Kit for diagnosis, treatment and / or prognosis evaluation of esophageal cancer

Abstract

The invention provides a kit for diagnosis, treatment and / or prognosis evaluation of esophageal cancer, and belongs to the technical field of biological medicine. The invention provides application of a reagent for detecting the expression quantity of an RHCG gene and / or an RHCG gene promoter region methylation region in preparation of products for esophageal cancer diagnosis, adjuvant therapy and / or prognosis evaluation. The RHCG gene and / or the RHCG gene promoter region methylation state has specificity to esophageal cancer tissues. Therefore, the methylation state of the RHCG gene and / or the RHCG gene promoter region can be selected as a detection marker to be used for diagnosis, auxiliary treatment and prognosis evaluation.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a kit for diagnosing, treating and / or evaluating the prognosis of esophageal cancer. Background Art

[0002] Esophageal cancer (EC) is the sixth leading cause of cancer-related death worldwide. Esophageal squamous cell carcinoma (ESCC), also known as esophageal squamous cell carcinoma, is the most common histological subtype of esophageal cancer, especially in East Asia, East and South Africa. Although the diagnosis and treatment of esophageal cancer have improved, 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. According to etiological and epidemiological studies, 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 of esophageal cancer.

[0003] The occurrence of esophageal cancer is caused by the combined effects of environmental and genetic factors. DNA methylation is the main epigenetic form of the human genome. In most types of cancer, DNA methylation is often abnormal. Methylation is a covalent modification of cytosine in genomic DNA, which mainly occurs in CpG dinucleotides in vertebrates and is often associated with long-term transcriptional repression. Existing technologies have found that in esophageal cancer, P16 and RASSF1A The promoter regions of genes are all in a hypermethylated state, and hypermethylation inhibits gene expression, thereby promoting the development of cancer. Studies have found that the methylation levels of miR-203 and miR-34a in esophageal squamous cell carcinoma tissues are significantly higher than those in adjacent normal tissues, and their hypermethylation and expression levels are significantly correlated. Analysis also found that their high methylation levels are significantly correlated with lymph node metastasis, indicating that their high methylation levels promote the development of esophageal squamous cell carcinoma. It was also found that low methylation in the promoter region of the PLCE1 gene regulates its high protein expression and promotes angiogenesis and proliferation of esophageal squamous cell carcinoma cells through the NFκB pathway and the expression of VEGF-C / Bcl-2. Therefore, exploring the methylation of genes in esophageal cancer is of great significance for the early diagnosis, accurate prognosis and personalized treatment of ESCC.

[0004] Rh type C glycoprotein (RHCG) is a member of the Rhesus (Rh) family and was originally identified as the Rh blood type antigen in human red blood cells. The Rh family is a transmembrane protein characterized by 12 transmembrane domains and intracytoplasmic N-termini and C-termini. Rh family proteins can be divided into two major categories based on their function: ammonia transporting Rh glycoproteins (RHAG, RHBG, and RHCG) and non-transporting Rh proteins (RHD and RHCE). As ammonia transporters, RHBG and RHCG are widely expressed in various tissues such as the kidney, liver, brain, and gastrointestinal tract, and play an important role in ammonia excretion and renal pH regulation. But about RHCG There are few studies on genes in esophageal cancer. Summary of the invention

[0005] The present invention provides a kit for diagnosing, treating and / or evaluating prognosis of esophageal cancer. RHCG Genes and / or RHCG The methylation region of the gene promoter region is a molecular marker, and the molecular marker can be used to perform specific detection of esophageal cancer simply and accurately.

[0006] The present invention provides a detection RHCG Genes and / or RHCG The use of a reagent for measuring the expression level of a methylation region in a gene promoter region in the preparation of products for diagnosis, adjuvant therapy and / or prognosis assessment of esophageal cancer.

[0007] In a preferred embodiment of the present invention, RHCG The nucleotide sequence of the methylated region where the methylated region of the gene promoter region is located is shown in SEQ ID No.1.

