Application of esophageal cancer molecular marker

Abstract

The invention discloses application of an esophageal cancer molecular marker. The esophageal cancer molecular marker comprises an IGFL1 gene and / or an expression product of the IGFL1 gene, and application of the IGFL1 gene and / or the expression product of the IGFL1 gene in preparation of an esophageal cancer diagnosis product. Experiments prove that the IGFL1 gene is an effective cancer suppressor gene of the esophageal squamous carcinoma, and the IGFL1 gene serving as a diagnosis biomarker of the esophageal squamous carcinoma is high in sensitivity and specificity and good in diagnosis efficiency. A rapid, accurate and non-invasive in-vitro detection mode or a new treatment target is provided for the esophageal squamous carcinoma, and the kit has huge clinical application value.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an application of an esophageal cancer molecular marker. Background Art

[0002] Esophageal cancer is one of the most aggressive tumors of the digestive tract, with a high incidence and low five-year survival rate. Esophageal cancer is considered the ninth most common cancer and the sixth most lethal cancer in the world. Esophageal squamous cell carcinoma is the most common subtype of esophageal cancer, accounting for 90% of esophageal cancer. Death in esophageal cancer patients is closely related to distant metastasis of cancer cells, so early diagnosis plays an important role in the patient's five-year survival rate. Simple and convenient screening methods can enable early diagnosis of esophageal cancer, which will greatly improve the patient's prognosis and reduce mortality.

[0003] The gold standard for diagnosing esophageal cancer in clinical practice is endoscopy, but it is not included in routine clinical screening tests due to its complex operation, invasiveness and poor patient compliance. Patients with esophageal squamous cell carcinoma have no obvious clinical symptoms in the early stages, and once discomfort occurs, the disease is often in the middle or late stages when diagnosed. This phenomenon has led to a significant increase in the mortality rate of esophageal cancer. Although endoscopy, as a major screening technology for esophageal cancer, can identify early esophageal squamous cell carcinoma, its invasiveness and serious side effects limit its widespread application.

[0004] Blood biomarkers mainly include DNA biomarkers, RNA biomarkers, protein biomarkers and metabolite biomarkers according to their sources. Circulating DNA in the blood mainly comes from programmed death and necrosis of tumor cells and surrounding normal cells. Its half-life is less than 2 hours, and its composition and content vary greatly. Circulating DNA usually accounts for only 0.01%-1% of DNA, which is lower than the detection range of conventional second-generation sequencing and qPCR. Therefore, it is not suitable for early diagnosis of cancer and is mostly used for monitoring postoperative recurrence. In addition, the cost of circulating DNA detection based on second-generation sequencing on the market is often tens of thousands, which is not conducive to promotion. Tumor circulating RNA mainly comes from active secretion of cells, mainly including mRNAs, miRNAs, piRNAs, snRNAs, snoRNAs, lncRNAs, lincRNAs, and circRNAs. Blood circulating RNA has low specificity and low signal-to-noise ratio; its composition is complex and forms a complex interaction network, but we know very little about it. Metabolite analysis in blood is based on mass spectrometry or nuclear magnetic resonance, and due to technical limitations, the sensitivity and specificity are not high. Moreover, metabolite analysis is easily affected by drugs and other diseases, and differences in clinical sampling operations and analysis methods can also affect experimental results. Proteins are relatively stable, and detection methods are mature. The ELISA method based on antigen-antibody reaction has good sensitivity, high specificity, and is simple to operate. It can be used for large-scale screening. Currently, most serological biomarkers used in clinical practice are proteins.

[0005] Serum protein markers have been widely used in clinical practice, but there are no reports on specific markers for esophageal squamous cell carcinoma. Although there are literature reports that some proteins such as SPP1, P53 antibody, CEA, CA19-9, SCC-Ag, midkine, VEGFR-3, etc. can be used as serological biomarkers for esophageal squamous cell carcinoma, these proteins lack sensitivity and specificity, and have also been reported to be associated with cancer in other types of cancer, so there is still a lack of specific serum protein markers for esophageal squamous cell carcinoma.

[0006] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0007] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of an esophageal cancer molecular marker, thereby providing a new molecular marker related to the diagnosis and prognosis of esophageal cancer and its application.

[0008] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0009] The first aspect of the present invention provides use of the IGFL1 gene and / or the expression product of the IGFL1 gene in preparing a product for diagnosing esophageal cancer.

