A circRNA related to the occurrence and development of non-small cell lung cancer and its application

Abstract

The present invention discloses a circRNA associated with the development and progression of non-small cell lung cancer and its application. The present invention provides the use of a substance for detecting the expression of the circular RNA circ_0035796 in the preparation of a product having any of the following functions: 1) diagnosis or auxiliary diagnosis of non-small cell lung cancer; 2) screening or auxiliary screening of non-small cell lung cancer; the nucleotide sequence of the circular RNA circ_0035796 is SEQ ID NO: 21-1163. The present invention found that the expression of the circRNA in the cancerous tissue of lung cancer patients was significantly upregulated compared with adjacent tissue. Overexpression or knockout experiments of circ_0035796 demonstrated that it can effectively promote the proliferation, invasion, and metastasis of lung cancer and is closely related to the development and progression of lung cancer. circ_0035796 is expected to become a new biomarker for the early screening and diagnosis of lung cancer and a new target for precision diagnosis and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a circRNA related to the occurrence and development of non-small cell lung cancer and an application thereof. Background Art

[0002] Lung cancer is one of the most common malignant tumors worldwide, characterized by high morbidity, high malignancy, poor drug treatment response, high mortality, and a poor prognosis. The development and progression of lung cancer is a complex, multi-stage process driven by genetic alterations in oncogenes and tumor suppressor genes. Furthermore, epigenetic changes are also widely present in the development and progression of tumors. Tumor cell genomes generally exhibit epigenetic alterations, such as DNA methylation, histone modifications, regulation of noncoding RNA, and upregulation of certain epigenetic factors. As a form of epigenetic regulation, noncoding RNA has garnered significant attention in current cancer research. Circular RNA (circRNA) is a closed circular RNA structure primarily produced by reverse splicing (a form of alternative RNA splicing) from pre-mRNA. The majority of circRNA is located in the cytoplasm, with only a small fraction localized in the nucleus. Compared to linear RNA, the covalent ring structure of circRNA is less susceptible to exonuclease degradation and exhibits greater structural stability.

[0003] In recent years, with the rapid development of next-generation sequencing technology and bioinformatics, circRNAs have been found to play a crucial role in the development and progression of tumors. CircRNAs are widely involved in tumor cell proliferation, apoptosis, differentiation, invasion, migration, and angiogenesis, and are closely linked to the development, progression, and prognosis of various tumors. In particular, circRNAs are differentially expressed in a variety of malignant tumors and normal tissues, and participate in the development and progression of a range of tumors, including lung cancer. The main biological functions of circRNAs include acting as miRNA sponges, interacting with RNA-binding proteins, and serving as templates for protein translation. CircRNA molecules act as miRNA sponges in cells, thereby relieving the inhibitory effects of miRNAs on target genes and enhancing their expression. This action is known as the competitive endogenous RNA mechanism (ceRNA). Summary of the Invention

[0004] One object of the present invention is to provide a method for detecting the expression of circular RNA circ_0035796.

[0005] The present invention provides the use of a substance for detecting the expression of circular RNA circ_0035796 in the preparation of a product having any of the following functions:

[0006] A1) Diagnosis or auxiliary diagnosis of non-small cell lung cancer;

[0007] A2) Differential diagnosis or differentiation of benign pulmonary nodules from non-small cell lung cancer;

[0008] A3) Screening or auxiliary screening for non-small cell lung cancer;

[0009] A4) diagnose or assist in diagnosing whether the patient is a non-small cell lung cancer patient;

[0010] A5) Screening or auxiliary screening of patients to be tested to determine whether they are patients with non-small cell lung cancer;

[0011] A6) Diagnosis or auxiliary diagnosis of whether the sample to be tested is derived from non-small cell lung cancer tissue;

[0012] A7) Screening or auxiliary screening of patients to be tested to determine whether their tissue originates from non-small cell lung cancer;

[0013] The nucleotide sequence of the circular RNA circ_0035796 is positions 21-1163 of sequence 1.

[0014] In the above application, the substance for detecting the expression of circular RNA circ_0035796 includes a probe that specifically binds to circ_0035796, or a primer that specifically amplifies circ_0035796.

