Application of reagent for detecting expression level of target protein in preparation of oral squamous cell carcinoma neck lymph node metastasis risk prediction kit and oral squamous cell carcinoma neck lymph node metastasis risk prediction kit

Abstract

The invention relates to an application of a reagent for detecting the expression level of a target protein in preparation of an oral squamous cell carcinoma neck lymph node metastasis risk prediction kit and the oral squamous cell carcinoma neck lymph node metastasis risk prediction kit. The key proteins for predicting the risk of oral squamous cell carcinoma neck lymph node metastasis are screened out, the neck metastasis of oral squamous cell carcinoma can be efficiently predicted by detecting the levels of the key proteins, the method has high accuracy and sensitivity, a scientific basis is provided for individualized implementation of neck cleaning and postoperative metastasis early warning mechanisms, and the method is suitable for popularization and application. The oral cancer neck operation complication rate can be reduced, and the survival rate and the life quality can be improved.

Description

Technical Field

[0001] The present application relates to the technical field of molecular typing detection, and in particular to the use of a reagent for detecting the expression level of a target protein in the preparation of a kit for predicting the risk of cervical lymph node metastasis of oral squamous cell carcinoma, and a kit for predicting the risk of cervical lymph node metastasis of oral squamous cell carcinoma. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors of the head and neck. It is prone to cervical lymph node metastasis. Even in cN0 OSCC patients, the rate of occult neck metastasis is still approximately 9%-40%, which is an important factor affecting the long-term prognosis of OSCC patients. Therefore, it is very important to screen for people at high risk of OSCC neck metastasis.

[0003] Traditional methods for predicting neck metastasis of oral squamous cell carcinoma include:

[0004] A nomogram model was developed to predict cervical lymph node metastasis in clinically early-stage oral squamous cell carcinoma (Cheng A, Wang Z, Yuan X, Liu H, Cao W, Wei W, Chang S, Han Z, Guo C, Feng Z. Development and validation of anomogram for the prediction of lymph node metastasis within 2-year postoperatively in cT1-T2N0 oral squamous cell carcinoma. Head Neck. 2023 Jan; 45(1): 103-114). This model incorporates all clinical parameters and new pathological indicators, combining the pathological grade, invasion depth, and tumor-infiltrating lymphocytes of early-stage oral squamous cell carcinoma to form a metastasis risk prediction model to predict cervical lymph node metastasis in clinically early-stage oral squamous cell carcinoma. Although the model is significantly better than the AJCC 8th T stage, further improvement is needed to improve diagnostic efficiency. Furthermore, because it does not provide metastasis-related molecular typing data, truly individualized diagnosis cannot be achieved.

[0005] CN108546761B describes a detection kit for predicting lymph node metastasis in oral squamous cell carcinoma. The gene profile detected by this invention patent is determined based on traditional transcriptome differential analysis. While this can reveal the mechanism of oral cancer metastasis to a certain extent, it cannot reflect the in situ state of living cells, making it difficult to achieve both temporal and spatial resolution. The research scope is narrow, and therefore this diagnostic method has significant shortcomings compared to the latest spatiotemporal omics, making it difficult to obtain optimal candidate molecules. Furthermore, the technical method used in the kit is a relatively quantitative real-time PCR method. Although this method has been successfully applied, subsequent verification and analysis have shown a low accuracy rate for diagnosing cervical metastasis in oral squamous cell carcinoma, with an area under the receiver operating characteristic (ROC) curve of less than 0.7. This method fails to meet the requirements for accurate diagnosis and therefore cannot be applied for market approval.

[0006] Overall, oral squamous cell carcinoma is highly heterogeneous and has obvious individual differences. Traditional clinical imaging, the American Joint Committee on Cancer (AJCC) / UICC TNM staging system for head and neck tumors, and molecular typing technology are unable to accurately identify patients at high risk of neck metastasis and guide treatment. There are also no molecular diagnostic products developed and marketed for the precise diagnosis of neck metastasis.

