Molecular markers for evaluating chemotherapy drug resistance of oral cancer and application of molecular markers

Abstract

The invention discloses a group of molecular markers for evaluating chemotherapy drug resistance of oral cancer, the molecular markers comprise lncRNA EUDAL, EGFR and STAT3, and the nucleotide sequence of the lncRNA EUDAL is as shown in SEO ID NO.1. The invention also discloses a kit for evaluating chemotherapy drug resistance of oral cancer, and the kit for evaluating chemotherapy drug resistance of oral cancer comprises lncRNA EUDAL, EGFR and STAT3. Experimental verification finds that the three markers including lncRNA EUDAL, EGFR and STAT3 provided by the invention have high identification accuracy for predicting whether the chemotherapy reactivity of a patient with oral cancer is good or poor, specifically, when the levels of lncRNA EUDAL, EGFR-pY1068 and STAT3-pY705 are low, the sensitivity of the patient to chemotherapy is evaluated to be good, and when the levels of lncRNA EUDAL, EGFR-pY1068 and STAT3-pY705 are low, the sensitivity of the patient to chemotherapy is evaluated to be good. And when the levels of the lncRNA EUDAL, the EGFR-pY1068 and the STAT3-pY705 are higher, the sensitivity of the patient to the chemotherapy is evaluated to be poorer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomarkers, and particularly relates to a set of molecular markers for evaluating the chemoresistance of oral cancer and their applications. Background Art

[0002] Oral cancer is a common head and neck malignant tumor, among which squamous cell carcinoma (OSCC) accounts for more than 90% of the incidence of oral cancer. The etiology of oral cancer is complex, the clinical incidence rate is relatively high, and it is prone to lymph node metastasis or systemic metastasis, resulting in poor prognosis of the disease and low survival rate of patients.

[0003] At present, the main strategy for treating oral cancer is surgical resection combined with a cisplatin-based chemotherapy regimen. However, clinical treatment has found that many patients have a poor response to chemotherapy, which in turn has adverse effects such as decreased treatment effect, increased treatment difficulty, and poor treatment prognosis. However, at present, there is a lack of a predictive evaluation method for the chemoresistance of patients before they receive medication, which often leads to the delay of the treatment opportunity for drug-resistant patients, bringing great mental pressure and economic burden to patients and their families.

[0004] Therefore, developing an accurate and reliable method for predicting the chemoresistance of oral cancer patients to a cisplatin-based chemotherapy regimen is an urgent problem to be solved in this field. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a set of molecular markers for evaluating the chemoresistance of oral cancer and their applications. The markers have high discrimination accuracy for predicting good or poor chemoresponsiveness of oral cancer patients, and can be used in the preparation of reagents and test kits for detecting the chemoresistance of oral cancer.

[0006] Based on the above, the present invention first provides a set of molecular markers for evaluating the chemoresistance of oral cancer, and the molecular markers include: lncRNA EUDAL, EGFR, and STAT3, wherein the nucleotide sequence of the lncRNA EUDAL is shown in SEQ ID NO.1.

[0007] The present invention also provides the application of the aforementioned molecular markers in the preparation of reagents or test kits for detecting the chemoresistance of oral cancer.

[0008] The present invention also provides a test reagent for evaluating the chemoresistance of oral cancer, and the reagent is used to detect the expression level of lncRNA EUDAL, as well as the phosphorylation levels of EGFR and STAT3 in a sample.

[0009] Preferably, the detection sites for the phosphorylation level of EGFR include any one or more of pY1045, pY1068, pY1086, and pY1173; the detection sites for the phosphorylation level of STAT3 include pY705 and / or pS727.

[0010] Preferably, the reagent is used to detect the expression level of lncRNA EUDAL, the phosphorylation level of EGFR-pY1068, and the phosphorylation level of STAT3-pY705 in a sample.

[0011] Preferably, the reagent detects the expression level of lncRNA EUDAL by RNA-FISH technology, and detects the phosphorylation levels of EGFR-pY1068 and STAT3-pY705 by immunohistochemistry technology.