[0010] Preferably, the sequence of the IGFL1 gene is the DNA sequence shown in SEQ ID NO.1.

[0011] Preferably, the expression product of the IGFL1 gene includes IGFL1 mRNA and / or IGFL1 protein.

[0012] Preferably, the esophageal cancer is esophageal squamous cell carcinoma.

[0013] Preferably, the product for esophageal cancer diagnosis includes an esophageal cancer detection-related RT-PCR kit or a real-time quantitative PCR kit or an in situ hybridization kit or an immunoassay kit or a gene chip or a protein chip or an application in a drug for treating esophageal cancer.

[0014] Preferably, the detection product detected by the RT-PCR kit or the real-time quantitative PCR kit or the in situ hybridization kit or the high-throughput sequencing platform is a product expressing the mRNA level of the IGFL1 gene.

[0015] Preferably, the in situ hybridization kit comprises a probe that hybridizes with the nucleic acid sequence of the IGFL1 gene; the gene chip comprises a probe that hybridizes with the nucleic acid sequence of the IGFL1 gene; and the protein chip comprises an antibody that specifically binds to the PARK2 protein.

[0016] Beneficial effects:

[0017] The present invention discloses an application of a molecular marker for esophageal cancer. The present invention statistically analyzes the IGFL1 gene expression in esophageal cancer tumor tissue and distal normal tissue, and finds that the IGFL1 expression in distal normal tissue is significantly higher than that in esophageal cancer tumor tissue. It is confirmed at the gene cellular level that IGFL1 can inhibit the occurrence and development of esophageal cancer, indicating that IGFL1 can be used as a target for clinical treatment of esophageal cancer. As a diagnostic biomarker for esophageal cancer, it has high sensitivity and specificity and good diagnostic efficiency. This provides a rapid, accurate, non-invasive in vitro detection method or a new therapeutic target for esophageal cancer, which has great clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a schematic diagram of the expression difference of IGFL1 in esophageal squamous cell carcinoma cells and normal esophageal epithelial cells detected by RT-qPCR experiment in Example 1 of the present invention.

[0019] Figure 2 This is a schematic diagram of the use of ELISA to verify the IGFL1 gene content level in the serum of esophageal squamous cell carcinoma patients and normal healthy people and the specificity and sensitivity for esophageal cancer diagnosis in Example 2 of the present invention. DETAILED DESCRIPTION

[0020] The present invention provides an application of esophageal cancer molecular markers. To make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail below. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0021] At present, there is still a lack of a rapid, accurate, non-invasive, and affordable in vitro detection method for esophageal squamous cell carcinoma. The current clinical application of esophageal cancer diagnosis technology is limited, mainly because: (1) tissue biopsy is highly invasive and not suitable for early cancer screening; (2) imaging detection technology (such as esophageal radiography and endoscopy) is limited in terms of equipment cost, operating technology, and invasiveness, and it is also difficult to promote it on a large scale as a cancer screening technology; (3) traditional serum protein markers (such as AFP, CEA, CA125, and CA199, etc.) have low sensitivity for esophageal cancer detection and cannot fully meet the requirements of esophageal cancer screening.

[0022] Insulin Growth Factor-Like Family Member 1 (IGFL1) is a member of the IGFL family discovered in 2006. It encodes a protein with a molecular weight of about 12KD and is located on chromosome 19q13.32 in the human body. Because of its specific expression pattern, mRNA is expressed to varying degrees in psoriasis and many cancers. The latest studies have shown that IGFL1 is closely related to a variety of solid tumors, including the regulation of tumor cell proliferation and apoptosis, malignant evolution and prognosis. Its high-affinity receptor IGFLR1 was found on the surface of mouse T cells, suggesting that the binding of IGFL1 to its receptor may affect related immune processes. However, there is currently no research on the expression of IGFL1 in esophageal cancer and its involvement in the pathogenesis of esophageal cancer.

[0023] Based on this, an embodiment of the present invention provides the use of the IGFL1 gene and / or the expression product of the IGFL1 gene in preparing a product for esophageal cancer diagnosis.