[0007] In view of this, this application is hereby filed. Summary of the Invention

[0008] The purpose of this application includes providing a risk prediction kit for cervical lymph node metastasis of oral squamous cell carcinoma, which can effectively predict the cervical metastasis of oral squamous cell carcinoma and provide a scientific basis for the individualized implementation of neck dissection and postoperative metastasis warning mechanism.

[0009] In a first aspect of the embodiments of the present application, there is provided a use of a reagent for detecting the expression level of a target protein in the preparation of a kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma;

[0010] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0011] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0012] In some embodiments of the present application, the kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

[0013] In a second aspect of the embodiments of the present application, a kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma is provided, the kit comprising: a reagent for detecting the expression level of a target protein;

[0014] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0015] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0016] In some embodiments of the present application, the kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

[0017] In a third aspect of the embodiments of the present application, a standard for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma is provided, wherein the standard comprises at least one of protein standards of FASN, DARS2, PTRH2 and OCIAD2.

[0018] In some embodiments of the present application, the standards include protein standards of FASN, DARS2, PTRH2 and OCIAD2.

[0019] In a fourth aspect of the embodiments of the present application, there is provided a use of a target protein in preparing a standard for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma;

[0020] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0021] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0022] Compared with traditional technologies, the advantages of this application include:

[0023] The inventors of this application creatively combined spatial transcriptomics technology with bio-orthogonal photocatalytic proteomics technology to analyze and screen out key proteins for predicting the risk of cervical lymph node metastasis of oral squamous cell carcinoma. By detecting the levels of these key proteins, the cervical metastasis of oral squamous cell carcinoma can be efficiently predicted with high accuracy and sensitivity, providing a scientific basis for the individualized implementation of neck dissection and postoperative metastasis warning mechanism, which will help reduce the complication rate of oral cancer neck surgery and improve survival rate and quality of life. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0025] Figure 1 、 Figure 2This is the CAT-Prox technology pattern diagram;

[0026] Figure 3 To verify the feasibility of applying CAT-Prox to oral squamous cell carcinoma cell lines;

[0027] Figure 4 is the result of protein enrichment analysis;

[0028] Figure 5 Flow chart for single cell suspension preparation;

[0029] Figure 6 、 Figure 7 The feasibility of bioorthogonal photocatalysis-based proteomics in the detection of oral cancer and adjacent tissues is high;

[0030] Figure 8 Western Blot was used to verify the protein expression of 8 pairs of samples;

[0031] Figure 9 Elisa was used to detect protein expression in 30 pairs of samples;

[0032] Figure 10 Receiver operating characteristic curve (ROC curve) for Elisa validation of molecular profiles to predict cervical metastasis of oral squamous cell carcinoma;

[0033] Figure 11 Tissue microarrays were used to verify the expression of 45 "metastatic" and 75 "non-metastatic" oral squamous cell carcinoma tissues;

[0034] Figure 12 Receiver operating characteristic curve (ROC curve) for tissue microarray validation of molecular profiling to predict cervical metastasis of oral squamous cell carcinoma. DETAILED DESCRIPTION

[0035] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the protection scope of this application. In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing embodiments and examples and are not intended to limit the present invention.

[0037] the term

[0038] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0039] The terms "and / or", "or / and", and "and / or" used herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical and" and also undoubtedly includes technical solutions connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution of all being connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

[0040] In the present invention, "plurality", "multiple", "multiple times", "multiple", etc., unless otherwise specified, refer to a number greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0041] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like include all suitable combinations of any two or more of the listed items.

[0042] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc. shall be based on the ability to implement the technical solution of the present invention, solve the technical problem of the present invention, and achieve the expected technical effect of the present invention.

[0043] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present invention.

[0044] Herein, “further”, “further”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be understood as limiting the scope of protection of the present invention.

[0045] As used herein, "optionally," "optional," and "optional" mean optional or dispensable, meaning that the option is selected from either of two parallel options: "optional" or "optional." If a technical solution contains multiple "optional" clauses, each "optional" clause is considered independent unless otherwise specified and there are no conflicts or constraints.

