Use of abcc1 inhibitors in the manufacture of a medicament for treating nasopharyngeal carcinoma

Abstract

This invention discloses the application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma. Immunohistochemical experiments revealed that ABCC1 protein expression in nasopharyngeal carcinoma tumor tissues is higher than in adjacent normal tissues. The positive rate of ABCC1 staining in advanced-stage patients is significantly higher than in early-stage patients, and the proportion of patients with high ABCC1 expression is also significantly higher in patients with poor prognosis than in patients with good prognosis. Therefore, this invention further designs ABCC1 inhibitors targeting ABCC1, finding that knocking down or silencing ABCC1 significantly inhibits the proliferation, migration, and invasion of nasopharyngeal carcinoma cells; simultaneously, inhibition of ABCC1 function effectively enhances CD8... + The immune effector function of T cells. Therefore, ABCC1 can be used in the diagnosis and treatment of nasopharyngeal carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to the application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma. Background Technology

[0002] Nasopharyngeal carcinoma (NPC) is a malignant tumor originating from the mucosal epithelium of the nasopharynx and is an important endemic subtype of head and neck squamous cell carcinoma. Among head and neck squamous cell carcinomas, NPC is one of the most prone to recurrence and distant metastasis, resulting in limited clinical benefit and poor prognosis associated with this cancer type. Furthermore, treatment options for patients with recurrent / metastatic NPC are limited, leading to a poor prognosis. Therefore, the discovery and identification of novel therapeutic targets has become crucial for improving the prognosis of NPC patients. Summary of the Invention

[0003] The first objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma.

[0004] A second objective of this invention is to provide the use of ABCC1 inhibitors in the preparation of medicaments for inhibiting the proliferation, and / or migration, and / or invasion of nasopharyngeal carcinoma cells.

[0005] The third objective of this invention is to provide the application of ABCC1 inhibitors in the preparation of drugs that enhance the killing effect of CD8+ T cells on nasopharyngeal carcinoma cells.

[0006] A fourth objective of this invention is to provide the use of reagents for detecting ABCC1 expression levels in the preparation of nasopharyngeal carcinoma diagnostic reagents, nasopharyngeal carcinoma diagnostic kits, or nasopharyngeal carcinoma diagnostic assessment kits.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma, wherein the ABCC1 inhibitor is si-ABCC1, and the target sequence of si-ABCC1 is CCTGTTTCCCCTTCTACTT (SEQ ID NO:1), which is located between bases 279 and 297 in the coding region of the ABCC1 gene (NM_004996).

[0009] The sequence of si-ABCC1 is as follows:

[0010] si-ABCC1 sense: 5′-CCUGUUUCCCCUUCUACUU-dTdT-3′ (SEQ ID NO: 2);

[0011] si-ABCC1 antisense: 5′-AAGUAGAAGGGGAAACAGG-dTdT-3′ (SEQ ID NO: 3).

[0012] The above-mentioned ABCC1 inhibitors are used in the preparation of drugs that inhibit the proliferation, and / or migration, and / or invasion of nasopharyngeal carcinoma cells.

[0013] The above-mentioned ABCC1 inhibitors are used in the preparation of drugs that enhance the killing effect of CD8+ T cells on nasopharyngeal carcinoma cells.

[0014] As a further preferred embodiment, the nasopharyngeal carcinoma cells include at least one of 5-8F, TW03, HK-1, and TW01 cells.

[0015] As a further preferred embodiment, the drug also includes pharmaceutically acceptable excipients.

[0016] Application of reagents for detecting ABCC1 expression levels in the preparation of nasopharyngeal carcinoma diagnostic reagents, nasopharyngeal carcinoma diagnostic kits, or nasopharyngeal carcinoma diagnostic assessment kits.

[0017] As a further preferred embodiment, the reagent for detecting ABCC1 expression levels includes a primer pair, Primer F (forward primer) and Primer R (reverse primer), whose nucleotide sequences are as follows:

[0018] Primer F: 5′-AGTGGAACCCCTCTCTGTTTAAG-3′ (SEQ ID NO: 4);

[0019] Primer R: 5'-CCTGATAGCTCTTGGTCTTCATC-3' (SEQ ID NO: 5).