[0015] In an embodiment of the present invention, the primers for specifically amplifying circ_0035796 are a primer pair consisting of sequence 2 and sequence 3.

[0016] The samples to be tested in the above applications come from plasma or tumor tissue.

[0017] Another object of the present invention is to provide a product.

[0018] The product provided by the present invention includes a substance for detecting the expression of circular RNA circ_0035796;

[0019] The product has any of the following functions:

[0020] A1) Diagnosis or auxiliary diagnosis of non-small cell lung cancer;

[0021] A2) Differential diagnosis or differentiation of benign pulmonary nodules from non-small cell lung cancer;

[0022] A3) Screening or auxiliary screening for non-small cell lung cancer;

[0023] A4) diagnose or assist in diagnosing whether the patient is a non-small cell lung cancer patient;

[0024] A5) Screening or auxiliary screening of patients to be tested to determine whether they are patients with non-small cell lung cancer;

[0025] A6) Diagnosis or auxiliary diagnosis of whether the sample to be tested is derived from non-small cell lung cancer tissue;

[0026] A7) Screening or auxiliary screening of patients to be tested to determine whether the cancer tissue originates from non-small cell lung cancer.

[0027] In the above, the subjects of screening or diagnosis are patients with lung tumors;

[0028] In the above, the patient to be tested is a patient suspected of having non-small cell lung cancer; the sample to be tested is derived from a sample of a patient suspected of having non-small cell lung cancer;

[0029] The use of the circular RNA circ_0035796 as a marker in the development or design of products for diagnosis, auxiliary diagnosis, screening or auxiliary screening of non-small cell lung cancer is also within the scope of protection of the present invention.

[0030] The use of substances that inhibit the expression of circular RNA circ_0035796 in any of the following applications is also within the scope of protection of the present invention:

[0031] B1) Treatment of non-small cell lung cancer;

[0032] B2) inhibiting the proliferation of non-small cell lung cancer cells;

[0033] B3) inhibiting the migration of non-small cell lung cancer cells;

[0034] B4) inhibiting the invasion of non-small cell lung cancer cells;

[0035] B5) inhibiting tumor formation of non-small cell lung cancer cells in vivo.

[0036] Another object of the present invention is to provide another product as follows.

[0037] The product provided by the present invention comprises the above-mentioned substance for inhibiting the expression of circular RNA circ_0035796;

[0038] The product has any of the following functions:

[0039] B1) Treatment of non-small cell lung cancer;

[0040] B2) inhibiting the proliferation of non-small cell lung cancer cells;

[0041] B3) inhibiting the migration of non-small cell lung cancer cells;

[0042] B4) inhibiting the invasion of non-small cell lung cancer cells;

[0043] B5) inhibiting tumor formation of non-small cell lung cancer cells in vivo.

[0044] The substance that inhibits the expression of circular RNA circ_0035796 is a substance that interferes with or silences the expression of circ_0035796.

[0045] The substance that interferes with or silences the expression of circ_0035796 is specifically a DNA molecule that interferes with the expression of circ_0035796 (sequence 4 in the embodiment of the present invention) or a vector that expresses the DNA molecule.

[0046] The use of the above-mentioned circular RNA circ_0035796 in preparing an animal model or cell model for screening drugs for treating non-small cell lung cancer is also within the scope of protection of the present invention.

[0047] The present invention discovered that circ_0035796 is upregulated in the plasma of patients with non-small cell lung cancer (NSCLC), demonstrating high sensitivity and specificity for diagnosing NSCLC. This circRNA is significantly more expressed in cancerous tissues compared to adjacent adjacent tissues. Overexpression and knockdown experiments of circ_0035796 have demonstrated its ability to effectively promote the proliferation, invasion, and metastasis of NSCLC, demonstrating its close association with the development and progression of lung cancer. circ_0035796 has the potential to become a novel biomarker for the early screening and diagnosis of NSCLC, and a new target for precision diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Figure 2 is a heat map of circ_0035796 expression in the plasma of benign patients, non-small cell lung adenocarcinoma patients, and non-small cell lung squamous cell carcinoma patients.

[0049] Figure 2 The expression of circ_0035796 in the plasma of different patients.

