Esophageal squamous carcinoma marker circRNA and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to an esophageal squamous cell carcinoma marker circRNA and application thereof, the marker is hsacirc0001701, and the sequence of the hsacirc0001701 is shown as SEQ ID NO.1. The invention also relates to an esophageal squamous cell carcinoma marker circRNA and application of the esophageal squamous cell carcinoma marker circRNA. The invention further discloses application of the marker in preparation of products of esophageal squamous cell carcinoma diagnosis and / or prognosis markers. The invention provides a new field of view for finding biomarkers and drug treatment targets for clinical diagnosis and prognosis evaluation of esophageal squamous carcinoma, lays a foundation for research and development of related drugs, has important significance for research and treatment of esophageal squamous carcinoma, and thus has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to an esophageal squamous cell carcinoma marker circRNA and its application. Background Art

[0002] The statements in this part merely provide background technical information related to the present invention and do not necessarily constitute prior art.

[0003] Esophageal squamous cell carcinoma is a common digestive tract malignant tumor. For patients with early and middle-stage esophageal squamous cell carcinoma, surgical treatment is the preferred method. For early esophageal squamous cell carcinoma, endoscopic submucosal dissection is the first-line treatment option for patients with cancer tissue limited to the esophageal mucosal layer or submucosal layer. For patients with advanced esophageal squamous cell carcinoma, radical surgery or neoadjuvant combined with radical surgery is widely accepted. Patients with advanced esophageal squamous cell carcinoma lose the opportunity for surgery. Although treatment options such as chemotherapy and immunotherapy have extended the survival period of patients with advanced esophageal squamous cell carcinoma, their 5-year survival rate is only 26%.

[0004] Due to its special circular structure without a 5' cap and 3' poly(A) tail, circRNA has higher stability and a longer half-life compared to linear RNA. At the same time, circRNA has tissue specificity, with different contents in different tissues, and has the advantage of being easily detectable, and circRNA can be detected from body fluids such as blood, urine, and saliva. CircRNA has the advantages of being a diagnostic marker and a therapeutic target. Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide an esophageal squamous cell carcinoma marker circRNA.

[0006] Another purpose of the present invention provides the application of the esophageal squamous cell carcinoma marker circRNA.

[0007] The present invention is achieved by the following technical solutions:

[0008] The first aspect of the present invention provides an esophageal squamous cell carcinoma marker circRNA, and the marker is: hsa_circ_0001701. Among them, the sequence of hsa_circ_0001701 is shown as SEQ ID NO.1.

[0009] The second aspect of the present invention provides the application of hsa_circ_0001701 in the preparation of products for diagnosing and / or prognosticating esophageal squamous cell carcinoma markers. By detecting the up-regulation of the expression of hsa_circ_0001701 in tissues or blood, it is beneficial to evaluate the relationship between the expression level of the hsa_circ_0001701 marker and esophageal squamous cell carcinoma.

[0010] The above applications include, but are not limited to, any of the following methods:

[0011] (1) By detecting the expression level of hsa_circ_0001701 in the patient's test sample, to confirm whether the patient is an esophageal squamous cell carcinoma patient;

[0012] (2) By detecting the expression level of hsa_circ_0001701 in the test sample of esophageal squamous cell carcinoma patients, to evaluate the prognosis of the patients;

[0013] (3) The application of the detection reagent for hsa_circ_0001701 in the preparation of diagnostic and / or prognostic detection reagents for esophageal squamous cell carcinoma.

[0014] In the application of the first aspect above, the test sample of the diagnostic marker is the patient's clinical biological sample, including but not limited to one or several of serum (plasma), whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, etc.

[0015] In the third aspect of the present invention, a reagent combination for the diagnosis and / or prognostic detection of esophageal squamous cell carcinoma is provided, and the reagent combination is used to detect the expression level of hsa_circ_0001701 in a test sample.

[0016] Preferably, a diagnostic kit suitable for detecting hsa_circ_0001701 by the PCR method is provided.

