CN104726448B - 一种用于肺癌组织分型的miRNA标志物及应用 - Google Patents
一种用于肺癌组织分型的miRNA标志物及应用 Download PDFInfo
- Publication number
- CN104726448B CN104726448B CN201410848918.1A CN201410848918A CN104726448B CN 104726448 B CN104726448 B CN 104726448B CN 201410848918 A CN201410848918 A CN 201410848918A CN 104726448 B CN104726448 B CN 104726448B
- Authority
- CN
- China
- Prior art keywords
- mirna
- lung tissue
- cancerous lung
- seq
- parting
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 210000004072 lung Anatomy 0.000 title claims abstract description 31
- 108091070501 miRNA Proteins 0.000 title claims abstract description 21
- 239000002679 microRNA Substances 0.000 title claims abstract description 19
- 239000003550 marker Substances 0.000 title claims abstract description 9
- 239000000523 sample Substances 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000000090 biomarker Substances 0.000 abstract description 4
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 20
- 206010028980 Neoplasm Diseases 0.000 description 17
- 201000011510 cancer Diseases 0.000 description 12
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 8
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 8
- 201000005202 lung cancer Diseases 0.000 description 8
- 208000020816 lung neoplasm Diseases 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
- 102100034343 Integrase Human genes 0.000 description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 3
- 201000005249 lung adenocarcinoma Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000001575 pathological effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000009007 Diagnostic Kit Methods 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000012937 correction Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 206010041823 squamous cell carcinoma Diseases 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000012151 immunohistochemical method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 208000003849 large cell carcinoma Diseases 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 208000000649 small cell carcinoma Diseases 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Abstract
本发明属于生物技术领域，涉及一种用于肺癌组织分型的miRNA标志物及应用。本发明提供了用于肺癌组织分型的miRNA标志物，包括miRNA‑205、miRNA‑21和miRNA‑26b；本发明还提供了序列如SEQ ID NO.1、SEQ ID NO.2、和SEQ ID NO.3所示的引物探针及其在制备肺癌组织分型试剂盒中的应用。本发明的有益效果在于首次发现了对肺癌组织分型具有极高诊断价值(AUC为1.000，敏感度和特异度分别为100％和100％)的生物标记物，并提供了能检测这些生物标记物在组织中含量的试剂盒。
Description
技术领域
本发明属于生物技术领域，涉及一种用于肺癌组织分型的 miRNA标志物及应用。
背景技术
肺癌是世界上最常见的恶性肿瘤之一,已成为我国城市人口恶性肿瘤死亡原因的第1位。非小细胞型肺癌包括鳞状细胞癌(鳞癌)、腺癌、大细胞癌，与小细胞癌相比其癌细胞生长分裂较慢,扩散转移相对较晚，非小细胞肺癌约占所有肺癌的80％，约75％的患者发现时已处于中晚期，5年生存率很低。其中，肺腺癌和肺鳞癌又是非小细胞肺癌中的两种最常见的肿瘤。各病理类型的肺癌在肿瘤的生物学行为、对放疗、化疗及分子靶向药物的敏感性等方面存在差异，各型预后也不相同。因此，在临床上对肺癌作出诊断时，明确其病理分型对制订治疗方案、分析预后非常重要。
目前医学界对肺癌的病理分型主要依靠微观形态学及免疫组织化学方法。传统的病理组织学方法是将获取的病变组织通过固定、脱蜡、染色等步骤，制作成切片在在显微镜下分辨其微观结构。该方法步骤繁多，需要白白等待一二天的时间，在病理学专业人员对切片进行判读时也难免主观性。因此，临床上迫切的需要一种快捷、有限、准确的肺癌的病理分型方法。
发明内容
本发明要解决的技术问题是：一种用于肺癌组织分型的miRNA 标志物。
为解决上述技术问题，本发明采用的技术方案如下：一种用于肺癌组织分型的miRNA标志物，所述标记物包括miRNA-205、 miRNA-21和miRNA-26b。
