Application of AKR1C2 protein or coding gene thereof or AKR1C2 gene promoter region methylation site in esophageal squamous cell carcinoma diagnosis

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of AKR1C2 protein or a coding gene thereof or an AKR1C2 gene promoter region methylation site in esophageal squamous cell carcinoma diagnosis. The invention detects the methylation level of the methylation site of the AKR1C2 gene promoter region and the expression level of AKR1C2 protein or the coding gene thereof in esophageal squamous cell carcinoma and para-carcinoma normal tissues, and finds that the average methylation level of the AKR1C2 gene promoter region and the expression level of the AKR1C2 gene are differentially expressed in esophageal squamous cell carcinoma and para-carcinoma normal tissues, and have statistical significance. The AKR1C2 protein or the coding gene thereof or the AKR1C2 gene promoter region methylation site can be used for diagnosis and / or adjuvant therapy of esophageal squamous carcinoma, and has strong specificity and high sensitivity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of AKR1C2 protein or its encoding gene or AKR1C2 gene promoter region methylation sites in the diagnosis of esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer (EC) is the sixth leading cause of death among cancers worldwide. Esophageal squamous cell carcinoma (ESCC), also known as esophageal squamous cell carcinoma, is the most common histological subtype of esophageal cancer. Although existing technologies have improved the diagnosis and treatment of esophageal cancer, the survival rate of patients with advanced esophageal squamous cell carcinoma is still very low due to early lymph node metastasis, and the 5-year survival rate of patients is still low. Etiological and epidemiological studies have shown that the occurrence of esophageal cancer is a disease caused by the combined effects of multiple factors. Therefore, it is particularly important to find the pathogenic factors and pathogenesis that lead to esophageal cancer. Existing technologies have found that in esophageal cancer, the promoter regions of the P16 and RASSF1A genes are both hypermethylated, and hypermethylation inhibits gene expression, thereby promoting the development of cancer.

[0003] AKR1C2 (Derl-like domain family member 1) is a member of the aldehyde / ketone reductase family 1, member C2. This gene encodes a member of the aldehyde / ketone reductase superfamily, which consists of more than 40 known enzymes and proteins. The differential expression of AKR1C2 in normal and tumor tissues is highly correlated with tumor characteristics and long-term survival of cancer patients. Its function of eliminating reactive oxygen species (ROS) further promotes chemotherapy resistance of tumor cells. Studies have shown that in the treatment of melanoma, BET inhibitors significantly enhance ferroptosis and improve immunotherapy response by targeting AKR1C2, revealing its key role in regulating the tumor microenvironment (Meng Y, Sun HY, He Y, et al. BET inhibitors potentiate melanoma ferroptosis and immunotherapy through AKR1C2 inhibition. Mil Med Res. 2023; 10(1): 61. Published 2023 Dec 4. doi: 10.1186 / s40779-023-00497-1). In gastric cancer, downregulated expression of AKR1C2 is not only closely related to immune response suppression, but has also been proven to be an independent prognostic biomarker (Liu W, Zhang F, Yang K, Yan Y. Comprehensive analysis regarding the prognostic significance of downregulated ferroptosis-related gene AKR1C2 in gastric cancer and its underlying roles in immune response. PLoS One. 2023; 18(1): e0280989. Published 2023 Jan 26. doi: 10.1371 / journal.pone.0280989). In thyroid cancer, AKR1C2 exhibits prognostic protective characteristics, and its high expression is significantly correlated with longer patient survival (JinYX, ZhouXF, ChenYY, et al. Up-Regulated AKR1C2 is correlated with favorable prognosis in thyroid carcinoma. J Cancer. 2019; 10(15): 3543-3552. Published 2019Jun 9. doi: 10.7150 / jca.28364).In contrast, this gene exhibits pro-metastasis properties in triple-negative breast cancer and reshapes the tumor molecular phenotype by regulating the androgen receptor signaling pathway (Li S, Lee W, Heo W, et al. AKR1C2 Promotes Metastasis and Regulates the Molecular Features of Luminal Androgen Receptor Subtype in Triple Negative Breast Cancer Cells. J Breast Cancer. 2023; 26(1): 60-76. doi: 10.4048 / jbc.2023.26.e1). Although AKR1C2 has shown significant clinical relevance in a variety of solid tumors, its research in esophageal cancer is still in its infancy and requires further exploration and research. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of AKR1C2 protein or its encoding gene or AKR1C2 gene promoter region methylation site in the diagnosis of esophageal squamous cell carcinoma, which has high specificity and sensitivity in the diagnosis of esophageal squamous cell carcinoma.