[0017] Furthermore, the primer is the primer for the hsa_circ_0001701 to be detected; in specific examples, the sequences of the circRNA primer and the internal reference primer are as follows:

[0018]

[0019] Beneficial effects

[0020] The circRNA of the present invention can not only be used for rapid and effective early detection of esophageal squamous cell carcinoma, but also provides a therapeutic target and an important basis for clinical applications such as gene therapy and drug therapy.

[0021] The present invention provides a new perspective for finding biomarkers and drug therapy targets for the clinical diagnosis and prognosis evaluation of esophageal squamous cell carcinoma, lays a foundation for the research and development of related drugs, and has important significance for the research and treatment of esophageal squamous cell carcinoma, so it has good practical application value. Brief description of the drawings

[0022] Figure 1 For qRT-PCR verification of the expression levels of has_circ_0001701 in esophageal squamous cell carcinoma and adjacent normal epithelial tissues;

[0023] Figure 2 It is the clinical correlation of the expression level of has_circ_0001701;

[0024] Figure 3 It is to verify the expression level of has_circ_0001701 in esophageal squamous cell carcinoma cell lines by qRT-PCR;

[0025] Figure 4 It is to verify the overexpression and knockdown efficiency of has_circ_0001701 in TE-1 cell line;

[0026] Figure 5 It is to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells by EdU assay;

[0027] Figure 6 It is to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells by CCK8 assay;

[0028] Figure 7 It is to verify that has_circ_0001701 promotes the proliferation of esophageal squamous cell carcinoma cells by colony formation assay;

[0029] Figure 8 It is the structural diagram of has_circ_0001701. Specific embodiments

[0030] The technical solutions of the present invention will be further explained and illustrated below through specific embodiments. All experimental supplies used in the present invention are commercially available.

[0031] Example 1

[0032] (1) Analysis of differential expression and clinical correlation of circ_0001701 in esophageal cancer. Clinical samples (Qilu Hospital of Shandong University, all breast cancer and adjacent tissue samples were obtained during surgery. All participants provided written informed consent. This study was approved by the Ethics Committee of Qilu Hospital of Shandong University)

[0033] Extract RNA from 45 pairs of esophageal squamous cell carcinoma and adjacent normal epithelial tissues, and verify has_circ_1701 (SEQ ID NO.1) which was found to be significantly highly expressed in cancer tissues in the sequencing by qRT-PCR.

[0034] The operation process is as follows:

[0035] (1) RNA extraction

[0036] The tissue RNA extraction kit used in this experiment is produced by Takara Company, and RNA extraction is carried out according to the instructions. The operation process is as follows:

[0037] ① Place a tissue the size of a soybean into a mortar, grind the tissue under liquid nitrogen conditions to prevent the specimen from melting, collect the tissue powder and transfer it to an enzyme-free EP tube.

[0038] ② Add 50x DDT solution to the lysis buffer, then add 350 μL of lysis buffer to each enzyme-free EP tube, pipette up and down repeatedly to mix thoroughly, ensure complete lysis, and let it stand at room temperature for 2 min.

[0039] ③ Place the gDNA adsorption column on the collection tube, then transfer the lysate to the gDNA adsorption column, centrifuge at 4 °C and 12,000 rpm for 1 min, and collect the filtrate.

[0040] ④ Add 350 μL of 70% ethanol to the filtrate and mix thoroughly.

[0041] ⑤ Transfer the mixed solution to the RNA adsorption column, centrifuge at 4 °C and 12,000 rpm for 1 min, and discard the filtrate.

[0042] ⑥ Add 500 μL of Buffer RWA to the RNA adsorption column, centrifuge at 4 °C and 12,000 rpm for 30 s, and discard the filtrate.

[0043] ⑦ Add 600 μL of Buffer RWB to the RNA adsorption column, centrifuge at 4 °C and 12,000 rpm for 30 s, and discard the filtrate.

[0044] ⑧ Prepare the DNase Ⅰ reaction solution. Take 41 μL of RNase free dH2O, 5 μL of 10x DNase I Buffer and 4 μL of Recombinant DNase Ⅰ, mix thoroughly and then pipette the mixture onto the center of the RNA adsorption column, let it stand at room temperature for 15 min.