[0012] Preferably, when the expression level of lncRNA EUDAL in the subject sample is low, and the phosphorylation levels of EGFR and STAT3 are low, it is evaluated that the subject has a better sensitivity to chemotherapy; when the expression level of lncRNA EUDAL is high, and the phosphorylation levels of EGFR and STAT3 are high, it is evaluated that the subject has a poor sensitivity to chemotherapy.

[0013] Preferably, the sample is a tumor tissue sample.

[0014] On the other hand, the present invention also provides a detection kit for evaluating the chemoresistance of oral cancer, and the kit includes the aforementioned reagent.

[0015] Compared with the prior art, the beneficial effects of the present invention at least include:

[0016] The present invention unexpectedly found that wild-type EGFR in oral squamous carcinoma cells under hypoxia showed spontaneous phosphorylation without ligand stimulation. Subsequently, through the omics technology of molecular interaction, the key molecule - lncRNA EUDAL that causes the continuous accumulation of phosphorylated EGFR in cells was found, and scientific experiments proved that this phenomenon led to the formation of tumor cell chemoresistance through the activation of the STAT3 pathway.

[0017] Furthermore, the present invention verified the predictive value of the levels of three markers, lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705, for the chemoresistance of oral cancer in a validation population. The results showed that when the levels of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 were all low, the patient had a better chemotherapy response; conversely, when the levels of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 were all high, the patient had a poor chemotherapy response.

[0018] The three biomarkers lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 provided by the present invention have high discrimination accuracy for predicting good or poor chemotherapy responsiveness in oral cancer patients. They can not only help doctors formulate more precise individualized treatment plans for patients, improve the treatment effect, but also reduce unnecessary pain for patients and waste of medical resources. Description of the Drawings

[0019] Figure 1 It is the result of detecting the expression level and phosphorylation level of EGFR protein during 48-hour hypoxic culture of 4 OSCC cell lines without ligand by WB; among them, EGFR-t represents the protein expression level.

[0020] Figure 2 It is the sequencing result of exons 18-21 of the EGFR gene in 4 OSCC cell lines, where:

[0021] A represents the sequencing result at G719 of exon 18;

[0022] B represents the sequencing result of exon 19;

[0023] C represents the sequencing result at T790 of exon 20;

[0024] D represents the sequencing result at L858 of exon 21.

[0025] Figure 3 It is the experimental result of detecting the half-life of pEGFR (i.e., phosphorylated EGFR) in OSCC cell lines, where:

[0026] A represents the degradation of pEGFR in 4 OSCC cell lines under the action of CHX;

[0027] B represents the gray-scale analysis result of the WB band in (A);

[0028] C represents the degradation of pEGFR in HN4 and HN6 cells after blocking lysosomes (Baf) or proteasomes (MG132) respectively;

[0029] D represents the gray-scale analysis result of the WB band in (C).

[0030] Figure 4 It is the enrichment and expression of lncRNA EUDAL on EGFR in OSCC cells, where:

[0031] A represents the RIP-seq result of HN4 and CAL-27 targeting pEGFR after hypoxic treatment;

[0032] B represents the result of bioinformatics analysis predicting the binding site of lncRNA EUDAL and EGFR;

[0033] C represents the enrichment degree of lncRNA EUDAL on pEGFR detected by RIP-qPCR;

[0034] D represents the expression levels of lncRNA EUDAL under normoxia and hypoxia conditions in OSCC cell lines;

[0035] Statistical test: ANOVA; ns, no statistical significance; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

[0036] Figure 5 It is for the activation status of downstream molecules of EGFR in OSCC cells under hypoxia.

[0037] Figure 6 It is for the effects of lncRNA EUDAL expression on the activation of EGFR and STAT3 in OSCC cells under hypoxia, where:

[0038] A represents the activation status of EGFR and STAT3 under hypoxia after overexpressing wild-type lncRNA EUDAL or mutant lncRNA EUDAL Del-mut in HN4 and HN6 cells;

[0039] B represents the grayscale analysis of the WB results in (A);

[0040] C represents the activation status of EGFR and STAT3 under hypoxia after knocking down the expression of lncRNA EUDAL in CAL-27 and HN30 cells;

[0041] D represents the grayscale analysis of the WB results in (C).

