Application of protein coded by circular RNA circVEGFC in esophageal squamous cell carcinoma diagnosis product

Abstract

The invention relates to an application of a protein coded by circular RNA circVEGFC in an esophageal squamous cell carcinoma diagnosis product. The invention provides a protein circVEGFC-182aa translated by a circular RNA (Ribonucleic Acid) molecule circVEGFC and application of the protein circVEGFC-182aa. It is found that the expression of a circular RNA molecule circVEGFC (namely hsacirc0004496) in esophageal squamous cell carcinoma tissue is remarkably increased, the circular RNA molecule circVEGFC participates in the malignant progression of esophageal squamous cell carcinoma, the circVEGFC can encode a protein containing 182 amino acids, namely circVEGFC-182aa, the protein is subjected to endogenous expression in esophageal squamous cell carcinoma, and the invasion and migration ability of esophageal squamous cell carcinoma is promoted; the result shows that the circVEGFC plays a biological function mainly through the specific protein circVEGFC-182aa coded by the circVEGFC, so that the circVEGFC-182aa can be used as a diagnosis marker of esophageal squamous cell carcinoma to assist diagnosis.

Description

Technical Field

[0001] The present invention belongs to the fields of molecular biotechnology, preventive medicine and clinical medicine technology, and specifically relates to the application of a protein encoded by circular RNA circVEGFC in a diagnostic product for esophageal squamous cell carcinoma. Background Art

[0002] Esophageal cancer is a common digestive tract malignancy, ranking eighth in the world, with approximately 403,000 new cases and 321,000 deaths each year. The incidence and mortality of esophageal squamous cell carcinoma in China are the highest in the world, and its pathological characteristics are different from those in Europe and the United States, mainly esophageal squamous cell carcinoma (ESCC).

[0003] Since esophageal squamous cell carcinoma has an insidious onset and mild early symptoms, many patients are already in the middle or late stages of the disease when they seek medical treatment, and distant metastasis has occurred; subsequent complications caused by tumor metastasis have become the main cause of death in patients with esophageal squamous cell carcinoma. Although there are some screening methods for esophageal squamous cell carcinoma, such as swallowing angiography and endoscopy, these methods are expensive and difficult to popularize, especially in some remote and rural areas, where the screening coverage is relatively low.

[0004] Currently, surgical treatment is still the preferred method for treating esophageal squamous cell carcinoma, but not all patients are suitable for surgical treatment. Some advanced patients may have distant metastasis or other diseases, which limits the range of treatment options. Targeted therapy (such as gefitinib and dovitinib) and immunotherapy (such as PD-1 / PD-L1 blockers) have achieved great success in treating a variety of malignant tumors including non-small cell lung cancer and melanoma, but only a small number of ESCC patients benefit from them. In addition, for high-risk, first-line advanced, recurrent esophageal squamous cell carcinoma patients, conventional treatment methods face a bottleneck effect. Therefore, in-depth exploration of the molecular mechanisms of the occurrence and development of ESCC and finding an effective early diagnosis and targeted treatment method will help doctors take more targeted treatment measures, which is crucial to improving patients' survival rates.

[0005] Circular RNA (circRNA) is a non-coding RNA that does not have a 5' end cap and a 3' end poly (A) tail, and forms a stable circular structure with covalent bonds. With the development of high-throughput sequencing technology and the deepening of the understanding of circRNA, circRNA is no longer considered to be a byproduct of shearing errors. A large number of circRNAs exist widely in a tissue-specific manner. Due to the special closed-loop structure of circRNA, it is not easily degraded by RNA exonucleases and has higher stability than linear RNA. Therefore, some circRNAs with high expression abundance in tumors are expected to become new clinical diagnostic and therapeutic targets.