[0008] In a preferred embodiment of the present invention, the methylated region includes at least one of the following: a methylated region CpG_50.70.78 consisting of the 1st to 6th CpG regions, a methylated region CpG_70.78.88 consisting of the 2nd to 8th CpG regions, a methylated region CpG_78.88.104 consisting of the 5th to 9th CpG regions, a methylated region CpG_88.104.121 consisting of the 7th to 11th CpG regions, a methylated region CpG_90.70.78 consisting of the 2nd to 8 ... The methylated region CpG_104.121.127 composed of the domain, the methylated region CpG_121.127.150 composed of the 10th to 14th CpG region, the methylated region CpG_127.150.169 composed of the 12th to 19th CpG region, the methylated region CpG_150.169.198 composed of the 14th to 20th CpG region and the methylated region CpG_169.198.224 composed of the 15th to 23rd CpG region.

[0009] In a preferred embodiment of the present invention, the methylated region includes at least one of the following: a methylated region CpG_104.121.127 consisting of the 9th to 12th CpG regions and a methylated region CpG_121.127.150 consisting of the 10th to 14th CpG regions.

[0010] In a preferred embodiment of the present invention, the detection RHCG Reagents for measuring the methylation level of gene promoter methylation regions include the EZ-96 DNA Methylation-Gold™ kit.

[0011] In a preferred embodiment of the present invention, the detection RHCG Reagents for detecting gene expression levels include gene detection reagents or immunohistochemical reagents.

[0012] The present invention provides a method for detecting RHCG The primer pair for detecting the expression level of the methylation region in the promoter region of a gene comprises an upstream primer having a nucleotide sequence as shown in SEQ ID No.2 and a downstream primer as shown in SEQ ID No.3.

[0013] The present invention provides a non-disease diagnosis purpose detection RHCG The method for determining the methylation level of the methylated region of the gene promoter region comprises the following steps: mixing the above primer pair and the genomic DNA treated with bisulfite to prepare a PCR amplification system for PCR amplification to obtain a PCR amplification product; The PCR amplification product was sequentially subjected to shrimp alkaline phosphatase treatment, transcription and enzyme digestion reaction, purification and methylation mass spectrometry analysis to obtain the sample RHCG Methylation levels of all CpG sites in gene promoter regions.

[0014] The present invention provides a kit for diagnosing, treating and / or evaluating the prognosis of esophageal cancer, comprising the above primer pair and a PCR amplification reagent.

[0015] In a preferred embodiment of the present invention, the esophageal cancer includes esophageal squamous cell carcinoma.

[0016] Beneficial effect: The present invention detects esophageal cancer tissue and adjacent normal tissue RHCG The expression level of the methylation region in the gene promoter region was analyzed statistically, and the results showed that the average methylation level in esophageal cancer tissue was higher than that in adjacent normal tissue, and the difference was statistically significant. RHCG The methylation status of the gene promoter region is specific to esophageal squamous cell carcinoma tissue. RHCG The methylation region of the gene promoter region can be used as a new molecular marker for the diagnosis, adjuvant therapy and prognosis evaluation of esophageal cancer. At the same time, the present invention also uses immunohistochemistry to detect the expression of RHCG protein in esophageal cancer tissues and adjacent normal tissues, and statistically analyzes the immunohistochemical scores of RHCG protein in esophageal cancer tissues and adjacent normal tissues. It was found that the expression of RHCG protein in esophageal squamous cell carcinoma was significantly lower than that in adjacent normal tissues, and the difference was statistically significant; at the same time, Kaplan-Meier survival analysis was used to compare the relationship between the expression status of RHCG protein in esophageal squamous cell carcinoma and the prognosis of patients. The results showed that the overall survival rate of patients with low expression of RHCG protein was lower (P=0.02), and the difference was statistically significant. Receiver Operating Characteristic (ROC) curve analysis showed that the sensitivity and specificity of RHCG protein for the diagnosis of esophageal squamous cell carcinoma were 95.1% and 75.0%, and the AUC value was 0.86, indicating that the diagnostic effect was good. Therefore, the expression of RHCG protein in esophageal squamous cell carcinoma is a good method for the diagnosis of esophageal squamous cell carcinoma. RHCG Genes can be used as detection markers for diagnosis, adjuvant therapy and prognosis assessment. RHCG Genes and / or RHCG The use of gene promoter methylation regions as detection markers in the preparation of products for diagnosis, adjuvant therapy and / or prognosis assessment of esophageal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 For Example 1 RHCG Illustration of the CpG sites of the primer-amplified sequences in the promoter region of the gene; Figure 2 For Example 1 RHCG Electrophoresis of gene PCR amplification products; Figure 3 This is a schematic diagram of the principle of methylation detection by MALDI-TOF MS method in Example 1; Figure 4 In Example 1, esophageal squamous cell carcinoma and adjacent normal tissues RHCG Comparison results of methylation levels of differentially methylated regions (DMRs) in gene promoter regions; Figure 5 For Example 1 RHCG The sensitivity and specificity of DMR methylation status in the gene promoter region in diagnosing esophageal squamous cell carcinoma; Figure 6 The RHCG protein immunohistochemical staining diagram of esophageal squamous cell carcinoma tissue and adjacent normal tissue in Example 2; Figure 7 This is a diagram showing the results of differential expression analysis of RHCG protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue in Example 2; Figure 8 This is a graph showing the relationship between RHCG protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue and patient prognosis in Example 2; Fig. 9 The sensitivity and specificity results of RHCG protein in esophageal squamous cell carcinoma tissue and adjacent normal tissue in Example 2 for diagnosing esophageal squamous cell carcinoma are shown in FIG. DETAILED DESCRIPTION