[0024] Application in the preparation of esophageal cancer diagnostic products: 1. For the development of Elisa kits, a large amount of patient serum can be collected for experimental verification to confirm the fluctuation range of IGFL1 content in esophageal squamous cell carcinoma patients, and compared with the IGFL1 content in the serum of healthy people, to determine the minimum detection limit and the upper limit of IGFL1 content that can distinguish esophageal squamous cell carcinoma patients from non-diseased people, thereby reducing diagnostic errors; ELISA kits can be used to monitor the fluctuation of IGFL1 protein content levels in patients, and then the effect of a certain treatment method used by the patient can be grasped. For example: During the treatment, the patient took anticancer drug A. In order to determine whether the drug is effective for the patient and whether the patient is resistant to the drug, blood is drawn from the patient regularly (determined according to the drug taking cycle), and serum is taken for ELISA experiments. The drug efficacy / patient recovery is determined by the concentration of IGFL1 protein in the serum; Early diagnosis of cancer: By collecting patient serum and using the ELISA kit to detect the IGFL1 protein content in the sample, it is possible to judge whether the patient has esophageal squamous cell carcinoma at an early stage. 2. Combined antibodies: The IGFL1 gene has differential expression in a variety of cancers. In order to improve the accuracy and specificity of esophageal squamous cell carcinoma diagnosis, it can also be tested in combination with other esophageal squamous cell carcinoma antibodies. That is, multiple target antibodies in the serum of the tested sample are verified by Elisa experiments to provide a better basis for diagnosis.

[0025] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and are only for illustrating the present invention but not limiting the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0026] Human esophageal cancer cell lines ECA109 and TE1, and human normal esophageal epithelial cells HEEC.

[0027] Example 1 The expression of IGFL1 gene in esophageal squamous cell carcinoma cells and esophageal normal epithelial cells was detected.

[0028] RT-qPCR

[0029] 1. TRIzol method to extract total cellular RNA

[0030] (1) Wash ECA109, TE1 and HEEC cells twice with PBS. Add 2 mL of TRIzol to a 6 cm culture dish, homogenize, and let stand at room temperature for 5 min.

[0031] (2) Add 200 μL of chloroform per mL of TRIzol, shake vigorously for 15-20 seconds, let stand at room temperature for 3 minutes, and centrifuge at 12,000 rpm at 4°C for 15 minutes;

[0032] (3) Carefully pipette the upper aqueous phase into a new 1.5 mL RNase-Free EP tube, add 500 μL of isopropanol per mL of TRIzol, gently invert up and down 6-8 times to mix, let stand at room temperature for 10 min, and centrifuge at 12,000 rpm at 4°C for 10 min;

[0033] (4) Discard the supernatant, add 1 mL of 75% ethanol prepared with DEPC water to wash the precipitate, and centrifuge at 7500 rpm at 4°C for 5 min;

[0034] (5) Gently aspirate the supernatant with a pipette and discard it. Let it stand at room temperature for 1-2 min to allow the ethanol to evaporate and dry. Add 40 μL RNase-Free Water and pipette to fully dissolve and mix the precipitate.

[0035] (6) Label the sample name, measure the RNA purity and concentration using a multifunctional microplate reader, and store at -80°C for future use.

[0036] 2. Reverse transcription of total cellular RNA

[0037] According to the instruction manual of SPARKscript II RT Plμs Kit (With gDNA Eraser), RNA was reverse transcribed and cDNA was synthesized. The reaction was carried out in two steps.

[0038] The first step is to remove the gDNA remaining in the total RNA extracted in the previous step. The reaction conditions are 42°C for 2 min. The reaction system is shown in Table 1.1 below.

[0039] Table 1.1 Reaction system for removing gDNA

[0040]

[0041] The second step is to reverse transcribe the total RNA, the reaction conditions are 50℃15min, 85℃5sec, the reaction system is shown in Table 1.2. The obtained reverse transcription product is labeled with the sample name and stored at -80℃ for future use.

[0042] Table 1.1 Reaction system for removing gDNA

[0043]

[0044] 3. RT-qPCR detection

[0045] Using the cDNA obtained in the previous step as a template, specific primers were used to perform RT-qPCR verification on IGFL1 and GAPDH, and ddH2O was used as a template as a blank control to exclude system contamination. Each sample was repeated three times, and the melting curve of the reaction product was analyzed. The reaction conditions are as follows: Stage 1: 94℃ pre-denaturation for 2min 30sec; Stage 2: 94℃10sec, 60℃30sec, a total of 40 cycles; Stage 3: melting curve 95℃15s, 60℃1min, 95℃15s, 60℃15s. The reaction system is shown in Table 1.3.