[0006] Studies have shown that circRNA is closely involved in the occurrence and development of tumors, and plays an important role in the proliferation, apoptosis, invasion, and metastasis of tumor cells. Current studies have shown that circRNA can play a role by acting as a microRNA sponge (miRNA sponge), competing for endogenous RNA (ceRNA), and acting as a protein scaffold or sponge by binding to RNA-binding protein (RBP). In addition, recent studies have pointed out that some circRNAs containing N6-methyladenosine (m6A) sites or ribosome entry sites (IRES) can play their biological roles by translating functional proteins or proteins. Such circRNAs are expected to become new biomarkers and therapeutic targets. At present, there are still many circular RNAs related to esophageal squamous cell carcinoma that have not been discovered. Research and search for these circular RNAs have important research value and clinical value for the prevention, treatment, and diagnosis of esophageal squamous cell carcinoma. Summary of the invention

[0007] In view of the deficiencies in the prior art, the object of the present invention is to provide a protein circVEGFC-182aa encoded by a circular RNA molecule circVEGFC as an esophageal squamous cell carcinoma diagnostic marker in the preparation of esophageal squamous cell carcinoma auxiliary diagnosis or esophageal squamous cell carcinoma prognosis prediction products.

[0008] To achieve the above object, the present invention adopts the following technical solutions: The use of circular RNA circVEGFC and the circVEGFC-182aa protein encoded thereby as diagnostic markers for esophageal squamous cell carcinoma in the preparation of auxiliary diagnosis of esophageal squamous cell carcinoma or prognosis prediction products for esophageal squamous cell carcinoma, the nucleotide sequence of the circular RNA circVEGFC is shown in SEQ ID NO.1, and the amino acid sequence of the circVEGFC-182aa protein is shown in SEQ ID NO.2.

[0009] The present invention conducted high-throughput sequencing of esophageal squamous cell carcinoma and adjacent tissues of patients with esophageal squamous cell carcinoma and found that has_circ_0004496 (named as circVEGFC) was significantly expressed in esophageal squamous cell carcinoma tissues and was involved in the malignant progression of esophageal squamous cell carcinoma. It was further found that circVEGFC can encode a protein containing 182 amino acids: circVEGFC-182aa, and experiments have shown that the protein is endogenously expressed in esophageal squamous cell carcinoma and promotes the invasion and migration ability of esophageal squamous cell carcinoma. This shows that circVEGFC mainly exerts its biological function through the encoded specific protein circVEGFC-182aa, so circVEGFC-182aa can be used as a diagnostic marker for esophageal squamous cell carcinoma to assist in diagnosis.

[0010] Another object of the present invention is to provide a product for detecting the expression level of circVEGFC-182aa protein and its application in auxiliary diagnosis of esophageal squamous cell carcinoma or prognosis prediction of esophageal squamous cell carcinoma.

[0011] To achieve the above object, the present invention adopts the following technical solutions: The use of a reagent for detecting the expression level of circVEGFC-182aa protein in the preparation of an auxiliary diagnosis product for esophageal squamous cell carcinoma or a prognosis prediction product for esophageal squamous cell carcinoma.

[0012] Furthermore, the auxiliary diagnosis includes assisting in determining the malignancy and clinical stage of tumors in patients with esophageal squamous cell carcinoma; the prognosis prediction includes predicting the treatment effect and five-year overall survival of esophageal squamous cell carcinoma.

[0013] Furthermore, the reagent includes a monoclonal antibody, a polyclonal antibody or a nanobody for detecting the expression level of circVEGFC-182aa protein. Preferably, the amino acid sequence of the immunogen sequence used to prepare an antibody specific for circVEGFC-182aa is shown in SEQ ID NO.3.

[0014] Furthermore, the product is a kit.

