A biomarker for diagnosing oral squamous cell carcinoma and its application

Abstract

The present invention discloses a biomarker for diagnosing oral squamous cell carcinoma and its application. The biomarker includes the DDX59-AS1 gene. The present invention discovers for the first time that there is a significant difference in the expression of the DDX59-AS1 gene between oral squamous cell carcinoma and normal tissues. The DDX59-AS1 gene can be used as a biomarker for diagnosing oral squamous cell carcinoma. Oral squamous cell carcinoma can be quickly diagnosed by detecting its expression level, and it has high sensitivity and specificity, effectively solving the technical problem of poor effect of diagnosing oral squamous cell carcinoma by eosin-hematoxylin (HE) staining and histomorphology.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a biomarker for diagnosing oral squamous cell carcinoma and an application thereof. Background Art

[0002] Oral squamous cell carcinoma (OSCC) is the most common oral cancer. Reportedly, approximately two-thirds of OSCC patients are diagnosed in the advanced stages of the disease. After undergoing comprehensive treatments including surgery, radiotherapy, and chemotherapy, these patients experience severe facial disfigurement and a significant decrease in quality of life. However, the five-year survival rate for early-stage OSCC patients is as high as 85.4%, and quality of life is significantly improved after treatment. Therefore, early diagnosis of OSCC is crucial for improving patient survival outcomes. Highly specific and sensitive biomarkers can significantly aid in the early diagnosis, treatment, and prognosis of OSCC patients.

[0003] Currently, the pathological diagnosis of oral squamous cell carcinoma mainly relies on eosin-hematoxylin (HE) staining and tissue morphology. However, when tumor tissue necrosis occurs or the tissue is squeezed, resulting in unclear structure, it is difficult to make a correct diagnosis based solely on HE staining and morphological observation.

[0004] To date, nearly 200 tumor markers are available for clinical use, categorized as cell surface tumor markers, serum tumor markers, and oncogene markers. Examples include embryonic antigens such as alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA), which are expressed during the embryonic period but not in healthy adults, but re-expressed with tumorigenesis. Tumor-associated glycoproteins include cancer antigen 50 (CA50), cancer antigen 125 (CA125), squamous cell carcinoma antigen (SCC-Ag), and epidermal growth factor receptor (EGFR). Furthermore, markers such as vascular endothelial growth factor (VEGF) and its receptor, and proliferating cell nuclear antigen (PCNA), are associated with tumor cell proliferation and differentiation. However, clinically specific and sensitive tumor markers are currently lacking.

[0005] Prognostic research on OSCC has primarily focused on protein-coding genes. However, with increasing research, cancer researchers have discovered that noncoding RNAs (ncRNAs), particularly long noncoding RNAs (lncRNAs), play a crucial role in the prognosis of many malignant tumors. lncRNAs are involved in diverse biological processes, including cell proliferation and apoptosis, growth and development, inflammatory responses, and the development and progression of various diseases, including cancer. lncRNAs regulate tumor development and progression and have long been considered a novel target for targeted anticancer therapy. However, limited research has examined the role of lncRNAs in OSCC prognosis.

[0006] In summary, the development of new markers related to oral squamous cell carcinoma and the expansion of the diagnostic criteria for oral squamous cell carcinoma are of great significance in the field of diagnosis and treatment of oral squamous cell carcinoma. Summary of the Invention

[0007] In view of the deficiencies of the prior art and the actual needs, the present invention provides a biomarker for diagnosing oral squamous cell carcinoma and its application. The present invention for the first time discovers that there is a significant difference in the expression of the DDX59-AS1 gene between oral squamous cell carcinoma and normal tissues. The DDX59-AS1 gene can be used as a new biomarker for the diagnosis of oral squamous cell carcinoma, solving the problem that the current diagnosis of oral squamous cell carcinoma mainly relying on eosin-hematoxylin (HE) staining and histomorphology has poor results.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a biomarker for diagnosing oral squamous cell carcinoma, and the biomarker includes the DDX59-AS1 gene.

