Application of circRNA (Ribonucleic Acid) coded protein as gastric cancer marker

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of circRNA (Ribonucleic Acid) coded protein as a gastric cancer marker. The circRNA disclosed by the invention is provided with an ORF (Open Reading Frame) capable of coding a microprotein circUBE2G1-99aa of 99 amino acids, and the amino acid sequence of the protein is shown as SEQ ID NO: 1. According to the present invention, the designed circUBE2G1-99aa targeting antibody is adopted to detect the expression level of circUBE2G1-99aa in the collected clinical sample, and the expression level of circUBE2G1-99aa in the stomach cancer patient tissue is down-regulated, and is negatively correlated with the large tumor, the lymph node metastasis and the advanced pathological stage of the stomach cancer patient.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to the application of a protein encoded by circRNA as a gastric cancer marker. Background Art

[0002] In recent years, the search for indicators that affect the occurrence, development and prognosis of gastric cancer at the epigenetic level and the development of targeted therapy have become a hot topic in gastric cancer treatment research. Further understanding of the molecular mechanism of gastric cancer pathogenesis and finding new therapeutic targets and prognostic biomarkers are the potential for realizing new treatment strategies in gastric cancer treatment, which has very important clinical significance and theoretical value for guiding treatment and improving prognosis of gastric cancer patients.

[0003] CircRNA is a type of non-coding RNA molecule that does not have a 5' end cap and a 3' end poly (A) tail, and forms a circular structure with a covalent bond. It has a stable structure, is tissue-specific, is not easily interfered by nucleases, and has a better half-life than linear RNA. Due to the lack of a 5' end cap structure, circRNA was initially believed to be unable to translate. According to current reports, circRNA can initiate translation by an intrinsic mechanism that is not cap-dependent. The translation of circRNA requires two basic elements, namely, a translation initiation element and an open reading frame. The translation initiation of traditional eukaryotic messenger RNA depends on the 5' end 7-methylguanine cap structure. Since circRNA is a covalent ring structure, it requires a special translation initiation element. At present, the translation modes of circRNA mainly include translation mediated by the ribosome entry site (Internal Ribosome Entry Site, IRES) and translation initiated by m6A modification, rolling circle amplification translation, and translation mediated by untranslated regions or other initiation elements.

[0004] With the development of high-throughput translation group sequencing technology, protein mass spectrometry technology and bioinformatics, some studies have shown that proteins encoded by circRNA can play an important regulatory role in the occurrence and development of tumors. Mechanistic studies have shown that the protein products encoded by circRNA can regulate the activity of signaling proteins (such as STAT3, β-catenin and mTOR, etc.), promote their activation or inhibition of downstream signaling pathways, and then affect tumor cell proliferation, apoptosis, invasion and metastasis, angiogenesis, metabolic reprogramming and immune escape and other biological functions, specifically regulate tumor occurrence, and are expected to become tumor-related diagnostic, prognostic and therapeutic targets. Summary of the invention

[0005] In view of the shortcomings of the existing problems, the purpose of the present invention is to provide a circRNA-encoded protein for use as a gastric cancer marker.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In the first aspect, the present invention protects the use of circUBE2G1 as a diagnostic or auxiliary diagnostic marker for gastric cancer. circUBE2G1 has an ORF that can encode a 99-amino acid microprotein circUBE2G1-99aa, and the amino acid sequence of the protein is: MKFITEIWHPNVDKNGDVCISILHEPGEDKYGYEKPEERWLPIHTVETIMISVISMLADPNG DSPANVDAAKEWREDRNGEFKRKVARCVRKSQETAFE, as shown in SEQ ID NO: 1.

[0008] In a second aspect, the present invention provides a reagent for detecting circUBE2G1 for use in preparing products for gastric cancer diagnosis or auxiliary diagnosis, screening or auxiliary screening.

[0009] In a specific embodiment, the product comprises a system for detecting circUBE2G1; preferably, the system comprises reagents and / or instruments for detecting circUBE2G1.