在本发明的第二方面，提供了上述miRNA标志物在制备肺癌组织分型试剂盒中的应用。
在本发明的第三方面，提供了一种肺癌组织分型试剂盒，其能够测定组织中的miRNA-205、miRNA-21和miRNA-26b的含量。
在本发明的一个优选例中，上述试剂盒含有miRNA-205、 miRNA-21和miRNA-26b的引物探针。
在本发明的一个优选例中，上述引物探针的序列如SEQ ID NO.1、SEQ IDNO.2和SEQ ID NO.3所示。
在本发明的第四方面，提供了核苷序列如SEQ ID NO.1、SEQ ID NO.2或SEQIDNO.3中的一种或多种组合物在制备肺癌组织分型试剂盒中的应用。
在本发明的一个优选例中，提供了序列如SEQ ID NO.1、SEQ ID NO.2和SEQIDNO.3所示引物探针的用途，其用于制备肺癌组织分型试剂盒。
所述诊断试剂盒还可以包括PCR反应常用试剂，如Taq酶，逆转录酶，缓冲液，dNTPs，MgCl2和DEPC水等；还可以含有标准品和/或对照品。
本发明的有益效果在于：首次发现了对肺癌组织分型具有极高诊断价值(AUC为1.000，敏感度和特异度分别为100％和100％)的生物标记物组合miRNA-205、miRNA-21和miRNA-26b；通过血浆 miRNA标志物和诊断试剂盒的研制和应用，可以使得肺癌组织分型更加方便易行，为临床医生快速准确掌握患者病情，为提高临床治疗效果奠定基础，并为发现具有潜在治疗价值的新型小分子药物靶标提供帮助。
附图说明
图1为肺腺癌与肺鳞癌肿瘤组织miRNA表达水平比较。
图2为肺腺癌与肺鳞癌肿瘤组织miRNA表达水平ROC曲线。
具体实施方式
一、研究对象
病例组为2012年11月至2014年3月在常州市第二人民医院收集的42例病例，均经病理组织学确诊，采血前未经过手术和放化疗。其中肺腺癌27例，肺鳞癌15例；按性别和年龄(±5岁)与病例进行频数匹配。用于研究的样本为同期收取，采样、分装、保存条件均一，通过对样品资料的整理。
二、研究方法
(1)肿瘤组织石蜡包埋，切片。
(2)按照石蜡组织总RNA提取试剂盒(miRNeasy FFPE Kit，货号NO.217504，德国Qiagen公司)说明书从石蜡组织中提取总 RNA。
(3)按照miRNA逆转录试剂盒(TaqManmicroRNA反转录试剂盒，货号NO.1302146R，美国ABI公司)说明书进行逆转录反应。反应总体积为15ul(总RNA5ul，TaqManMicroRNAAssay 3ul,核酸酶自由水4.16ul，RNase抑制剂0.19ul，缓冲液1.5ul，Multiscribe逆转录酶1.00ul和dNTP0.15ul)，在不同温度下(16℃，42℃，85℃，4 ℃)进行不同时长(30mins，30mins，5mins，10mins)反应。使用 RNU44为内参，进行数据归一化校正。
(4)实时荧光定量PCR按照TaqMan试剂盒(TaqMan通用混合试剂盒II，货号NO.121207，美国ABI公司)说明书进行。PCR扩增体系总体积为10ul，反应共40个循环。PCR反应Ct值通过7900实时荧光定量PCR仪(美国ABI公司)测定，每个反应重复三次。
三、研究结果
肺腺癌和肺鳞癌两组病例的肿瘤组织中miRNA-205，miRNA-21，miRNA-26b表达水平有显著差异，其中，miRNA-205在肺鳞癌中比在肺腺癌中显著上调；miRNA-21，miRNA-26b在肺鳞癌中比在肺腺癌中显著下调。具体数据如图1和表1所示：
表1：肺腺癌与肺鳞癌肿瘤组织miRNA表达水平比较(ΔCT)(X±S)
表2：miRNA引物探针信息及目标序列
ROC曲线分析显示，3种miRNA作为生物标记物对肺癌分型具有极高诊断价值(AUC为1.000，敏感度和特异度分别为100％和 100％)；详细结果图2及表3所示。
表3:ROC分析结果
a.在非参数假设下
b.零假设：实面积＝0.5
使用SPSS 19.0软件包进行数据分析，对于两组之间的比较，首先进行方差齐性检测，对于方差齐性的两组数据，采用Student’s t-test 进行比较分析；对于方差不齐的两组数据，采用Welch校正分析。 P<0.05被认为差异具有统计学意义。将具有统计学差异的数据进行二元logistic回归，得出预测概率值，并将预测概率值用于后续ROC曲线分析。ROC曲线分析用于评价miRNA在肺癌组织分型中的价值，曲线下(AUC)面积越接近1，则说明该指标做为肺癌组织分型的价值越高。
用于组织分型的试剂盒包括如SEQ ID NO.1、SEQ ID NO.2和 SEQ ID NO.3所示的引物探针，可由Life Technologies公司购得；还可以有可以有相应PCR技术所需的常用试剂，如：Taq酶，逆转录酶，缓冲液，dNTPs，MgCl2和DEPC水等，这些都是本领域技术人员熟知的；另外还可以含有标准品和/或对照品。
本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。
Claims (5)
1.一种用于肺癌组织分型miRNA标志物，所述标记物由miRNA-205、miRNA-21和miRNA-26b组成。
2.一种如权利要求1所述的用于肺癌组织分型的miRNA标志物在制备肺癌组织分型试剂盒中的应用。
3.一种肺癌组织分型试剂盒，其能够测定组织中的miRNA-205、miRNA-21和miRNA-26b的含量。
4.如权利要求3所述的肺癌组织分型试剂盒，其特征在于所述试剂盒含有miRNA-205、miRNA-21和miRNA-26b的引物探针。
5.一种如权利要求4所述的肺癌组织分型试剂盒，其特征在于引物探针的序列如SEQID NO.1、SEQ ID NO.2和SEQ ID NO.3所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410848918.1A CN104726448B (zh) | 2014-12-29 | 2014-12-29 | 一种用于肺癌组织分型的miRNA标志物及应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410848918.1A CN104726448B (zh) | 2014-12-29 | 2014-12-29 | 一种用于肺癌组织分型的miRNA标志物及应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104726448A CN104726448A (zh) | 2015-06-24 |
| CN104726448B true CN104726448B (zh) | 2018-03-30 |
Family
ID=53450864
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410848918.1A Active CN104726448B (zh) | 2014-12-29 | 2014-12-29 | 一种用于肺癌组织分型的miRNA标志物及应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104726448B (zh) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039554A (zh) * | 2015-08-06 | 2015-11-11 | 浙江省中医药研究院 | 一种肺癌检测试剂盒及其用途 |
