Application of markers based on the level of single exosomal transcription factor TAZ in serum in the preparation of diagnostics for lung cancer

Abstract

The present invention provides a use of a marker based on the level of a single exosomal transcription factor TAZ in serum for the preparation and treatment of lung cancer diagnosis. The present invention discovers for the first time that the level of a single exosomal transcription factor TAZ is significantly reduced in lung cancer serum and can also serve as a negative marker for lung cancer diagnosis. The detection method comprises: ultracentrifuging serum to obtain exosomes, resuspending the exosomes in phosphate-buffered saline, and performing a concentration test. Equal amounts of exosomes are lysed and the TAZ content in the equal amounts of exosomes is detected, and the obtained value is divided by the number of exosomes to obtain the TAZ protein level in a single exosome. The exosome marker of the present invention is based on the level of transcription factor TAZ protein in a single exosome, and analysis based on equal amounts of exosomes can improve the reliability of the marker. The exosomal TAZ protein level, as a negative marker, can be used in combination with other positive markers or negative markers in serum to improve the accuracy of disease detection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a method for detecting exosome protein levels from blood as a marker for lung cancer diagnosis, and specifically relates to the content of transcription factor TAZ in a single exosome. Background Art

[0002] Lung cancer is currently the malignant tumor with the highest morbidity and mortality in China and worldwide. Lung cancer is often insidious at onset, with early symptoms often subtle and easily overlooked. Furthermore, lung cancer progresses rapidly, with approximately 30% of patients already having distant metastases by the time they present for treatment. Of patients who present with noticeable symptoms, 75% are already metastatic or locally advanced, and 50-60% develop distant metastases during treatment. Furthermore, treatment outcomes for lung cancer have remained largely unchanged, with a five-year survival rate of only 18%. This is fundamentally due to a lack of early diagnosis. Current diagnostic methods for lung cancer include imaging tests such as CT scans and tissue biopsies. Imaging tests have a high probability of missing tumors in the early stages, when the tumor is small. Tissue biopsies, due to their invasive nature, are painful and typically performed after an initial imaging diagnosis, resulting in a delay and detrimental treatment outcomes.

[0003] The emergence of liquid biopsy technology has addressed these issues and accelerated the diagnosis of cancer. As a branch of in vitro diagnostics, liquid biopsy uses blood or urine to diagnose diseases like cancer. Its advantages lie in its ability to reduce the risks of biopsies through non-invasive sampling, effectively prolong patient survival, and offer a high cost-effectiveness. It is currently the most promising non-invasive tumor diagnostic method, possessing significant clinical value and market potential.

[0004] Exosomes are double-membrane vesicles with a diameter of 30-150 nm, secreted by cells. They encapsulate cell-specific biomolecules such as proteins, nucleic acids, and lipids. Almost all cells, including tumor cells, secrete exosomes, and they are distributed throughout various bodily fluids. Because the contents of exosomes secreted by tumor cells can reflect the characteristics of the tumor from which they originate, serum exosomes can serve as biomarkers for cancer and are an important research and development area for liquid biopsies.

[0005] Currently, the existing markers in the serum exosomes of lung cancer patients can be roughly divided into the following categories: RNA: including Exosome Diagnostics' product ExoDx Lung (ALK) detection kit, miRNA (patent application number: PCT / CN2018 / 090043), circRNA (application number: 201810112800.0), etc.; and protein: Caveolin-1 (application number: 201910293336.4), LBP (application number: 201810318885.8), GCC2 (application number: 201880072074.2), AHSG and / or ECM-1 (application number: 201810561202.1), etc., each with its own advantages and disadvantages.

[0006] Currently, there are numerous RNA biomarkers (RNA, miRNA, circRNA, etc.), and multiple biomarkers are generally required in combination to achieve ideal sensitivity and diagnostic efficacy. Currently, no single biomarker can be used alone for clinical disease diagnosis. Protein biomarkers are relatively rare, and most are positive markers for lung cancer. They also suffer from poor sensitivity and specificity. More importantly, the amount of exosomes in serum varies from patient to patient, and the extraction process can result in varying degrees of exosome loss. Simply comparing the exosomal protein content in equal amounts of serum between healthy individuals and patients is inaccurate.