[0015] The beneficial effects of the present invention are: The present invention provides a circular RNA molecule circVEGFC translation protein circVEGFC-182aa and a use thereof; the present invention finds that the expression of the circular RNA molecule circVEGFC (i.e., hsa_circ_0004496) is significantly increased in esophageal squamous cell carcinoma tissue and is involved in the malignant progression of esophageal squamous cell carcinoma; circVEGFC can encode a protein containing 182 amino acids: circVEGFC-182aa, which is endogenously expressed in esophageal squamous cell carcinoma and promotes the invasion and migration ability of esophageal squamous cell carcinoma; high expression of circVEGFC in esophageal squamous cell carcinoma tissue is positively correlated with the poor prognosis of esophageal squamous cell carcinoma and is significantly correlated with the shorter five-year overall survival of patients; circVEGFC-182aa can be used as a diagnostic marker for esophageal squamous cell carcinoma to assist in diagnosis.

[0016] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 Schematic diagram of high-throughput sequencing and biological analysis results, screening out circVEGFC that is highly expressed in esophageal squamous cell carcinoma and has translational potential; Figure 2 This is the tissue FISH result, showing that circVEGFC is highly expressed in esophageal squamous cell carcinoma tissue; Figure 3 Schematic diagram of qRT-PCR demonstrating increased expression of circVEGFC in esophageal squamous cell carcinoma cells; Figure 4 This is a schematic diagram of Kaplan-Meier survival curve analysis for patients with esophageal squamous cell carcinoma; Figure 5 The circular schematic diagram and identification diagram of circVEGFC, circVEGFC is formed by reverse splicing of exons 2-4; Figure 6 Schematic diagram of the verification of circRNA stability by actinomycin D and RNase digestion experiments; Figure 7 This is a schematic diagram of FISH results of esophageal squamous cell carcinoma cells; Figure 8 This is a schematic diagram of the results of the cytoplasm-nucleus separation experiment; Fig. 9 Schematic diagram of the Transwell experiment results of circVEGFC in vitro, verifying that circVEGFC can promote the invasion and migration of esophageal squamous cell carcinoma cells; Fig.10 Schematic diagram of the coding potential of circVEGFC; Fig.11 Schematic diagram of Western blot detection of circVEGFC-182aa in esophageal squamous cell carcinoma cell lines; Fig.12 Schematic diagram of mass spectrometry detection of the unique amino acid sequence of circVEGFC-182aa; Fig.13 The results of IRSE dual luciferase reporter gene assay indicate that circVEGFC drives translation initiation through IRES; Fig.14 A schematic diagram of the translation vector for constructing the mutated start codon ATG; Fig.15 Schematic diagram for in vitro verification that circVEGFC promotes the invasion and migration of esophageal squamous cell carcinoma by encoding circVEGFC-182aa; Fig.16 Schematic diagram of in vivo imaging to verify that circVEGFC promotes lung metastasis of esophageal squamous cell carcinoma by encoding circVEGFC-182aa in mice; Fig.17 Schematic diagram of HE staining of metastases in mice to verify that circVEGFC promotes lung metastasis of esophageal squamous cell carcinoma by encoding circVEGFC-182aa. DETAILED DESCRIPTION

[0019] In order to better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0020] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0021] 1.1 This study first performed high-throughput sequencing on 5 pairs of esophageal squamous cell carcinoma and adjacent normal tissues to detect the expression of circular RNA, and performed bioinformatics analysis using IRES finder and EMBOSS software. It was found that circVEGFC (hsa_circ_0004496) was upregulated in esophageal squamous cell carcinoma and had translation potential.

[0022] 1.2 FISH was used to detect the expression of circVEGFC in esophageal squamous cell carcinoma tissue microarray, and the correlation between circVEGFC and the clinical pathological characteristics of esophageal squamous cell carcinoma patients was analyzed.

[0023] Tissue FISH experimental steps and scoring criteria: a) Tissue dewaxing: Tissue sections were baked at 65°C for 2 hours, immersed in xylene I and II for 20 minutes, immersed in anhydrous ethanol I and II for 20 minutes, immersed in 95% ethanol for 10 minutes, immersed in 80% ethanol for 10 minutes, washed with water 3 times, and washed with PBS 3 times.