| CN106568936B (zh) * | 2016-10-12 | 2018-04-24 | 宁波大学 | 基于多功能化二硫化钼的miRNA-21电化学发光免疫传感器的制备方法及其应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770561A (zh) * | 2009-12-24 | 2012-11-07 | 复旦大学 | 用于诊断不同亚型肺癌的基于组织的微-rna方法 |
| CN103993082A (zh) * | 2006-01-05 | 2014-08-20 | 俄亥俄州立大学研究基金会 | 用于肺癌的诊断、预后和治疗的基于微小rna 的方法和组合物 |
-
2014
- 2014-12-29 CN CN201410848918.1A patent/CN104726448B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993082A (zh) * | 2006-01-05 | 2014-08-20 | 俄亥俄州立大学研究基金会 | 用于肺癌的诊断、预后和治疗的基于微小rna 的方法和组合物 |
| CN102770561A (zh) * | 2009-12-24 | 2012-11-07 | 复旦大学 | 用于诊断不同亚型肺癌的基于组织的微-rna方法 |
Non-Patent Citations (2)
| Title |
|---|
| "Diagnostic Assay Based on has-miR-205 Expression Distinguishes Squamous From Nonsquamous Non-Small-Cell Lung Carcinoma;Lebanony D等;《Journal of Clinical Oncology》;20090420;第27卷(第12期);第2030-2037页 * |
| microRNA在非小细胞肺癌组织中的表达及其临床应用研究;王亚南等;《临床检验杂志》;20111031;第29卷(第7期);第516-519页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104726448A (zh) | 2015-06-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Dieci et al. | Discordance in receptor status between primary and recurrent breast cancer has a prognostic impact: a single-institution analysis | |
| Bansal et al. | Feasibility of microRNAs as biomarkers for Barrett's esophagus progression: a pilot cross-sectional, phase 2 biomarker study | |
| Hou et al. | Circulating microRNAs as novel prognosis biomarkers for head and neck squamous cell carcinoma | |
| CN102971435A (zh) | 用于内分泌治疗下的乳腺癌复发预测的方法 | |
| de Carvalho et al. | Accuracy of microRNAs as markers for the detection of neck lymph node metastases in patients with head and neck squamous cell carcinoma | |
| Martens et al. | DNA methylation as a biomarker in breast cancer | |
| CN106834462A (zh) | 一组胃癌基因的应用 | |
| US20110070582A1 (en) | Gene Expression Profiling for Predicting the Response to Immunotherapy and/or the Survivability of Melanoma Subjects | |
| Xiaofei | Expressions of miR-21 and miR-210 in breast cancer and their predictive values for prognosis | |
| Gao et al. | Prognostic value of microRNAs in colorectal cancer: a meta-analysis | |
| Zhou et al. | Identification of microRNAs as biomarkers for cholangiocarcinoma detection: A diagnostic meta-analysis | |
| Li et al. | Expression patterns of microRNA-329 and its clinical performance in diagnosis and prognosis of breast cancer | |
| Almeida et al. | Topoisomerase II-alfa gene as a predictive marker of response to anthracyclines in breast cancer | |
| Lawson et al. | Tissue banking of diagnostic lung cancer biopsies for extraction of high quality RNA | |
| Werdich et al. | A review of advanced genetic testing for clinical prognostication in uveal melanoma | |
| Tekcham et al. | Epigenetic downregulation of PTEN in gallbladder cancer | |
| CN101613748A (zh) | 一种检测胰腺癌血清标志物的方法 | |