[0020] As a further preferred embodiment, the kit also includes TB Green Premix, ddH2O, cDNA template, etc.

[0021] The present invention has the following advantages and effects compared with the prior art:

[0022] 1. This invention, through immunohistochemical experiments, shows that the expression of ABCC1 protein in nasopharyngeal carcinoma tumor tissue is higher than that in adjacent normal tissue, and the positive rate of ABCC1 staining in advanced patients is significantly higher than that in early-stage patients. The proportion of patients with high ABCC1 expression in patients with poor prognosis is also significantly higher than that in patients with good prognosis. This indicates that ABCC1 plays an important role in the pathogenesis and immunotherapy of nasopharyngeal carcinoma. In other words, ABCC1 can serve as a novel molecular marker and drug target for the diagnosis and treatment of nasopharyngeal carcinoma, thereby guiding clinical intervention and treatment, and improving patient survival rates and quality of life.

[0023] 2. This invention, through experiments, found that knocking down or silencing ABCC1 can significantly inhibit the proliferation, migration, and invasion of nasopharyngeal carcinoma cells; simultaneously, the inhibition of ABCC1 function can effectively enhance CD8. + The immune effector function of T cells is enhanced, thereby increasing their killing activity against tumor cells. Therefore, ABCC1 inhibitors can not only directly inhibit the malignant progression of nasopharyngeal carcinoma, but also enhance the body's anti-tumor immune response by reversing immune escape, showing promising application prospects in the diagnosis and treatment of nasopharyngeal carcinoma and in improving the sensitivity of existing immunotherapies. Attached Figure Description

[0024] Figure 1 The graph shows the expression results of ABCC1 in nasopharyngeal carcinoma. A represents the expression of ABCC1 in NPC samples from the GEO database; B represents the survival curves of nasopharyngeal carcinoma patients with high / low ABCC1 expression based on the TARGET database; C represents the RNA expression level of ABCC1 in normal nasopharyngeal epithelial cells and four types of nasopharyngeal carcinoma cells detected by qRT-PCR; D represents the protein expression level of ABCC1 in normal nasopharyngeal epithelial cells and four types of nasopharyngeal carcinoma cells detected by protein immunoblotting; E represents the immunohistochemical analysis of ABCC1 expression in nasopharyngeal carcinoma tissue; and F represents the survival curves of patients with high / low ABCC1 expression based on immunohistochemistry.

[0025] Figure 2 Figure 1 shows the results of ABCC1 promoting the proliferation, migration, and invasion of nasopharyngeal carcinoma cells (5-8F, TW03). In this figure, A shows the knockdown efficiency of ABCC1 in nasopharyngeal carcinoma cells as detected by protein immunoblotting; B and C show the effects of ABCC1 knockdown on the proliferation of nasopharyngeal carcinoma cells as detected by CCK-8 assay and colony formation assay; and D shows the effects of ABCC1 knockdown on the migration and invasion of nasopharyngeal carcinoma cells as detected by Transwell assay.

[0026] Figure 3 The diagram shows the experimental results of ABCC1 regulating CD8+ T cell function. A represents a schematic diagram of the co-culture experiment of activated CD8+ T cells and nasopharyngeal carcinoma cells; B and D represent the levels of immune effector factors IFN-γ, IL-2, and TNF-α secreted by CD8+ T cells in the co-culture supernatant, detected by ELISA, respectively; E represents the amount of granzyme B secreted in the co-culture supernatant, detected by ELISA; and F represents the cytotoxicity of CD8+ T cells against nasopharyngeal carcinoma cells, detected by an LDH release assay. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to embodiments. Obviously, the described embodiments are only a part of the embodiments in this application, and the implementation of the present invention is not limited thereto. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are all commercially available.

[0028] This invention recruited 112 patients with nasopharyngeal carcinoma who were first diagnosed at the Affiliated Cancer Hospital of Guangxi Medical University between 2015 and 2018. None of the patients received preoperative radiotherapy, chemotherapy, or interventional therapy. All patients signed informed consent forms. All procedures were approved by the Medical Research Ethics Committee of the Affiliated Cancer Hospital of Guangxi Medical University in accordance with the Declaration of Helsinki (ethics number: KY-2022-310).