[0042] Figure 7 It indicates that the lncRNA EUDAL-EGFR-STAT3 axis positively regulates cell autophagy activity, where:

[0043] A represents the cell autophagy activity in HN4 and HN6 cells after overexpressing wild-type lncRNA EUDAL or mutant lncRNA EUDAL Del-mut or directly inhibiting the activation of STAT3 while overexpressing wild-type lncRNA EUDAL;

[0044] B represents the cell autophagy activity in CAL-27 and HN30 cells after knocking down lncRNA EUDAL or directly inhibiting the activation of STAT3;

[0045] C represents the drug resistance of cells in HN4 and HN6 cells after overexpressing wild-type lncRNA EUDAL and directly inhibiting STAT3 or autophagy activity on this basis;

[0046] D represents the drug resistance of CAL-27 and HN30 cells after knocking down lncRNA EUDAL, directly inhibiting STAT3, or autophagy activity;

[0047] Among them, Figure 6 and Figure 7 EUDAL wt represents overexpression of wild-type lncRNA EUDAL, EUDAL Del-mut represents overexpression of a mutant of lncRNA EUDAL, which is a mutant lncRNA EUDAL designed after bioinformatics analysis to "lose the ability to bind to EGFR", and EUDAL KD represents knocking down lncRNA EUDAL.

[0048] Figure 8 It is indicated that the lncRNA EUDAL-EGFR-STAT3 axis reflects the chemotherapeutic responsiveness of OSCC patients, where:

[0049] A represents the CT examination results of patients with good and poor chemotherapy effects;

[0050] B represents the HE staining of tumor tissues, EUDAL FISH, pEGFR, and pSTAT3 immunohistochemical results of patients with good and poor chemotherapy effects;

[0051] C represents the differences in the levels of EUDAL, pEGFR, and pSTAT3 in tumor tissues of patients with good and poor chemotherapy effects; error bars: median and interquartile range; Mann-Whitney U test was used;

[0052] D represents the correlation of the levels of EUDAL, pEGFR, and pSTAT3 in patients' tumor tissues; n = 45, ****: P < 0.0001; Spearman rank correlation test was used. Detailed implementation manners

[0053] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below in combination with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] Based on the information contained in the present invention, various changes can be easily made to the precise description of the present invention by those skilled in the art without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined processes, properties or components, as these embodiments and other descriptions are only for illustrative purposes of specific aspects of the present invention. In fact, all various changes that can be made by those skilled in the art in the present field or related fields are covered within the scope of the appended claims.

[0055] Term Explanation

[0056] lncRNA: Long non-coding RNA, a class of non-coding RNA molecules with a length exceeding 200 nucleotides. It does not encode proteins but plays a key role in many biological processes such as gene expression regulation, cell differentiation, development, and disease occurrence and development. It can regulate gene expression at multiple levels such as the transcriptional level and post-transcriptional level by interacting with DNA, RNA, or proteins.

[0057] The "lncRNAEUDAL" gene described in the present invention is located at positions 230,280,312 - 230,281,893 on chromosome 1. The transcript lncRNAEUDAL is distributed in the nucleus and cytoplasm, and its nucleotide sequence is as shown in SEO ID NO.1. The specific transcript number of the lncRNA EUDAL in the Ensembl database is ENST00000414640.

[0058] The specific sequence of SEO ID NO.1 is:

[0059]

[0060] EGFR: Epidermal Growth Factor Receptor, a transmembrane protein receptor that plays a crucial role in processes such as cell growth, proliferation, and differentiation.

[0061] STAT3: Signal Transducer and Activator of Transcription 3, a protein that plays a crucial role in intracellular signal transduction and gene expression regulation, and is of great significance in cell growth, differentiation, immune response, and tumor development.

[0062] As mentioned above, in view of the deficiencies of the prior art, through a large number of studies and experiments, the present invention finally provides a set of molecular markers for evaluating the chemoresistance of oral cancer. The markers include: lncRNA EUDAL, EGFR, and STAT3. Among them, the nucleotide sequence of the lncRNA EUDAL is as shown in SEQ ID NO.1.

[0063] On the other hand, the present invention also provides the application of the aforementioned molecular markers in the preparation of reagents or kits for detecting the chemoresistance of oral cancer.

[0064] On the other hand, the present invention also provides a detection reagent for evaluating the chemoresistance of oral cancer. The reagent is used to detect the expression level of lncRNA EUDAL in a sample, as well as the phosphorylation levels of EGFR and STAT3.