[0018] The present invention provides a detection RHCG Genes and / or RHCG The use of a reagent for measuring the expression level of a methylation region in a gene promoter region in the preparation of products for diagnosis, adjuvant therapy and / or prognosis assessment of esophageal cancer.

[0019] The present invention RHCG The nucleotide sequence of the methylation region where the methylation site in the gene promoter region is located is shown in SEQ ID No. 1, specifically: 5'-gaggttggtgttccaggccatgctgcaggggtgcctggcCGggctggcagCGggCGgttCGgaCGctCGgaggcCGgCGgggcttttgagaccCGggcaggggggCGgggCGgccaCGtggggcaCGgagcagctgactCGggcacCGccCGCGCGctCGcccaactggcaggggcccaacccaagcCGagggccccaaaagattcccCGCGtCGggctgggggacagCGgcaggagcagaggctaaggcagtgggcagga-3'. The methylation region of the present invention is located at RHCGThe methylated region at 4800bp~5060bp upstream of the transcription start point of the gene (Gene ID: 51458) was transcribed into RNA fragments, and 13 CpG units were obtained after RNase A digestion, among which CpG_13 was not detected. Therefore, a total of 12 CpG units were obtained, including CpG_1, CpG_2.3.4, CpG_5.6, CpG_7.8, CpG_9, CpG_10.11, CpG_12, CpG_14, CpG_15.16.17.18.19, CpG_20, CpG_21.22.23 and CpG_24. The methylation region was based on three adjacent CpG sites as a window, and a total of 9 methylation regions (DMRs) were obtained, including: CpG_50.70.78, CpG_70.78.88, CpG_78.88.104, CpG_88.104.121, CpG_104.121.127, CpG_121.127.150, CpG_127.150.169, CpG_150.169.198 and CpG_169.198.224. In particular, CpG_104.121.127 and CpG_121.127.150 were used as markers with better detection effect.

[0020] Table 1 DMRs and their corresponding CpG sites

[0021] In the present invention, the Mann-Whitney U test was used to find that the average methylation levels of the 9 DMRs in esophageal squamous cell carcinoma tissues were higher than those in adjacent normal tissues, especially CpG_104.121.127 and CpG_121.127.150 had differences in methylation levels in esophageal squamous cell carcinoma tissues and adjacent normal tissues. Receiver operating characteristic (ROC) curve analysis was performed. RHCG The AUC value of CpG_104.121.127 and CpG_121.127.150 was 0.731, and the sensitivity and specificity were 95.5% and 46.2%, respectively, with statistically significant differences (P=0.024). RHCG The methylation region of the gene promoter region is used as a detection target for preparing reagents for diagnosis, adjuvant therapy and prognosis evaluation of esophageal cancer, especially the methylation frequency of CpG_104.121.127 and CpG_121.127.150 as a biomarker for screening and diagnosis of esophageal cancer.