[0029] Example 1

[0030] Cellular experiments confirmed that the ABCC1 inhibitor (si-ABCC1) can target and inhibit the expression of the ABCC1 gene, and verified this effect by inhibiting the proliferation, migration, and invasion of nasopharyngeal carcinoma cells.

[0031] I. To investigate the expression of ABCC1 in nasopharyngeal carcinoma tumor tissue

[0032] To screen for differentially expressed genes between nasopharyngeal carcinoma and normal tissues, two public datasets, GSE53819 and GSE102349, in the GEO (Gene Expression Omnibus) database were analyzed. The results are as follows: Figure 1 As shown, the results indicate that the expression of ABCC1 mRNA is significantly increased in nasopharyngeal carcinoma tumor tissue. Figure 1 A), and patients with high expression of it often have a poor prognosis (A). Figure 1 (B in the original text). Immunohistochemical analysis was used for further verification, and the specific process is as follows:

[0033] Tissue microarray sections (derived from CoreBio: human nasopharyngeal epithelial cells NP69, human nasopharyngeal carcinoma cells TW03, TW01, 5-8F, HK-1) ​​were routinely dewaxed and gradually hydrated with a gradient of alcohols. Incubation with 3% (v / v) H2O2 at room temperature for 10-15 minutes was performed to eliminate endogenous peroxidase activity. The sections were washed and soaked in PBS for 5 minutes, followed by antigen retrieval using Tris-EDTA antigen retrieval solution (G1203, Seville) under high temperature and autoclave. Blocking with 3% (v / v) bovine serum albumin (BSA) at room temperature for 30 minutes was performed. Primary antibody was added at an appropriate dilution, and the mixture was incubated overnight at 4°C. Secondary antibody was then incubated at 37°C for 30-60 minutes. DAB visualization was performed for 3-15 minutes, with microscopic observation and timely termination of the reaction. Counterstaining with hematoxylin was performed, followed by differentiation with 1% (v / v) hydrochloric acid alcohol, and bluing with warm water. Routine dehydration and clearing were performed, followed by mounting with neutral resin and microscopic analysis. The positive rate and intensity of staining were calculated. The primary antibody used was anti-ABCC1 (1:100, #72202, Cell Signaling Technology); the secondary antibody used was goat anti-rabbit IgG (H+L) (1:2000, #7074, Cell Signaling Technology), which was diluted with PBST containing 0.1% Tween-20 and incubated in the dark before use.

[0034] Cell lines and cell culture: Human nasopharyngeal carcinoma cell lines 5-8F, TW03, HK-1, and TW01, and normal nasopharyngeal epithelial cells NP69 were all standard cell lines used in this study. The cells used were provided by the Key Laboratory of Early Prevention and Treatment of Regional High-Incidence Tumors, Ministry of Education, Guangxi Medical University. All cells were cultured and expanded in RPMI-1640 medium (Gibco), supplemented with a mixture of 10% (v / v) fetal bovine serum (Gibco) and 1% (v / v) penicillin-streptomycin. The culture conditions were a 37°C incubator with 5% CO2. Cell proliferation, colony formation, and Transwell assays were also performed using this medium.

[0035] qRT-PCR and protein immunoblotting showed that the mRNA and protein expression levels of ABCC1 in nasopharyngeal carcinoma cells were significantly higher than those in normal nasopharyngeal epithelial cells. Figure 1 (C and D in the text).

[0036] The expression of ABCC1 protein in nasopharyngeal carcinoma tumor tissue was higher than that in adjacent normal tissue, and patients with high ABCC1 expression had a worse prognosis than those with low ABCC1 expression. Figure 1 (E and F in the table). In addition, the positive rate of ABCC1 staining in late-stage patients was significantly higher than that in early-stage patients (Table 1, *P<0.05).

[0037] Table 1. Correlation analysis between clinical characteristics of nasopharyngeal carcinoma patients and ABCC1 expression.