[0046] Table 1.3 RT-qPCR amplification system

[0047]

[0048] The 2-ΔΔCt method was used to analyze the relative expression of IGFL1 at the cellular mRNA level.

[0049] Schematic diagram of the difference in IGFL1 expression between esophageal cancer tumor tissue and distal normal tissue Figure 1 shown. Figure 1 The results presented in Figure 2 showed that the expression of IGFL1 in esophageal squamous cell carcinoma cells was significantly downregulated compared with that in normal esophageal epithelial cells (P<0.01), indicating that IGFL1 is lowly expressed in esophageal cancer cells.

[0050] Example 2 The IGFL1 gene content in the serum of patients with esophageal squamous cell carcinoma and normal healthy subjects was detected.

[0051] ELISA

[0052] Specimen collection and reagent preparation

[0053] (1) Serum samples should be collected using disposable pyrogen-free and endotoxin-free test tubes (EDTA, citrate, and heparin anticoagulants are all acceptable). Serum should avoid hemolysis. For hyperlipidemia specimens, suspended matter in the specimens should be removed by centrifugation to make the specimens clear and transparent. The specimens to be tested should be tested as soon as possible and stored at 2-8°C for 48 hours. For longer periods of time, they must be frozen (-20°C or -80°C) to avoid repeated freezing and thawing.

[0054] (2) Preparation of washing solution: dilute with distilled water 1:30 (e.g., add 29 mL of distilled water to 1 mL of concentrated washing solution);

[0055] (3) Preparation of standard: Take 8 1.5mL centrifuge tubes and label them S1, S2, S3, S4, S5, S6, S7, blank. Add 900μL of standard / sample diluent to the first tube S1, and add 200μL of standard / sample diluent to the second to eighth tubes respectively. Add 100μL of standard solution (400.0ng / mL) to the first tube and mix well on a vortex mixer. Use a pipette to aspirate 200μL and transfer to the second tube. Repeat this process to make double dilutions to S7. The eighth tube is a blank control. The concentrations of the configured standard curve are: 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, 0.625, 0ng / mL (the amount of standard and the range of the standard curve can also be configured according to your needs);

[0056] (4) Preparation of biotinylated antibody working solution: 20 minutes before use, dilute 100X biotinylated antibody into 1X working solution with biotinylated antibody diluent, prepare according to the required amount, use on the same day, and discard the remaining;

[0057] (5) Preparation of SABC working solution: 20 minutes before use, dilute 100X SABC into 1X working solution with SABC diluent, prepare according to the required amount, use it on the same day, and discard the remaining;

[0058] (6) Preparation of TMB colorimetric solution: 10 minutes before use, mix TMB colorimetric solution A and solution B by 1:1 and place in a dark place for later use.

[0059] Testing procedures

[0060] (1) Sample addition: Add 50 μL of standard / sample diluent to the blank well, and add 50 μL of standard or sample to be tested to each of the remaining wells. Mix the reaction plate and place it at 37°C for 50 minutes.

[0061] (2) Washing: Wash the reaction plate three times with 1X washing solution. Add 300 μL of 1X washing solution to each well, shake / diffuse for 1-2 minutes each time, and blot dry on filter paper.

[0062] (3) Add 100 μL of biotinylated antibody diluent to the blank well, and add 100 μL of 1X biotinylated antibody working solution to each of the remaining wells, mix well, and incubate at 37°C for 50 minutes;

[0063] (4) Washing: Wash the reaction plate three times with 1X washing solution. Add 300 μL of 1X washing solution to each well, shake / soak for 1-2 minutes each time, and blot dry on filter paper.

[0064] (5) Add 100 μL of SABC working solution to each well, mix well, and incubate at 37°C for 30 minutes;

[0065] (6) Washing: Wash the reaction plate three times with 1X washing solution. Add 300 μL of 1X washing solution to each well, shake / diffuse for 1-2 minutes each time, and blot dry on filter paper.

[0066] (7) Add 100 μL of the pre-prepared TMB mixture to each well, mix well, and incubate at 37°C in the dark for 20 minutes;

[0067] (8) Add 50 μL of stop solution to each well, mix well, and measure the absorbance at 450 nm using an ELISA reader within 30 minutes.

[0068] Result judgment and calculation

[0069] (1) It is recommended that all OD values ​​be subtracted from the blank well values ​​before calculation. If the blank well OD is lower than 0.1, it can also be calculated directly.