[0024] B) Prehybridization: Add 200 μl Blocking Solution to the slide and incubate in a humidified chamber at 37°C for 1 hour.

[0025] c) Hybridization: Add the prepared probes onto the glass slide and incubate in a humidified box at 37°C overnight.

[0026] D) The next day, add Cy5-Streptavidin Conjugate to label the has_circ_0004496 fluorescence signal on the slide and incubate at room temperature away from light for 1 hour.

[0027] e) Add 100 μl of DAPI dye and stain for 10 min in a dark place.

[0028] F) Seal the slides with mounting medium and observe under confocal microscope. The appearance of cy5 red fluorescence in the cytoplasm was defined as positive. Five fields of view were randomly selected from each tissue to evaluate the staining intensity of positive cells. The criteria were as follows: 0 points: most cells had no red fluorescence or only weak expression; 1 point: most cells had low expression or medium intensity expression in <50% of the cells; 2 points: most cells had medium or high intensity expression. Definition: ≤1 point was low expression, >1 point was high expression.

[0029] 3) qRT-PCR was used to detect circVEGFC in esophageal squamous cell carcinoma cell lines and normal esophageal epithelium. All esophageal squamous cell carcinoma cells were purchased from Wuhan Pronocell Biotechnology Co., Ltd.

[0030] 4) The expression level of circVEGFC and clinical pathological data of 114 patients with esophageal squamous cell carcinoma were analyzed, and it was found that circVEGFC was positively correlated with lymph node metastasis, clinical stage and shorter five-year overall survival.

[0031] Table 1 Correlation analysis between circVEGFC expression and clinicopathological features of 114 patients with esophageal squamous cell carcinoma

[0032] Figure 1-3 The results suggest that circVEGFC is highly expressed in esophageal squamous cell carcinoma. Figure 4 Table 1 showed that high expression of circVEGFC was positively correlated with patients' shorter five-year overall survival and lymph node metastasis.

[0033] This example confirmed that circVEGFC was upregulated in cancer tissues of patients with esophageal squamous cell carcinoma and was significantly associated with poor prognosis.

[0034] 2.1 To confirm the existence of circVEGFC, we used agarose gel electrophoresis in KYSE150 cells and Sanger sequencing to obtain the correct reverse splicing site, confirming that circVEGFC was reverse spliced ​​from exons 2 to 4. Actinomycin D assay and RNase digestion assay were used to confirm the stability of the closed ring structure of circVEGFC.

[0035] 2.2 Construction of circVEGFC probes and detection of subcellular localization of circVEGFC using fluorescence in situ hybridization. The cytoplasmic-nuclear separation kit was used to separate the components of esophageal squamous cell carcinoma cells, and cytoplasmic and nuclear RNA were extracted. The subcellular localization of circVEGFC was further verified by PCR detection.

[0036] Circularity verification and subcellular localization detection of circVEGFC Figure 4-7 shown.

[0037] in, Figure 5 The circular schematic diagram and identification diagram of circVEGFC. Agarose gel electrophoresis confirmed the circularity of circVEGFC, and Sanger sequencing confirmed that it was indeed formed by reverse splicing of exons 2-4.

[0038] Figure 6 This is a schematic diagram of the results of the actinomycin D experiment and the RNase enzyme digestion experiment. As shown in the figure, circVEGFC has a longer half-life than VEGFC mRNA and is more tolerant to RNase enzymes.

[0039] Figure 7Schematic diagram of FISH results of esophageal squamous cell carcinoma cells, demonstrating that circVEGFC is mainly localized in the cytoplasm.

[0040] Figure 8 This is a schematic diagram of the cytoplasmic-nuclear separation qRT-PCR results, which further confirmed the FISH results and proved that circVEGFC is mainly located in the cytoplasm.