[0065] Preferably, the detection sites for the phosphorylation level of EGFR include any one or more of pY1045, pY1068, pY1086, and pY1173; the detection sites for the phosphorylation level of STAT3 include pY705 and / or pS727.

[0066] Preferably, the reagent is used to detect the expression level of lncRNA EUDAL, the phosphorylation level of EGFR-pY1068, and the phosphorylation level of STAT3-pY705 in a sample.

[0067] Preferably, the reagent detects the expression level of lncRNA EUDAL by RNA-FISH technology, and detects the phosphorylation levels of EGFR-pY1068 and STAT3-pY705 by immunohistochemistry technology.

[0068] Preferably, the sample is a tumor tissue sample.

[0069] On the other hand, the present invention also provides a detection kit for evaluating the chemoresistance of oral cancer, and the kit includes the aforementioned reagents.

[0070] It has been experimentally demonstrated in the present invention that when the expression level of lncRNA EUDAL in the subject sample is low, and the phosphorylation levels of EGFR-pY1068 and STAT3-pY705 are low, the subject has better sensitivity to chemotherapy; when the expression level of lncRNA EUDAL is high, and the phosphorylation levels of EGFR and STAT3 are high, the subject has poor sensitivity to chemotherapy.

[0071] The experimental exploration process of the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0072] Example 1

[0073] In order to find a sensitive method that can accurately and reliably predict the response of oral cancer patients to cisplatin-based chemotherapy, the present invention has conducted a large number of studies, including:

[0074] As Figure 1 shown, the present invention first purchased four oral cancer cell lines (HN4, HN6, HN30, and CAL-27) from ATCC, and placed these four oral cancer cells under the same hypoxic and ligand-free stimulation conditions for 48 hours. After the culture ended, the protein expression level of EGFR and the phosphorylation level of EGFR (phosphorylation sites include pY1068, pY1086, pY1045, pY845, pY1148) in each group of cells were detected by Western blot (WB). Unexpectedly, it was found that under hypoxic and ligand-free stimulation conditions, EGFR showed spontaneous and continuous activation, and the activation levels (i.e., phosphorylation levels) of different cell lines were inconsistent. Among them, the activation levels of CAL-27 and HN30 were relatively high, while the activation levels of HN4 and HN6 were relatively low. Moreover, the protein expression level of EGFR in the two cell lines with lower activation levels was not significantly different or significantly reduced compared with the other two cell lines.

[0075] The above experimental phenomenon has attracted great interest from the research group of the present invention, and a comparative study has been continued on this phenomenon to explore the reasons for the differences between the two types of cells with higher and lower activation levels, so as to find the molecular mechanism of continuous activation of EGFR under hypoxia.

[0076] As Figure 2 shown, first, exon 18-21 of the EGFR gene in four oral cancer cell lines was sequenced by first-generation sequencing. After comparing the sequencing results, the present invention excluded the possibility that EGFR activation was caused by mutations.

[0077] As Figure 3As shown, the molecular mechanism of EGFR activation was further studied. Four OSCC (oral squamous cell carcinoma) cell lines were co-cultured with cycloheximide (CHX) for 24 h respectively. CHX can block protein synthesis and directly observe the protein degradation half-life. The results showed that the protein degradation half-life of EGFR was significantly longer in CAL-27 and HN30 cells, while it was significantly shorter in HN4 and HN6 cells ( Figure 3 A-B); HN4 and HN6 were continuously co-cultured with CHX and Baf (or MG-132) respectively. Baf was used to block lysosomal acidification, and MG-132 is a proteasome inhibitor. The results showed that the activation level of EGFR in cells was significantly increased after co-culture with CHX and Baf. In summary, by comparing the differences between the two types of cells, the present invention found that EGFR activation was mainly caused by abnormally increased EGFR stability, which was caused by lysosomal inhibition of EGFR. This conclusion continued to guide the present invention to explore "the key molecular events that initiate the EGFR lysosomal degradation mechanism".

