CN112575083A - Application of miRNA-374b-5p rich in exosome as marker for diagnosing endometrial cancer - Google Patents
Application of miRNA-374b-5p rich in exosome as marker for diagnosing endometrial cancer Download PDFInfo
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Abstract
The invention provides application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer, and belongs to the technical field of cancer diagnosis. The results of the embodiment of the invention show that miRNA-374b-5p rich in peripheral plasma exosomes can be used as a marker for diagnosing endometrial cancer, and an experimental foundation is laid for further developing early-stage endometrial cancer detection products, so that contribution is made to reducing the harm of endometrial cancer.
Description
Technical Field
The invention relates to the technical field of cancer diagnosis, in particular to application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer.
Background
The incidence of endometrial cancer is second place in gynecological malignancies and the mortality is third place. Most endometrial cancers are diagnosed early and with a good prognosis. However, about 20% of endometrial cancers are diagnosed with local metastasis and 10% are diagnosed with distant metastasis. In addition, the 5-year survival rate for patients with in-situ endometrial cancer is about 86% -95%, the survival rate for patients with local metastasis will be 48% -71%, and the 5-year survival rate for patients with distant metastasis is only 9% -17%. Accurate early diagnosis and prognosis analysis are particularly important. At present, early diagnosis of endometrial cancer is mainly based on early symptoms, but only a part of people have early symptoms. Finding markers for diagnosing endometrial cancer is therefore of great importance.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer, and the miRNA-374b-5p rich in exosome can be used as the marker for diagnosing endometrial cancer.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer.
Preferably, the area under the working characteristic curve of the diagnostic subject for the exosome-enriched miRNA-374b-5p is 0.9543; the 95% confidence interval is 0.9184-0.9901; p < 0.0001.
Preferably, the diameter of the exosome is 30-150 nm.
Preferably, the surface markers of the exosomes include HSP70, CD63, CD81 and CD 9.
The invention provides application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer, and the miRNA-374b-5p rich in exosome can be used as the marker for diagnosing endometrial cancer. And an experimental foundation is laid for further developing early-stage endometrial cancer detection products, so that contribution is made to reducing the harm of endometrial cancer. In the present invention, AUC of miRNA-374b-5p enriched in the exosome is 0.9543; 95% confidence intervals 95% 0.9184-0.9901; p < 0.0001.
Drawings
FIG. 1 is a diagnostic subject working profile analysis of plasma exosomes enriched with miR-374b-5p as a diagnostic marker for endometrial cancer;
FIG. 2 is a diagnostic subject work characteristic curve analysis of plasma exosomes enriched with miR-194-5p as a diagnostic marker of endometrial cancer;
FIG. 3 is a diagnostic subject working characteristic curve analysis of plasma exosomes enriched with miR-424-5p as a diagnostic marker of endometrial cancer;
FIG. 4 is an analysis of the working profile of a diagnosed subject whose plasma exosomes are rich in LGALS3BP as a diagnostic marker for endometrial cancer.
Detailed Description
The invention provides application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer. In the present invention, the area under the working characteristic curve AUC of the diagnostic subject of miRNA-374b-5p enriched in exosomes is 0.9543; 95% CI: 0.9184-0.9901; p < 0.0001. In the invention, the nucleotide sequence of the miRNA-374b-5p is 5 'AUAUAAUACAACCUGCUAAGUG-3' (SEQ ID No. 1).
In the invention, the diameter of the exosome is preferably 30-150 nm; surface markers for the exosomes preferably include HSP70, CD63, CD81 and CD 9.
In order to further illustrate the present invention, the following detailed description of the invention is given in conjunction with examples, which should not be construed to limit the scope of the invention.
Example 1
1. Plasma exosome separation: plasma exosomes were extracted from 42 healthy persons, 80 early stage endometrial cancers, and 7 patients with metastatic endometrial cancer using a commercial kit (QIAGEN, Germany). 1ml of plasma sample was mixed with 1ml of XBP reagent at a volume ratio of 1: 1 and incubated at room temperature for 5 min. The mixture was then applied to an exoEasy adsorption column, centrifuged at 500g for 1min, and the filtrate was discarded. The residual liquid was washed with 10ml XWP, centrifuged at 5000g for 10min, and the supernatant was discarded. Finally, 200. mu.l of Buffer XE elution reagent was added to the exoEasy adsorption column, incubated for 5min, centrifuged at 5000g for 5min, and the filtrate rich in exosomes was collected.
2. And (3) identifying exosomes: and (3) identifying the diameter of the exosome in the filtrate between 30 and 150nm by electron microscopy and particle size analysis, and determining exosome surface markers HSP70, CD63, CD81 and CD9 by using a protein immunoblotting method.