[0010] The present invention statistically analyzes the sequencing results of oral squamous cell carcinoma and oral normal tissues in the TCGA database, and a total of 32 cases of oral normal tissues and 331 cases of oral squamous cell carcinoma tissues are included. It is found that there is a significant difference in the expression of the DDX59-AS1 gene between oral squamous cell carcinoma and normal tissues, which can be used as a new biomarker suitable for the diagnosis of oral squamous cell carcinoma. Subsequently, the in-situ hybridization technique is used to detect the expression of DDX59-AS1 in the pathological paraffin sections of oral squamous cell carcinoma tissues, confirming the positive expression of this gene in oral squamous cell carcinoma and its application value in diagnosis and prognosis evaluation.

[0011] In the second aspect, the present invention provides the application of the biomarker for diagnosing oral squamous cell carcinoma described in the first aspect in the preparation of products for detecting oral squamous cell carcinoma.

[0012] In the third aspect, the present invention provides a kit for detecting oral squamous cell carcinoma, and the kit includes reagents for detecting the expression level of the biomarker for diagnosing oral squamous cell carcinoma described in the first aspect.

[0013] Preferably, the reagent includes a specific probe for detecting the DDX59-AS1 gene.

[0014] In the present invention, reagents for detecting the gene expression level commonly used in the art are applicable to the present invention without special limitations.

[0015] In the present invention, specific probes can be designed according to the nucleic acid sequence of the DDX59-AS1 gene as needed.

[0016] Preferably, the kit further comprises dewaxing clearing solution, anhydrous ethanol, hydrogen peroxide, antigen retrieval solution, hybridization buffer, serum blocking solution, DAB color developing solution and neutral gum.

[0017] In a fourth aspect, the present invention provides an early diagnosis model for oral squamous cell carcinoma, wherein the input variables of the early diagnosis model for oral squamous cell carcinoma include the expression level values of the biomarkers for diagnosing oral squamous cell carcinoma described in the first aspect.

[0018] Preferably, the output variable of the oral squamous cell carcinoma early diagnosis model includes the differential expression fold, and the calculation formula of the differential expression fold is shown in equation (1):

[0019]

[0020] Preferably, the criterion for determining oral squamous cell carcinoma positivity is that the differential expression fold is ≥1.5.

[0021] In a fifth aspect, the present invention provides an early diagnosis device for oral squamous cell carcinoma, comprising a detection unit and an analysis unit, wherein the detection unit is configured to perform the following steps: detecting the expression level value of the DDX59-AS1 gene in a sample of an individual to be tested; and the analysis unit is configured to perform the following steps: inputting the detected expression level value of the DDX59-AS1 gene into the early diagnosis model for oral squamous cell carcinoma described in the fourth aspect for data analysis, outputting the differential expression multiple, and determining whether the patient is positive for oral squamous cell carcinoma.

[0022] In the present invention, gene expression levels can be determined using standard methods known in the art. Conventional methods for determining gene expression levels use RNA levels as a standard. Similarly, in the present invention, the RNA level of the DDX59-AS1 gene can be used as a standard to determine gene expression levels.

[0023] Preferably, the individual sample to be tested is selected from any one or a combination of at least two of oral tissue, blood, plasma, body fluid, cells or saliva.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention discovered for the first time that there is a significant difference in the expression of the DDX59-AS1 gene between oral squamous cell carcinoma and normal tissue. The DDX59-AS1 gene can be used as a marker for diagnosing oral squamous cell carcinoma. By detecting its expression level, oral squamous cell carcinoma can be quickly diagnosed, and it has certain advantages in specificity and sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1The figure shows the expression levels of the DDX59-AS1 gene in oral squamous cell carcinoma and normal tissues;

[0027] Figure 2 The figure shows the expression results of the DDX59-AS1 gene in oral squamous cell carcinoma tissues with paired normal tissues;

[0028] Figure 3 The figure is the ROC curve of the DDX59-AS1 expression level;