[0022] The present invention uses immunohistochemistry to detect the expression of RHCG protein in esophageal cancer tissues and adjacent normal tissues, and statistically analyzes the immunohistochemical scores of RHCG protein in esophageal cancer tissues and adjacent normal tissues. It was found that the expression of RHCG protein in esophageal cancer was significantly lower than that in adjacent normal tissues, and the difference was statistically significant. At the same time, Kaplan-Meier survival analysis was used to compare the relationship between the expression status of RHCG protein in esophageal squamous cell carcinoma and the prognosis of patients. The results showed that the overall survival rate of patients with high expression of RHCG protein was lower (P=0.020), and the difference was statistically significant. Receiver operating characteristic (ROC) curve analysis showed that the sensitivity and specificity of RHCG protein for the diagnosis of esophageal squamous cell carcinoma were 95.1% and 75.0%, and the AUC value was 0.86, indicating that the diagnostic effect was good. Therefore, based on the expression of RHCG protein in esophageal cancer, RHCG Genes can be used as detection markers for diagnosis, adjuvant therapy and prognostic evaluation of esophageal cancer.

[0023] The product of the present invention includes a detection RHCG Reagents and / or detection of methylation levels in gene promoter methylation regions RHCG The reagents for detecting gene expression, including the reagents for detecting methylation levels, are not particularly limited in the art, such as the EZ-96 DNA Methylation-Gold™ kit used in the examples.

[0024] The detection method of the present invention RHCG The reagents for gene expression include gene detection reagents and / or immunohistochemical reagents. The present invention has no special limitation on the immunohistochemical reagents. RHCG Immunohistochemical reagents for detecting gene expression all belong to the protection scope of the present invention. The gene detection reagents of the present invention may include detection RHCG Primer pairs and PCR amplification reagents for methylation sites in gene promoter regions.

[0025] The present invention provides a method for detecting RHCG The primer pair for the expression level of the methylation region of the gene promoter region includes an upstream primer whose nucleotide sequence is shown as SEQ ID No.2 and a downstream primer shown as SEQ ID No.3.

[0026] SEQ ID No.2: 5'-aggaagagag GAGGTTTGGTGTTTTAGGTTATGTTG-3'; SEQ ID No. 3: 5'-CATCTCCAATTCCATCACCCATCCTtcggaagagggatatcactcagcataatgac-3'.

[0027] The present invention provides a non-disease diagnosis purpose detection RHCG The method for determining the methylation level of the methylated region of the gene promoter region comprises the following steps: mixing the above primer pair and the genomic DNA treated with bisulfite to prepare a PCR amplification system for PCR amplification to obtain a PCR amplification product; The PCR amplification product was sequentially subjected to shrimp alkaline phosphatase treatment, transcription and enzyme digestion reaction, purification and methylation mass spectrometry analysis to obtain the sample RHCG Methylation levels of all CpG sites in gene promoter regions.

[0028] Treating the DNA of the sample to be tested with bisulfite to obtain bisulfite-treated DNA; Performing PCR amplification on the bisulfite-treated DNA using a primer pair for detecting the methylation site in the promoter region of the RHCG gene to obtain a PCR amplification product; The PCR amplification product was sequentially subjected to shrimp alkaline phosphatase treatment, transcription and enzyme digestion reaction, resin purification and methylation mass spectrometry analysis to obtain the methylation level of all CpG sites in the promoter region of the RHCG gene of the sample.

[0029] The present invention extracts genomic DNA from tissue samples, such as esophageal squamous cell carcinoma tissue as a sample for DNA extraction in the embodiment, and genomic DNA is used as a template. The present invention does not specifically limit the method of DNA extraction, and conventional DNA extraction methods in the art can be used for extraction, such as using QIAamp DNA FFPE TissueKit to extract genomic DNA in the embodiment.

[0030] The present invention performs bisulfite treatment on the extracted genomic DNA, and the treatment method is not particularly limited, such as using a conventional bisulfite modification method, or using the method in the embodiment to perform bisulfite modification using EZ-96 DNAMethylation-GoldTM Kit. The DNA obtained by the present invention after the bisulfite treatment can be stored at -20°C if not used immediately.

[0031] The present invention uses the primer pair shown in SEQ ID No.2 and SEQ ID No.3 to amplify the target fragment from the DNA treated with bisulfite to obtain a PCR amplification product. The PCR amplification system of the present invention includes 10 μL of DNA treated with bisulfite, 0.2 μL of upstream primer with a concentration of 200 pmoL / μL, 0.2 μL of downstream primer with a concentration of 200 pmoL / μL, 0.2 μL of PCR Enzyme with a concentration of 0.4~1U / μL, 1.0 μL of dNTPs with a final reaction concentration of 200 μM, 1.0 μL of 10×PCR Buffer with a final reaction concentration, and ddH2O to make up the volume to 10 μL. The procedure of the PCR amplification reaction of the present invention includes: 94°C pre-denaturation for 4 minutes; 94°C denaturation for 20 seconds, 56°C annealing for 30 seconds, 72°C extension for 1 minute, 45 cycles; 72°C extension for 3 minutes.