[0045] ⑨ Take 350 μL of Buffer RWB and add it to the center of the RNA adsorption column, centrifuge at 4 °C and 12,000 rpm for 30 s, and discard the filtrate.

[0046] ⑩ Repeat step ⑦.

[0047] Place the RNA adsorption column back into the collection tube, centrifuge at 4 °C and 12,000 rpm for 2 min.

[0048] Transfer the RNA adsorption column to a new 1.5 mL enzyme-free EP tube, pipette 50 μL of enzyme-free water onto the center of the RNA adsorption column, and let it stand at room temperature for 5 min.

[0049] Centrifuge at 4 °C and 12,000 rpm for 2 min to obtain RNA, which can be frozen or used for the next experiment.

[0050] (2) circRNA Reverse Transcription PCR

[0051] The circRNA reverse transcription kit used in this experiment was produced by Shanghai Yisheng Biotechnology Co., Ltd. After measuring the RNA concentration, reverse transcription of RNA was carried out according to the instructions. The operation process is as follows:

[0052] ① Remove gDNA. Prepare the following reaction solution in a 200 μL enzyme-free EP tube, mix well and incubate at 42 °C for 2 min.

[0053]

[0054] ② Prepare a 20 μL reverse transcription system.

[0055]

[0056] ③ After the reverse transcription system is prepared, mix well and carry out reverse transcription according to the following program.

[0057]

[0058]

[0059] (3) qRT-PCR

[0060] The reverse transcription kit used in this experiment was produced by Shanghai Yisheng Biotechnology Co., Ltd.

[0061] ① Prepare a 20 μL reaction system on ice. The specific components are as follows:

[0062]

[0063] ② Add the prepared reaction system to the eight-well strip, and set three replicate wells for each sample. Amplify according to the three-step method according to the instructions.

[0064]

[0065] ③ β-catin was used as an internal reference gene, and the relative expression level of the gene was calculated according to the 2 -ΔCT formula.

[0066] The sequences of the circRNA primers and the internal reference primers are as follows:

[0067]

[0068]

[0069] Figure 1Among them, blue represents the relative expression level of circRNA in normal esophageal epithelial tissues, and red represents the relative expression level of circRNA in esophageal squamous cell carcinoma samples. β-actin is used as an internal reference. Cancer: Esophageal squamous cell carcinoma samples; Normal: Normal esophageal epithelial tissues. The results suggest that the expression level of has_circ_0001701 in esophageal squamous cell carcinoma tissues is significantly higher than that in adjacent tissues( Figure 1 , P = 0.0011). P < 0.05 is considered to be statistically significant.

[0070] Example 2

[0071] Taking the median of the relative expression level of has_circ_0001701 in esophageal squamous cell carcinoma samples as the dividing line, the first 23 patients with relatively high expression levels were divided into the has_circ_0001701 high-expression group, and the remaining patients were the has_circ_0001701 low-expression group. Clinical data of the patients, including age, gender, and pathological results, were obtained from the hospital information management system.

[0072] Figure 2 The expression level of has_circ_0001701 in affects the prognosis of patients with esophageal squamous cell carcinoma. Low: has_circ_0001701 relatively low-expression group; High: has_circ_0001701 relatively high-expression group. Survival analysis shows that the survival period of patients in the has_circ_0001701 high-expression group is significantly lower than that in the has_circ_0001701 low-expression group.

[0073] Example 3

[0074] To further verify the expression levels of has_circ_0001701 in normal esophageal epithelial cells and esophageal squamous cell carcinoma cell lines, RNA of HET-1A, Eca-109, TE-1, KYSE-410, KYSE-150, and KYSE-30 cells was extracted respectively, and the relative expression level of has_circ_0001701 was detected by qRT-PCR after reverse transcription.

[0075] Figure 3 For the relative expression levels of has_circ_0001701 in normal esophageal epithelial cells and esophageal squamous cell carcinoma cell lines, the results show that the expression of has_circ_0001701 is significantly up-regulated in all detected esophageal squamous cell carcinoma cell lines. The TE-1 cell line with the most obvious up-regulation of expression was selected for subsequent experiments.