[0078] Based on the above, as Figure 4 shown, HN4 and CAL-27 cell lines were respectively cultured under hypoxic and ligand-free stimulation conditions. After the culture, RNA Immunoprecipitation Sequencing was used to study the interaction between intracellular RNA and phosphorylated EGFR protein. As a result, lncRNA ENST00000414640 (i.e., lncRNA EUDAL, the nucleotide sequence is shown in SEQ ID NO.1) enriched on phosphorylated EGFR protein that was significantly increased under hypoxia was found ( Figure 4 A, C and D), and the binding site of lncRNA EUDAL to EGFR was predicted by bioinformatics analysis. lncRNA EUDAL can bind to the site on EGFR where the protein that can degrade EGFR is recruited, so it can prevent these degradation-related proteins Grb2 and c-Cbl from binding to EGFR, thus protecting EGFR from degradation ( Figure 4 B).

[0079] As Figure 5 shown, the present invention detected the activation of EGFR downstream molecules (including Akt, ERK1 / 2, STAT1, STAT3) in OSCC cells under hypoxia by WB, and the results showed that the phosphorylation trend of STAT3 was highly consistent with the activation trend of EGFR.

[0080] As Figure 6As shown, by further intervening in the expression of lncRNA EUDAL in OSCC cells, such as overexpressing wild-type lncRNA EUDAL, overexpressing mutant lncRNA EUDAL Del-mut, or knocking down wild-type lncRNA EUDAL, etc., the changes in the activation of downstream STAT3 protein were observed. The results showed that when wild-type lncRNA EUDAL was overexpressed in HN4 and HN6 cells (i.e., the EUDAL wt group), the phosphorylation level of STAT3-pY705 increased significantly, while when the lncRNA EUDAL Del-mut mutant was overexpressed (i.e., the EUDAL Del-mut group), the phosphorylation level of STAT3-pY705 did not change significantly ( Figure 6 A-B of). Thus, it can be seen that the binding of wild-type lncRNA EUDAL to EGFR will cause subsequent various biological effects; when wild-type lncRNA EUDAL was knocked down in CAL-27 and HN30 cells (i.e., the EUDAL KD group), the phosphorylation level of STAT3-pY705 decreased significantly ( Figure 6 C-D of). In summary, the above results indicate that there is a significant positive correlation between the expression level of wild-type lncRNA EUDAL and the activation level of STAT3 protein.

[0081] Continuing to observe the changes in the self-protection mechanism - autophagy activity by intervening in lncRNA EUDAL and STAT3, it was found that there was a significant positive correlation between the activation degrees of lncRNA EUDAL, EGFR, and STAT3 and autophagy activity: after overexpressing wild-type lncRNA EUDAL, the phosphorylation levels of EGFR and STAT3 increased significantly, the expression of p62 protein decreased, and autophagy activity in cells increased significantly; after knocking down lncRNA EUDAL or inhibiting the activation of STAT3 with the STAT3-IN-1 inhibitor in the group with high expression of lncRNA EUDAL, autophagy activity was significantly inhibited ( Figure 7 A, B of). In addition, the present invention also co-cultured OSCC cells with cisplatin drugs and simultaneously included different groups, and performed different interventions on the lncRNA EUDAL expression level, STAT3 activation degree, or autophagy activity of the cells. After the culture ended, by detecting IC 50Evaluate the sensitivity of tumor cells to chemotherapeutic drugs under different intervention measures. The results showed that significantly increasing the sensitivity of tumor cells to chemotherapeutic drugs could be achieved by targeting and reducing the expression of wild-type lncRNA EUDAL with siRNA (the siRNA target sequence was GCAAAGGATTCGTCAGAAA), inhibiting STAT3 activation with the STAT3-IN-1 inhibitor (purchased from MCE, HY-100753), or directly targeting and inhibiting autophagy activity with CQ (chloroquine, purchased from MCE, HY-17589A). Figure 7 C and D).

[0082] Based on the above research results, the present invention clarified that EGFR continuously activated by lncRNA EUDAL could trigger the activation of the downstream STAT3 pathway, and STAT3 activation could lead to the formation of tumor cell drug resistance by inducing autophagy activity in tumor cells.

[0083] Example 2

[0084] To verify the cellular and molecular mechanisms discovered in the research of Example 1, 45 patients with oral squamous cell carcinoma who received preoperative induction chemotherapy were included in this example. The changes in tumor size were obtained from imaging data, and the patients were divided into two cohorts: those with better chemotherapy response (22 cases) and those with poorer chemotherapy response (23 cases). Figure 8 A).