[0041] This example confirms the circular nature of circVEGFC and its primary localization in the cytoplasm.

[0042] 3.1 circVEGFC overexpression plasmid and siRNA were constructed and transfected into KYSE30 cells with low circVEGFC expression and KYSE150 cells with high circVEGFC expression, respectively. The circVEGFC overexpression plasmid was purchased from Guangzhou Jisai Biotechnology Co., Ltd., siRNA was purchased from Guangzhou Ruibo Biotechnology Co., Ltd., and the transfection reagent used in the experiment was purchased from Promega.

[0043] 3.2 Transwell experiment verified the effect of circVEGFC on the invasion and migration ability of esophageal squamous cell carcinoma cells.

[0044] Transwell assay: Take 4×10 cells transfected in 3.1 4 The cells were resuspended in 200 μl of empty culture medium and inoculated into the upper chamber of a Transwell chamber with a pore size of 8.0 μm containing or without matrix gel. 600 μl of complete culture medium was added to the lower chamber, which was then incubated in a 37°C incubator for 48 hours. After being taken out, the cells in the upper chamber were wiped off and fixed, and the cells on the membrane of the lower chamber were stained with crystal violet, observed under a microscope, photographed, and the results analyzed.

[0045] The experimental results are as follows Fig. 9 Shown Fig. 9 The figure is a schematic diagram of the migration and invasion results of KYSE30 cells and KYSE150 cells, where the control group is the empty plasmid transfected group, the circVEGFC is the overexpression plasmid group, the si-NC is the small interfering RNA transfected control group, and the si-circVEGFC is the circVEGFC interference transfected group. The results showed that circVEGFC promoted the invasion and migration ability of esophageal squamous cell carcinoma cells.

[0046] This example demonstrates that circVEGFC promotes the invasion and migration of esophageal squamous cell carcinoma cells.

[0047] According to the circRNADb and Tanscirc databases, circVEGFC has an open reading frame (ORF) of 549 nt in length, encoding a protein with an amino acid sequence of 182aa, which we named circVEGFC-182aa. Amino acid comparison revealed that the amino acid sequence of the protein encoded by circVEGFC is mostly homologous to VEGFC, and only the 16 amino acids at the C-terminus are specific sequences. Therefore, we used commercial VEGFC antibodies for protein immunoblotting experiments to detect the synthesized new protein, and used mass spectrometry experiments to detect the unique amino acid sequence.

[0048] In addition to ORF sequences, circular RNA encoding proteins also needs to follow its unique translation initiation mechanism. It has been found that circRNA translation initiation mainly depends on IRES sites or m6A modification. The IRES sequence predicted by the database is inserted into the dual fluorescent reporter gene vector to detect its activity.

[0049] 4.1 Western blot: 4.1.1 After extracting total cell protein by RIPA method, the protein concentration of each group was detected by BCA method, and the loading amount of each group was ensured to be consistent. The protein was denatured at 100℃ before use, cooled to room temperature, and then the subsequent experiments were carried out; 4.1.2 Slowly add each group of protein samples and markers into the electrophoresis channel, and adjust the voltage to 80V for electrophoresis; after the marker enters the separation gel, adjust the voltage to 120V and stop when the bromophenol blue reaches the bottom; 4.1.3 Cut a PVDF membrane of appropriate size and activate it with methanol for 3 minutes before covering it on the gel. Be careful not to have any air pockets between the gel and the membrane. Then cover it with filter paper and sponge. After making a "sandwich" model, place it in the transfer tank and transfer the membrane at a constant current of 330 mA in an ice bath for 25 minutes. 4.1.4 After washing the transferred membrane with TBST, place it in 5% skim milk powder blocking solution and block it on a shaker for 1.5 hours at room temperature; 4.1.5 After washing again with TBST, place in the prepared primary antibody solution at 4°C overnight; 4.1.6 On the next day, remove the membrane from the primary antibody solution and wash it three times with TBST, then incubate it with the secondary antibody corresponding to the species of the primary antibody at room temperature on a shaker for 1.5 hours; 4.1.7 Take out the membrane again, wash it three times with TBST, develop it by ECL method, take pictures and perform grayscale analysis.