[0007] It is well known that YAP and TAZ proteins are core molecules of the Hippo signaling pathway. First discovered 20 years ago in Drosophila, the Hippo pathway is a conserved signaling pathway composed of a series of kinases, transcriptional coactivators, and DNA-binding proteins. When the pathway is activated, pMST1 / 2 and pLATS1 / 2 further phosphorylate YAP / TAZ, trapping them in the cytoplasm and inhibiting their nuclear import. When the Hippo pathway is inhibited, unphosphorylated YAP / TAZ enter the nucleus, promoting the transcription of numerous downstream genes involved in cell proliferation, survival, and migration, ultimately triggering tumorigenesis in various tissues. Therefore, overactivation of YAP and TAZ is common in cancer, as is gene amplification and epigenetic regulation of YAP and TAZ loci. YAP- and TAZ-mediated transcriptional activity is crucial for tumor development and progression, primarily affecting tumor proliferation, survival, and stemness. Currently, there are no reports of exosomal TAZ proteins in malignant tumors, both in existing technologies and in the literature. Summary of the Invention

[0008] Existing techniques for identifying serum exosome markers for lung cancer, even when expanded to other diseases, fail to account for individual differences in serum exosome counts and the varying degrees of exosome loss in different samples during the exosome extraction process. Using the amount of exosome RNA or protein in equal amounts of serum as a marker to distinguish healthy individuals from patients results in a loss of sensitivity and specificity. Currently, no literature or related technologies have found that lung cancer exosomes contain TAZ, nor have any reports found that exosome TAZ can be used as a negative marker for lung cancer diagnosis. Therefore, the primary objective of this invention is to identify new serum exosome markers for lung cancer, focusing on the amount of protein contained in equal amounts of serum exosomes. Specifically, the present invention uses the TAZ protein level in equal amounts of exosomes to distinguish between healthy individuals and lung cancer patients. The results indicate that the TAZ protein content in individual exosomes from lung cancer patients is significantly reduced compared to that in healthy individuals, making it a suitable negative marker for lung cancer diagnosis.

[0009] The second object of the present invention is to provide a method for detecting the level of the above-mentioned single exosomal transcription factor TAZ.

[0010] In order to achieve the above primary purpose, the solution of the present invention is:

[0011] Application of a marker based on the level of a single exosomal transcription factor TAZ in serum for the preparation and treatment of lung cancer diagnosis.

[0012] Preferably, the level of a single exosomal transcription factor TAZ in the serum is significantly reduced in the serum of lung cancer patients, that is, the TAZ protein content is significantly reduced. The key point is that TAZ protein is a cancer-promoting molecule in cells, but is secreted into exosomes and becomes a negative marker for lung cancer. This is because cells prefer to retain TAZ inside the cells to exert a cancer-promoting effect rather than secrete it outside the body, that is, in exosomes. Therefore, the content of TAZ protein in a single exosome in the serum of lung cancer patients is significantly reduced.

[0013] Specifically, a marker based on the level of a single exosomal transcription factor TAZ in serum can be used alone as a negative marker for lung cancer diagnosis. In addition, a single exosomal transcription factor TAZ can also be used in combination with other TAZ positive or negative markers other than exosomal transcription factors in serum, thereby making it possible to use other positive markers as a combination marker based on the TAZ protein marker. Among them, other positive markers or negative markers include existing and undiscovered lung cancer markers other than TAZ protein, and other positive markers include but are not limited to one or more of miR-21, miR-205, miR-126 and miR-486-5p.