[0032] After obtaining the PCR amplification product, the present invention sequentially performs shrimp alkaline phosphatase treatment, transcription and enzyme digestion reaction, purification and methylation mass spectrometry analysis on the PCR amplification product to obtain the sample RHCG The present invention has no special restrictions on the specific process and method of shrimp alkaline phosphatase treatment, transcription and enzyme digestion reaction, purification and methylation mass spectrometry analysis, and conventional analysis processes and methods in the art can be used, such as the purification method can be resin purification.

[0033] Obtain the sample RHCG After determining the methylation level of all CpG sites in the gene promoter region, the present invention further comprises the step of RHCG The methylation levels of all CpG sites in the gene promoter region were compared by Mann-Whitney U test. RHCG The methylation levels of CpG sites in the gene promoter region vary among different samples. RHCG When the methylation level of the CpG site in the gene promoter region is significantly lower than that of normal tissue, it indicates that the sample to be tested is an esophageal squamous cell carcinoma sample.

[0034] The present invention provides a kit for diagnosing, treating and / or evaluating the prognosis of esophageal cancer, comprising the above primer pair and a PCR amplification reagent.

[0035] In a preferred embodiment of the present invention, the esophageal cancer includes esophageal squamous cell carcinoma.

[0036] To further illustrate the present invention, a kit for diagnosing, treating and / or evaluating the prognosis of esophageal cancer provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0037] The raw materials and reagents used in the examples of the present invention can be commercially available products in the art unless otherwise specified: the paraffin tissue DNA extraction kit was purchased from QIAamp DNA FFPE Tissue Kit; the bisulfite and purification kit was purchased from EZ-96 DNA Methylation-Gold TM Kit).

[0038] The immunohistochemical method in the examples of the present invention refers to Wang Guanghui's 2013 Identification of MXRA5 as a novel tissue biomarker for colorectal cancer progression.

[0039] The graphing software used in the embodiments of the present invention is SPSS 26.0 and GraphPad prism 9.

[0040] The reagents used in the embodiments of the present invention are shown in Table 2, the main experimental instruments and consumables are shown in Table 3, and the prepared reagents used in the experiments are shown in Table 4.

[0041] Table 2 Experimental reagents

[0042] Table 3 Experimental instruments and consumables

[0043] Table 4 Reagent preparation

[0044] Example 1 Esophageal squamous cell carcinoma RHCG Gene methylation changes and their clinicopathological significance 1. DNA preparation: 50 tumor tissues and their corresponding adjacent normal tissues from patients with esophageal squamous cell carcinoma were selected, fixed with formalin, and embedded in paraffin. The adjacent normal tissues were all wax blocks of normal esophageal tissues and were selected from samples at a distance of more than 2 cm from the esophageal squamous cell carcinoma tissues. The genomic DNA was extracted using the QIAamp DNA FFPE Tissue Kit, and the absorbance was measured by UV spectrophotometer to determine its content and purity. Gel electrophoresis was used to ensure the quality of the extracted DNA.

[0045] 2. Bisulfite treatment: Use EZ-96 DNA Methylation-GoldTM Kit for bisulfite modification. Use the obtained DNA immediately or store it at -20℃ for future use.

[0046] 3. PCR amplification reaction: design RHCG The primers for amplifying the gene promoter region are SEQ ID No. 2 and SEQ ID No. 3. The amplified fragment is as follows Figure 1 As shown, it is 261 bp, of which, Figure 1 The sequence is a reverse sequence, and CpG sites are represented by dots; dark colors mark CpG sites that can be detected, and light colors mark CpG sites that cannot be detected due to sequence problems.

[0047] 10 μL PCR reaction system: ddH2O 6.4 μL, 10×PCR Buffer 1.0 μL, dNTPs 200 μM each 1.0 μL, PCR polymerase (5 U / μL) 0.2 μL, upstream primer (10 pmol / μL) 0.2 μL, downstream primer (10 pmol / μL) 0.2 μL and DNA template 1.0 μL; PCR reaction program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 45 cycles; final extension at 72°C for 5 min.