[0029] Figure 4A The figure is a nomogram for predicting the 1-year, 3-year, and 5-year overall survival rates of patients with oral squamous cell carcinoma;

[0030] Figure 4B The figure is the calibration plot of the nomogram for predicting the overall survival rate;

[0031] Figure 4C The figure is a nomogram for predicting the 1-year, 3-year, and 5-year specific survival rates of patients with oral squamous cell carcinoma;

[0032] Figure 4D The figure is the calibration plot of the nomogram for predicting the specific survival rate;

[0033] Figure 5 The figure shows the in-situ hybridization staining results of the cancer tissues and paired adjacent cancer tissues of OSCC patients. Detailed implementation methods

[0034] To further elaborate on the technical means and effects adopted by the present invention, the present invention will be further described below in combination with embodiments and drawings. It can be understood that the specific implementation methods described herein are only used to explain the present invention and not to limit the present invention.

[0035] For those not specifying specific technologies or conditions in the embodiments, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through regular channels.

[0036] Example 1

[0037] In this example, the expression analysis of the DDX59-AS1 gene in oral squamous cell carcinoma (squamous carcinoma) and oral normal tissues was carried out.

[0038] First, sequencing data of oral squamous cell carcinoma and normal oral tissues were obtained from the TCGA database, including data of 32 normal oral tissues and 331 oral squamous cell carcinoma tissue samples. The RNAseq data in FPKM (Fragments Per Kilobase per Million) format was converted into TPM (transcripts per million reads) format for expression comparison between samples. Through the Wilcoxon rank sum test statistical method, it was analyzed that the DDX59-AS1 gene was significantly highly expressed in OSCC samples of oral squamous cell carcinoma, and the result was statistically significant (p < 0.001, Figure 1 ). The expression of DDX59-AS1 in 32 oral squamous cell carcinoma tissues with paired normal tissues was also analyzed, and the results showed that DDX59-AS1 was also significantly highly expressed in cancer tissues (P < 0.001, Figure 2 ). According to the receiver operating characteristic (ROC) curve, the effectiveness of the DDX59-AS1 expression level in distinguishing normal tissues from cancer tissues was analyzed. The area under the curve (AUC) of DDX59-AS1 was 0.732, and the results indicated that DDX59-AS1 showed high sensitivity and specificity and might be a potential diagnostic molecule ( Figure 3 ).

[0039] Example 2

[0040] In this example, in situ hybridization experiments were carried out on clinical samples of DDX59-AS1 in paired oral squamous cell carcinoma and normal tissues for verification.

[0041] To further determine the importance of DDX59-AS1 expression, in situ hybridization staining was performed on cancer tissues and paired adjacent cancer tissues of 20 OSCC patients. The steps of the in situ hybridization experiment are as follows:

[0042] (1) Probe design and synthesis: The FISH probe for DDX59-AS1 was designed and synthesized by Wuhan Sevier (Shanghai Branch) Biotechnology Co., Ltd., and the sequence was: 5’-CTTACTGGATCTTGTGCCTAAGAAGCCCAA-3’;

[0043] (2) Tissue fixation: After the oral squamous cell carcinoma tissues and normal tissues were taken out, washed, and immediately put into the fixative (prepared with DEPC water) for fixation for 48 h;

[0044] (3) Dehydration: After tissue fixation, dehydration was carried out with gradient alcohol, followed by wax immersion and embedding;

[0045] (4) Sectioning: Paraffin was sectioned by a microtome, the sections were picked up by a spreading machine, and baked in an oven at 62 °C for 2 h;

[0046] (5) Dewaxing of paraffin sections: Place the sections in dewaxing clearing solution I for 15 min, dewaxing clearing solution II for 15 min, anhydrous ethanol I for 5 min, and anhydrous ethanol II for 5 min, air-dry, and soak in DEPC water;

[0047] (6) Digestion: Depending on the length of tissue fixation, the sections were boiled in the repair solution for 10 min, cooled naturally, and then circled with a gene pen. According to the different characteristics of different tissues, proteinase K (20 μg / mL) was added dropwise and digested at 37°C for 20 min. After rinsing with pure water, the sections were washed with PBS for 5 min × 3 times;