[0046] In this document, the terms "first," "second," "third," and "fourth," etc., are used for descriptive purposes only and should not be understood as indicating or implying relative importance or quantity, nor as implicitly indicating the importance or quantity of the technical features indicated. Furthermore, "first," "second," "third," and "fourth," etc., serve only as non-exhaustive enumeration and description and should not constitute a closed-ended limitation on quantity.

[0047] In the present invention, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0048] Herein, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, which means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0049] The temperature parameters herein, unless otherwise specified, allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the precision range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0050] As used herein, % (w / w) and wt% both refer to weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0051] All documents mentioned herein are cited as references in this application, just as each document is cited as a reference individually. Unless they conflict with the invention purpose and / or technical solution of this application, the cited documents to which this invention relates are cited in their entirety and for all purposes. When cited documents are involved in this invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When cited documents are involved in this invention, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, this application shall prevail or it shall be modified adaptively according to the description in this application.

[0052] In a first aspect of the embodiments of the present application, there is provided a use of a reagent for detecting the expression level of a target protein in the preparation of a kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma;

[0053] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0054] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0055] In some embodiments of the present application, the kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

[0056] In a second aspect of the embodiments of the present application, a kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma is provided, the kit comprising: a reagent for detecting the expression level of a target protein;

[0057] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0058] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0059] In some embodiments of the present application, the kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

[0060] In a third aspect of the embodiments of the present application, a standard for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma is provided, wherein the standard comprises at least one of protein standards of FASN, DARS2, PTRH2 and OCIAD2.

[0061] In some embodiments of the present application, the standards include protein standards of FASN, DARS2, PTRH2 and OCIAD2.

[0062] In a fourth aspect of the embodiments of the present application, there is provided a use of a target protein in preparing a standard for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma;

[0063] The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

[0064] In some embodiments of the present application, the target proteins include FASN, DARS2, PTRH2 and OCIAD2.

[0065] The inventor creatively determined the molecular profile based on the combined analysis of spatial transcriptomics and bio-orthogonal photocatalytic proteomics, and verified through multiple sets of transcript level and protein level analysis that it can effectively predict high-risk patients with oral squamous cell carcinoma cervical metastasis. On the one hand, these two technologies have their own unique advantages: (1) Orthogonal photocatalytic proteomics technology is developed based on the bio-orthogonal reaction of the 2022 Nobel Prize in Chemistry. The research and development project team of this application is the first international team to apply this technology. This application is also the first to apply this technology to clinical tumor detection. This molecular profile is the first to apply it to the prediction of oral squamous cell carcinoma cervical lymph node metastasis. It has high spatiotemporal accuracy. The bio-orthogonal photocatalytic proteomics technology CAT-Ex can restore the true activity state of cells in a complex biological environment, in multiple dimensions, and to the greatest extent. It contains the time and space information that is key to disease progression and difficult to capture, thereby screening out key proteins closely related to oral squamous cell carcinoma metastasis. (2) DSP spatial transcriptome can visualize the complex gene expression in a specific space inside the tumor, obtain transcriptome data and analyze the location of cells in the tissue, conduct a more detailed study of gene expression in a spatial context, and explore the heterogeneity of gene expression between different regions within the tumor; thereby discovering more information about the tumor at different times and in different spaces, and obtaining spatial molecular information about the interaction between tumor cells and the immune microenvironment that is lacking in conventional high-throughput sequencing. On the other hand, the protein spectra obtained by the combined analysis of the two technologies in this application have higher accuracy than the spectra selected by a single technology. Compared with using only one technology, the accuracy and sensitivity of the protein spectra selected by this kit can be better guaranteed, which enables us to screen out the optimal molecules for predicting neck metastasis of oral squamous cell carcinoma.