[0048] Steps: (1) Prepare the PCR reaction solution on ice according to the above reaction system to prevent inactivation due to long-term exposure to high temperature; (2) Seal the microplate with a plate sealing film, centrifuge the microplate at 3000 g for 1 minute, and incubate the samples in the microplate using a PCR instrument according to the above reaction conditions; (3) Mix 3 μL of PCR product with 1 μL of 6× loading buffer and perform electrophoresis on 1.5% agarose gel at 160V for 20 min to observe the results. Figure 2 As shown, the results are good and subsequent experiments can be continued.

[0049] 4. Shrimp alkaline phosphatase treatment PCR amplification product (SAP): 7μL reaction system: ddH2O 1.70μL, Shrimp alkaline phosphotase (SAP) Enzyme 0.30μL and PCR amplification product 5.00μL.

[0050] The reaction conditions were as follows: incubation at 37°C for 20 min, further incubation at 85°C for 5 min, and storage at 4°C.

[0051] Steps: (1) Add 7 μL of SAP reaction solution to each well of the 384-well reaction plate, carefully cover the well with a 384-well sealing film, and press each well firmly to prevent evaporation during the PCR process; (2) Mix the plate and centrifuge at 3000 g for 1 min. After centrifugation, proceed with the subsequent procedures according to the above reaction conditions.

[0052] 5. T-cut / RNase A digestion reaction: 7μL reaction system: ddH2O 3.21μL, 5×T7 Polymerase Buffer 0.89μL, T Cleavagemix 0.22μL, DTT (100μmol / L) 0.22μL, T7 RNA&DNA polymerase 0.40μL, RNase A 0.06μL, SAP and PCRMix 2.0μL.

[0053] Reaction conditions: incubate at 37°C for 3 h and store at 4°C.

[0054] Steps: (1) Prepare a mixed solution according to the above reaction system; (2) Take 2 μL of PCR product and 5 μL of T transcription / RNase A mixture to a new 384-well plate; (3) Cover the microplate with sealing film and centrifuge the microplate at 3000g for 1 minute; (4) Incubate the samples in the microplate in a PCR instrument according to the above reaction conditions.

[0055] The principle of MALDI-TOF MS methylation detection is as follows Figure 3 As shown, it was found through testing RHCG The gene promoter contains 24 CpG sites, and after T cleavage, there are 13 CpG units (of which CpG unit CpG_13 cannot be covered and cannot be analyzed), and actually contains 12 CpG units and 23 CpG sites. The total number of CpG units analyzed in 100 samples in this embodiment is 2300. The methylation region is a window with 3 adjacent CpG sites, and its corresponding relationship with the CpG site is shown in Table 1. The methylation level of the methylation region is the average of the methylation rates of the corresponding 3 CpG sites.

[0056] SPSS 26.0 software was used to analyze the esophageal squamous cell carcinoma and adjacent normal tissues. RHCG Mann-Whitney U test for gene methylation level comparison RHCG The methylation levels of 9 methylation regions of the gene in the two groups of samples were different, and differentially methylated regions (DMRs) were screened. Figure 4 As shown in Table 5, RHCG The methylation distribution of two DMRs (CpG_104.121.127 and CpG_121.127.150) of the gene was different between the two groups (P<0.05).

[0057] Table 5 Comparison of RHCG DMR methylation rates in esophageal squamous cell carcinoma and adjacent normal tissues

[0058] In Table 5, ESCC stands for esophageal squamous cell carcinoma, and Normal stands for normal tissue adjacent to the cancer. P <0.05,** indicates P <0.01, *** indicates P <0.001. The values ​​are expressed as medians (P25, P75). The methylation level of each CpG site and the calculated value of the area under the curve (AUC) were analyzed by receiver operating characteristic (ROC) curve. RHCG The AUC value of CpG_104.121.127 / CpG_121.127.150 was 0.731 (P=0.024), and the sensitivity and specificity were 95.5% and 46.2%, respectively, with statistically significant differences ( Figure 5 ). Screening the methylation frequency of CpG_104.121.127 / CpG_121.127.150 as a biomarker for screening and diagnosis of esophageal cancer survival prognosis.

[0059] Example 2 Esophageal squamous cell carcinoma tissue and adjacent normal tissue RHCG Protein expression Immunohistochemical analysis of 143 cases of esophageal squamous cell carcinoma and 105 cases of adjacent normal tissues RHCG Protein expression.

