Application of plasma exosomes A1BG and TF in diagnosis of cervical cancer lymph node metastasis

Abstract

The invention relates to application of plasma exosomes A1BG and TF in diagnosis of cervical cancer lymph node metastasis. Detection of lymph node metastasis is achieved through the plasma exosome A1BG and the plasma exosome TF, the ratio of the abundance of the plasma exosome A1BG to the mass of the total exosome is limited to be smaller than or equal to 14.2690 ng / mg, the ratio of the abundance of the exosome TF to the mass of the total exosome is limited to be smaller than or equal to 96.2011 ng / mg, and combined detection is conducted through the plasma exosome A1BG and the plasma exosome TF, so that the sensitivity is 44%, the specificity is 100%, and the detection time is shortened. The preoperative joint detection of the A1BG and the TF can be prompted to detect nearly half of the patient without lymph node metastasis, and unnecessary pelvic lymph node cleaning operation is avoided.

Description

Technical Field

[0001] The present invention belongs to the technical field of lymph node metastasis detection, and specifically relates to the application of plasma exosomes A1BG and TF in diagnosing lymph node metastasis of cervical cancer. Background Art

[0002] Cervical cancer is one of the most common malignant tumors of the female reproductive system. The key to its successful treatment lies in early detection and accurate assessment of lymph node metastasis. Pelvic lymph node dissection is an important part of surgical treatment of cervical cancer. It can help doctors determine whether the tumor has spread to the pelvic lymph nodes, thereby determining the subsequent treatment plan. However, the current lymph node dissection has some problems, such as large surgical trauma, high risk of complications, and long recovery period.

[0003] However, not all patients with cervical cancer who seek medical treatment have pelvic lymph node metastasis. Therefore, how to accurately predict which patients do not have lymph node metastasis and do not need lymph node dissection is of great significance for reducing unnecessary surgical trauma and improving patients' quality of life. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a plasma exosome protein marker with the ability to diagnose cervical cancer lymph node metastasis.

[0005] The technical solution of the present invention is as follows:

[0006] Application of plasma exosomal A1BG and TF as markers in the diagnosis of lymph node metastasis of cervical cancer.

[0007] Furthermore, the ratio of the abundance of exosome A1BG to the total exosome mass is ≤14.2690 ng / mg.

[0008] Furthermore, the ratio of the exosome TF abundance to the total exosome mass is ≤96.2011 ng / mg.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1. The present invention realizes the detection of lymph node metastasis through plasma exosome A1BG and plasma exosome TF, by limiting the ratio of plasma exosome A1BG abundance to total exosome mass to ≤14.2690 ng / mg and the ratio of exosome TF abundance to total exosome mass to ≤96.2011 ng / mg, and through combined detection of plasma exosome A1BG and plasma exosome TF, the sensitivity is 44% and the specificity is 100%, indicating that preoperative combined detection of A1BG and TF can detect nearly half of the patients without lymph node metastasis, avoiding unnecessary pelvic lymph node dissection. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a diagram for identifying exosomes in a small sample of plasma according to the present invention;

[0012] Figure 2 This is a diagram of the Label-Free whole proteomics detection results of the present invention;

[0013] Figure 3 It is a protein immunoblot of A1BG, TF, APOA1, SAA4, C1S and PHLDB2 in the plasma exosomes of the present invention;

[0014] Figure 4 This is a diagram showing the clinical application value of plasma exosomes A1BG and TF of the present invention. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] like Figures 1 to 4 As shown, the application of plasma exosome A1BG and TF in the diagnosis of cervical cancer lymph node metastasis;

[0017] 1. Test Reagents

[0018] 1. The plasma samples of patients in the cervical cancer lymph node metastasis group and the non-metastasis group of the present invention were obtained from the First Affiliated Hospital of Zhengzhou University. In this embodiment, plasma was collected from 4 patients with cervical cancer lymph node metastasis and 4 patients with cervical cancer lymph node non-metastasis, and stored at -80°C in time. Other reagents or consumables used were commonly available on the market or could be obtained by those skilled in the art through public channels.

[0019] 2. Test methods

[0020] 1. Collect human peripheral blood using EDTA anticoagulant blood collection tubes, centrifuge at 3000 rpm for 10 min, transfer the supernatant to a new 15 mL centrifuge tube, centrifuge at 10000 rpm for 10 min to obtain the supernatant, transfer the obtained supernatant (plasma sample) to a new 5 mL centrifuge tube, and freeze in a -80 °C refrigerator for subsequent exosome extraction.