[0014] In order to achieve the above second purpose, the solution of the present invention is:

[0015] A method for detecting a marker of a single exosomal transcription factor TAZ level in serum, comprising the following steps:

[0016] (1) Collect blood from subjects (lung cancer patients and healthy subjects) in a vacuum blood collection tube, place it at room temperature for 15 ± 0.1 min, wait for blood to coagulate, and centrifuge it. The supernatant, which is serum, is collected and centrifuged again to remove impurities such as fat on the surface of the liquid.

[0017] (2) ultracentrifuging the supernatant after centrifugation in step (1), resuspending the precipitate in phosphate buffered saline (PBS), and ultracentrifuging again to obtain the precipitate that is the exosome;

[0018] (3) After enrichment, the exosomes were resuspended in 30 μL PBS, 1 μL was taken and diluted 10,000 times with PBS, and the exosome concentration was detected;

[0019] (4) After obtaining the exosome concentrations of lung cancer patients and healthy subjects, equal amounts of exosomes (2×10 8 -3×10 8 The cells were lysed by adding loading buffer to denature the proteins, and proteins of different sizes were separated. The proteins were incubated with TAZ protein antibodies to develop color. The TAZ protein levels in equal amounts of exosomes were compared, and the color depth was quantified using ImageJ software. The obtained value was divided by the number of exosomes to obtain the TAZ protein level in a single exosome.

[0020] Preferably, in step (1), the centrifugal speed after blood coagulation is 3000±10 rpm, and the time is 10±0.1 min; the centrifugal speed after the supernatant is drawn is (3000±10)×g, and the time is 15±0.1 min.

[0021] Preferably, in step (2), the method for extracting exosomes is selected from one or more of the following methods: ExtraPEG method, SBI ExoQuick method, magnetic bead method, size exclusion chromatography, ultrafiltration method, microfluidics method and ultracentrifugation method.

[0022] Preferably, in step (2), the speed of ultracentrifugation is (100000±100)×g, and the time is 70±0.1 min.

[0023] Preferably, in step (3), the method for detecting the exosome concentration is selected from one or more of nanoflow analyzer, dynamic light scattering, flow cytometry, exosome protein quantification and nanoparticle tracking analysis.

[0024] Preferably, in step (4), the method for measuring the expression level of TAZ protein is selected from one or more of protein chip analysis, immunoassay, ligand binding analysis, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, surface-enhanced laser desorption-ionization time-of-flight mass spectrometry, radioimmunoassay, radial immunodiffusion, immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry / mass spectrometry, Western blotting, enzyme-linked immunosorbent assay and Western blot.

[0025] Preferably, in step (4), the cracking temperature is 95±1°C and the cracking time is 5±0.1 min.

[0026] The present invention originally discovered that the transcription factor TAZ can be selectively secreted into exosomes by lung cancer cells, and compared with the healthy control group, lung cancer patients sort less TAZ into exosomes, that is, the TAZ protein content in individual exosomes in the serum of lung cancer patients is significantly reduced. If the equal amount of exosomes is not strictly limited, but the detection is based on the equal amount of serum, the difference in the TAZ content of individual exosomes may be covered by the individual differences in the number of exosomes in the patient's serum, resulting in reduced sensitivity. Therefore, the present invention intends to protect the protein content in a single exosome as a marker, which is different from the prior art of extracting exosomes with equal amounts of serum and then detecting the protein content. Therefore, the step of detecting the number of exosomes is added in the present invention. The key point is to ensure that the number of exosomes is consistent, thereby detecting the protein content.

[0027] Due to the adoption of the above solution, the beneficial effects of the present invention are:

[0028] The lung cancer serum exosome marker of the present invention is based on the level of the transcription factor TAZ protein in a single exosome. First, the number of exosomes can be accurately quantified, and the analysis based on equal amounts of exosomes can improve the reliability of the marker. Second, the level of exosomal TAZ protein is reduced in lung cancer serum patients. Therefore, it can be used alone as a negative marker or in combination with other positive markers or negative markers in serum, thereby improving the accuracy of disease detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is an electron micrograph of exosomes in an embodiment of the present invention.