[0005] The present invention provides the use of a reagent for detecting a biomarker in one or more of the following:

[0006] (1) Prepare products for diagnosing esophageal squamous cell carcinoma;

[0007] (2) preparing products for the adjuvant treatment of esophageal squamous cell carcinoma;

[0008] The biomarkers include one or more of the AKR1C2 gene, the AKR1C2 protein, and the methylation site in the promoter region of the AKR1C2 gene;

[0009] The methylation site in the promoter region of the AKR1C2 gene includes CpG_1; the CpG_1 is located at 36 bp in the promoter region of the AKR1C2 gene.

[0010] Preferably, the methylation sites in the promoter region of the AKR1C2 gene further include one or more of CpG_2, CpG_3, CpG_4 and CpG_5.6;

[0011] The CpG_2 is located at 105 bp in the promoter region of the AKR1C2 gene;

[0012] The CpG_3 is located at 130 bp in the promoter region of the AKR1C2 gene;

[0013] The CpG_4 is located at 202 bp in the promoter region of the AKR1C2 gene;

[0014] The CpG_3 is located at 225bp and 234bp in the promoter region of the AKR1C2 gene.

[0015] Preferably, the nucleotide sequence of the methylation region where the methylation site of the AKR1C2 gene promoter region is located is shown in SEQ ID NO: 1; the CpG_1 is the first methylation site of the nucleotide sequence shown in SEQ ID NO: 1, located at 26 bp of the nucleotide sequence shown in SEQ ID NO: 1.

[0016] Preferably, the reagents for detecting biomarkers include primers and / or antibodies; the primers include upstream primers and downstream primers;

[0017] The upstream primer comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0018] The downstream primer includes the nucleotide sequence shown in SEQ ID NO: 3.

[0019] Preferably, the product comprises one or more of a kit, a gene chip and a device containing a detection module.

[0020] Preferably, the kit includes one or more of a gene detection kit, an immunohistochemistry detection kit and a DNA methylation detection kit.

[0021] Preferably, the expression level of the AKR1C2 gene and / or AKR1C2 protein is significantly upregulated in patients with esophageal squamous cell carcinoma.

[0022] Preferably, the methylation level of the methylation site in the promoter region of the AKR1C2 gene is significantly downregulated in patients with esophageal squamous cell carcinoma.

[0023] The present invention also provides a kit for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, comprising an upstream primer and a downstream primer;

[0024] The upstream primer comprises the nucleotide sequence shown in SEQ ID NO: 2;

[0025] The downstream primer includes the nucleotide sequence shown in SEQ ID NO: 3.

[0026] The present invention also provides a device for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, comprising a detection module and an evaluation module;

[0027] The detection module is used to detect one or more of the expression level of the AKR1C2 gene, the expression level of the AKR1C2 protein, and the methylation level of the methylation site in the promoter region of the AKR1C2 gene; the methylation site in the promoter region of the AKR1C2 gene includes CpG_1; the CpG_1 is located at 36bp in the promoter region of the AKR1C2 gene;

[0028] The evaluation module is used to determine whether the subject has esophageal squamous cell carcinoma based on one or more of the AKR1C2 gene expression level, the AKR1C2 protein expression level, and the methylation level of the AKR1C2 gene promoter region methylation site.

[0029] Beneficial effects:

[0030] The present invention provides the use of a reagent for detecting a biomarker for one or more of the following: (1) preparing a product for diagnosing esophageal squamous cell carcinoma; (2) preparing a product for adjuvant treatment of esophageal squamous cell carcinoma; the biomarker comprises one or more of the following: the AKR1C2 gene, the AKR1C2 protein, and a methylation site in the AKR1C2 gene promoter region; the methylation site in the AKR1C2 gene promoter region comprises CpG_1; the CpG_1 is located at 36 bp in the AKR1C2 gene promoter region. The present invention detects the expression levels of the methylation sites in the AKR1C2 gene promoter region in esophageal squamous cell carcinoma tissue and adjacent normal tissue, and performs statistical analysis. The results show that the average methylation level in esophageal squamous cell carcinoma tissue is lower than that in adjacent normal tissue, and the difference is statistically significant. The methylation status of the AKR1C2 gene promoter region is specific for esophageal squamous cell carcinoma tissue and has high sensitivity. The present invention uses immunohistochemistry to detect AKR1C2 protein expression in esophageal squamous cell carcinoma tissue and adjacent normal tissue. Statistical analysis of the immunohistochemical scores for AKR1C2 protein in these tissues revealed that AKR1C2 protein expression in esophageal squamous cell carcinoma tissue was significantly higher than in adjacent normal tissue. The difference was statistically significant, with strong specificity and sensitivity. Therefore, using one or more of the KR1C2 gene, AKR1C2 protein, and methylation sites in the AKR1C2 gene promoter region as new markers can be used for the diagnosis and adjuvant treatment of esophageal squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0032] Figure 1 Schematic diagram of the CpG sites in the primer amplification sequence of the AKR1C2 gene promoter region in Example 1;