[0085] Paraffin sections of oral cancer tumor samples from the two cohorts were taken and stained with HE; in addition, the lncRNA EUDAL level in tumor tissues was detected by RNA-FISH, and the probe was designed and synthesized by Ribobio according to the lncRNA EUDAL target sequence; the levels of EGFR-pY1068 and STAT3-pY705 were detected by immunohistochemistry, and the detection antibodies used were 3777S (pEGFR) and 9145S (pSTAT3) from CST, respectively.

[0086] As Figure 8 shown, the results found that the levels of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 were all lower in the tumor tissues of patients with better chemotherapy response; conversely, in patients with poorer chemotherapy response, the levels of these three indicators in tumor tissues were all higher. Figure 8 B and C). There was a good positive correlation among the three indicators of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 in tumor tissues. Figure 8 D). This further verified the predictive value of the lncRNA EUDAL-EGFR-STAT3 positive regulatory pathway for the chemotherapy response of oral cancer patients.

[0087] In summary, the present invention provides a set of molecular markers for evaluating the chemoresistance of oral cancer. The molecular markers include: lncRNA EUDAL, EGFR, and STAT3. Among them, the nucleotide sequence of the lncRNA EUDAL is shown as SEQ ID NO.1. It is found through experimental verification of the present invention that the three markers lncRNA EUDAL, EGFR, and STAT3 provided by the present invention have high discrimination accuracy for predicting good or poor chemotherapeutic responsiveness of oral cancer patients. Specifically, when the levels of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 are all low, the sensitivity of the patient to chemotherapy is better; when the levels of lncRNA EUDAL, EGFR-pY1068, and STAT3-pY705 are all high, the sensitivity of the patient to chemotherapy is poor.

[0088] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and alternatives to the present invention will be obvious. Therefore, the protection scope of the present invention should be defined by the appended claims.

Claims

1. A group of molecular markers for evaluating oral cancer chemotherapy resistance, characterized in that: The molecular markers include: lncRNA EUDAL, EGFR and STAT3, wherein the nucleotide sequence of the lncRNA EUDAL is shown as SEO ID NO.

1.

2. Use of the molecular marker according to claim 1 in the preparation of a reagent or kit for detecting oral cancer chemotherapy resistance.

3. A detection reagent for evaluating oral cancer chemotherapy resistance, characterized in that: The reagent is used to detect the expression level of lncRNAEUDAL in a sample, as well as the phosphorylation level of EGFR and the phosphorylation level of STAT3.

4. The detection reagent for evaluating oral cancer chemotherapy resistance according to claim 3, characterized in that: The phosphorylation level detection sites of EGFR include any one or more of pY1045, pY1068, pY1086 and pY1173; the phosphorylation level detection sites of STAT3 include pY705 and / or pS727.

5. The detection reagent for evaluating oral cancer chemotherapy resistance according to claim 3, characterized in that: The reagent is used to detect the expression level of lncRNAEUDAL, the phosphorylation level of EGFR-pY1068 and the phosphorylation level of STAT3-pY705 in a sample.

6. The detection reagent for evaluating oral cancer chemotherapy resistance according to claim 5, characterized in that: The reagent detects the expression level of lncRNA EUDAL by RNA-FISH technology, and detects the phosphorylation level of EGFR-pY1068 and the phosphorylation level of STAT3-pY705 by immunohistochemistry technology.

7. The detection reagent for evaluating oral cancer chemotherapy resistance according to claim 3, characterized in that: When the expression level of lncRNA EUDAL and the phosphorylation levels of EGFR and STAT3 in the subject's sample is low, the subject's sensitivity to chemotherapy is evaluated to be good; when the expression level of lncRNA EUDAL and the phosphorylation levels of EGFR and STAT3 are high, the subject's sensitivity to chemotherapy is evaluated to be poor.

8. The detection reagent for evaluating oral cancer chemotherapy resistance according to claim 3, characterized in that: The sample is a tumor tissue sample.

9. A detection kit for evaluating oral cancer chemotherapy resistance, characterized in that: The kit comprises the reagent according to any one of claims 3-8.

Images