CN113755587A - Marker for real-time evaluation of tumor tissue CD47 expression level and application thereof - Google Patents

Marker for real-time evaluation of tumor tissue CD47 expression level and application thereof Download PDF

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CN113755587A
CN113755587A CN202111010311.2A CN202111010311A CN113755587A CN 113755587 A CN113755587 A CN 113755587A CN 202111010311 A CN202111010311 A CN 202111010311A CN 113755587 A CN113755587 A CN 113755587A
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cervical cancer
expression level
zeb1
expression
tumor
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王薇
陈晓静
梁莉
吴砂
郭楚鸿
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First Affiliated Hospital of Guangzhou Medical University
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Abstract

The invention belongs to the field of tumor immunotherapy evaluation, and particularly relates to a marker for evaluating the expression level of tumor tissue CD47 in real time and application thereof. The invention discovers that ZEB1 is an important transcription factor for regulating and controlling the expression of the CD47 of the cervical cancer cell based on in vitro experimental research, and discovers that ZEB1 is enriched in exosomes in an anoxic cervical cancer cell for the first time; the expression level of the plasma exosome ZEB1 of the patient is further verified to be in positive correlation with the expression level of the tumor tissue CD47 in a cervical cancer clinical sample, and the expression level of the plasma exosome ZEB1 of the cervical cancer patient is detected, so that the expression level of the CD47 in the cervical cancer tissue can be reflected, and the method has important guiding significance for the evaluation of the cervical cancer CD47 immunotherapy.

Description

Marker for real-time evaluation of tumor tissue CD47 expression level and application thereof
Technical Field
The invention belongs to the field of tumor immunotherapy evaluation, and relates to a marker for evaluating the expression level of tumor tissue CD47 in real time and application thereof.
Background
With the intensive research of natural immune checkpoints, the field of tumor immunology has led to a new era in the treatment of cancer, and inhibitory immune checkpoint blockade is one of the most important advances in anticancer therapy. CD47 plays an important role in immune response and tumor immune escape as a novel immune checkpoint molecule. CD47 initiates "eat me" inhibitory signals by binding to signal-regulatory protein alpha (sirpa) on the macrophage surface, thereby evading tumor cells from phagocytosis by macrophages and affecting specific tumoricidal T cell activation, mediating tumor progression. The CD47-SIRP alpha axis immune blocker is one of the most promising new targets of tumor immunology in the last decade, but the immunotherapy is not applicable to all people, and it is important how to effectively screen out tumor patients with high expression of CD47 and make the tumor patients benefit from the immunotherapy. The current mode for detecting CD47 is mainly an immunohistochemical method, but has the following problems: 1. the expression of the CD47 protein in the tumor tissue is visually detected by adopting an immunohistochemical method, but real-time detection cannot be realized; 2. immunohistochemical antibody diversity, assessment of heterogeneous hierarchy. Therefore, how to efficiently and accurately judge the expression level of the CD47 in the tumor tissue in real time is important for the evaluation of the patient immunotherapy.
Liquid biopsy refers to a detection technique that non-invasively obtains biomarkers from bodily fluids and analyzes them to reflect information about the tissue from which they are derived. With the progress of society and the continuous development of medical technology, the requirement for accurate diagnosis and treatment of malignant tumors is continuously increased. With the increasingly intensive study of the molecular mechanisms of tumors, the research involved in liquid biopsy has progressed from classical tumor circulating cells (CTCs) and tumor circulating dna (ctdna) to tumor cell-derived exosomes.
Exosomes (exosomes) derived from tumor cells are rich in materials characteristic of tumor cells, especially proteins, are ubiquitous in patient body fluids, can transfer these genetic information or tumor signaling pathways into the tumor microenvironment, promote tumor progression, and have significant advantages in early screening and clinical diagnosis of tumors. Therefore, the exosome which is derived from the tumor tissue and can reflect the expression quantity of the CD47 is searched, the expression quantity of the CD47 in the tumor tissue is accurately judged in real time, and the exosome has important significance for the evaluation of the immunotherapy of the cervical cancer.