[0010] In a third aspect, the present invention protects a kit for diagnosing or assisting diagnosis, screening or assisting screening of gastric cancer, wherein the kit contains a reagent for detecting circUBE2G1.

[0011] In a specific embodiment, the reagent for detecting circUBE2G1 is an antibody, antibody fragment or a modified substance thereof for detecting circUBE2G1.

[0012] In a fourth aspect, the present invention also protects the use of a drug that promotes the expression of circUBE2G1 in the preparation of a drug for the treatment or adjuvant treatment of gastric cancer.

[0013] In a specific embodiment, the drug that promotes the expression of circUBE2G1 is an expression vector containing the circUBE2G1 coding sequence.

[0014] In a more specific embodiment, said vector is a CMV vector.

[0015] In a specific embodiment, the medicament further comprises a pharmaceutically acceptable carrier.

[0016] Beneficial Effects

[0017] The present invention uses the designed antibody targeting circUBE2G1-99aa to detect the expression level of circUBE2G1-99aa in the collected clinical samples, and combined with the analysis of pathological data, it is found that the expression level of circUBE2G1-99aa in the tissues of gastric cancer patients is downregulated and negatively correlated with larger tumors, lymph node metastasis and advanced pathological stage in gastric cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 circUBE2G1 is downregulated in gastric cancer. (A) Venn diagram analysis of circRNAs with coding potential in GEO dataset, RNC-seq data and TransCirc database (|log2(FC)|≥1, FDR<0.05). (B) qRT-PCR quantification results of circCFL1, circFAM114A2, circPCCA, circZFAND6, and circUBE2G1 in 28 gastric cancer tissues and adjacent normal tissues. (C) qRT-PCR detection of circUBE2G1 expression levels in various gastric cancer cell lines. (D) Correlation between circUBE2G1 expression levels and clinicopathological characteristics in 28 gastric cancer patients. (*P<0.05, **P<0.01).

[0019] Figure 2 circUBE2G1-99aa is endogenously present in gastric cancer cells and tissues. (A) Schematic diagram of ORF and IRES software prediction of circUBE2G1-99aa (left), and schematic diagram of the epitope recognition region (red) of the custom antibody (right). (B) Schematic diagram of the construction of the full-length IRES vector and the truncated vector in the dual luciferase experiment (left), and the detection of IRES activity in HEK-293FT (right). (C) Western blot detection of the protein expression level of circUBE2G1-99aa in gastric cancer cells. (D) Schematic diagram of the recognition sites of si-circUBE2G1 and si-linear UBE2G1. (E) qRT-PCR and Western blot verification of the independent coding of circUBE2G1-99aa. (F) Mass spectrometry identification of the endogenous presence of circUBE2G1-99aa in gastric cancer tissues and cells. (ns means no statistical significance, *P<0.05, ***P<0.001).

[0020] Figure 3circUBE2G1-99aa is downregulated in gastric cancer and is associated with the progression of gastric cancer. (A) Expression levels of circUBE2G1-99aa protein in 60 pairs of tumor tissues (T) and matched adjacent tissues (ANT) (12 pairs are shown). (B) ImageJ software was used to scan the protein grayscale value to quantify the protein expression level of circUBE2G1-99aa in gastric cancer tissues and adjacent tissue specimens. (C, D, E) Combined with the analysis of clinical pathological data, high expression of circUBE2G1-99aa was negatively correlated with larger tumor size, advanced tumor classification, and lymph node metastasis in gastric cancer patients. (F) ROC curves showed that circUBE2G1-99aa has the potential to be used as a diagnostic indicator for gastric cancer. (*P<0.05, **P<0.01).