[0033] Figure 2This is the electrophoresis diagram of the PCR amplification product of the AKR1C2 gene in Example 1;

[0034] Figure 3 This is a schematic diagram of the principle of methylation detection by MALDI-TOF MS in Example 1;

[0035] Figure 4 This is a comparison of the methylation levels of CpG differential sites in the promoter region of the AKR1C2 gene in esophageal squamous cell carcinoma and adjacent normal tissues in Example 1; where * indicates P < 0.05;

[0036] Figure 5 The sensitivity and specificity of the CpG unit methylation status of the AKR1C2 gene promoter region in diagnosing esophageal squamous cell carcinoma in Example 1;

[0037] Figure 6 The CpG unit methylation status of the promoter region of the AKR1C2 gene in esophageal squamous cell carcinoma tissue and the prognosis of the patient in Example 1;

[0038] Figure 7 This is the immunohistochemical staining of AKR1C2 protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue in Example 2;

[0039] Figure 8 This is the differential expression analysis of AKR1C2 protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue in Example 2; where **** indicates P < 0.0001;

[0040] Figure 9 Example 2: AKR1C2 protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue and patient prognosis;

[0041] Figure 10 This is Example 2: The sensitivity and specificity of AKR1C2 protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue for diagnosing esophageal squamous cell carcinoma. DETAILED DESCRIPTION

[0042] The present invention provides the use of a reagent for detecting a biomarker in one or more of the following:

[0043] (1) Prepare products for diagnosing esophageal squamous cell carcinoma;

[0044] (2) preparing products for the adjuvant treatment of esophageal squamous cell carcinoma;

[0045] The biomarkers include one or more of the AKR1C2 gene, the AKR1C2 protein, and the methylation site in the promoter region of the AKR1C2 gene;

[0046] The methylation site in the promoter region of the AKR1C2 gene includes CpG_1; the CpG_1 is located at 36 bp in the promoter region of the AKR1C2 gene.

[0047] As an embodiment, the methylation site in the AKR1C2 gene promoter region of the present invention further includes one or more of CpG_2, CpG_3, CpG_4 and CpG_5.6; the CpG_2 is located at 105bp in the AKR1C2 gene promoter region; the CpG_3 is located at 130bp in the AKR1C2 gene promoter region; the CpG_4 is located at 202bp in the AKR1C2 gene promoter region; the CpG_3 is located at 225bp and 234bp in the AKR1C2 gene promoter region.

[0048] As an embodiment, the nucleotide sequence of the methylation region where the methylation site in the promoter region of the AKR1C2 gene of the present invention is located is shown in SEQ ID NO: 1, specifically: 5'-tggagtttaagcccaagctggagtgCGatggtgcaatcccaactcactgcaacctctgcctcccaggttcaagctattt tcctggcttagcctcCGgagtagctggaattacagatgtgCGcccccatgaccagctaattttttctatttttagtagaaata gggtttcaccatgttagccaggctggtctCGaactcctgacctcaggtgatcCGcctgcctCGgctcccaaaatctctt gttatttaaagattatctctttgtctggcttttagagttgg-3'; the methylation region of the present invention is located in the AKR1C2 gene (Gene ID: 1646, updated on 18-Sep-2024) The methylated region at 2267bp to 2548bp upstream of the transcription start point was transcribed into an RNA fragment, which was cleaved by RNaseA to obtain 5 CpG units, specifically including CpG_1, CpG_2, CpG_3, CpG_4, and CpG_5.6, among which CpG_1 is the first methylation site appearing in the 5'-3' direction of the sequence shown in SEQ ID NO: 1, located at 26bp of the nucleotide sequence shown in SEQ ID NO: 1.