[0048] (7) Block endogenous peroxidase: add 3% methanol-H2O2 dropwise, incubate at 25°C in the dark for 15 min, place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times, 5 min each time;

[0049] (8) Prehybridization: Add prehybridization solution and incubate at 37°C for 1 h;

[0050] (9) Hybridization: Pour off the pre-hybridization solution and add the hybridization solution containing the probe DDX59-AS1 at a concentration of 500 nM. Hybridize overnight in a constant temperature incubator at 42°C.

[0051] (10) Post-hybridization washing: remove the hybridization solution, wash with 2×SSC at 37°C for 10 min, wash with 1×SSC at 37°C for 5 min twice, and wash with 0.5×SSC at 25°C for 10 min.

[0052] (11) Imaging oligo (DIG) incubation: add hybridization solution containing imaging oligo (DIG) at a dilution ratio of 1:400; incubate at 42°C for 3 h, wash at 37°C for 10 min in 2×SSC, wash twice at 37°C for 5 min, and wash at 37°C for 10 min in 0.5×SSC.

[0053] (12) Add blocking solution: add blocking serum (normal rabbit serum) and block at 25°C for 30 min;

[0054] (13) Add mouse anti-digoxigenin-labeled peroxidase (anti-DIG-HRP): Pour off the blocking solution, add anti-DIG-HRP, incubate at 37°C for 50 min, and wash with PBS for 5 min × 4 times;

[0055] (14) DAB color development: After the slices are slightly dried, freshly prepared DAB color development solution is added to the circle. The color development time is controlled under a microscope. The positive color is brown-yellow. Rinse the slices with pure water to stop the color development.

[0056] (15) Counterstaining of cell nuclei: Counterstain with Harris hematoxylin for about 3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, wash with tap water, turn blue with ammonia solution, and rinse with running water;

[0057] (16) Dehydration and sealing: Place the sections in 75% alcohol for 6 min, 85% alcohol for 6 min, 100% alcohol I for 6 min, 100% alcohol II for 6 min, n-butanol for 6 min, and dewaxing transparent liquid in sequence. Take the sections out of the dewaxing transparent liquid and let them dry slightly, then seal them with super clean quick-drying sealing glue.

[0058] (17) Microscopic examination, image acquisition and analysis.

[0059] According to the staining results, representative images are shown in Figure 2. Figure 5 , AB are squamous cell carcinoma tissues taken from the palate, CD are normal tissues taken from the palate; EF are squamous cell carcinoma tissues taken from the tongue, GH are normal tissues taken from the tongue, indicating that DDX59-AS1 staining is positive in the cytoplasm of cancer tissues, but negative in adjacent tissues.

[0060] Example 3

[0061] This example analyzes the sensitivity and specificity of the detection results of Example 2.

[0062] Sensitivity and specificity formula:

[0063] Sensitivity = number of true positive cases / (number of true positive cases + number of false negative cases) × 100%, the rate of correctly judging positives (Table 1);

[0064] Specificity = number of true negative cases / (number of true negative cases + number of false positive cases)) × 100%, the rate of correctly judging non-positive cases (Table 2).

[0065] Table 1

[0066]

[0067] Table 2

[0068]

[0069] Example 4

[0070] This example analyzes the prognostic performance of the DDX59-AS1 gene in the clinicopathological aspects of OSCC.