[0021] Figure 4 In vitro and in vivo experiments showed that circUBE2G1-99aa inhibited the proliferation of gastric cancer cells. (A) Schematic diagram of the construction of Flag-circUBE2G1, circUBE2G1-ATG mut and Linear circUBE2G1-99aa vectors. (B) qRT-PCR and Western blot detected the successful construction of overexpression stable transgenic lines in gastric cancer cell lines HGC-27 and AGS. (CE) HGC-27 and AGS were divided into four groups: Empty vector, circUBE2G1-OE, circUBE2G1-ATG mut and Linear UBE2G1-99aa, and CCK8, colony formation and Edu staining (scale bar is 100μm) experiments were performed to detect the proliferation and metastasis ability of gastric cancer cells. (F) The above cell models were used to construct CDX models, and the tumor volume and tumor weight were detected to analyze the effect of circUBE2G1-99aa on the proliferation ability of gastric cancer cells. (ns means no statistical significance, **P<0.01, ***P<0.001). DETAILED DESCRIPTION

[0022] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0023] Example 1 circUBE2G1 is downregulated in gastric cancer

[0024] 1.1 Implementation Methods

[0025] (1) Extraction of total RNA, concentration determination and integrity testing

[0026] a. Extraction of RNA from gastric cancer tissue

[0027] The gastric cancer tissue samples were quickly ground into powder in liquid nitrogen to ensure that the tissue was fully broken to release intracellular RNA. 50 mg of the ground tissue powder was added to 1 mL of pre-cooled TRIzol and homogenized with a homogenizer. 200 μl of chloroform was added to the above homogenate, mixed and allowed to stand for 5 minutes, and centrifuged at 4°C, 12000 rpm for 10 minutes to separate the organic phase from the aqueous phase. After separation, 300 μl of supernatant was taken, transferred to a new centrifuge tube, 300 μl of isopropanol was added and mixed, and allowed to stand on ice for 10 minutes for RNA precipitation. After frozen incubation, centrifuged at 4°C, 12000 rpm for 10 minutes, the supernatant was discarded, and the RNA precipitate was left. After mixing with 75% ethanol and centrifuged at 4°C, 12000 rpm for 10 minutes, the RNA precipitate was washed to remove salts and other impurities, and after centrifugation and drying again, 20 μl of RNase-free water was added to dissolve the RNA.

[0028] b. Extraction of RNA from cell culture

[0029] First, remove the old culture medium from the cell culture plate, scrape the cells with a cell scraper, wash them twice with PBS, discard the supernatant, and add 11mL TRIzol. The subsequent process is the same as extracting RNA from tissues.

[0030] c. RNA integrity and concentration detection

[0031] Take 1 μg of RNA sample and mix it with RNA Loading Buffer, and perform electrophoresis on 1% agarose gel. Observe the 18S and 28S rRNA bands. Normally, they should show a bright and clear 2:1 ratio, indicating that the RNA integrity is good. Then use NanoDrop ultra-micro spectrophotometer to measure the RNA sample concentration. (A260 / A280 between 1.8 and 2.1 meets the experimental requirements).

[0032] (2) Reverse transcription experiment

[0033] Take out the required number of RNase-free reverse transcription reaction tubes and add 1 μg of total RNA to them. Add Oligo(dT), 5×RT Buffer, dNTP Mix, RT Enzyme Mix and gDNA Remover according to the ratio and concentration provided by the reverse transcription kit (Vazyme, R323-01) and mix evenly. After instant centrifugation, place the mixture on ice for 5 minutes. Incubate the reaction system at 42°C for 5 minutes for reverse transcription reaction, and then incubate at 85°C for 5 seconds to terminate the reaction. Finally, add the cDNA product obtained by reverse transcription to RNase-free water and dilute it 5 times, and store it in a -20°C or -80°C refrigerator for subsequent PCR amplification or other experiments. All operations are performed in an RNase-free working environment to ensure the integrity of RNA and the accuracy of experimental results.