| Zhang et al. | MicroRNAs in sputum specimen as noninvasive biomarkers for the diagnosis of nonsmall cell lung cancer: An updated meta-analysis | |
| Park et al. | ΔNp63 to TAp63 expression ratio as a potential molecular marker for cervical cancer prognosis | |
| Jiang et al. | Polymer nanofiber-based microchips for EGFR mutation analysis of circulating tumor cells in lung adenocarcinoma | |
| CN104726448B (zh) | 一种用于肺癌组织分型的miRNA标志物及应用 | |
| Kosari et al. | Shared gene expression alterations in prostate cancer and histologically benign prostate from patients with prostate cancer | |
| Nakajima et al. | Ribonucleic acid microarray analysis from lymph node samples obtained by endobronchial ultrasonography-guided transbronchial needle aspiration | |
| CN104774925B (zh) | Brca2的3′非翻译区在制备肿瘤诊断、治疗和预后试剂中的应用 | |
| Zhuang et al. | c-Met and RON expression levels in endometrial adenocarcinoma tissue and their relationship with prognosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190712 Address after: 213164 Fourth Floor, Building A, Tianrun Science and Technology Building, 18 Changwuzhong Road, Wujin District, Changzhou City, Jiangsu Province Patentee after: CHANGZHOU JIEAO MEDICAL INSPECTION INSTITUTE Co.,Ltd. Address before: 213164 Fourth Floor, Building A, Tianrun Science and Technology Building, 801 Changwu Middle Road, Changzhou Science and Education City, Changzhou City, Jiangsu Province Patentee before: CHANGZHOU GOPATH PATHOLOGICAL DIAGNOSIS TECHNOLOGY CO.,LTD. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20200609 Address after: 213000 4 / F, building a, Tianrun technology building, Changzhou science and Education City, No. 801, Changwu Middle Road, Wujin District, Changzhou City, Jiangsu Province Patentee after: CHANGZHOU GOPATH PATHOLOGICAL DIAGNOSIS TECHNOLOGY Co.,Ltd. Address before: 213164 Fourth Floor, Building A, Tianrun Science and Technology Building, 18 Changwuzhong Road, Wujin District, Changzhou City, Jiangsu Province Patentee before: CHANGZHOU JIEAO MEDICAL INSPECTION INSTITUTE Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210510 Address after: 226200 building 16, 899 Jinke Road, Qidong life and Health Science Park, Qidong City, Nantong City, Jiangsu Province Patentee after: Kini immune (Nantong) Biomedical Technology Co.,Ltd. Address before: 4 / F, block a, Tianrun science and technology building, Changzhou science and Education City, No. 801, middle Changwu Road, Wujin District, Changzhou City, Jiangsu Province, 213000 Patentee before: CHANGZHOU GOPATH PATHOLOGICAL DIAGNOSIS TECHNOLOGY Co.,Ltd. |
|
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230718 Address after: 213164 Floor 4, Block A, Tianrun Science and Technology Building, Changzhou Science and Education City, No. 801, Middle Changwu Road, Wujin District, Changzhou City, Jiangsu Province Patentee after: CHANGZHOU GOPATH PATHOLOGICAL DIAGNOSIS TECHNOLOGY CO.,LTD. Address before: 226200 building 16, 899 Jinke Road, Qidong life and Health Science Park, Qidong City, Nantong City, Jiangsu Province Patentee before: Kini immune (Nantong) Biomedical Technology Co.,Ltd. |