[0049] The present invention found, through the Mann-Whitney U test, that the average methylation levels of the five CpG units in esophageal squamous cell carcinoma tissue were significantly lower than those in adjacent normal tissue. In particular, there was a difference in the methylation levels of the single CpG unit CpG_1 between esophageal squamous cell carcinoma tissue and adjacent normal tissue. The methylation levels of the methylation sites in the promoter region of the AKR1C2 gene were significantly downregulated in esophageal squamous cell carcinoma patients. Kaplan-Meier survival analysis revealed that the expression levels of the AKR1C2 gene and / or AKR1C2 protein were significantly upregulated in esophageal squamous cell carcinoma patients. The present invention uses immunohistochemistry to detect AKR1C2 protein expression in esophageal squamous cell carcinoma tissue and adjacent normal tissue. Statistical analysis of the immunohistochemical scores for AKR1C2 protein in these tissues revealed that AKR1C2 protein expression was significantly higher in esophageal squamous cell carcinoma than in adjacent normal tissue, with the difference being statistically significant. The expression levels of the AKR1C2 gene and / or AKR1C2 protein were significantly upregulated in patients with esophageal squamous cell carcinoma. Therefore, one or more of the AKR1C2 gene, AKR1C2 protein, and methylation sites in the AKR1C2 gene promoter region can be used as detection targets for the diagnosis and adjuvant treatment of esophageal squamous cell carcinoma. In particular, the methylation frequency of the CpG_1 site can be used as a biomarker for screening and diagnosis of esophageal squamous cell carcinoma, with high specificity and sensitivity.

[0050] As an embodiment, the reagent for detecting biomarkers of the present invention includes primers and / or antibodies; the primers include upstream primers and downstream primers; the upstream primer includes the nucleotide sequence shown in SEQ ID NO: 2; the downstream primer includes the nucleotide sequence shown in SEQ ID NO: 3.

[0051] In one embodiment, the product of the present invention comprises one or more of a kit, a gene chip, and a device containing a detection module. In another embodiment, the kit of the present invention comprises one or more of a gene detection kit, an immunohistochemistry detection kit, and a DNA methylation detection kit. The present invention does not specifically limit the composition of the kit; any reagent capable of detecting AKR1C2 genes, AKR1C2 proteins, and methylation sites in the AKR1C2 gene promoter region may be used as a component of the kit.

[0052] The present invention also provides a kit for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, comprising an upstream primer and a downstream primer; the upstream primer comprises the nucleotide sequence shown in SEQ ID NO: 2; the downstream primer comprises the nucleotide sequence shown in SEQ ID NO: 3.

[0053] As an embodiment, the kit of the present invention further comprises PCR amplification reagents. As an embodiment, the PCR amplification reagents of the present invention include, but are not limited to, dNTPs, PCR buffer, and Tap polymerase. In the specific implementation process, other reagents can be conventionally selected according to actual needs.

[0054] The present invention also provides a device for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, comprising a detection module and an evaluation module;

[0055] The detection module is used to detect one or more of the expression level of the AKR1C2 gene, the expression level of the AKR1C2 protein, and the methylation level of the methylation site in the AKR1C2 gene promoter region; the methylation site in the AKR1C2 gene promoter region includes CpG_1; the CpG_1 is located at 36bp in the AKR1C2 gene promoter region, or the CpG_1 is the first methylation site of the nucleotide sequence shown in SEQ ID NO:1, and is located at 26bp in the nucleotide sequence shown in SEQ ID NO:1.

[0056] The evaluation module is used to determine whether the subject has esophageal squamous cell carcinoma based on one or more of the AKR1C2 gene expression level, the AKR1C2 protein expression level, and the methylation level of the AKR1C2 gene promoter region methylation site.

[0057] To further illustrate the present invention, the application of the AKR1C2 protein or its encoding gene or the methylation site of the AKR1C2 gene promoter region in the diagnosis of esophageal squamous cell carcinoma provided by the present invention is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0058] The reagents used in the examples of the present invention are shown in Table 1, the main experimental instruments and consumables are shown in Table 2, and the prepared reagents used are shown in Table 3.