[0066] In terms of molecular spectra: As far as spectra screening is concerned, the protein spectra selected in this application are obtained based on the joint analysis of two technologies: spatial transcriptomics and bio-orthogonal photocatalytic proteomics, and have undergone multiple verifications at the transcriptional and protein levels. In terms of the diagnostic ability of the selected spectra, after verification at the transcriptome level of 30 pairs of oral cancer metastasis and non-metastasis samples, the AUROC of this molecular spectra for diagnosing oral cancer metastasis is 0.7733 (higher than the differentiation between metastasis and non-metastasis within the same tumor group reported in most previous literature). This molecular spectra combination is the latest, unique, efficient, and diagnostically effective. This kit is expected to become the first molecular diagnostic product for the precise diagnosis of cervical metastasis of oral squamous cell carcinoma.

[0067] The kit of this application involves screening "high-risk metastasis" proteins based on the mechanism of tumor metastasis, rather than starting from the manifestations of tumors such as clinical parameters and pathological indicators. It can reveal the mechanism of tumor metastasis to a certain extent, and proteins directly determine the function and phenotype of the body, making diagnosis more personalized. Therefore, the preparation of a protein diagnostic kit is the most direct, critical and important.

[0068] In terms of application, the test kit of the present application: ① can detect tumor tissues through Elisa reagents, antibody chips, etc., which is simple to operate and does not require additional genetic manipulation. The detection time can be shortened to 1 day, the research scope is also wider, and it can be applied to clinical samples. ② It can realize the simultaneous detection of protein spectra of multiple patient samples in one kit analysis, significantly reducing the detection cost and reagents, and the operation is simple and the results are reliable. ③ The detection adopts a protein chip method that is simpler to operate, has a wider range of applications, and is more efficient. It does not involve genetic manipulation, avoids the tedious and harsh conditions of genetic manipulation, shortens the detection time, and is low in cost. Tumor samples from multiple patients can be tested at the same time, improving detection efficiency.

[0069] The embodiments of the present invention will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. For experimental methods in the following examples where specific conditions are not specified, reference is made to the guidance provided in the present invention, and may also be made according to experimental manuals or conventional conditions in the art, or according to conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0070] In the following specific examples, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operational accuracy are allowed.

[0071] Example 1

[0072] 1. To verify the feasibility of applying the new technology CAT-Prox to the cervical metastasis of oral squamous cell carcinoma (applied in living tissue).

[0073] 1.1 Principle of CAT-Prox technology, such as Figure 1 and Figure 2 As shown: ① The iridium photosensitizer excited by blue light specifically targets the mitochondria of living cells; ② The azidoaminophenyl-protected methylene quinone probe is added; ③ Blue light irradiation is turned on; ④ The iridium photosensitizer is excited to trigger the removal of the azidoaminophenyl; ⑤ The active methylene quinone probe is released, labeling nearby mitochondrial proteins; ⑥ The labeled proteins are enriched and analyzed by tandem mass spectrometry.

[0074] CAT-Prox was applied to oral squamous cell carcinoma cell lines and found to be well compatible with oral squamous cell carcinoma Cal27 cell lines, with high specificity for specific cell subspace identification and accurate spatiotemporal positioning. Figure 3 As shown in the figure, CAT-Prox increases the recognition rate of mitochondrial specific proteins from less than 6% of the existing technology to 60%, and increases the efficiency of organelle protein detection by 10 times, proving that this project has good feasibility. This application will conduct enrichment analysis on the detected mitochondrial proteins, such as Figure 4 As shown, these proteins are mainly related to mitochondrial gene expression, cellular respiration, mitochondrial organization, and mitochondrial transmembrane transport.

[0075] 1.2 CAT-Prox was used to examine the primary lesions and adjacent tissues of patients with oral squamous cell carcinoma cervical metastasis, and differential proteomics results at the organelle level were obtained. This is the first time internationally that this technology has been demonstrated to be highly feasible for use in living tissues and to be capable of locally capturing differentially expressed proteins at the subcellular organelle level.

[0076] like Figure 6 As shown in the figure, this technology was used to detect the primary lesions and adjacent tissues of patients with oral squamous cell carcinoma cervical metastasis, and differential proteomics results at the organelle level were obtained. From the WB results, it can be seen that differential proteins can be labeled and enriched in both tumor tissues and normal tissues.