[0034] (3) Real-time fluorescence quantitative PCR

[0035] Extract and quantify the total RNA of the sample to be tested, and perform reverse transcription to generate cDNA. Purchase the full-form gold fluorescent quantitative PCR Master Mix, which contains a thermostable DNA polymerase, an optimized buffer, dNTPs, and other necessary components. For each sample, add the components to the PCR reaction tube in the following order: SYBR Green I, PCR Master Mix, upstream and downstream primers (10μM), cDNA sample (1μg), and add RNase-free water to the specified total volume. Load the prepared PCR reaction system into the 96-well plate of the fluorescent quantitative PCR instrument, and set the corresponding cycle parameters, including: pre-denaturation at 94°C, usually for 30s, to fully denature the DNA template. Cycling stage: denaturation at 94°C, 5s; binding / annealing at 53°C, 15s; extension at 72°C, 10s. Start the PCR reaction, the fluorescent quantitative PCR instrument will collect the fluorescent signal after each cycle, and the software will record the Ct value (threshold cycle number). After the PCR reaction is completed, the relative quantitative data is calculated by the analysis software. The calculation of the relative expression of the gene adopts 2 -ΔΔCt The expression level of GAPDH was used as the internal reference. The primer sequences are as follows:

[0036]

[0037] 1.2 Implementation Results

[0038] In order to explore the differentially expressed circRNAs in gastric cancer, this study screened out candidate genes with significant differential expression between the cancer group and the normal group by analyzing the gastric cancer circRNA sequencing dataset in the GEO database (GSE89143 / GSE100170). Then, we performed ribosome-nascent peptide chain complex sequencing (RNC-seq) on the RNA of gastric cancer cells, and drew a Venn diagram by intersecting the above analysis results with the Transcirc database for predicting the translation potential of circRNAs. Finally, five circRNAs with translation potential and differential expression of more than 2.0 times were screened out, namely circCFL1, circFAM114A2, circPCCA, circZFAND6, circUBE2G1( Figure 1 A). To verify the above sequencing results, this study collected 28 gastric cancer tissues and 28 adjacent normal tissues. qRT-PCR experiments showed that the expression levels of the five circRNAs in gastric cancer tissues were all downregulated, which was consistent with the sequencing results. Among them, circUBE2G1 (circ_0003239) was the most significantly downregulated ( Figure 1 B). CircUBE2G1 was quantitatively analyzed by qRT-PCR in human gastric mucosal cell lines (GES-1) and gastric cancer cell lines (MKN-45, MKN-28, HGC-27, and AGS). The results showed that compared with GES-1 cells, circUBE2G1 was downregulated in gastric cancer cell lines ( Figure 1 C). To analyze the clinical correlation between the expression level of circUBE2G1 and gastric cancer patients, combined with the analysis of the clinical pathological data of patients, the expression level of circUBE2G1 was significantly downregulated in gastric cancer tissues (P=0.0013). Based on the above results, it was shown that circUBE2G1 was downregulated in gastric cancer tissues.

[0039] Example 2 circUBE2G1 encodes microprotein circUBE2G1-99aa

[0040] 2.1 Implementation Methods

[0041] (1) Protein extraction and concentration determination

[0042] 1) Extraction of total protein

[0043] Precool the centrifuge to 4°C in advance, aspirate the culture medium, wash once with precooled PBS buffer, and aspirate. Add 1mL PBS buffer, scrape the cells with a cell scraper, and transfer the cell suspension to a 1.5mL sterile EP tube. If there are cells remaining in the dish, add a small amount of PBS to rinse and transfer to the EP tube. Centrifuge at 4500rpm for 5min at 4°C and discard the supernatant. Add RIPA lysis buffer (prepare and use immediately, add 1μL protease inhibitor to 100μLRIPA lysis buffer) to the cell pellet, blow slowly and evenly, shake at 4°C for more than 1h to allow the lysis buffer and cells to fully contact. Centrifuge at 12000rpm for 15min at 4°C, collect the supernatant in a sterilized EP tube, which is the total protein extract.