[0059] Table 1 Reagents

[0060]

[0061]

[0062] Table 2 Experimental instruments and consumables

[0063] name company Origin optical microscope Olympus Japan 4℃ refrigerator Haier China -20℃ Refrigerator Haier China constant temperature box Beijing Yongguangming Medical Instrument Factory China Electric blast drying oven Beijing Yongguangming Medical Instrument Factory China Micropipette Eppendorf Germany Micro-wave oven Midea China 1.5mL EP tube Axygen USA Tip Axygen USA slides Jiangsu Shitai Experimental Equipment Co., Ltd. China Cover glass Jiangsu Shitai Experimental Equipment Co., Ltd. China Wet box Haimen Kangtai Experimental Equipment Factory China Staining rack Haimen Kangtai Experimental Equipment Factory China

[0064] Table 3 Reagent preparation

[0065]

[0066] Example 1

[0067] 1. Sample Collection

[0068] Tumor tissues from 50 patients with esophageal squamous cell carcinoma and their corresponding 50 adjacent normal tissues were collected. The samples were fixed in formalin and embedded in paraffin. The adjacent normal tissues were selected from the area more than 2 cm away from the esophageal squamous cell carcinoma tissues.

[0069] 2. DNA Preparation

[0070] The genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit, and the content and purity were determined by measuring the absorbance with an ultraviolet spectrophotometer. The quality of the extracted DNA was ensured by gel electrophoresis.

[0071] 3. Bisulfite treatment

[0072] The extracted DNA was modified with bisulfite using the EZ-96 DNA Methylation-Gold™ Kit. The modified DNA was used immediately or stored at -20°C for later use.

[0073] 4. PCR amplification reaction

[0074] (1) Design upstream primers and downstream primers for the amplified fragment of the AKR1C2 gene promoter region, and perform PCR amplification using the modified DNA from step 3 as a template; wherein, the schematic diagram of the CpG site of the amplified fragment of the AKR1C2 gene promoter region is as follows Figure 1 As shown, Figure 1 The sequence is a reverse sequence, and CpG sites are represented by dots; dark marks are detectable CpG sites, and light marks are undetectable CpG sites;

[0075] Upstream primer: 5'-aggaagagagTGGAGTTTAAGTTTAAGTTGGAGTG-3' (SEQ ID NO: 2); Downstream primer: 3'-cagtaatacgactcactatagggagaaggctCCAACTCTAAAAAC CAAACAAAAAA-5' (SEQ ID NO: 3);

[0076] PCR amplification system: 1.0 μL 10× PCR Buffer, 1.0 μL dNTPs, 0.2 μL PCR polymerase (5 U / μL), 0.2 μL 10 pmol / μL upstream primer, 0.2 μL 10 pmol / μL downstream primer, 1.0 μL template, and 6.4 μL ddH2O. The reaction system preparation process was completed on ice to prevent inactivation due to prolonged exposure to high temperature.

[0077] PCR amplification program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 45 cycles; extension at 72°C for 5 min; storage at 4°C.

[0078] (2) Take 3 μL of PCR product and mix it with 1 μL of 6× loading buffer. Then, perform electrophoresis on 1.5% agarose gel at 160V for 20 min and observe the results. Figure 2 As shown. Figure 2 It can be concluded that the results are good and subsequent experiments can be continued.

[0079] 5. Treatment of PCR amplification products with shrimp alkaline phosphatase (SAP):

[0080] (1) Add 7 μL of SAP reaction solution to each well of a 384-well reaction plate, carefully cover with a 384-well sealing film, and press each well firmly to prevent evaporation during the PCR process; the SAP reaction solution includes 1.70 μL of ddH2O, 0.30 μL of Shrimp alkalinephosphotase (SAP) Enzyme, and 5.00 μL of PCR amplification product;

[0081] (2) After mixing, centrifuge at 3000×g for 1 min. After centrifugation, proceed with the following procedures according to the following reaction conditions: incubate at 37°C for 20 min, incubate at 85°C for 5 min, and store at 4°C.

[0082] 6. T-cut / RNase A digestion reaction

[0083] (1) T transcription / RNaseA mixture: 0.89 μL 5× T7 Polymerase Buffer, 0.22 μL TCleavage mix, 0.22 μL 100 μmol / L DTT, 0.40 μL T7 RNA & DNA polymerase, 0.06 μL RNaseA, 2.0 μL shrimp alkaline phosphatase-treated PCR amplification product from step 5, and 3.21 μL ddH2O;

[0084] (2) Transfer 2 μL of PCR product and 5 μL of Transcription / RNase A mixture to a new 384-well plate;

[0085] (3) Cover with sealing film and centrifuge the microplate at 3000 g for 1 min;

[0086] (4) Incubate at 37°C for 3 h in a PCR instrument and store at 4°C.