[0060] The prepared esophageal cancer tissue chip wax block was sliced ​​into 4 μm sections and the immunohistochemistry Envision two-step method was used. The specific steps are as follows: (1) Before the experiment, place the prepared 4 μm white slides into a slide box and store them in a 4°C refrigerator for subsequent experiments; (2) Before the experiment, insert the white slice of the tissue chip stored in a 4°C refrigerator into a metal basket and bake it in a 60°C oven for 1.5 to 2 hours to prevent the tissue from falling off; (3) Place the paraffin tissue sections baked in a 60°C oven in the xylene I solution, xylene II solution, and xylene III solution tanks in turn to perform tissue dewaxing. After dewaxing, place the sections in the anhydrous ethanol solution, 90% by volume ethanol solution, 80% by volume ethanol solution, and 70% by volume ethanol solution in turn 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 3 times and place in tap water to prevent the sections from drying out. (4) Take a repair box, pour 230 mL of the prepared citric acid buffer solution (pH 6.0) into the repair box, heat the liquid to 100°C in a microwave oven at high 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. When 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°C, and then rinse it with clean water 3 times; (5) Prepare 3 ml of H2O2 solution with 30% H2O2, place the slices in it to block endogenous peroxidase activity, incubate at room temperature in the dark for 10 min, and rinse with clean water three times; (6) Place the slices in a washing tank filled with PBS and wash on a horizontal shaker for three times, 5 minutes each time, replacing the PBS liquid each time; (7) Prepare 5% fetal bovine serum blocking solution with BSA powder in advance, take the slices out of 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.

[0061] (8) Take out the wet box and place it at room temperature. Wipe off the excess fetal bovine serum blocking solution from each slice. Add 200 μL of the pre-prepared primary antibody to each slice (PBS is used instead of primary antibody in the negative control group). Place the slice horizontally in the wet box and store it in a 4°C refrigerator overnight. (9) The next day, take the wet box containing the slices out of the 4°C refrigerator and place it in an incubator to warm up for 30 minutes. (10) Repeat step (6); (11) Wipe off excess PBS with absorbent paper, add 150 μL of secondary antibody to each slice, gently smear the liquid surface with a toothpick to ensure that the secondary antibody evenly covers the tissue, and place in the incubator for half an hour; (12) Repeat step (6); (13) Prepare DAB colorimetric reagent in advance, solution B: solution C = 1:100 (v / v), and place in a 4°C refrigerator for later use; (14) Dry the slices and place them on a wet box, wipe off the excess PBS liquid, add about 100 μL of DAB colorimetric reagent to each slice, observe the tissue coloring intensity and coloring location under a microscope, and control the coloring time according to the coloring intensity; (15) After reaching the optimal color development effect, place it in room temperature water to terminate the color development and record the color development time; (16) After all the sections have developed color, place the sections in hematoxylin solution for counterstaining for 30 seconds, acidify in acid alcohol for 3-5 seconds, rinse 5 times, and return to blue in tap water for 5 minutes; (17) Place the white slices in a 70% by volume ethanol solution, an 80% by volume ethanol solution, a 90% by volume ethanol solution, and an anhydrous ethanol tank for 5 minutes (the purpose of this step is to dewax. If you want to dewax better, you can extend the time to 10 minutes). Then, place them in xylene I, xylene II, and xylene III in turn. Place the slices in each solution tank for 5 minutes (the purpose of this step is to remove alcohol) and dehydrate the slices until they are transparent. After the slices were dried, they were sealed with a neutral gum coverslip and observed under a microscope.

[0062] Results analysis: Olympus optical microscope was used at 200 times magnification, and the double scoring semi-quantitative method was used for scoring: <5% positive cells were 0 points; 6%-25% positive cells were 1 point; 26%-50% positive cells were 2 points; 51%-75% positive cells were 3 points; >75% positive cells were 4 points. The staining intensity was scored according to the presence and depth of cell coloration: no cell coloration was 0 points; light yellow was 1 point; brownish yellow was 2 points; and brownish brown was 3 points. Then, the percentage of positive cells and the staining intensity were multiplied and graded. When the score was 0-1, it was graded as (-); 2-4 was (+); 5-8 was (++); 9-12 was (+++). Among them, - was negative; + was weakly positive; ++ / +++ was strongly positive. The range was 0~12.