[0044] 2) Protein concentration determination

[0045] Prepare BCA working solution at a ratio of 50:1 and vortex to mix. Add 200 μL BCA working solution to each well of a 96-well plate. Add 16 μL PBS to every 4 μL protein supernatant and dilute 5 times. Add 20 μL of the diluted protein sample to the BCA working solution and incubate at 37°C for 30 minutes. After cooling to room temperature, use an ELISA reader to measure the absorbance value of each well at a wavelength of 562 nm. Calculate the protein concentration based on the standard curve fitted by the software, which is the final concentration of the sample protein.

[0046] 3) Western blot

[0047] Prepare 12% SDS-PAGE gel using SDS-PAGE gel kit (Beyotime, P0012A). After instant centrifugation, protein samples and markers were loaded into the SDS-PAGE gel wells for constant voltage electrophoresis. During electrophoresis, the upper gel was charged at 80V, and the voltage was changed to 120V after bromophenol blue entered the lower gel. Electrophoresis was stopped near the bottom of the gel when bromophenol blue reached the bottom of the gel. Cut the PVDF membrane and soak it in methanol for 3s to activate it. Transfer the membrane at 4°C, 250mA constant current, and time for 60min. At room temperature, add 5% skimmed milk powder to the square fresh-keeping box to cover the membrane to block the PVDF membrane, and incubate it on a shaker for 2h while slowly shaking. After washing the membrane with TBST, incubate with the primary antibody and shake it overnight at 4°C. The next day, the membrane was washed three times with TBST (each time TBST covered the membrane, and the shaker was shaken at low speed for 10 minutes), then incubated with HRP-secondary antibody (1:5000) at room temperature for 2 hours, and then washed three times with TBST. Finally, the luminescent solution (Beyotime, P0018FM, A solution: B solution = 1:1) was prepared in a black square fresh-keeping box, and the PVDF membrane was immersed for a few seconds before exposure on the ECL luminometer and image collection.

[0048] (2) Dual luciferase reporter gene assay

[0049] This experiment uses the dual luciferase reporter gene detection kit of Yisheng Biotechnology to perform the following operations. The full-length, truncated and IRES-deficient plasmids were transfected into HEK-293FT cells, that is, the reporter gene and the internal reference pGL3-TK (10ng) were transferred into the cells. After 24 hours, 100μL of cell lysis solution was added and incubated on ice for 5 minutes to fully lyse. Then, fluorescence detection was performed, and 20μL of lysis solution was taken and added to the culture plate. Set up 5 wells for replication. Prepare 1X firefly luciferase reaction working solution and 1X sea renilla luciferase reaction solution. Add 100μL of firefly luciferase reaction solution, shake the plate to mix, put it into the microplate reader, and detect the activity of firefly luciferase at a wavelength of 480nm. The detection was completed within 30min. Add 100μL of sea renilla luciferase reaction solution, shake the plate to mix, and detect the activity of sea renilla luciferase at a wavelength of 480nm. The detection was completed within 30min. Calculation results: experimental group ratio = (experimental group F-background F) / (experimental group R-background R). Control group ratio = (control group F-background F) / (control group R-background R). Expression multiple = experimental group ratio / control group ratio.

[0050] 2.2 Implementation Results

[0051] The present invention first analyzed the RNA sequence of circUBE2G1. The prediction by CircPrimer2.0 tool and the analysis of CircBase database showed that circUBE2G1 had three ORFs, namely ORF1 (+65-+364, 300nt / 99aa), ORF2 (+96-+215, 120nt / 39aa), and ORF3 (+246-+335, 90nt / 29aa). Since the full-length sequence of ORF1 contains both ORF2 and ORF3, the present invention selected ORF1 to verify its coding potential, and the protein encoded by it was temporarily named circUBE2G1-99aa. It was predicted that there was an IRES structure between -381nt and +153nt, and the existence of this structure further provided the possibility for the protein translation of circUBE2G1 ( Figure 2 A left).