[0038]

[0039] II. Investigating the effects of ABCC1 inhibitors on the proliferation, migration, and invasion of nasopharyngeal carcinoma cells.

[0040] (1) Cell lines and cell culture

[0041] Human nasopharyngeal carcinoma cell lines 5-8F and TW03, and normal nasopharyngeal epithelial cells NP69, were provided by the Key Laboratory of Early Prevention and Treatment of Regional High-Incidence Tumors, Ministry of Education, Guangxi Medical University. All cells were cultured and expanded in RPMI-1640 medium (Gibco) supplemented with a mixture of 10% (v / v) fetal bovine serum (Gibco) and 1% (v / v) penicillin-streptomycin. The culture conditions were a 37°C incubator with 5% CO2. Cell proliferation, colony formation, and Transwell assays were also performed using this medium.

[0042] (2) siRNA transfection

[0043] a. Based on the ABCC1 gene sequence, design siRNA to inhibit ABCC1 gene expression. The target sequence of the ABCC1 siRNA is CCTGTTTCCCCTTCTACTT (SEQ ID NO:1), which is located between bases 279 and 297 in the coding region (NM_004996) of the ABCC1 gene. Then, commission a biotechnology company to synthesize the following sequence:

[0044] Justice Chain (si-ABCC1 sense): 5′-CCUGUUUCCCCUUCUACUU-dTdT(3' overhang)-3′(SEQ IDNO:2);

[0045] Antisense chain (si-ABCC1 antisense): 5′-AAGUAGAAGGGGAAACAGG-dTdT-3′ (SEQ ID NO:3);

[0046] siNC: 5'-AAGTAGAAGGGGAAA-dTdT-3' (SEQ ID NO: 6).

[0047] b. First, culture cells (5-8F, TW03) in 6-well plates, add culture medium to a final volume of 2 ml, and incubate the cells in a 37℃, 5% CO2 incubator. When the cell density reaches 60%, transfect si-ABCC1 or siNC (as control NC) respectively. Gently wash the inoculation wells twice with PBS, remove the PBS, add 1000 μL of Opti-MEM medium, add 500 μL of Opti-MEM medium to an EP tube, add 6.25 μL of 20 μM siRNA, add 5 μL of lipo3000, gently pipette to mix, and incubate at room temperature for 20 min to prepare the transfection complex. Add the mixture to the inoculation plate, mix gently, and transfect for 24-72 h. Set up 3 replicates for each experiment.

[0048] (3) RNA isolation and qRT-PCR analysis

[0049] Total RNA was extracted using Trizol reagent (Solarbio, China) as recommended by the manufacturer. The extracted RNA (2000 ng) was then reverse transcribed using the PrimeScript™ RT kit (Takara) according to the manufacturer's instructions to obtain a cDNA template. RT-qPCR was performed on a real-time PCR instrument using TB Green Premix (Takara), with GAPDH as a standard control. Primer sequences are shown in Table 4.

[0050] Prepare the PCR reaction solution on ice according to the table below (20 μL reaction volume, with 3 replicates). The reaction volume (20 μL) is shown in Table 2, the reaction conditions are shown in Table 3, and the qRT-PCR primer sequences are shown in Table 4.

[0051] Table 2 Reaction System

[0052]

[0053] Table 3 Reaction conditions

[0054]

[0055] Table 4 qRT-PCR primer sequences

[0056]

[0057] (4) Western blot

[0058] The collected samples were placed in RIPA buffer (Beyotime) containing 1:100 protease and phosphatase inhibitor (Beyotime) to extract proteins. The prepared protein samples were then added to a 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) system and electrophoresed at a constant voltage (200 V). Subsequently, the proteins were transferred to a PVDF membrane at a constant current of 400 mA for 120 minutes. After blocking the membrane with 5% (w / v) skim milk powder for 1 hour, the membrane was incubated with primary antibodies diluted accordingly for 14–16 hours. Then, the membrane was incubated with secondary antibodies at room temperature for 1 hour. After incubation, the PVDF membrane strips were washed and developed. Finally, the relative protein abundance was analyzed using ImageJ software. The experiment was performed in triplicate. The primary antibodies used for detection were: anti-Tubulin (#11224–1-AP, 1:2000, Wuhan Sanying) and anti-ABCC1 (#72202, 1:1000, CST). The secondary antibody was HRP-labeled goat anti-rabbit IgG (#SA00001-2, 1:5000, Wuhan Sanying). After incubation, it was developed using ECL high-sensitivity luminescent solution (#PK10003, Wuhan Sanying). Finally, the relative protein abundance was analyzed using ImageJ software.