[0030] Figure 2 This is a graph showing the detection results of the nanoparticle tracking analysis system (NTA) on the concentration and particle size analysis of exosomes in an embodiment of the present invention (the horizontal axis Size is the particle size, and the vertical axis Concentration is the concentration).

[0031] Figure 3This is a diagram showing the identification of exosome protein markers using Western blot technology in an embodiment of the present invention.

[0032] Figure 4 This is a graph showing TAZ levels in equal amounts of exosomes in the serum of healthy subjects and lung cancer patients detected by Western blot technology in an embodiment of the present invention.

[0033] Figure 5 This is a statistical graph comparing the TAZ content of individual exosomes in the serum of healthy subjects and lung cancer patients in the examples of the present invention. The ordinate is the grayscale value of the Western blot image statistically calculated by ImageJ software divided by the number of exosomes measured by NTA.

[0034] Figure 6 This is a receiver operating characteristic (ROC) curve for evaluating the content of single exosome TAZ in serum as a negative marker for lung cancer in the examples of the present invention. DETAILED DESCRIPTION

[0035] The marker based on the level of a single exosomal transcription factor TAZ in serum of the present invention can be used in the preparation and treatment of lung cancer diagnosis, and the level of a single exosomal transcription factor TAZ is reduced in the serum of lung cancer patients. TAZ (Transcriptional coactivator with PDZ-binding motif) is a transcriptional coactivator with a PDZ binding motif in the Hippo signaling pathway, and the gene is named WWTR1 (WW domain-containing transcription regulator protein 1).

[0036] In addition, markers based on the level of a single exosomal transcription factor TAZ in serum can also be used alone as negative markers for the diagnosis of lung cancer, or can be used in combination with other TAZ positive or negative markers in serum other than exosomal transcription factors. Downregulated lung cancer negative markers include but are not limited to miR-152, Let-7a or miR-148a, and other upregulated positive markers include but are not limited to miR-21, miR-205, miR-126 or miR-486-5p.

[0037] The method for detecting a marker of a single exosomal transcription factor TAZ level of the present invention comprises the following steps:

[0038] (1) Collect blood from subjects (lung cancer patients and healthy subjects) in a vacuum blood collection tube, place it at room temperature for 15 ± 0.1 min, wait for blood to coagulate, and centrifuge it. The supernatant, which is serum, is collected and centrifuged again to remove impurities such as fat on the surface of the liquid.

[0039] (2) ultracentrifuging the supernatant after centrifugation in step (1), resuspending the precipitate in phosphate buffered saline (PBS), and ultracentrifuging again to obtain the precipitate that is the exosome;

[0040] (3) After the enriched exosomes were resuspended in 30 μL PBS, 1 μL was diluted 10,000 times with PBS, and the exosome concentration was detected using the nanoparticle tracking analysis system (NTA);

[0041] (4) After obtaining the exosome concentrations of lung cancer patients and healthy subjects, equal amounts of exosomes (2×10 8 -3×10 8 The cells were lysed by adding loading buffer to denature the proteins. Proteins of different sizes were separated by western blot and incubated with TAZ protein antibodies to develop color. The TAZ protein levels in equal amounts of exosomes were compared, and the color depth was quantified using ImageJ software. The obtained value was divided by the number of exosomes to obtain the TAZ protein level in a single exosome.

[0042] In step (1), the centrifugal speed after coagulation can be 3000±10 rpm, preferably 3000 rpm; the time can be 10±0.1 min, preferably 10 min; the centrifugal speed after aspirating the supernatant can be (3000±10)×g, preferably 3000×g; the time can be 15±0.1 min, preferably 15 min.

[0043] In step (2), the speed of ultracentrifugation can be (100,000±100)×g, preferably 100,000×g; the time can be 70±0.1 min, preferably 70 min.

[0044] In step (2), in addition to the ultracentrifugation method used in the present invention, there are other alternative exosome extraction methods, including but not limited to the ExtraPEG method, SBIExoQuick method, magnetic bead method, size exclusion chromatography, ultrafiltration method and microfluidics method.