[0021] 2. Extract exosomes from plasma by the following two methods: ultracentrifugation and exosome isolation kit (CD63 affinity method);

[0022] 2.1 Extraction of exosomes for Label-Free whole proteomics detection by ultracentrifugation:

[0023] S11: Rapidly thaw the plasma sample at 37°C, transfer the plasma sample to a new centrifuge tube, and centrifuge at 2000×g, 4°C, for 30 min;

[0024] S12: Carefully transfer the supernatant to a new centrifuge tube, centrifuge again at 10,000×g, 4°C, 45 min, take the supernatant, filter through a 0.45 μm filter membrane, and collect the filtrate;

[0025] S13: Transfer the filtrate to a new centrifuge tube, select an ultraspeed rotor, centrifuge at 4°C, 100,000×g for 70 min, remove the supernatant, resuspend with 10 mL of pre-cooled 1× PBS, select an ultraspeed rotor, and ultracentrifuge again at 4°C, 100,000×g for 70 min. Remove the supernatant, resuspend with 200 μL of pre-cooled 1× PBS, extract 10 μL of protein, and store the remaining exosomes at -80°C for subsequent label-free proteomics quantification.

[0026] 2.2. Extract exosomes using an exosome isolation kit:

[0027] Exosomes were extracted for Western blotting according to the manufacturer's instructions on the exosome isolation kit;

[0028] The specific steps are as follows: the frozen plasma samples were taken out, and the thawed plasma samples were incubated with magnetic beads treated with extraction reagents at 4°C. After 4 hours, the supernatant was removed, and the magnetic beads were washed twice with 1×PBS. Subsequently, 100 μL of elution solution was added to the magnetic beads, the mixture was thoroughly mixed with a pipette, and the supernatant was then collected. The elution step was repeated three times, and exosomes were present in the supernatant. The collected supernatant was stored at -80°C for subsequent immunoblotting.

[0029] The obtained exosome morphology was identified by electron microscopy, and the obtained exosome particle size and specific markers were analyzed and identified by nanoparticle tracking analysis (NTA) and protein immunoblotting (WB). Figure 1 : The plasma exosomes extracted by ultracentrifugation were identified by electron microscopy (A), NTA (B) and WB (C). The identification results showed that the extract was exosomes). The results showed that the extracted vesicles met the characteristics of exosomes in terms of morphology, particle size distribution and specific marker labeling.

[0030] 3. Perform Label-Free whole proteomics detection on the exosomes obtained above:

[0031] 3.1 Protein extraction:

[0032] Exosomes were lysed with 8M urea, and protease inhibitors were added at 10% of the lysate volume. The supernatant was collected after centrifugation at 14000rcf at 4°C for 20 min, and the protein concentration of the supernatant was determined by the Bradford method and stored at -80°C.

[0033] 3.2. Enzymatic desalting:

[0034] 1) Take an appropriate amount of supernatant protein and add DTT with a final concentration of 5 mM, incubate at 37°C for 1 hour, then return to room temperature, add iodoacetamide with a final concentration of 10 mM, and incubate at room temperature in the dark for 45 minutes to obtain a protein sample;

[0035] 2) Dilute the protein sample 4 times with 25 mM ammonium bicarbonate, add trypsin at a ratio of 50:1 between protein and trypsin, incubate at 37°C overnight, and add formic acid the next day to adjust the pH to less than 3 to terminate the enzyme digestion;

[0036] 3) Use a C18 desalting column to desalt the protein sample after enzyme digestion, activate the desalting column with 100% acetonitrile, balance the column with 0.1% formic acid, load the protein sample after enzyme digestion onto the column, then wash the column with 0.1% formic acid to wash away impurities, and finally elute with 70% acetonitrile, collect the flow-through, and lyophilize to form a lyophilized powder.

[0037] 3.3 Mass spectrometry

[0038] Mobile phase A (100% water, 0.1% formic acid) and B (80% acetonitrile, 0.1% formic acid) were prepared, 10 μL of A was used to dissolve the lyophilized powder, centrifuged at 14000 g for 20 min at 4°C, 1 μg of the supernatant was injected, and the liquid mass spectrometer was used for liquid-mass spectrometry. The proportion of mobile phase B was increased from 6% to 12% in 18 minutes, and from 12% to 30% B gradient separation in 79 minutes, and increased to 40% in 16 minutes, and then washed at 95% B gradient at a speed of 600 nL / min for 10 minutes. ORBITRAP ECLIPSE mass spectrometer with FAIMS Pro was used. TM Interface, the compensation voltage CV is switched between -45 and -65 every 1S, Nanospray Flex TMThe (NSI) ion source was used, the ion spray voltage was set to 2.0 kV, the ion transfer tube temperature was set to 320°C, the mass spectrometry adopted data-dependent acquisition mode, the full scan range of the mass spectrometry was m / z 350-1500, the primary mass spectrometry resolution was set to 120000 (m / z), the AGC was 4×105, and the maximum C-trap injection time was 50 ms; the secondary mass spectrometry detection adopted the "Top Speed" mode, the secondary mass spectrometry resolution was set to 15000 (200 m / z), the AGC was 5×104, the maximum injection time was 22 ms, the peptide fragmentation collision energy was set to 33%, and the mass spectrometry detection raw data (.raw) were generated.