[0087] (5) MALDI-TOF MS method was used for methylation mass spectrometry detection. The specific detection principle is as follows: Figure 3As shown, the results show that the AKR1C2 gene promoter contains 6 CpG sites, and after T cleavage, it becomes 5 CpG units, which actually contains 5 CpG units and 6 CpG sites. The total number of CpG units analyzed in the 50 samples in this example is 255.

[0088] 7. SPSS 26.0 software was used to perform the Mann-Whitney U test on the methylation level of the AKR1C2 gene in esophageal squamous cell carcinoma and adjacent normal tissues. The methylation differences of the five CpG units of the AKR1C2 gene were compared between the two groups of samples, and the CpG units with differential methylation were screened. The results showed that the methylation distribution of one CpG unit (CpG_1) of the AKR1C2 gene was different between the two groups (P<0.05), as shown in Table 4 and Figure 4 .

[0089] Table 8 Comparison of methylation rates of each CpG site of AKR1C2 in esophageal squamous cell carcinoma and adjacent normal tissues

[0090] CpGUnit Normal (N=23) ESCC (N=28) Z P CpG_1 0.19(0.00，0.29) 0.37(0.07，0.64) -2.263 0.024* CpG_2 0.87(0.00，1.00) 0.88(0.62，0.98) -0.112 0.911 CpG_3 0.97(0.84，1.00) 0.95(0.78，1.00) -0.608 0.543 CpG_4 0.98(0.89，1.00) 0.92(0.73，0.99) -1.220 0.223 CpG_5.6 0.92(0.80，0.96) 0.91(0.83，0.95) -0.502 0.615

[0091] Note: ESCC denotes esophageal squamous cell carcinoma; Normal denotes adjacent normal tissue. * indicates P < 0.05. Values are medians.

[0092] Receiver operating characteristic (ROC) curve analysis was performed on the methylation levels of each CpG site and the calculated area under the curve (AUC). The results showed that the AUC value of AKR1C2CpG_1 was 0.684 (P=0.025), and the sensitivity and specificity were 60.7% and 82.6%, respectively, with statistically significant differences ( Figure 5 ).

[0093] Based on the expression levels of CpG-1 detected in 50 esophageal squamous cell carcinoma tumor tissues and 50 corresponding adjacent normal tissues, the cut-off value was calculated using the ROC curve to be 0.305. Therefore, some esophageal squamous cell carcinoma patients were divided into a low methylation group (methylation rate ≤ 30.5%, n = 10) and a high methylation group (methylation rate ≥ 30.5%, n = 13). The Kaplan-Meier survival analysis was used to compare the relationship between the methylation level of AKR1C2 CpG_1 in the samples and the patient's diagnosis and prognosis. The results are as follows Figure 6 As shown. Figure 6As can be seen, the survival rate of the hypermethylated group was slightly higher than that of the hypomethylated group at certain time points, but this difference was not statistically significant (P = 0.27). The survival curves of the two groups were relatively close throughout the observation period, with no significant difference. This may mean that methylation level is not the main factor affecting the survival rate of these patients, or that a larger sample size is needed to detect potential differences.

[0094] Example 2

[0095] Expression of AKR1C2 protein in esophageal squamous cell carcinoma tissues and adjacent normal tissues.

[0096] 1. Sample Collection

[0097] A total of 233 cases of esophageal squamous cell carcinoma tissues and 184 cases of adjacent normal tissues were collected. The samples were fixed in formalin and embedded in paraffin to obtain tissue microarray wax blocks. The adjacent normal tissues were selected from the area more than 2 cm away from the esophageal squamous cell carcinoma tissues.

[0098] 2. Take the prepared tissue microarray wax block and make 4μm serial sections. Use the two-step immunohistochemistry Envision method for detection. The specific steps are as follows:

[0099] (1) Place the prepared 4 μm white slides into a slide box and store them in a 4°C refrigerator for subsequent experiments.

[0100] (2) Before the experiment, the white slices of the tissue chip stored in the 4°C refrigerator were placed in a metal basket and baked in a 60°C oven for 1.5 to 2 hours to prevent the tissue from falling off;

[0101] (3) Place the paraffin tissue sections baked in a 60°C oven in a xylene I solution, a xylene II solution, and a xylene III solution tank in sequence to perform tissue dewaxing. After dewaxing, place the sections in an absolute ethanol solution, a 90% by volume ethanol solution, an 80% by volume ethanol solution, and a 70% by volume ethanol solution in sequence to wash off the xylene on the paraffin tissue white sections. The sections are immersed in each solution tank for 5 minutes. Rinse with tap water three times and place in tap water to prevent drying.