[0059] (5) CCK-8 experiment and clonogenesis experiment

[0060] Nasopharyngeal carcinoma cell lines were seeded in 6-well plates. When the cell density reached 60%, transfection was performed according to the method described in step (2) above, transfecting siNC and si-ABCC1 cells into the cells respectively. After 24 hours of transfection, nasopharyngeal carcinoma cells were seeded at 2.0 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates and cultured. At 0 h, 24 h, 48 h, 72 h, and 96 h after culture, 10 μL of CCK-8 reagent was added to each well, and incubation was continued for 1 hour. Immediately afterwards, absorbance at 450 nm was measured using a microplate reader to assess cell viability. For colony formation assays, nasopharyngeal carcinoma cells were seeded at 800 cells per well into 6-well plates and cultured at 37°C for 14 days. Colonies were then fixed with 4% paraformaldehyde for 30 minutes and stained with crystal violet for 30 minutes. Colonies with ≥50 cells were counted using ImageJ software. The experiment was performed in triplicate.

[0061] (6) Transwell experiment

[0062] Transfect siNC and si-ABCC1 into cells using the same method described above. 24 hours after transfection, add 2×10⁻⁶ cells... 4Cells were placed in the upper chamber of a Transwell chamber containing serum-free 1640 medium, while the lower chamber contained 1640 medium with 15% (v / v) fetal bovine serum (FBS). For invasion assays, matrix gel was added to the upper chamber before cell seeding; 600 μl of 1640 medium containing 15% (v / v) FBS was added to the lower chamber. After incubation for 36 hours, cells on the surface of the upper chamber were gently wiped away. Subsequently, cells that migrated or invaded into the lower chamber were fixed with 4% paraformaldehyde, stained with 1% crystal violet, and counted and imaged under four randomly selected microscopic fields.

[0063] (7) Experimental results

[0064] The biological role of ABCC1 in nasopharyngeal carcinoma development was evaluated by knocking down ABCC1 expression in human nasopharyngeal carcinoma carcinoma lines (5-8F and TW03). Western blot analysis showed that si-ABCC1 effectively reduced ABCC1 expression. Figure 2 (A) Knockdown of ABCC1 can inhibit the proliferation, migration, and invasion of osteosarcoma cells. Figure 2 (B, C, and D in the original text).

[0065] III. Investigating the effect of ABCC1 inhibition on the killing activity of T cells against nasopharyngeal carcinoma cells.

[0066] (1) Extraction and co-culture of CD8+ T cells

[0067] CD8+ T cell samples were obtained from fresh blood extracted from healthy donors and negatively screened using a magnetic bead-based method following standard procedures using a STEMCELL sorting kit. Sorted CD8+ T cells were activated with CD3 / CD28 antibody (STEMCELL) for 48 hours, and then cultured in T cell-specific medium (STEMCELL) supplemented with 20 ng / ml IL-2 (STEMCELL). siNC and si-ABCC1 were transfected into tumor cells (5-8F and TW03) using the same method described above. 24 hours post-transfection, 2×10⁻⁶ cells were cultured. 4 The cells were placed in the upper chamber of a Transwell co-culture chamber containing T-cell-specific culture medium, with a CD8+ T-cell to tumor cell ratio of 10:1 (cell number ratio). After co-incubation for 48 h, the cells and supernatant were collected.