[0071] The Kaplan-Meier analysis was used to evaluate the predictive value of DDX59-AS1 for overall survival and specific survival in clinicopathological subgroups. Cox regression analysis was performed in specific subgroups. In terms of overall survival rate, DDX59-AS1 was statistically significant in the T2&T3&T4 (HR = 1.435 (1.026 - 2.006), p = 0.035) subgroup of T stage, the Stage IV (HR = 1.654 (1.057 - 2.589), p = 0.028) subgroup of Clinical stage, the Male (HR = 1.526 (1.019 - 2.285), p = 0.040) subgroup of Gender, the subgroup of Age, the Yes (HR = 1.507 (1.036 - 2.193), p = 0.032) subgroup of Smoker, the subgroup of Alcohol history, the No (HR = 2.616 (1.523 - 4.494)), p < 0.001) subgroup of Lymphovascular invasion, the No (HR = 2.008 (1.010 - 3.992), p = 0.047) subgroup of Perineural invasion, the WT (HR = 2.023 (1.037 - 3.946), p = 0.039) subgroup of TP53 status, the WT (HR = 1.653 (1.146 - 2.383), p =  0.007) subgroup of PIK3CA status, the subgroup of Primary therapy outcome, the G1&G2 (HR = 1.615 (1.102 - 2.367), p = 0.014) subgroup of Histologic grade, the White (HR = 1.429 (1.011 - 2.019), p = 0.043) subgroup of Race, and the N2&N3 (HR = 1.889 (1.026 - 3.475), p = 0.041) subgroup of N stage (Table 3). The prognostic performance of specific survival in subgroups is shown in Table 4. Among them, HR: hazard ratio; CI: confidence interval; WT: wild type; Mut: mutation. The expression level of DDX59-AS1 was higher in elderly men, smokers, and drinkers. The higher the expression of DDX59-AS1, the higher the T stage and clinicopathological stage of patients, and the lower the overall survival rate and specific survival rate of patients, and the worse the prognosis.

[0072] Table 3

[0073]

[0074]

[0075]

[0076] Table 4

[0077]

[0078]

[0079]

[0080] Example 5

[0081] In this example, a nomogram based on DDX59-AS1 expression was constructed and validated.

[0082] The nomogram can predict the prognosis of OSCC patients and test the effectiveness of the nomogram through a calibration curve. This nomogram integrates independent clinical characteristics determined by multivariate analysis of Primary therapy outcome, Perineural invasion, and DDX59-AS1 for overall survival (C-index = 0.699, Figure 4A ) and specific survival (C-index = 0.761, Figure 4C ). The calibration curve made ideal predictions for the three nomograms of 1-year, 3-year, and 5-year clinical outcomes, and the bias-corrected line was close to the ideal curve (i.e., the 45-degree line), indicating good consistency between the predicted values and the observed values ( Figure 4B and Figure 4D ).

[0083] In summary, through statistical analysis of the sequencing results of oral squamous cell carcinoma and oral normal tissues in the TCGA database, the present invention found that the expression of DDX59-AS1 was significantly different between oral squamous cell carcinoma and normal tissues, which was suitable for use in the diagnosis of oral squamous cell carcinoma. The expression of DDX59-AS1 was detected in the pathological paraffin sections of oral squamous cell carcinoma tissues by in situ hybridization technology, and the positive expression of this gene in oral squamous cell carcinoma and its application value in diagnosis and prognosis evaluation were determined.

[0084] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the present invention product, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

Claims

1. An early diagnosis device for oral squamous cell carcinoma, characterized in that: The device includes a detection unit and an analysis unit; The detection unit is used to perform the following steps: detecting the expression level of the DDX59-AS1 gene in the individual sample to be tested; The analysis unit is used to perform the following steps: inputting the detected expression level value of the DDX59-AS1 gene into the oral squamous cell carcinoma early diagnosis model for data analysis, outputting the differential expression multiple, and determining whether the oral squamous cell carcinoma is positive.

2. The early diagnosis device for oral squamous cell carcinoma according to claim 1, characterized in that: The calculation formula for the differential expression fold of the oral squamous cell carcinoma early diagnosis model is shown in equation (1):

3. The early diagnosis device for oral squamous cell carcinoma according to claim 1, characterized in that: The criterion for determining oral squamous cell carcinoma as positive is that the differential expression fold is ≥1.

5.

4. The early diagnosis device for oral squamous cell carcinoma according to claim 1, characterized in that The individual sample to be tested is selected from any one of oral tissue, body fluid or cells, or a combination of at least two of them.

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