[0076] Example 4

[0077] To further verify the effect of the expression of has_circ_0001701 on the biological functions of esophageal squamous carcinoma cells, the overexpression plasmid of has_circ_0001701, the blank control plasmid (Vector), as well as siRNA and siRNA-NC were transfected into the TE-1 and KYSE-150 cell lines respectively.

[0078] Figure 4 In A. Verification of the overexpression efficiency of has_circ_0001701; B. Verification of the knockdown efficiency of has_circ_0001701. has_circ_0001701: the overexpression plasmid group of has_circ_0001701; Vector: the blank plasmid control group; has_circ_0001701 siRNA#1: the small interfering sequence 1 group; has_circ_0001701 siRNA#2: the small interfering sequence 2 group; siRNA-NC: the small interfering control group. The results showed that after transfection with the overexpression plasmid or recombinant lentivirus of has_circ_0001701, qRT-PCR showed that the relative expression level of has_circ_0001701 increased significantly; while after transfection with the shRNA recombinant lentivirus, qRT-PCR showed that the relative expression level of has_circ_0001701 decreased significantly, and the expression level of FNDC3B did not change significantly.

[0079] After transfection with the overexpression plasmid and siRNA of has_circ_0001701 in the TE-1 cell line, its effect on the proliferation of esophageal squamous carcinoma cells was verified by EdU, CCK8 and colony formation assays.

[0080] Example 5

[0081] EdU assay: EdU is a thymidine analogue that can be taken up by cells. During cell proliferation, it can replace normal thymidine and integrate into new DNA to form an EdU-DNA complex, which can be visualized under a fluorescence microscope after binding to a fluorescent dye, thereby realizing the detection of cell proliferation ability. In this study, an EdU kit produced by Shanghai Beyotime Biotechnology Co., Ltd. was used, and the experiment was carried out according to the instructions. The operation procedure is as follows:

[0082] (1) Reagent preparation

[0083]

[0084]

[0085] (2) Experimental procedure

[0086] ① Count the cells to be experimented and seed them at 2×10 5Inoculate cells at a density of cells per well into a 96-well plate. After shaking well, incubate overnight in an incubator.

[0087] ② EdU labeling. Dilute the EdU solution at a ratio of 1:1000 to 10 μM. According to the volume of 100 μL of medium per well, prepare an appropriate amount of EdU working solution and preheat the prepared working solution in a 37 °C water bath. Discard the old medium and add 100 μL of the prepared EdU working solution to each well. Incubate in an incubator for 2 h.

[0088] ③ After the EdU labeling is completed, discard the medium, rinse the cells once with an appropriate amount of PBS buffer, add 50 μL of 4% paraformaldehyde for tissues, and fix at room temperature for 15 min.

[0089] ④ Aspirate and discard the fixing solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times.

[0090] ⑤ Aspirate and discard the washing solution, add 100 μL of permeabilization solution to each well, and incubate at room temperature for 15 min. Remove the permeabilization solution, then add an appropriate amount of washing solution to rinse the cells for 3 min, and repeat 2 times.

[0091] ⑥ Add 50 μL of Click reaction solution to each well, shake well, and incubate in the dark at room temperature for 30 min.

[0092] ⑦ Aspirate and discard the Click reaction solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times.

[0093] ⑧ Aspirate and discard the washing solution, add 100 μL of Hoechst working solution to each well, shake well, and incubate in the dark at room temperature for 10 min.

[0094] ⑨ Aspirate and discard the Hoechst working solution, add an appropriate amount of washing solution to each well to rinse the cells for 3 min, and repeat 3 times. Then take pictures under a fluorescence microscope. Red fluorescence represents EdU-positive cells, and blue fluorescence represents DAPI-labeled cell nuclei. Randomly select 3 fields of view per well for observation and photography. EdU positive rate = (red fluorescence cells / blue fluorescence cells) x 100%.