[0068] (2) Elisa experiment

[0069] The levels of granzyme B, IL-2, TNF-α, and IFN-γ in the supernatant of the co-culture system were measured by ELISA according to the manufacturer's instructions. Granzyme B levels were detected using a high-sensitivity human granzyme B ELISA kit (SKU: E-EL-H1617) according to the experimental protocol. IL-2 levels were detected using a high-sensitivity human IL-2 ELISA kit (SKU: E-EL-H0099) according to the experimental protocol. TNF-α levels were detected using a high-sensitivity human TNF-α ELISA kit (SKU: E-EL-H0109) according to the experimental protocol. Interferon-γ levels were detected using a high-sensitivity human interferon-γ ELISA kit (SKU: E-EL-H0108) according to the experimental protocol. Quantitative analysis was performed using a microplate reader (Multiskan™ FC, ThermoFisher, MA, USA) to measure absorbance at 450 nm. The experiments were performed in triplicate.

[0070] (3) LDH experiment

[0071] Nasopharyngeal carcinoma cells (5-8F and TW03) (10) 3 (10 cells / well) were seeded into 96-well plates, with an effector / target ratio of 10:1. The cells were then co-cultured with CD8+ T cells (prepared using the same method as step (1) above) for two days. The supernatant was collected, and the release of lactate dehydrogenase (LDH) was detected using an LDH cytotoxicity assay kit (SKU: C0016) according to the manufacturer's instructions. The experiment was performed in triplicate.

[0072] (4) The experimental results are as follows:

[0073] To investigate the regulatory effect of ABCC1 on CD8+ T cell function, a co-culture system of activated CD8+ T cells and nasopharyngeal carcinoma cells was first constructed. Figure 3 (A) Compared with siNC group nasopharyngeal carcinoma cells, CD8+ T cells co-cultured with ABCC1 knockdown nasopharyngeal carcinoma cells showed significantly increased levels of immune effector cytokines (IFN-γ, IL-2, and TNF-α). Figure 3 (B, C, and D in the text). Furthermore, the results of granzyme B detection in the co-culture system showed that, compared with the siNC group of nasopharyngeal carcinoma co-culture groups, the CD8+ T cells in the ABCC1 silencing group secreted significantly more granzyme B (B, C, and D in the text). Figure 3 The cytotoxicity assay based on LDH release further showed that the amount of LDH released in the co-culture supernatant increased after ABCC1 silencing, indicating that it can enhance the killing effect of CD8+ T cells on tumor cells. Figure 3(F in the text). The above results indicate that ABCC1 can inhibit the effector function of CD8+ T cells, which may mediate immune escape from nasopharyngeal carcinoma.

[0074] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of ABCC1 inhibitors in the preparation of drugs for treating nasopharyngeal carcinoma, characterized in that: The ABCC1 inhibitor is si-ABCC1, and the target sequence of si-ABCC1 is CCTGTTTCCCCTTCTACTT, which is located between 279 and 297 bases in the coding region of the ABCC1 gene.

2. The application according to claim 1, characterized in that, The sequence of si-ABCC1 is as follows: si-ABCC1 sense: 5′-CCUGUUUCCCUUCUACUU-dTdT-3′; si-ABCC1 antisense: 5′-AAGUAGAAGGGGAAACAGG-dTdT-3′.

3. The use of the ABCC1 inhibitor as described in claim 1 or 2 in the preparation of a medicament for inhibiting the proliferation, and / or migration, and / or invasion of nasopharyngeal carcinoma cells.

4. The use of the ABCC1 inhibitor as described in claim 1 or 2 in the preparation of a drug that enhances the killing effect of CD8+ T cells on nasopharyngeal carcinoma cells.

5. The application according to claim 4, characterized in that: The nasopharyngeal carcinoma cells include at least one of 5-8F, TW03, HK-1, and TW01 cells.

6. Application of reagents for detecting ABCC1 expression levels in the preparation of nasopharyngeal carcinoma diagnostic reagents, nasopharyngeal carcinoma diagnostic kits, or nasopharyngeal carcinoma diagnostic assessment kits.

7. The application according to claim 6, characterized in that: The reagent for detecting ABCC1 expression levels includes primer pair Primer F and Primer R, whose nucleotide sequences are as follows: Primer F: 5′-AGTGGAACCCCTCTCTGTTTAAG-3′; Primer R: 5′-CCTGATAGCTCTTGGTCTTCATC-3′.

8. The application according to claim 6, characterized in that: The kit also includes TB Green Premix and / or ddH2O.

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