3. Three groups of plasma exosome RNAs are respectively extracted for miRNA sequencing analysis: and carrying out differential analysis on the miRNA sequencing result to find out differential miRNA, and carrying out enrichment analysis on biological information GO and KEGG by using a DAVID database, namely carrying out clustering analysis according to the difference of the functions of the regulatory genes of the miRNAs with differential expression and the difference of the involved signal paths, so as to be convenient for searching meaningful target miRNAs subsequently.
4. Selecting plasma exosome miRNAs as endometrial cancer diagnosis markers: collecting 42 healthy persons, 80 early-stage endometrial cancers and 7 plasma exosomes of patients with metastatic endometrial cancers, extracting RNA, screening out miRNAs with high expression in the plasma exosomes of the patients with the endometrial cancers according to miRNA sequencing analysis results, verifying the screened high-expression miRNAs by detecting the miRNAs in the plasma exosomes of the patients with the endometrial cancers of 42 healthy persons, 80 early-stage endometrial cancers and 7 patients with the metastatic endometrial cancers by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction), analyzing by using a diagnostic test object working characteristic curve (ROC curve), and selecting the miRNAs with high expression in the plasma of the patients with the endometrial cancers as endometrial cancer diagnostic markers, namely miRNA-374b-5p, wherein the results are shown in figure 1.
Figure 1 shows that miRNA-374b-5p, which is exosome-rich, is a diagnostic marker for endometrial cancer, diagnosing the area under the working characteristic curve, AUC, of a subject as 0.9543; 95% confidence interval 95% CI: 0.9184-0.9901; p < 0.0001. Therefore, the miRNA-374b-5p rich in exosome as the endometrial cancer diagnostic marker has high sensitivity, specificity and accuracy.
Comparative example 1
1. Plasma exosome separation: plasma exosomes were extracted from 42 healthy persons, 80 early stage endometrial cancers, and 7 patients with metastatic endometrial cancer using a commercial kit (QIAGEN, Germany). 1ml of plasma sample was mixed with 1ml of XBP reagent at a volume ratio of 1: 1 and incubated at room temperature for 5 min. The mixture was then applied to an exoEasy adsorption column, centrifuged at 500g for 1min, and the filtrate was discarded. The residual liquid was washed with 10ml XWP, centrifuged at 5000g for 10min, and the supernatant was discarded. Finally, 200. mu.l of Buffer XE elution reagent was added to the exoEasy adsorption column, incubated for 5min, centrifuged at 5000g for 5min, and the filtrate rich in exosomes was collected.
2. And (3) identifying exosomes: and (3) identifying the diameter of the exosome in the filtrate between 30 and 150nm by electron microscopy and particle size analysis, and determining exosome surface markers HSP70, CD63, CD81 and CD9 by using a protein immunoblotting method.
3. Three groups of plasma exosome RNAs are respectively extracted for miRNA sequencing analysis: and carrying out differential analysis on the miRNA sequencing result to find out the differential miRNA, and carrying out enrichment analysis on the biological information GO and KEGG by using a DAVID database, namely carrying out cluster analysis according to the difference of the functions of the regulating genes of the miRNAs with differential expression and the difference of the involved signal paths.
4. Selecting plasma exosome miRNAs as endometrial cancer diagnosis markers: collecting 42 healthy persons, 80 early-stage endometrial cancers and 7 plasma exosomes of patients with metastatic endometrial cancers, extracting RNA, screening out miRNAs with high expression in the plasma exosomes of the patients with the endometrial cancers according to miRNA sequencing analysis results, verifying the screened high-expression miRNAs by detecting the miRNAs in the plasma exosomes of the patients with the endometrial cancers of 42 healthy persons, 80 early-stage endometrial cancers and 7 patients with the metastatic endometrial cancers by utilizing qRT-PCR (quantitative reverse transcription-polymerase chain reaction), analyzing by using a diagnostic test object working characteristic curve (ROC curve), and selecting the miRNAs with high expression in the plasma of the patients with the endometrial cancers as endometrial cancer diagnostic markers, wherein the results are shown in figures 2 and 3.
As can be seen from fig. 2 and fig. 3, miRNA-194-5p enriched in exosomes is used as a diagnostic marker for endometrial cancer, and AUC 0.8902 is the area under the working characteristic curve of the diagnostic subject; 95% confidence interval 95% CI 0.8309-0.9495; p < 0.0001. miRNA-424-5p rich in exosome is used as a endometrial cancer diagnostic marker, and the AUC (area under a working characteristic curve) of a diagnosed subject is 0.8140; 95% confidence interval 95% CI 0.7412-0.8869; p < 0.0001.