[0045] In step (3), in addition to the nanoparticle tracking analysis system (NTA) used in the present invention, other alternatives to the exosome concentration detection method include other instruments that can detect the concentration of particles around 100 nanometers, such as exosomes, including but not limited to nanoflow analyzers, dynamic light scattering, flow cytometry, and exosome protein quantification.

[0046] In step (4), in addition to the western blot technology used in the present invention, there are other alternative methods for detecting protein content in the exosomal protein TAZ, including but not limited to protein chip analysis, immunoassay, ligand binding analysis, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, surface-enhanced laser desorption-ionization time-of-flight mass spectrometry, radioimmunoassay, radial immunodiffusion, immunodiffusion, rocket immunoelectrophoresis, immunohistochemical staining, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry / mass spectrometry, Western blot and enzyme-linked immunosorbent assay, etc.

[0047] In step (4), the pyrolysis temperature may be 95±1°C, preferably 95°C; and the pyrolysis time may be 5±0.1 min, preferably 5 min.

[0048] The present invention will be further described below with reference to the examples.

[0049] Example:

[0050] Unless otherwise specified, the experimental methods used in the examples are conventional methods.

[0051] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0052] In the embodiment, the serum may be one or more selected from blood, serum, and plasma, and is preferably blood, but is not limited thereto.

[0053] The nanoparticle tracking system (NTA) in the embodiment is ZetaView from Particle Metrix, Germany. However, non-limiting examples of methods for detecting the number of exosomes include: nanoflow analyzer, dynamic light scattering, flow cytometry, and exosome protein quantification.

[0054] The ultracentrifuge in the examples is from Backman, USA. Non-limiting examples of serum exosome extraction methods include: ExtraPEG method, SBI ExoQuick method, magnetic bead method, size exclusion chromatography, ultrafiltration method and microfluidics method.

[0055] Non-limiting examples of methods for measuring or comparing the expression level of TAZ protein in the embodiments include: protein chip analysis, immunoassay, ligand binding analysis, matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, surface-enhanced laser desorption-ionization time-of-flight mass spectrometry, radioimmunoassay, radial immunodiffusion, immunodiffusion, rocket immunoelectrophoresis, immunohistostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry, liquid chromatography-mass spectrometry / mass spectrometry, Western blot and enzyme-linked immunosorbent assay, etc.

[0056] In this embodiment, the method for detecting a marker of the level of a single exosome transcription factor TAZ comprises the following steps:

[0057] (1) Collect blood from subjects (lung cancer patients and healthy people) in vacuum blood collection tubes and place them at room temperature for 15 minutes. After blood coagulation, centrifuge at 3000 rpm for 10 minutes and aspirate the supernatant, which is serum. The serum is then centrifuged at 3000 × g for 15 minutes to remove impurities such as fat on the surface of the liquid.

[0058] (2) Exosomes were extracted from the treated serum by ultracentrifugation. The serum was centrifuged at 100,000 × g for 70 min, the precipitate was collected, resuspended in PBS, and centrifuged again at 100,000 × g for 70 min. The precipitate obtained was the exosomes.

[0059] (3) Figure 1 As shown in Figure 2, transmission electron microscopy (TEM) morphological analysis showed that exosomes exhibited a classic cup-shaped structure with a diameter of less than 200 nm. Figure 2 As shown in Figure 2, most particles have a diameter of less than 200 nm, which is consistent with the size distribution of exosomes. Figure 3 As shown, the enriched exosomes were protein-marked, showing that the extracted exosome samples contained exosome protein markers (ALIX, TSG101, CD63, CD9, YAP) and the target protein TAZ.

[0060] (4) After enrichment, the exosomes were resuspended in 30 μL PBS, and 1 μL was diluted 10,000 times with PBS. The exosome concentration was detected using a nanoparticle tracking analysis system (NTA).