[0050] 4.2 Dual luciferase reporter gene experiment: 4.2.1 The cells transfected with each IRES dual luciferase reporter gene vector were plated into 96-well plates and the experiment was started after the cells adhered to the plate; 4.2.2 Detection of firefly cocoon luciferase activity: Use a multichannel pipette to add an equal volume (75 μl) of Dual-Glo° luciferase detection reagent to each well and mix well; 4.2.3 Wait for 20 minutes until the cells are fully lysed, and then detect the luminescent signal on a luminescence detector or a multi-function microplate reader with a luminescence module; 4.2.4 Detection of Renilla luciferase activity: Add the same volume of Dual-Glo° Stop&Glo° detection reagent as the initial culture medium (75 μl) to each well and mix well; 4.2.5 Wait for 20 minutes until the cells are fully lysed, and then detect the luminescent signal on a luminescence detector or a multi-function microplate reader with a luminescence module; 4.2.6 Calculate the ratio of reporter gene signals in each well and normalize the ratio of each experimental group using the ratio of the control group.

[0051] The results are as follows Figure 10-13 shown.

[0052] Fig.10 This is a schematic diagram of the coding potential of circVEGFC, with the ORF and IRES locations marked, the specific sequence of the translatable protein, the unique amino acid sequence, and the location of the amino acid sequence encoded by circVEGFC in the VEGFC antibody recognition region.

[0053] Fig.11 Schematic diagram of circVEGFC-182aa detection in four esophageal squamous cell carcinoma cell lines, demonstrating the endogenous existence of circVEGFC-182aa.

[0054] Fig.12 This is a schematic diagram of mass spectrometry detection. After Western blot electrophoresis, the gel strip was sent for mass spectrometry detection. The mass spectrometry showed that 8 peptides could be matched with circVEGFC-182aa. The figure lists the mass spectrometry secondary spectrum of the unique sequence.

[0055] Fig.13 The results of IRSE dual luciferase reporter gene assay indicate that circVEGFC drives translation initiation through IRES.

[0056] This example confirms that circVEGFC can indeed encode a protein with an amino acid sequence length of 182 amino acids, which is named circVEGFC-182aa.

[0057] 5.1 Construct an overexpression plasmid of circVEGFC with a mutated start codon ATG, and transfect it and the normal circVEGFC overexpression plasmid into KYSE150 cells.

[0058] 5.2 Transwell experiment verified the effect of circVEGFC-182aa on the invasion and migration ability of esophageal squamous cell carcinoma cells.

[0059] Transwell assay: Take 4×10 cells transfected in 5.1 4 Resuspend each cell in 200 μl of empty medium and inoculate it into the upper chamber of a Transwell chamber with a pore size of 8.0 μm containing or without matrix gel. Add 600 μl of complete medium to the lower chamber, and then incubate it in a 37°C incubator for 48 hours. After taking it out, wipe off the cells in the upper chamber and fix it. Use crystal violet to stain the cells on the membrane of the lower chamber. Observe under a microscope, take pictures, and analyze the results.

[0060] The results are as follows Figure 14-15 shown. Fig.14 Schematic diagram of the construction of circVEGFC overexpression vector and the circVEGFC overexpression vector with mutated translation start codon ATG. Fig.15 The figure is a schematic diagram of the migration and invasion results of KYSE150 cells after transfection with the above vectors, where the control group is the empty plasmid transfected group, the circVEGFC is the overexpression plasmid group, and the circVEGFC-mut-ATG group is the transfected mutated start codon group. The results showed that compared with the circVEGFC overexpression group, the invasion and migration ability of KYSE150 cells was significantly reduced after the start codon ATG mutation, suggesting that circVEGFC exerts biological functions through the specific protein circVEGFC-182aa it encodes.