[0052] According to literature reports, the IRES element is one of the most important structures for circRNA to initiate protein translation. Therefore, the present invention speculates whether the IRES element of circUBE2G1 also has the function of initiating protein translation. In order to verify the ability of IRES to promote translation, the present invention first constructed a dual-luciferase reporter gene vector, amplified the full-length IRES sequence (IRS-WT) of circUBE2G1 and two IRES truncated sequences (IRES-Del-1, IRES-Del-2) by PCR, and inserted them into the pGL3-Basic vector respectively. Subsequently, the pGL3-Basic empty vector was transiently transfected into HEK-293FT cells respectively, and the fluorescence intensity of Fluc and Rluc was detected by an ELISA instrument. The activity of IRES was verified by calculating the ratio of Fluc / Rluc. The results showed that compared with the pGL3-Basic empty vector, the full-length IRES can effectively promote the expression of fluorescent protein, while the fluorescent protein translation ability of the truncated IRES was inhibited to a certain extent ( Figure 2 B).

[0053] Based on the above results, the present invention has proved that the IRES element of circUBE2G1 can initiate the translation of circUBE2G1, and will then further explore whether circUBE2G1-99aa exists endogenously in gastric cancer cells and tissues. Since the recognition epitope of the commercialized UBE2G1 antibody avoids the circUBE2G1-99aa protein sequence and cannot recognize the circUBE2G1-99aa protein, the present invention customized an antibody that can recognize the circUBE2G1-99aa (10kD) protein epitope (commissioned by Wuhan Abotek Biotechnology Co., Ltd. to prepare). Sequence alignment shows that circUBE2G1-99aa is completely homologous to the parent protein UBE2G1-170aa sequence, so the anti-circUBE2G1-99aa antibody can simultaneously identify circUBE2G1-99aa (10kD) and UBE2G1-170aa (20kD) ( Figure 2 A right). Western blot was used to detect the expression level of endogenous circUBE2G1-99aa in gastric cancer cell lines. The results showed that circUBE2G1-99aa was downregulated in gastric cancer cell lines ( Figure 2 C).

[0054] In order to ensure that the identified microprotein is indeed produced by direct translation of circUBE2G1 itself, rather than the result of different reading frames or different start codons of linear UBE2G1 generated by the UBE2G1 gene. The present invention designs siRNAs targeting the reverse splicing site region of circUBE2G1 and the non-homologous region of circUBE2G1 and linear UBE2G1 respectively. Figure 2 D), transfected gastric cancer cell line MKN-28, the results showed that after specific knockdown of circUBE2G1, the expression level of circUBE2G1-99aa was downregulated but did not affect the expression of UBE2G1-170aa protein; after specific knockdown of linearUBE2G1, its protein level was downregulated but did not affect the expression level of circUBE2G1-99aa microprotein encoded by circUBE2G1 ( Figure 2 E). In order to further clarify the endogenous expression of circUBE2G1-99aa in gastric cancer tissues and gastric cancer cells, the total protein of gastric cancer tissues and gastric cancer cells MKN-28 was detected by mass spectrometry. The protein lysates of gastric cancer tissues and MKN-28 cells were separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and the corresponding molecular weight bands (10kD) were cut for protein spectrum analysis. The peptide sequence of circUBE2G1-99aa was successfully identified, confirming the independent endogenous existence of circUBE2G1-99aa in gastric cancer tissues and cells ( Figure 2 F).

[0055] Example 3 circUBE2G1-99aa as a potential marker for diagnosis and prognosis of gastric cancer