[0061] (5) After obtaining the exosome concentrations of lung cancer patients and healthy subjects, equal amounts of exosomes (2×10 8 Then, the cells were lysed by adding loading buffer and heated at 95℃ for 5 min to denature the proteins. Proteins of different sizes were separated by western blot and incubated with TAZ protein antibodies to develop color. The levels of TAZ protein in equal amounts of exosomes were compared (e.g. Figure 4 As shown in the figure, since the number of exosomes extracted from different samples is different, in order to ensure the detection of TAZ, the amount of exosomes loaded should be as large as possible. Therefore, the sample with high concentration should be loaded with a large amount, and the sample with low concentration should be loaded with a small amount. Figure 4 The number of exosomes measured by NTA is also shown in the figure: the content of TAZ in the serum of healthy people is higher than that in lung cancer patients.

[0062] (6) ImageJ software was used to quantify the color depth, and the obtained value was divided by the number of exosomes to obtain the protein level of TAZ in a single exosome. GraphPad software was used for statistical analysis. All data were given as the mean plus standard deviation. The data were evaluated using the unpaired t test analysis method. A P value of less than 0.05 was considered statistically significant. Figure 5 As shown in the results, the content of single exosome TAZ in the serum of healthy subjects was significantly higher than that in lung cancer patients, and the statistical difference p value was less than 0.01. Figure 6 As shown, the results showed that the area under the curve (AUC) was 0.8958, proving that this lung cancer negative marker has a good diagnostic efficacy in the diagnosis of lung cancer. When the diagnostic threshold is 0.002001, the sensitivity and specificity are 91.67%.

[0063] The above description of the embodiments is intended to facilitate understanding and use of the present invention by those skilled in the art. Those skilled in the art will readily be able to make various modifications to these embodiments and apply the general principles described herein to other embodiments without resorting to creative effort. Therefore, the present invention is not limited to the above-described embodiments. Any improvements or modifications made by those skilled in the art based on the principles of the present invention that do not depart from the scope of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Use of a reagent for detecting a marker based on the level of a single exosomal transcription factor TAZ in serum in the preparation of a lung cancer diagnostic product, characterized in that: The level of the single exosomal transcription factor TAZ is reduced in the serum of lung cancer patients, and the detection method of the marker based on the level of the single exosomal transcription factor TAZ in the serum comprises the following steps: (1) Collect the blood of the subject and place it at room temperature for 15±0.1 min. After blood coagulation, centrifuge it and aspirate the supernatant. (2) Ultracentrifuging the supernatant after centrifugation in step (1), resuspending the precipitate in phosphate buffered saline, and ultracentrifuging again to obtain exosomes; (3) resuspending the exosomes in phosphate buffered saline, diluting the solution, and detecting the exosome concentration; (4) Take an equal amount of exosomes from step (3) 2×10 8 -3×10 8 The cells were lysed by adding loading buffer to denature the proteins, which were then separated and incubated with antibodies against TAZ protein to develop color. The color depth was quantified using ImageJ software, and the obtained value was divided by the number of exosomes to obtain the level of the transcription factor TAZ in a single exosome.

2. The use according to claim 1, characterized in that: The level of a single exosomal transcription factor TAZ was used alone as a negative marker in serum, or was used in combination with other markers other than the level of the exosomal transcription factor TAZ in serum.

3. The use according to claim 1, characterized in that: In step (1), the centrifugal speed after blood coagulation is 3000±10 rpm, and the time is 10±0.1 min; the centrifugal speed after the supernatant is drawn is (3000±10)×g, and the time is 15±0.1 min.

4. The use according to claim 1, characterized in that: In step (3), the method for detecting the exosome concentration is selected from one of nanoflow cytometry, dynamic light scattering, flow cytometry, exosome protein quantification and nanoparticle tracking analysis.

5. The use according to claim 1, characterized in that: In step (4), the cracking temperature is 95±1°C and the time is 5±0.1 min.

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