[0077] like Figure 7 The protein expression intensity in normal tissues detected was 80% of that in cancer tissues. Mitochondrial protein species detected in tumor tissues accounted for 22% of the total protein, while mitochondrial protein species detected in normal tissues accounted for 23% of the total protein.

[0078] 2. Use bioorthogonal photocatalytic proteomics technology to screen the protein spectrum related to oral squamous cell carcinoma neck metastasis and perform preliminary verification using Western Blot.

[0079] 2.1 Sample inclusion criteria

[0080] ① Age: ≥18 years old; ② Gender: Any; ③ Lesion location: Tongue, buccal mucosa, upper and lower gums, floor of mouth, hard palate mucosa; ④ Oral squamous cell carcinoma patients: Patients with primary oral squamous cell carcinoma confirmed by preoperative biopsy, clinical stage cT1-4N0-2M0 (AJCC 8th), and planned to undergo concurrent neck dissection; ⑤ General condition: Routine examinations such as three routine tests, blood biochemical tests, and electrocardiograms to exclude surgical contraindications, and examinations such as chest X-rays and / or abdominal ultrasound and / or enhanced CT and / or MRI and / or PET / CT to exclude distant metastases.

[0081] 2.2 Detection

[0082] 2.2.1 Eight pairs of oral squamous cell carcinoma primary lesions (metastatic vs. non-metastatic) and their metastatic lymph node specimens were obtained from the Department of Oral and Maxillofacial Head and Neck Oncology, Beijing Stomatological Hospital, Capital Medical University. The expression of mitochondrial proteins in these samples was detected using the bioorthogonal photocatalytic CAT-Prox technique.

[0083] (1) The proteins detected in the metastatic and non-metastatic groups were counted, with the protein up-regulation threshold being 2 and the protein down-regulation threshold being 0.5.

[0084] (2) Proteins with up-regulation / down-regulation repetition rate ≥ 50% were screened in the transfer group.

[0085] (3) The protein combination obtained in step (2) is further screened in the non-metastatic group to screen out proteins whose expression trends are inconsistent with those of the metastatic group.

[0086] Finally, a "high-risk" profile for oral squamous cell carcinoma neck metastasis was obtained, consisting of five molecules: FASN, DARS2, PTRH2, OCIAD2, and HSPD1. The screening process and results are shown in Table 1.

[0087] Table 1. Mitochondrial protein expression of 8 pairs of samples screened using CAT-Prox

[0088] Uniprot ID Transfer group up Transfer group downgraded Upregulation in the non-metastatic group Down-regulation in the non-metastatic group Whether it meets the conditions Gene name P10809 6 1 3 2 yes HSPD1 O95202 5 1 4 0 no Q6P148 5 1 0 0 yes DARS2 P04179 4 1 5 1 no P05141 4 2 4 4 no P49327 4 0 0 1 yes FASN P53007 4 2 6 0 no Q56VL3 4 1 1 0 yes OCIAD2 Q9Y3E5 4 1 2 0 yes PTRH2 P30041 1 6 0 6 no P12235 2 5 0 6 no P32119 1 5 0 5 no P04040 1 4 0 3 no P07195 1 4 0 7 no P21796 1 4 0 7 no

[0089] The following steps are involved in the CAT-Prox test:

[0090] (1) Preparation of proteomics-labeled cells

[0091] 1) Preparation of cell membrane-targeted iridium catalyst and p-azidobenzyl-protected methylenequinone-biotin probe for proteomic labeling.

[0092] 2) Digest and dissociate the tumor tissue obtained in 2.1.1 to prepare the corresponding cell suspension ( Figure 5 ).

[0093] 3) The single cells were transferred to a culture medium containing 150 nM cell surface-targeted iridium catalyst and incubated in the dark at room temperature for 15 minutes.

[0094] 4) The culture medium containing the catalyst was discarded by centrifugation and the cells were washed once with PBS.

[0095] 5) Transfer the single cell to a culture medium containing 100 μM biotin probe, and irradiate the cell culture medium with blue light for 10-20 minutes.