[0056] To explore the expression of circUBE2G1-99aa in clinical samples of gastric cancer, this study first detected the expression level of circUBE2G1-99aa in another 60 pairs of gastric cancer tissue specimens (60 gastric cancer tissues and 60 adjacent tissues) by Western blot (12 pairs are shown) ( Figure 3 A), and then the grayscale value was scanned by ImageJ software to quantify the protein expression level of circUBE2G1-99aa in gastric cancer tissue and adjacent tissue specimens, and the clinical pathological data of gastric cancer patients were analyzed. The results showed that the expression of circUBE2G1 in gastric cancer tissue was significantly lower than that in paired adjacent tissues ( Figure 3 B) and was associated with tumor size in gastric cancer patients (P = 0.0302) ( Figure 3 C), lymph node metastasis (P = 0.0115) ( Figure 3 D) and pathological clinical stage (P=0.0203) ( Figure 3E) was negatively correlated. ROC curve analysis showed that circUBE2G1-99aa has the potential to be used as a diagnostic marker for gastric cancer ( Figure 3 F). The above results indicate that the micropeptide circUBE2G1-99aa encoded by circUBE2G1 is downregulated in gastric cancer and is closely related to the malignant progression of gastric cancer, and has the potential to serve as a potential indicator for the diagnosis and prognosis of gastric cancer.

[0057] Example 4. In vitro and in vivo experiments showed that circUBE2GQ-99aa inhibited the proliferation of gastric cancer cells

[0058] 4.1 Implementation Methods

[0059] (1) CCK-8

[0060] HGC-27 and AGS cells transfected for 24 hours were inoculated into 5 96-well plates (2000 / well), with 6 replicate wells for each sample. After inoculation, one plate was measured every day for 5 days. The cell growth status was detected with an inverted microscope, and the wells with good growth status, uniform cell distribution and density were photographed and recorded. Under the light-proof condition of the workbench, 10 μL of CCK-8 solution was added to each well. The operation should be quick to avoid long-term exposure of cells to the outdoors. Incubate for 2 hours at 37°C, 5% CO2, and 90% humidity. Measure the absorbance at 450nm with an enzyme reader. Use Excel and Graphpad Prism to process and analyze the results.

[0061] (2) Clone formation

[0062] After counting the HGC-27 and AGS cells transfected for 24 hours, inoculate them into six-well plates (1000 / well) and place them in a cell culture incubator for 9 to 14 days. Change the medium every 3 days and observe the cell status. When clone spots are formed, take pictures of the cells under a microscope. Discard the supernatant, wash once with 1×PBS, add 1mL of 4% paraformaldehyde to each well for 30 to 60 minutes, and wash once with PBS. Add 1mL of 0.1% crystal violet stain to each well and stain the cells for 10 to 20 minutes. Wash the cells carefully several times with PBS, dry them, and finally take pictures and count them using an inverted microscope.

[0063] (3) Edu cell proliferation assay

[0064] This experiment uses the Biyuntian EdU-555 cell proliferation detection kit for operation, and the steps are as follows. Plate an appropriate number of cells in a 24-well plate. After the cells are cultured overnight and return to normal, add 1μL EdU working solution to each well and continue to incubate the cells for 2h; after labeling, remove the culture medium and add 1mL fixative solution, fix at room temperature for 15min; remove the fixative solution, wash the cells 3 times with 1mL washing solution per well, 3-5min each time; remove the washing solution, use 1mL permeabilization solution per well, incubate at room temperature for 10-15min; remove the permeabilization solution, wash the cells 2 times with 1mL washing solution per well, 5min each time; dissolve the ClickAdditive Solution in deionized water. Prepare 2.5mL Click reaction solution according to the ratio (must be used within 15min of preparation); remove the washing solution in the previous step, add 0.1mL Click reaction solution to each well, gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample, and incubate at room temperature in the dark for 30min; remove the Click reaction solution, and wash 3 times with washing solution, 5min each time. Use Hoechst33342 to stain the nucleus, prepare 1×Hoechst 33342 solution: dilute Hoechst33342 with PBS at a ratio of 1:1000; after removing the above washing solution, add 1mL of 1×Hoechst 33342 solution to each well, incubate at room temperature and away from light for 10 minutes; remove the 1×Hoechst 33342 solution, wash 3 times with washing solution, 3-5 minutes each time; then perform fluorescence detection (Hoechst33342 is blue fluorescence, with a maximum excitation wavelength of 346nm and a maximum emission wavelength of 460nm). Be careful to avoid light and use a fluorescence inverted microscope for photography.