Disclosure of Invention
In view of the problems in the prior art, the invention aims to provide a marker for evaluating the expression level of CD47 in tumor tissues in real time and application thereof. The plasma exosome protein derived from the cervical cancer cells and closely related to the expression level of the CD47 of the tumor tissue is screened out, and the expression level of the plasma exosome protein is detected in real time, so that the expression level of the CD47 in the tumor tissue is reflected in real time, and the real-time evaluation on whether a cervical cancer patient is suitable for a CD47 immunotherapy is facilitated.
In a first aspect, the invention provides a marker for real-time assessment of expression level of CD47 in tumor tissue, the marker is a plasma exosome ZEB1, and the plasma exosome ZEB1 is derived from tumor cells.
Further, the expression level of the plasma exosome ZEB1 was positively correlated with the expression level of tumor tissue CD 47.
Further, the tumor is cervical cancer.
According to the invention, by detecting the expression levels of ZEB1 and CD47 in the cervical cancer cell lines cultured under the conditions of normal oxygen and oxygen deficiency, the ZEB1 and CD47 are highly expressed in the oxygen deficiency cervical cancer cells compared with the normal oxygen cervical cancer cells, the expression of CD47 is reduced or increased by using lentivirus to knock down or over express the ZEB1 of the cervical cancer cell lines, and the dual luciferase confirms that ZEB1 is an upstream transcription factor of CD47 and transcriptionally up-regulates the expression of CD 47.
Extracting exosomes secreted by the cervical cancer cell line by a gradient ultracentrifugation method, and finding that the ZEB1 protein is enriched in exosomes derived from an anoxic cervical cancer cell line; further verified in a cervical cancer clinical sample, the result shows that compared with an early cervical cancer patient, the expression of the plasma exosome ZEB1 of a late cervical cancer patient is obviously increased; the relative expression level of the ZEB1 in the plasma exosomes of the patients is positively correlated with the relative expression level of the CD47 in the tumor tissues, so that the real-time evaluation on the expression level of the CD47 in the tumor tissues can be realized by detecting the expression level of the ZEB1 in the plasma exosomes of the cervical cancer patients in real time.
In a second aspect, the invention provides the use of plasma exosomes ZEB1 in the assessment of cervical cancer CD47 immunotherapy.
According to the above application, the high expression of the plasma exosome ZEB1 indicates that the cervical cancer patient is suitable for the CD47 immunotherapy.
The experiment proves that the expression level of the plasma exosome ZEB1 is in positive correlation with the expression level of the CD47 of the cervical cancer tissue, the expression level of the CD47 in the cervical cancer tissue is indirectly reflected in real time by detecting the expression level of the ZEB1 in the exosome of the cervical cancer patient, and then the exosome can be used for evaluating whether the cervical cancer patient is suitable for the CD47 immunotherapy.
In a third aspect, the present invention provides a method for assessing cervical cancer CD47 immunotherapy using plasma exosomes ZEB1, comprising the steps of:
whether the cervical cancer patient is suitable for the CD47 immunotherapy is evaluated by detecting the expression level of the plasma exosome ZEB1 which indirectly reflects the expression level of CD47 in tumor tissues.
Further, the numerical relationship between the expression level of plasma exosome ZEB1 and the expression level of tumor tissue CD47 was as follows:
y＝0.2273x+0.7739(R20.5191), wherein y represents the expression level of tumor tissue CD47 and x represents the expression level of plasma exosome ZEB 1.
Further, high expression of plasma exosome ZEB1 suggests that cervical cancer patients are suitable for CD47 immunotherapy.
Compared with the prior art, the beneficial effects of this application are as follows:
the application discovers that ZEB1 is an important transcription factor for regulating and controlling the expression of CD47 of the cervical cancer cell based on in-vitro experimental research, and discovers that ZEB1 is enriched in exosomes in an anoxic cervical cancer cell for the first time; further verifies that the expression level of the plasma exosome ZEB1 of the cervical cancer patient is positively correlated with the expression level of the tumor tissue CD47 in the cervical cancer clinical sample. The expression level of the plasma exosome ZEB1 of the cervical cancer patient is detected in real time, so that the expression level of CD47 in a cervical cancer tissue can be reflected in real time, and the method has important guiding significance for the evaluation of the cervical cancer CD47 immunotherapy.