[0102] (4) Take a repair box, pour 230mL of the prepared citric acid buffer solution (pH 6.0) into the repair box, heat the liquid to 100℃ in a microwave oven, heat for 3 minutes, place the slice below the boiling repair liquid level, place it in a pressure cooker, turn on the power and increase it to 1000W for heating. After the pressure cooker valve sprays air, adjust the power from 1000W to 800W and perform high-pressure repair for 8 minutes. After the time is up and the pressure cooker is vented, take out the repair box and cool it at room temperature until the temperature of the citric acid buffer solution drops to about 40℃. Then rinse it with clean water 3 times;

[0103] (5) Prepare 3 mL of 30% H2O2 solution and place the slices in it to block the endogenous peroxidase activity. Incubate at room temperature in the dark for 10 min and rinse with clean water three times.

[0104] (6) Place the slices in a washing tank filled with PBS and wash them on a horizontal shaker three times for 5 minutes each, replacing the PBS solution each time.

[0105] (7) Prepare 5% fetal bovine serum blocking solution with BSA powder in advance, remove the slices from the washing tank, place them on a wet box, absorb the excess PBS solution with absorbent paper, add about 200 μL of the prepared 5% fetal bovine serum blocking solution to each slice, and place the wet box horizontally in a 37°C constant temperature incubator for half an hour.

[0106] (8) Remove the wet box and place it at room temperature. Wipe off excess fetal bovine serum blocking solution from each slice. Add 200 μL of the pre-prepared primary antibody to each slice (PBS was used instead of primary antibody in the negative control group). Place the slice horizontally in the wet box and store in a refrigerator at 4°C overnight.

[0107] (9) The next day, remove the wet box containing the sections from the 4°C refrigerator and place it in an incubator to rewarm for 30 minutes;

[0108] (10) Repeat step (6);

[0109] (11) Wipe off excess PBS with absorbent paper, add 150 μL of secondary antibody to each section, gently smear the liquid surface with a toothpick to ensure that the secondary antibody evenly covers the tissue, and place in an incubator for half an hour;

[0110] (12) Repeat step (6);

[0111] (13) Prepare DAB colorimetric reagent (solution B:solution C = 1:100 (v / v)) in advance and store in a refrigerator at 4°C.

[0112] (14) Dry the slices and place them on a wet box. Wipe off excess PBS liquid. Add about 100 μL of DAB colorimetric reagent to each slice and observe the tissue coloring intensity and coloring location under a microscope. Control the coloring time according to the coloring intensity.

[0113] (15) After the optimal color development effect is achieved, place the mixture in room temperature water to terminate the color development and record the color development time;

[0114] (16) After all sections have developed color, place the sections in hematoxylin solution for counterstaining for 30 seconds, acidify in acid alcohol for 3–5 seconds, rinse five times, and return to blue in tap water for 5 minutes;

[0115] (17) Place the white slices in 70% ethanol solution, 80% ethanol solution, 90% ethanol solution, and anhydrous ethanol for 5 minutes (the purpose of this step is to dewax. If you want to dewax better, you can extend it to 10 minutes). Then put them in xylene I, xylene II, and xylene III in turn. Place the slices in each solution for 5 minutes (the purpose of this step is to remove alcohol) and dehydrate the slices until they are transparent.

[0116] (18) After the slices were dried, they were sealed with a neutral gum cover slip and observed under an Olympus optical microscope. Figure 7 Figures A (40×) and B (200×) show AKR1C2 protein in esophageal squamous cell carcinoma tissue, while images C (40×) and D (200×) show AKR1C2 in adjacent normal tissues. AKR1C2 protein is primarily expressed in the cytoplasm and nucleus of esophageal squamous cell carcinoma cells.

[0117] (19) Under an Olympus optical microscope with a magnification of 200 times, a double-scoring semi-quantitative method was used for scoring: <5% cells were positive for 0 points; 6% to 25% cells were positive for 1 point; 26% to 50% cells were positive for 2 points; 51% to 75% cells were positive for 3 points; >75% cells were positive for 4 points. The staining intensity was scored according to the presence and depth of cell coloration: no cell coloration for 0 points; light yellow for 1 point; brownish yellow for 2 points; and brownish brown for 3 points. Then, the percentage of positive cells and the staining intensity were multiplied and graded. When the score was 0 to 1, it was graded as (-); 2 to 4 points were (+); 5 to 8 points were (++); 9 to 12 points were (+++). Among them, - is negative; + is weakly positive; ++ / +++ is strongly positive. The range is 0 to 12. The results are as follows. Figure 8 As shown in the figure, the expression of AKR1C2 protein in esophageal squamous cell carcinoma was significantly higher than that in adjacent normal tissues, and the difference was statistically significant.