[0039] 3.4 Data processing:

[0040] The raw data files were processed and quantified using Proteome Discoverer software (version 2.4.1.15) (Thermo Fisher Scientific, Massachusetts, USA), and the SEQUEST algorithm was used to search [RefSeq Human Protein Database (24,078 sequences, release 2017_03)];

[0041] The search parameters used are as follows: the type of quantification method is Precursor Quantification, the enzyme cleavage type is Trypsin, the quantification mode is intensity, the maximum number of enzyme missed cleavage sites allowed is 2, the mass deviation tolerance range when searching the precursor ion library is 20ppm, the mass deviation tolerance range when searching the fragment ion library is 0.05Da, the specific variable modification type is set to Oxidation / +15.995Da(M), the N-terminal modification type is Acetyl / +42.011Da(N-Terminal), and the specific fixed modification type is set to Carbamidomethyl / +57.021Da(C).

[0042] Comparison of plasma exosome proteomic characteristics between the two groups of patients showed that 435 proteins were identified, of which 405 were quantified. Statistical analysis showed that 14 proteins were significantly changed in the lymph node metastasis group compared with the non-lymph node metastasis group (P < 0.05, FC ≥ 1.5 times), of which 7 proteins were up-regulated and 7 proteins were down-regulated ( Figure 2 : There were 14 protein molecules in the plasma exosomes of cervical cancer patients with lymph node metastasis and those without metastasis with a difference multiple greater than 1.5 and statistically significant, among which 7 molecules were upregulated and downregulated in the plasma exosome protein levels of the lymph node metastasis group compared with the non-metastasis group).

[0043] Based on Label-Free whole proteomics detection and previous research results, 12 exosomal proteins were selected for large-sample validation. The 12 proteins were: RPSA, SAA4, PHLDB2, C1S, TF, A1BG, RAB39A, CNDP1, EHD3, APOA1, C1QC and GNAI2.

[0044] 3.5. Verify the above exosome proteins using Western blotting:

[0045] Plasma was collected from another 20 patients with cervical cancer lymph node metastasis and 20 patients without lymph node metastasis and stored promptly at -80°C.

[0046] A total of 24 patients with lymph node metastasis and 24 patients without lymph node metastasis were included. The mean age of patients in the metastasis group and the non-metastasis group was 48.96±12.31 (28-72 years) and 47.79±12.31 (28-73 years), respectively. There was no significant difference in age between the two groups (P=0.796).

[0047] According to the 2023 FIGO cervical cancer staging system, all patients in the metastatic group were diagnosed with stage IIIC cervical cancer, including 18 cases (75.0%) of stage IIIC1 and 6 cases (25.0%) of stage IIIC2. In the non-metastatic group, 20 patients (83.3%) were diagnosed with stage I cervical cancer and 4 patients (16.7%) were diagnosed with stage II cervical cancer. In addition, the lymph node status of patients in the non-metastatic group was determined by postoperative pathological examination, while in the metastatic group, 54.2% (13 / 24) of patients had their lymph node status determined by postoperative pathological examination, and 45.8% (11 / 24) of patients had their lymph node status determined by preoperative imaging examination. Table 1 shows the basic clinical characteristics of the subjects.

[0048]

[0049]

[0050] Table 1

[0051] Exosomes for Western blotting were extracted using an exosome isolation kit (CD63 affinity method). Thawed plasma samples were incubated with magnetic beads treated with the extraction reagent at 4°C according to the manufacturer's instructions on the exosome isolation kit. The supernatant was removed after 4 h and the magnetic beads were washed twice with 1× PBS. Subsequently, 100 μL of elution buffer was added to the magnetic beads and the mixture was mixed thoroughly with a pipette. The supernatant was then collected. The elution step was repeated three times. Exosomes were present in the supernatant. The collected supernatant was stored at -80°C for subsequent Western blotting.