[0095] Figure 5 Among them, the representative figure and result statistical chart of the EdU experiment, scale bar = 100 μm. has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA#1: small interfering sequence 1 group; has_circ_0001701 siRNA#2: small interfering sequence 2 group; siRNA-NC: small interfering control group. The EdU experiment shows that the has_circ_0001701 overexpression group has a higher proportion of cells in the proliferation phase, while knockdown reduces the proportion of cells in the proliferation phase.

[0096] Example 6

[0097] CCK8 assay: The CCK-8 assay is a commonly used method for detecting cell viability and proliferation ability. Its active ingredient is a tetrazolium salt that can be metabolized by living cells into yellow cresol salt. The metabolite is positively correlated with cell viability and quantity, and the viability and quantity of the measured cells can be evaluated by detecting the optical density of the metabolite. The experimental procedure is as follows:

[0098] (1) Count the cells to be experimented, inoculate them into a 96-well plate at a density of 2000 cells / well, make 5 replicate wells for each well, and make 4 replicate plates for each plate. Incubate overnight in a cell culture incubator.

[0099] (2) Prepare the CCK-8 solution.

[0100]

[0101] (3) Discard the culture medium, add 100 μL of 10% CCK-8 staining solution to each well, and incubate in the incubator in the dark for 2 h.

[0102] (4) After incubation, use a multi-functional microplate reader to detect the absorbance value (Optical Density, OD value) at 450 nm.

[0103] (5) Measure one replicate plate every 24 h and draw a curve.

[0104] Figure 6 The CCK8 assay result curve graph, has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA#1: small interfering sequence 1 group; has_circ_0001701 siRNA#2: small interfering sequence 2 group; siRNA-NC: small interfering control group. The curve drawn by the CCK8 assay shows that overexpression of has_circ_0001701 increases the cell growth rate and viability, and the results of the knockdown group are opposite.

[0105] Example 7

[0106] Colony formation assay:

[0107] (1) Count the cells to be experimented, inoculate them into a 6-well plate at a density of 1.5×10^3 cells / well, set 3 replicate wells for each group, shake well and culture in the incubator for 10 - 12 days. Observe the cell morphology during this period and change the medium as needed.

[0108] (2) After obvious cell clusters visible to the naked eye are formed, aspirate and discard the culture medium. After rinsing with PBS buffer, add 1 mL of tissue fixative to each well and fix at room temperature for 20 min.

[0109] (3) Aspirate and discard the tissue fixative. After rinsing with an appropriate amount of PBS buffer, add 1 mL of 0.1% crystal violet solution and stain at room temperature for 30 min.

[0110] (4) Recover the crystal violet, rinse 3 times with an appropriate amount of PBS buffer and 2 times with an appropriate amount of double-distilled water. Discard the liquid and let it dry naturally.

[0111] (5) Place it under a shadowless bottom lamp to take pictures and count the number of colonies formed.

[0112] Figure 7 Representative figure of colony formation experiment and result statistical chart, has_circ_0001701: has_circ_0001701 overexpression plasmid group; Vector: blank plasmid control group; has_circ_0001701 siRNA#1: small interfering sequence 1 group; has_circ_0001701 siRNA#2: small interfering sequence 2 group; siRNA-NC: small interfering control group. The colony formation experiment shows that overexpression of has_circ_0001701 results in the formation of more cell clone clusters, while the number of clone clusters decreases after knocking down has_circ_0001701.

[0113] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An esophageal squamous cell carcinoma marker circRNA, characterized in that, The biomarker is: hsa_circ_0001701, and the sequence of hsa_circ_0001701 is shown as SEQ ID NO.

1.

2. Application of hsa_circ_0001701 in the product for preparing a diagnostic and / or prognostic biomarker for esophageal squamous cell carcinoma.

3. A reagent combination for the diagnosis and / or prognosis detection of esophageal squamous cell carcinoma, characterized in that, The reagent combination is used to detect the expression level of hsa_circ_0001701 in a sample.

4. The reagent combination for esophageal squamous cell carcinoma diagnosis and / or prognosis detection according to claim 3, wherein The reagent combination is a diagnostic kit, and the primers of the diagnostic kit are:

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