[0061] This example confirms that circVEGFC promotes the invasion and migration of esophageal squamous cell carcinoma cells through the specific protein circVEGFC-182aa it encodes.

[0062] 6.1 Tail vein injection of esophageal squamous cell carcinoma cells to establish a lung metastasis model 6.1.1 Use 4-week-old male nude mice and prepare for inoculation after acclimating to the experimental environment for one week; 6.1.2 Stable cell lines established after transfection with empty vector, overexpression of circVEGFC, and overexpression of circVEGFC-mut-ATG were digested, washed, and resuspended in PBS to adjust the cell concentration to 5×10 4 pcs / 100μl; 6.1.3 Inject 100 μl of cell suspension into the tail vein of nude mice. Four weeks later, observe lung metastasis by small animal in vivo imaging; 6.1.4 After 8 weeks, the lung metastasis was recorded by in vivo imaging of small animals, and the animals were euthanized to obtain lung tissue samples; 6.1.5 The lung tissue samples were fixed with tissue fixative and made into wax blocks. After sectioning, they were stained with HE and photographed.

[0063] The above experimental results are as follows Figure 16-17 shown.

[0064] Fig.16 Schematic diagram and fluorescence intensity statistics of the lung metastasis model constructed by tail vein injection of esophageal squamous cell carcinoma cells. The in vivo imaging of mice showed that compared with the empty vector group, the lung fluorescence intensity of the circVEGFC overexpression group was stronger, but the fluorescence intensity was weakened after the start codon ATG mutation, suggesting that circVEGFC promotes esophageal squamous cell carcinoma lung metastasis in mice mainly through the protein circVEGFC-182aa it encodes.

[0065] Fig.17 HE staining images of lung tissues of mice in each group. Compared with the control group, the circVEGFC overexpression group had more lung metastatic nodules, while the lung metastatic nodules were reduced after the start codon ATG mutation.

[0066] In summary, the present invention provides a circular RNA molecule circVEGFC translation protein circVEGFC-182aa and its use; the present invention found that the expression of the circular RNA molecule circVEGFC (i.e., hsa_circ_0004496) was significantly increased in esophageal squamous cell carcinoma tissue and was involved in the malignant progression of esophageal squamous cell carcinoma. circVEGFC can encode a protein containing 182 amino acids: circVEGFC-182aa, which is endogenously expressed in esophageal squamous cell carcinoma and promotes the invasion and migration ability of esophageal squamous cell carcinoma; the above results indicate that circVEGFC mainly exerts its biological function through the protein circVEGFC-182aa it encodes. Therefore, circVEGFC-182aa can be used as a diagnostic marker for esophageal squamous cell carcinoma to assist in diagnosis.

[0067] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of circVEGFC-182aa protein encoded by circular RNA circVEGFC as a diagnostic marker for esophageal squamous cell carcinoma in the preparation of an auxiliary diagnosis product for esophageal squamous cell carcinoma or a prognosis prediction product for esophageal squamous cell carcinoma, characterized in that: The nucleotide sequence of the circular RNA circVEGFC is shown in SEQ ID NO.1, and the amino acid sequence of the circVEGFC-182aa protein is shown in SEQ ID NO.

2.

2. Use of a reagent for detecting the expression level of the circVEGFC-182aa protein described in claim 1 in the preparation of a product for auxiliary diagnosis of esophageal squamous cell carcinoma or prognosis prediction of esophageal squamous cell carcinoma.

3. The use according to claim 2, characterized in that The reagent is an antibody for detecting the expression level of circVEGFC-182aa protein, and the amino acid sequence of the immunogenic sequence of the antibody is shown in SEQ ID NO.

3.

4. The use according to claim 1 or 2, characterized in that: The product is a test kit.

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