[0052] 10 μg of each exosome sample was taken as the sample amount and electrophoresed using 10% and 12% SDS-PAGE gels. The whole process was carried out according to the standard Western blotting experimental procedure;

[0053] The primary antibodies used in this experiment include anti-SAA4 (20398-1-AP), anti-PHLDB2 (27940-1-AP), anti-C1S (14554-1-AP), anti-TF (17435-1-AP), anti-A1BG (66260-1-1g), anti-APOA1 (14427-1-AP), anti-CNDP1 (17759-1-AP), anti-RAB39A (13355-1-AP), anti-RPSA (14533-1-AP), anti-EHD3 (25320-1-AP), anti-C1QC (66268-1-AP), anti-GNAI2 (11136-1-AP), anti-CD63 (ab134045), anti-CD81 (ab109201), and anti-Flotillin 2 (ab181988). The total protein content was estimated using the signal intensity of Flotillin 2 as a reference.

[0054] 3. Analysis Method

[0055] 1. SPSS (version 25.0) was used for data analysis. igraph was used to draw network diagrams. The t-test was used to identify proteins with significant expression differences (P<0.05, FC≥1.5). For continuous numerical variables, the differences between three or more groups were analyzed by one-way analysis of variance. The chi-square test was used to analyze the differences between groups for categorical variables. The receiver operating characteristic (ROC) curves of these proteins were drawn, and the areas under the curves (AUCs) of these proteins were compared to evaluate the diagnostic efficacy of exosomes. P<0.05 was considered statistically significant.

[0056] 2. Use igraph and SPSS25.0 software for statistical analysis and graphing, and calculate the area under the curve to evaluate the diagnostic efficacy of exosomal A1BG.

[0057] 3. It was found that compared with the non-metastasis group, the abundance of plasma exosomes A1BG, TF, APOA1, SAA4, C1S and PHLDB2 in the lymph node metastasis group was upregulated, among which the abundance of A1BG and TF increased most significantly ( Figure 3 ).

[0058] 4. Therefore, we focused on plasma exosomes A1BG and TF to explore their clinical application value in preoperative lymph node assessment of cervical cancer. Except for 8 samples used for Label-Free whole proteomics detection (insufficient remaining samples), the remaining 40 samples (20 cases with lymph node metastasis and 20 cases without lymph node metastasis) were quantified using BCA, and then plasma exosomes A1BG (KE00130) and TF (KE00300) were detected using enzyme-linked immunosorbent assay (ELISA) kits. ELISA was performed according to the instructions on the corresponding kits, and the test data were statistically analyzed;

[0059] The results showed that the A1BG and TF in plasma exosomes in the lymph node metastasis group were significantly higher than those in the non-metastasis group, and the fold change (FC) was about 2 times ( Figure 4 A and B), the ROC curves showed that the AUCs of A1BG and TF were 0.798 and 0.935, respectively ( Figure 4 C and D), the specificity was set to 100%, and the corresponding value with the highest sensitivity was set as the cutoff value to ensure that all patients with lymph node metastasis were not missed.

[0060] The attached Figure 4 A and B in the figure are the contents of A1BG and TF in plasma exosomes of cervical cancer patients detected by ELISA method, and the detection data were statistically analyzed to compare the differences in the contents of A1BG and TF in plasma exosomes between patients with cervical cancer complicated with lymph node metastasis and patients without lymph node metastasis.

[0061] C and D are ROC curve analyses of A1BG and TF ELISA results.

[0062] E is the sensitivity and specificity of plasma exosomal A1BG and TF in patients with cervical cancer without lymph node metastasis at different cutoff points.

[0063] The results showed that the critical value of A1BG in exosomes was 14.2690. When the ratio of A1BG in exosomes to total exosomes (ratio of exosome A1BG abundance to total exosome mass, ng / mg) was ≤14.2690, the sensitivity was 20% and the specificity was 100% ( Figure 4 E), the critical value of TF in exosomes was 96.2011. When TF in exosomes / total exosomes (ratio of exosome TF abundance to total exosome mass, ng / mg) was ≤96.2011, the sensitivity was 30% and the specificity was 100% ( Figure 4 E).

[0064] Combined detection of A1BG and TF in exosomes was used to screen more patients without lymph node metastasis. The results showed that when A1BG / exosome ≤14.2690 or TF / exosome ≤96.2011, the sensitivity was 44% and the specificity was 100% ( Figure 4 E), indicating that preoperative combined detection of A1BG and TF can detect nearly half of patients without lymph node metastasis and avoid unnecessary pelvic lymph node dissection.

[0065] Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of plasma exosome A1BG and TF as markers in the diagnosis of lymph node metastasis of cervical cancer.

2. The use of plasma exosomes A1BG and TF as markers in diagnosing cervical cancer lymph node metastasis according to claim 1, characterized in that: The ratio of the exosome A1BG abundance to the total exosome mass is ≤14.2690 ng / mg.

3. The use of plasma exosomes A1BG and TF as markers in diagnosing cervical cancer lymph node metastasis according to claim 1, characterized in that: The ratio of the exosome TF abundance to the total exosome mass is ≤96.2011 ng / mg.

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