[0063] The results are as follows Figure 6 As shown in the figure, A (40×) and B (200×) are RHCG staining in normal tissues adjacent to esophageal cancer, and C (40×) and D (200×) are RHCG staining in esophageal squamous cell carcinoma tissues. It can be seen that RHCG protein is mainly expressed in the cell membrane of esophageal squamous cell carcinoma cells.

[0064] The immunohistochemical scores of RHCG protein in esophageal squamous cell carcinoma tissues and adjacent normal tissues were statistically analyzed. Figure 7 As shown in the figure, the expression of RHCG protein in esophageal squamous cell carcinoma was significantly lower than that in adjacent normal tissues, and the difference was statistically significant.

[0065] ROC curve and Kaplan-Meier survival analysis were used to compare the relationship between RHCG protein expression in esophageal squamous cell carcinoma and the diagnosis and prognosis of ESCC. Figures 8 and 9 As shown in the figure, the sensitivity and specificity of RHCG protein for diagnosing esophageal squamous cell carcinoma were 95.1% and 75.0%, and the AUC value was 0.86, indicating that the diagnostic effect was good ( Figure 8 ), patients with low RHCG protein expression had a poor prognosis, and the difference was statistically significant ( Fig. 9 ).

[0066] In conclusion, the RHCG gene in esophageal cancer can be used as a detection marker for diagnosis, auxiliary treatment and prognosis evaluation of esophageal cancer.

[0067] Although the above embodiment describes the present invention in detail, 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 protection scope of the present invention.

Claims

1. A test RHCG Genes and / or RHCG The use of a reagent for measuring the expression level of a methylation region in a gene promoter region in the preparation of products for diagnosis, adjuvant therapy and / or prognosis assessment of esophageal cancer.

2. The application according to claim 1, characterized in that: Said RHCG The nucleotide sequence of the methylated region where the methylated region of the gene promoter region is located is shown in SEQ ID No.

1.

3. The application according to claim 2, characterized in that: The methylated region includes at least one of the following: a methylated region CpG_50.70.78 consisting of the 1st to 6th CpG regions, a methylated region CpG_70.78.88 consisting of the 2nd to 8th CpG regions, a methylated region CpG_ 78.88.104, the methylated region CpG_88.104.121 composed of the 7th to 11th CpG regions, the methylated region CpG_104.121.127 composed of the 9th to 12th CpG regions, the methylated region CpG_121.127.150 composed of the 10th to 14th CpG regions, the methylated region CpG_127.150.169 composed of the 12th to 19th CpG regions, the methylated region CpG_150.169.198 composed of the 14th to 20th CpG regions and the methylated region CpG_169.198.224 composed of the 15th to 23rd CpG regions.

4. The use according to claim 2 or 3, characterized in that: The methylated region includes at least one of the following: a methylated region CpG_104.121.127 consisting of the 9th to 12th CpG regions and a methylated region CpG_121.127.150 consisting of the 10th to 14th CpG regions.

5. The use according to claim 2 or 3, characterized in that: The detection RHCG Reagents for measuring the methylation level of gene promoter methylation regions include the EZ-96 DNA Methylation-Gold™ kit.

6. The use according to claim 1, characterized in that: The detection RHCG Reagents for detecting gene expression levels include gene detection reagents or immunohistochemical reagents.

7. A method for detecting RHCG A primer pair for detecting the expression level of a methylated region in a gene promoter region, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID No.2 and a downstream primer as shown in SEQ ID No.

3.

8. A test for non-disease diagnosis purposes RHCG The method for measuring the methylation level of a methylated region in a gene promoter region is characterized in that: The method comprises the following steps: mixing the primer pair according to claim 7 and the genomic DNA treated with bisulfite to prepare a PCR amplification system, performing PCR amplification, and obtaining a PCR amplification product; The PCR amplification products were sequentially subjected to shrimp alkaline phosphatase treatment, transcription and enzyme digestion reactions, purification and methylation mass spectrometry analysis to obtain the sample RHCG Methylation levels of all CpG sites in gene promoter regions.

9. A kit for diagnosis, treatment and / or prognosis assessment of esophageal cancer, characterized in that: It comprises the primer pair described in claim 7 and a PCR amplification reagent.

10. The kit according to claim 9, characterized in that: The esophageal cancer includes esophageal squamous cell carcinoma.

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