In addition, compared with the method for evaluating the expression level of CD47 in the cervical cancer tissue by the existing immunohistochemical method, the expression level of CD47 in the cervical cancer tissue is indirectly reflected by the expression level of the plasma exosome ZEB1, and due to the minimally invasive property and the real-time property of the plasma exosome of the patient during acquisition, the method has the advantages of high real-time property and high accuracy, is convenient to operate, and has higher popularization and application values, and the expression level of CD47 in the cervical cancer tissue can be reflected in real time.
Drawings
FIG. 1 is a diagram showing the expression distribution and regulation of ZEB1 and CD47 in cervical cancer cells;
FIG. 2 is a graph of the relation between the relative expression amount of the plasma exosome ZEB1 and the expression amount of tumor tissue CD47 of a cervical cancer patient and an ROC curve chart for evaluating the cervical cancer CD47 immunotherapy by using the expression level of the plasma exosome.
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples. It will be understood by those skilled in the art that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.
Example 1
This example will search for the relationship between exosome ZEB1 and cervical cancer tissue CD47 from the following two sections.
A first part: hypoxic cervical cancer cells highly express ZEB1 and promote transcriptional regulation of CD 47.
The expression levels of ZEB1 and CD47 in the cervical cancer cell lines (SiHa and C33a) cultured under the normoxic and hypoxic conditions were respectively detected by immunofluorescence, Western Blot and fluorescent quantitative PCR, and the results indicate that the hypoxic cervical cancer cells highly express ZEB1 and CD47 compared with the normoxic cervical cancer cells (FIG. 1A).
The expression level of CD47 is reduced or increased by using lentivirus to knock down or over express the expression level of the cervical cancer cell line ZEB1 (FIG. 1B and FIG. 1C). The dual luciferase results confirmed that ZEB1 is an upstream transcription factor of CD47, transcriptionally upregulating expression of CD47 (fig. 1D).
A second part: the relative expression amount of the exosome ZEB1 in the plasma of the cervical cancer patient is effective for evaluating the expression of the CD47 in tumor tissues, and is used for evaluating whether the cervical cancer patient is suitable for the CD47 immunotherapy.
Exosomes secreted by normoxic and hypoxic cervical cancer cell lines were extracted by gradient ultracentrifugation, morphology of exosomes was identified by transmission electron microscopy (fig. 2A), expression of exosome marker proteins CD63, TSG101, HSP70 and ZEB1 was identified by Western Blot, and it was found that ZEB1 protein was enriched in exosomes derived from hypoxic cervical cancer cell lines (fig. 2C). Further verified in cervical cancer clinical samples, 20 cases of peripheral blood samples of early-stage cervical cancer (Figo 2018, stages I and II) and late-stage cervical cancer patients (Figo 2018, stages III and IV) are respectively collected, plasma exosome extraction kit (Ex5022-03) is used for extracting plasma exosomes (figure 2B) of clinical cervical cancer patients and detecting the expression of ZEB1 (figures 2C-D), and the result shows that the expression of the plasma exosomes ZEB1 of the late-stage cervical cancer patients is obviously increased compared with the early-stage cervical cancer patients. CD47 expression was also detected by immunohistochemistry on pathological sections of the cervix matched with clinical blood samples, scored using H-score: h-score ═ Σ pi (i +1), where pi represents the number of positive cells as a percentage of the number of all cells in the section; i represents the staining intensity. The results showed that the tumor tissue CD47 expression was significantly increased in patients with advanced cervical cancer compared to patients with early cervical cancer (fig. 2D). Further, the spaerman correlation analysis shows that the expression level of the exosome ZEB1 in the plasma of the cervical cancer patient is obviously and positively correlated with the expression level of the tumor tissue CD47 (r is 0.5191, P is<0.001) (fig. 2E). Expression of plasma exosome ZEB1The numerical relationship between the amount and the expression amount of CD47 in cervical cancer tissue is: y-0.2273 x +0.7739 (R)20.5191), wherein y represents the expression level of tumor tissue CD47 and x represents the expression level of plasma exosome ZEB 1.