Compared with miRNA-374b-5p (AUC: 0.9543; 95% CI: 0.9184-0.9901; p <0.0001) rich in plasma exosomes, miRNA-194-5p rich in exosomes and miRNA-424-5p have poor diagnostic sensitivity, specificity and accuracy.
Comparative example 2
1. Plasma exosome separation: plasma exosomes were extracted from healthy humans, early stage endometrial cancer, and patients with distant metastatic endometrial cancer using a commercial kit (exotquick, SBI, USA). Plasma samples (1ml) were mixed with thrombin (60 μ l, 611U/m, SBI) for 5min at room temperature, then centrifuged at 10000rpm for 5min and the fibrin pellet discarded. Then, 250. mu.l of ExoQuick solution was added to the supernatant in a ratio of 1: 4. After incubation at 4 ℃ for 1h, the remaining exosome suspension was centrifuged at 1500g for 30min, and finally the exosomes were resuspended in 1ml PBS and filtered through a filter with a diameter of 0.22 μm.
2. And (3) identifying exosomes: electron microscopy and particle size analysis identified exosomes with diameters between 30-150nm, and the exosome surface markers HSP70, CD63, CD81 and CD9 were determined using Western immunoblotting.
3. Three groups of plasma exosome proteins are respectively extracted to perform proteomic mass spectrometry to analyze the content of different proteins: and performing differential analysis on the proteomic results to find differential protein, and performing biological information GO and KEGG biological information enrichment analysis by using a DAVID database.
4. Selecting plasma exosome proteins as endometrial cancer diagnosis markers: collecting 42 healthy persons, 80 early-stage endometrial cancer and 7 plasma exosomes of metastatic endometrial cancer patients, extracting proteins, screening the proteins with high expression of the plasma exosomes of the endometrial cancer patients according to the proteomic mass spectrum result, carrying out large sample verification on the proteins by using ELISAs, and analyzing by using a diagnostic test object working characteristic curve (ROC) curve. Finally, the LGALS3BP protein was screened as a diagnostic marker for endometrial cancer (fig. 4) (see reference 1), and the area under the working characteristic curve AUC of the exosome-rich LGALS3BP protein was 0.7406; 95% confidence interval 95% CI 0.6506-0.8305; p < 0.0001.
The exosome-rich LGALS3BP protein was less sensitive, specific and accurate in diagnosis compared to plasma exosome-rich miRNA-374b-5p (AUC ═ 0.9543; 95% CI: 0.9184-0.9901; p < 0.0001).
Reference 1:
Song Y,Wang M,Tong H,Tan Y,Hu X,Wang K,Wan X.Plasma exosomes from endometrial cancer patients contain LGALS3BP to promote endometrial cancer progression.Oncogene.2020Nov 18.doi:10.1038/s41388-020-01555-x.Epub ahead of print.PMID:33208911.
the foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.
Sequence listing
<110> first women and infants health care institute in Shanghai City
<120> application of miRNA-374b-5p rich in exosome as marker for diagnosing endometrial cancer
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
auauaauaca accugcuaag ug 22
Claims (4)
1. Application of miRNA-374b-5p rich in exosome as a marker for diagnosing endometrial cancer.
2. The use according to claim 1, wherein the exosome-enriched miRNA-374b-5p has an area under the working characteristic curve for a diagnostic subject of 0.9543; the 95% confidence interval is 0.9184-0.9901; p < 0.0001.
3. The use according to claim 1, wherein the exosomes have a diameter of 30-150 nm.
4. The use according to claim 1, wherein the surface markers of the exosomes comprise HSP70, CD63, CD81 and CD 9.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140148350A1 (en) * | 2010-08-18 | 2014-05-29 | David Spetzler | Circulating biomarkers for disease |
| CN109609634A (en) * | 2018-12-24 | 2019-04-12 | 朱伟 | One kind circulation miRNA marker relevant to carcinoma of endometrium auxiliary diagnosis and its application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20140148350A1 (en) * | 2010-08-18 | 2014-05-29 | David Spetzler | Circulating biomarkers for disease |
| CN109609634A (en) * | 2018-12-24 | 2019-04-12 | 朱伟 | One kind circulation miRNA marker relevant to carcinoma of endometrium auxiliary diagnosis and its application |
Non-Patent Citations (4)
| Title |
|---|
| QIAN WANG ET AL.: "Novel miRNA markers for the diagnosis and prognosis of endometrial cancer", 《J CELL MOL MED.》 * |
| 罗荣城等: "《生物标志物与精准医学》", 31 March 2020 * |
| 赵婉等: "miRNA与子宫内膜癌的相关性及临床应用可能性的研究进展", 《世界复合医学》 * |
| 高艳霞等: "miRNA-519a在子宫内膜癌中的诊断及预后价值及机制探寻", 《解剖科学进展》 * |
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