country of ref document: EP

Kind code of ref document: A1