According to the H-score of the tumor tissue CD47 expression, the definition of H-score is more than or equal to 8, and the expression is high in CD 47; definition H-score <8 is low expression of CD 47. The ROC curve is combined to further discover that the relative expression quantity of the plasma exosome ZEB1 of the cervical cancer patient can effectively evaluate the expression of the tumor tissue CD47 (the area under the ROC curve is 0.901, and P is less than 0.001), and Youden's index indicates that the optimal boundary value of the plasma exosome ZEB1 of the cervical cancer patient for evaluating the expression quantity of the tumor tissue CD47 is 2.350, namely the relative expression quantity of the plasma exosome ZEB1 of the cervical cancer patient is more than or equal to 2.350, and the tumor tissue CD47 is highly expressed, thereby prompting that the cervical cancer patient is suitable for the CD47 immunotherapy; compared with the relative expression level of <2.350 and the low expression level of CD47 in tumor tissues, the plasma exosome ZEB1 of the cervical cancer patient indicates that the cervical cancer patient is not suitable for the CD47 immunotherapy (figure 2F).
Therefore, the expression level of CD47 in the tissues of the cervical cancer patients can be reflected by detecting the expression of ZEB1 in plasma exosomes; therefore, by sampling and detecting the expression quantity of ZEB1 in the plasma exosome of the cervical cancer patient in real time, the real-time evaluation of the expression level of CD47 in the tumor tissue can be realized, and the method has extremely important significance for evaluating whether the cervical cancer patient is suitable for the CD47 immunotherapy. Due to the real-time property and the convenient operation of the exosome detection, the exosome detection method has higher popularization and application values.
In conclusion, in the embodiment, in-vitro experimental research shows that the ZEB1 is an important transcription factor for regulating the expression of the CD47 of the cervical cancer cell, and the ZEB1 is found to be enriched in the exosome in the hypoxic cervical cancer cell for the first time, so that the expression amount of the ZEB1 in the plasma exosome of the cervical cancer patient is positively correlated with the expression amount of the CD47 in the cervical cancer tumor tissue in a clinical cervical cancer sample. The expression level of the plasma exosome ZEB1 of the cervical cancer patient can be detected, the expression level of CD47 in the cervical cancer tissue can be reflected, and the real-time expression level of CD47 in the tumor tissue can be reflected in real time through the real-time detection of the plasma exosome ZEB1, so that the real-time expression level has important guiding significance for the evaluation of the cervical cancer CD47 immunotherapy.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims (7)

1. A marker for real-time assessment of the expression level of CD47 in tumor tissues, wherein the marker is a plasma exosome ZEB1, and the plasma exosome ZEB1 is derived from tumor cells.
2. The marker according to claim 1, wherein the expression level of the plasma exosome ZEB1 is positively correlated with the expression level of tumor tissue CD 47.
3. The marker of claim 1 or 2, wherein the tumor is cervical cancer.
4. Use of plasma exosome ZEB1 in cervical cancer CD47 immunotherapy evaluation.
5. A method for assessing cervical cancer CD47 immunotherapy using plasma exosome ZEB1, comprising the steps of:
whether the cervical cancer patient is suitable for the CD47 immunotherapy is evaluated by detecting the expression level of the plasma exosome ZEB1 which indirectly reflects the expression level of CD47 in tumor tissues.
6. The method according to claim 5, wherein the numerical relationship between the expression level of the plasma exosome ZEB1 and the expression level of tumor tissue CD47 is as follows:
y＝0.2273x+0.7739(R20.5191), wherein y denotes the expression level of tumor tissue CD47 and x denotes the plasma exosome ZEB1The expression level of (a).
7. The method of claim 6, wherein high expression of the plasma exosome ZEB1 is indicative of suitability for CD47 immunotherapy in cervical cancer patients.
CN202111010311.2A 2021-08-31 2021-08-31 Marker for real-time evaluation of tumor tissue CD47 expression level and application thereof Withdrawn CN113755587A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Application publication date: 20211207