[0096] 6) After irradiation, wash once with PBS.

[0097] 7) Transfer the cells to a test tube placed on ice. Centrifuge the cells at 850 g and 4°C for 3 minutes and discard the supernatant.

[0098] 8) Store at -80°C or perform subsequent experiments

[0099] (2) Lyse cells and enrich labeled proteins using streptavidin magnetic beads

[0100] 1) Treat the centrifuged cells with an appropriate amount of RIPA strong lysis buffer supplemented with protease inhibitors and perform ultrasonic treatment to obtain a clear lysate.

[0101] 2) Add MeOH / CHCl3 to precipitate the protein and centrifuge at 10,000 g and 4°C for 20 min.

[0102] 3) Wash three times with ice-cold methanol.

[0103] 4) Use an appropriate amount of PBS containing 0.2 wt% SDS to re-dissolve the precipitated protein and place it in a 15 mL test tube.

[0104] 5) Wash the streptavidin resin beads three times with PBS for equilibrium.

[0105] 6) Add an appropriate amount of magnetic bead suspension to each test tube and incubate with gentle rotation at room temperature for 3 hours.

[0106] 7) Centrifuge at 1400 g for 3 min, resuspend in PBS containing 0.2 wt% SDS, and incubate at room temperature for 10 min.

[0107] 8) Wash 6 times with 5 mL PBS.

[0108] (3) Protease cleavage and dimethylation labeling on magnetic beads

[0109] 1) Centrifuge the protein-bound magnetic beads and discard the supernatant.

[0110] 2) Add an appropriate amount of 6 M urea in PBS solution and 200 mM dithiothreitol, resuspend the magnetic beads, and incubate at 37°C for 30 min.

[0111] 3) Add an appropriate amount of 400 mM iodoacetamide, and incubate the mixture obtained in step 2) again in the dark at 37° C. for 30 min.

[0112] 4) Wash four times with 0.1 M trimethylammonium bicarbonate buffer, centrifuge and discard the supernatant.

[0113] 5) Add appropriate amount of 0.1 M TEAB buffer and trypsin, incubate at 37°C for 16 h to digest the protein to obtain a peptide solution.

[0114] 6) Add appropriate amount of NaBH3CN and 4% (v / v) stable isotope formaldehyde (CH2O or 13 CD2O), mix well and incubate at room temperature for 30 min.

[0115] 7) Add a certain amount of 1% (v / v) ammonia water to quench the reaction, and then add a certain amount of formic acid.

[0116] 8) After vortexing, the heavy-labeled and light-labeled samples were combined in a 1:1 (v / v) ratio to obtain the dimethyl-labeled sample, and the sample was separated at C according to the procedure. 18 Desalt on a desalting column and evaporate the solvent to dryness in a vacuum concentrator.

[0117] (4) LC-MS / MS analysis of samples and data processing (all mass spectrometry data were processed using MaxQuant v1.6.10 software)

[0118] 1) The dimethyl-labeled peptides were analyzed using high performance liquid chromatography coupled with an LTQ orbitrap mass spectrometer to obtain peptide information for each sample.

[0119] 2) Protein quantification was performed by calculating the ratio of light labeling to heavy labeling.

[0120] 3) The secondary mass spectra of the samples were searched in the UniProt database to identify the proteins.

[0121] 4) Statistical analysis of the identified proteins was performed to evaluate the degree of cell membrane enrichment achieved by the method and to discover new protein types or protein differences in different cell types.

[0122] 2.2.2 Preliminary verification of the screened protein profile using Western Blot (COXIV as internal reference)

[0123] The results are as follows Figure 8 As shown in the figure, the expression levels of the proteins screened in the metastatic group were higher than those in the non-metastatic group.

[0124] 3. Validate the molecular profile associated with oral squamous cell carcinoma cervical metastasis composed of the above five proteins.