[0118] (20) ROC curve and Kaplan-Meier survival analysis were used to compare the relationship between AKR1C2 protein expression in samples and patient diagnosis and prognosis. The results are as follows: Figures 9-10 shown.

[0119] according to Figure 9 It can be seen that patients with high expression of AKR1C2 protein have a slightly worse prognosis, but the difference is not statistically significant. Figure 10 It can be seen that the sensitivity and specificity of AKR1C2 protein for diagnosing esophageal squamous cell carcinoma are 83.4% and 90.1%, and the AUC value is 0.923, indicating a good diagnostic effect.

[0120] Based on the above content, it can be seen that one or more of the KR1C2 gene, AKR1C2 protein and AKR1C2 gene promoter region methylation sites can be used for the diagnosis and adjuvant treatment of esophageal squamous cell carcinoma with high specificity and sensitivity.

[0121] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Use of reagents for detecting biomarkers in one or more of the following: (1) Prepare products for diagnosing esophageal squamous cell carcinoma; (2) preparing products for the adjuvant treatment of esophageal squamous cell carcinoma; The biomarkers include one or more of the AKR1C2 gene, the AKR1C2 protein, and the methylation site in the promoter region of the AKR1C2 gene; The methylation site in the promoter region of the AKR1C2 gene includes CpG_1; the CpG_1 is located at 36 bp in the promoter region of the AKR1C2 gene.

2. The use according to claim 1, characterized in that The methylation sites in the promoter region of the AKR1C2 gene further include one or more of CpG_2, CpG_3, CpG_4 and CpG_5.6; The CpG_2 is located at 105 bp in the promoter region of the AKR1C2 gene; The CpG_3 is located at 130 bp in the promoter region of the AKR1C2 gene; The CpG_4 is located at 202 bp in the promoter region of the AKR1C2 gene; The CpG_3 is located at 225bp and 234bp in the promoter region of the AKR1C2 gene.

3. The use according to claim 1, characterized in that The nucleotide sequence of the methylation region where the methylation site of the AKR1C2 gene promoter region is located is shown in SEQ ID NO: 1; the CpG_1 is the first methylation site of the nucleotide sequence shown in SEQ ID NO: 1, located at 26 bp of the nucleotide sequence shown in SEQ ID NO:

1.

4. The use according to claim 1, characterized in that The reagents for detecting biomarkers include primers and / or antibodies; the primers include upstream primers and downstream primers; The upstream primer comprises the nucleotide sequence shown in SEQ ID NO: 2; The downstream primer includes the nucleotide sequence shown in SEQ ID NO:

3.

5. The use according to claim 1, characterized in that The product includes one or more of a kit, a gene chip, and a device containing a detection module.

6. The use according to claim 5, characterized in that The kit includes one or more of a gene detection kit, an immunohistochemistry detection kit and a DNA methylation detection kit.

7. The use according to any one of claims 1 to 6, characterized in that The expression level of the AKR1C2 gene and / or AKR1C2 protein is significantly upregulated in patients with esophageal squamous cell carcinoma.

8. The use according to any one of claims 1 to 6, characterized in that: The methylation level of the methylation site in the promoter region of the AKR1C2 gene is significantly downregulated in patients with esophageal squamous cell carcinoma.

9. A kit for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, characterized in that: Includes upstream primers and downstream primers; The upstream primer comprises the nucleotide sequence shown in SEQ ID NO: 2; The downstream primer includes the nucleotide sequence shown in SEQ ID NO:

3.

10. A device for diagnosing and / or assisting in the treatment of esophageal squamous cell carcinoma, characterized in that: Includes detection module and evaluation module; The detection module is used to detect one or more of the expression level of the AKR1C2 gene, the expression level of the AKR1C2 protein, and the methylation level of the methylation site in the promoter region of the AKR1C2 gene; the methylation site in the promoter region of the AKR1C2 gene includes CpG_1; the CpG_1 is located at 36bp in the promoter region of the AKR1C2 gene; The evaluation module is used to determine whether the subject has esophageal squamous cell carcinoma based on one or more of the AKR1C2 gene expression level, the AKR1C2 protein expression level, and the methylation level of the AKR1C2 gene promoter region methylation site.

Images