[0065] (4) Transwell experiment

[0066] Migration: After counting the HGC-27 and AGS cells transfected for 24 hours, single cell suspensions were prepared, with 20,000 cells per group, and resuspended in 200 μL DMEM medium. The prepared cell suspension was added to the upper chamber of the Transwell, and a medium with a high serum concentration of 20% FBS was added to the lower chamber. The cell plate was placed in a 37°C incubator for 24 hours. Wash with PBS, invert and dry, fix with 4% paraformaldehyde for 30 minutes, wipe off the cells in the upper chamber, wash and dry, and stain with crystal violet for 30 minutes, and take pictures under a microscope. Invasion: Dilute Matrigel high-concentration matrix gel and DMEM medium at a ratio of 1:8 on ice, take 50 μL of the diluted matrix gel and spread it on the upper chamber surface of the Transwell chamber, and place it in a 37°C, 5% CO2 cell culture incubator for 2 hours to air dry and solidify. After counting the HGC-27 and AGS cells transfected for 24 hours, single cell suspensions were prepared. 30,000 cells per group were resuspended in 200 μL DMEM medium. The prepared cell suspension was added to the upper chamber of the Transwell, and the medium with 20% FBS high serum concentration was added to the lower chamber. Subsequent operations were the same as migration.

[0067] 4.2 Implementation Results

[0068] Four kinds of tagged vectors (empty vector; circUBE2G1-OE: the tagged circUBE2G1 sequence was cloned into a CMV-inducible expression vector containing flanking loop sequences; circUBE2G1-ATG mut: the circUBE2G1 sequence tagged with a start codon mutant (ATG→ATT) was cloned into a CMV-inducible expression vector containing flanking loop sequences; linearcircUBE2G1-99aa: a linear fusion expression vector of circUBE2G1-99aa with a Flag tag was constructed using the PLVX-Puro empty vector without loop sequences) were transfected into HGC-27 and AGS cells ( Figure 4 A). When tumor cells were transfected with empty vector and ATG mutant plasmid, circUBE2G1 lost its coding capacity ( Figure 4 B). The results of CCK8 and colony formation assays showed that overexpression of circUBE2G1-99aa inhibited cell proliferation ( Figure 4 C, D and E).

[0069] To further evaluate whether circUBE2G1-99aa plays an anti-tumor role in vivo, a xenograft mouse model was established by subcutaneous injection of HGC-27 cells. The results showed that the growth rate and tumor weight of the circUBE2G1-OE group and the LinearcircUBE2G1-99aa group were significantly lower than those of the empty vector group and the circUBE2G1-ATG mutant group ( Figure 4F). The above results showed that circUBE2G1-99aa inhibited the proliferation level of gastric cancer cells.

[0070] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

1. Application of circUBE2G1 as a diagnostic or auxiliary diagnostic marker for gastric cancer.

2. Use of reagents for detecting circUBE2G1 in the preparation of products for diagnosis or auxiliary diagnosis, screening or auxiliary screening of gastric cancer.

3. The use according to claim 2, characterized in that: The product includes a system for detecting circUBE2G1, and the system includes reagents and / or instruments for detecting circUBE2G1.

4. A kit for gastric cancer diagnosis or auxiliary diagnosis, screening or auxiliary screening, characterized in that: The kit contains reagents for detecting circUBE2G1.

5. The kit according to claim 4, characterized in that The reagent for detecting circUBE2G1 is an antibody, antibody fragment or modified product thereof for detecting circUBE2G1.

6. Application of circUBE2G1 in the preparation of drugs for the treatment or adjuvant treatment of gastric cancer.

7. Use of drugs that promote the expression of circUBE2G1 in the preparation of drugs for the treatment or adjuvant treatment of gastric cancer.

8. The use according to claim 7, characterized in that: The drug for promoting the expression of circUBE2G1 is an expression vector containing a circUBE2G1 coding sequence.

9. The use according to claim 8, characterized in that: The drug also contains a pharmaceutically acceptable carrier.

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