[0125] Thirty pairs of metastatic and non-metastatic oral squamous cell carcinoma specimens were obtained from the Department of Head and Neck Oncology, Beijing Stomatological Hospital (inclusion criteria were the same as those described under item 2). The expression levels of spectral proteins were verified by Western Blot and ELISA. During the testing, the result of "HSPD1" was abnormal, and the four genes "FASN", "DARS2", "PTRH2" and "OCIAD2" were finally screened.

[0126] The results of Elisa detection of protein expression in 30 pairs of samples are as follows Figure 9 As shown in the figure, the expression levels of all proteins screened in the metastatic group were higher than those in the non-metastatic group, and except for DARS2, the differences were statistically significant.

[0127] By constructing a regression equation based on the relative expression levels of four genes in 30 pairs of specimens, the Y1 value representing the risk of oral squamous cell carcinoma metastasis was calculated: Y1 = 1.173*FASN + 1.011*PTRH2 + 0.937*DARS2 + 1.010*OCIAD2. A receiver operating characteristic (ROC) curve was generated, and the results showed that the area under the ROC curve (AUROC) for this spectrum in predicting neck "metastasis" and "non-metastasis" within the oral cancer group reached 0.7733 (higher than the discrimination between metastasis and non-metastasis within similar tumor groups reported in most previous literature). The receiver operating characteristic (ROC) curve is shown in the figure below. Figure 10 shown.

[0128] 4. Verification of protein levels using tissue microarray technology

[0129] 45 metastatic and 75 non-metastatic oral squamous cell carcinoma specimens were obtained from the Department of Head and Neck Oncology, Beijing Stomatological Hospital (inclusion criteria are the same as those described in item 2), and the spectrum was verified at the protein level using tissue microarray technology. Figure 11 ) Based on the positive staining intensity and staining range of each site, the protein was divided into four levels: negative (-), weakly positive (+), positive (++), and strongly positive (+++). A regression equation was constructed based on the protein expression and lymph node metastasis, and the Y2 value representing the risk of oral cancer metastasis was calculated: Y2 = 0.107*FASN+14.712*PTRH2+4.092*DARS2+5.158*OCIAD2+0.105. The receiver operating characteristic curve (ROC curve) was constructed, and the results showed that the area under the ROC curve (AUROC) for the protein chip produced by this spectrum type to predict neck "metastasis" and "non-metastasis" in the oral cancer group reached 0.8114 ( Figure 12). The above-described embodiments only express several implementation methods of the present invention, which are convenient for understanding the technical solutions of the present invention in a specific and detailed manner, but they cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several variations and improvements can be made, which all fall within the scope of protection of the present invention. In addition, it should be understood that after reading the above-mentioned teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided by the present invention are all within the scope of protection of the claims attached to the present invention. Therefore, the scope of protection of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. Application of a reagent for detecting target protein expression levels in the preparation of a kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma; The target protein detected by the reagent includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

2. The use according to claim 1, characterized in that The target proteins detected by the reagent include FASN, DARS2, PTRH2 and OCIAD2.

3. The use according to claim 1 or 2, characterized in that The kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

4. A kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma, characterized in that: The kit includes: a reagent for detecting the expression level of a target protein; The target protein detected by the reagent includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

5. The kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma according to claim 4, characterized in that: The target proteins detected by the reagent include FASN, DARS2, PTRH2 and OCIAD2.

6. The kit for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma according to claim 4 or 5, characterized in that: The kit includes Elisa reagents and / or a biochip; optionally, the kit includes Elisa reagents.

7. A standard for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma, characterized in that: The standard comprises at least one of protein standards of FASN, DARS2, PTRH2 and OCIAD2.

8. The standard product for predicting the risk of cervical lymph node metastasis in oral squamous cell carcinoma according to claim 7, characterized in that: The standards include protein standards of FASN, DARS2, PTRH2 and OCIAD2.

9. Application of target protein in the preparation of risk prediction standards for cervical lymph node metastasis in oral squamous cell carcinoma; The target protein includes at least one of FASN, DARS2, PTRH2 and OCIAD2.

10. The use according to claim 9, characterized in that The target proteins include FASN, DARS2, PTRH2 and OCIAD2.

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