[0070] (2) Use the concentration of the standard as the horizontal axis and the OD value as the vertical axis to draw a standard curve manually or using software. Calculate the corresponding content based on the sample OD value and then multiply it by the dilution factor.

[0071] Schematic diagram of the IGFL1 gene content in the serum of patients with esophageal squamous cell carcinoma and normal healthy people and its specificity and sensitivity in diagnosing esophageal cancer Figure 2 shown.

[0072] Figure 2Results: Compared with the content in normal healthy human serum, the content of IGFL1 in the serum of patients with esophageal squamous cell carcinoma was lower (P<0.0001), and the levels of IGFL1 (I, II, III, IV) in the serum of patients with esophageal cancer at different TNM stages were significantly lower than those in the serum of normal healthy human serum (P<0.0001). IGFL1 has a high diagnostic performance, AUC=0.767, 95%CI=0.689-0.845, sensitivity 62.3%, specificity 86.3% (P<0.0001). This shows that IGFL1 can be used as a good serum biomarker for the diagnosis of esophageal cancer.

[0073] At present, the diagnosis of esophageal cancer generally adopts pathological histological examination and traditional medical imaging technology. Due to its own limitations, traditional imaging technology cannot provide sufficient clinical basis for the early diagnosis of esophageal cancer. Although pathological histological examination is the "gold standard" for the diagnosis of tumor diseases, it is more traumatic, requires more accurate tissue positioning, and requires the tumor to grow to a considerable number of cancer cells before it can be detected. Therefore, it is impossible to understand the dynamic changes of the condition of esophageal cancer patients in real time. There have been many research reports on serum protein markers, such as SPP1, P53 antibody, CEA, CA19-9, SCC-Ag, midkine, VEGFR-3, etc., which can be used as serum protein markers for esophageal squamous cell carcinoma, but these proteins lack sensitivity and specificity. There are also reports that they are associated with cancer in other types of cancer. There is still a lack of specific serum protein markers for esophageal squamous cell carcinoma, especially markers that can be used for early diagnosis.

[0074] The present invention statistically analyzed the IGFL1 gene expression in esophageal cancer tumor tissue and distal normal tissue, and found that the IGFL1 expression in distal normal tissue was significantly higher than that in esophageal cancer tumor tissue. It was confirmed at the gene cellular level that IGFL1 can inhibit the occurrence and development of esophageal cancer, suggesting that IGFL1 can be used as a target for clinical treatment of esophageal cancer.

[0075] The IGFL1 gene can also be overexpressed in cancer cells to simulate normal human esophageal cells. In vitro cell functional experiments have shown that overexpression of the IGFL1 gene can inhibit the proliferation and migration of esophageal cancer cells. At the molecular level of the gene, it has been confirmed that IGFL1 can inhibit the occurrence and development of esophageal cancer, suggesting that IGFL1 can be used as a target for clinical treatment of esophageal cancer.

[0076] It should be understood that the application of the present invention is not limited to the above examples. For ordinary technicians in this field, improvements or changes can be made based on the above description. All these improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. Use of the IGFL1 gene and / or the expression product of the IGFL1 gene in the preparation of a product for esophageal cancer diagnosis.

2. The use according to claim 1, characterized in that: The sequence of the IGFL1 gene is the DNA sequence shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that: The expression product of the IGFL1 gene includes IGFL1 mRNA and / or IGFL1 protein.

4. The use according to claim 1, characterized in that: The esophageal cancer is esophageal squamous cell carcinoma.

5. The use according to claim 1, characterized in that: The product for esophageal cancer diagnosis includes an esophageal cancer detection-related RT-PCR kit or a real-time quantitative PCR kit or an in situ hybridization kit or an immunoassay kit or a gene chip or a protein chip or an application in a drug for treating esophageal cancer.

6. The use according to claim 5, characterized in that: The detection product detected by the RT-PCR kit or the real-time quantitative PCR kit or the in situ hybridization kit or the high-throughput sequencing platform is a product expressing the IGFL1 gene mRNA level.

7. The use according to claim 5, characterized in that: The in situ hybridization kit comprises a probe hybridizing with the nucleic acid sequence of the IGFL1 gene; the gene chip comprises a probe hybridizing with the nucleic acid sequence of the IGFL1 gene; and the protein chip comprises an antibody specifically binding to the PARK2 protein.

Images