[0050] Figure 3 Figure 2 is the ROC curve of the relative expression level of circ_0035796 in plasma.

[0051] Figure 4 The expression of circ_0035796 in tumor tissues and adjacent adjacent tissues of patients with non-small cell lung cancer.

[0052] Figure 5 The expression of circ_0035796 overexpression vector and siRNA in cells NCI-H3255.

[0053] Figure 6 The growth curves of circ0035796 overexpressing cells and circ0035796 knockout cells were measured using CCK8.

[0054] Figure 7The results of the proliferation experiment using Edu to detect circ0035796 high-expressing cells and circ0035796 knockout cells.

[0055] Figure 8 Figure 2 shows the migration experiment results of circ0035796 overexpressing cells and circ0035796 knockout cells.

[0056] Figure 9 Figure 2 shows the invasion experiment results of circ0035796 overexpressing cells and circ0035796 knockout cells.

[0057] Figure 10 The results of inhibiting tumor cell proliferation in vivo in circ0035796 overexpressing cells and circ0035796 knockout cells are shown. DETAILED DESCRIPTION

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0059] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0060] The source of the blood samples in the following examples: This research group collaborated with the Department of Thoracic Surgery at the General Hospital of the Chinese People's Liberation Army to select all patients with non-small cell lung cancer and benign lung nodules admitted to the hospital for treatment as research subjects. All patients were fully informed and signed informed consent forms before admission. Subsequently, preoperative blood samples were collected from the patients and separated into three parts: plasma, PBMC, and RBC. The blood samples were then packaged into RNase-free EP tubes and frozen for storage. At the same time as the samples were collected, detailed medical records of the patients were recorded and retained, including information such as gender, age, smoking status, smoking amount, years of smoking, whether they had infectious viral infections, and whether they had a history of cancer.

[0061] The sample selection criteria and grouping basis in the following examples are:

[0062] Based on the patients' clinical data, blood samples from patients with a history of cancer or infectious viral infections (such as infectious hepatitis, syphilis, and HIV) were excluded. Plasma samples from patients with non-small cell lung adenocarcinoma and non-small cell lung squamous cell carcinoma (NSCLC) who had not undergone any surgery, radiotherapy, chemotherapy, or drug treatment, as well as plasma samples from patients with benign lung nodules (BENIGN, including alveolar epithelial hyperplasia, chronic granulomas, fibrous hyperplasia, and polyps) who had not undergone any surgery, radiotherapy, chemotherapy, or drug treatment, were used as experimental subjects (Table 1) for screening and detection of differentially expressed genes.

[0063] Table 1 is a list of the patients' clinical data

[0064]

[0065]

[0066] Note: P values ​​were calculated using the chi-square test or Fisher's exact test for categorical variables and the T test or Wilcoxon rank-sum test for continuous variables.

[0067] Some of the experimental methods in the following examples are as follows:

[0068] 1. Separation of plasma

[0069] 10 mL of fasting venous blood was collected from the patient before surgery and placed in a blood collection tube containing EDTA as an anticoagulant. The blood was separated within 2 hours after collection.

[0070] 1) Centrifuge freshly collected whole blood at 400 × g for 15 minutes at room temperature;

[0071] 2) After centrifugation, the upper yellow clear liquid is plasma, which is transferred to a 1.5 mL centrifuge tube and stored at -80°C until use.

[0072] 2. Extraction of total RNA from plasma

[0073] When using the miRNeasy Serum / Plasma Kit (QIAGEN) for the first time, prepare Buff RWT and Buff RPE by adding anhydrous ethanol as specified in the instructions.

[0074] 1) Take 250 μL of plasma separated from blood;

[0075] 2) Centrifuge at 16,000 × g for 10 minutes at 4°C;

[0076] 3) Carefully pipette 200 μL of the upper plasma into a 1.5 mL centrifuge tube;

[0077] 4) Add 1 mL of QIAzol Lysis Buffer, mix thoroughly by pipetting, and let stand at room temperature for 5 minutes;

[0078] 5) Add chloroform equal to the original volume of the plasma sample (200 μL), cover tightly, and vortex to mix for 15 seconds;

[0079] 6) Let stand at room temperature for 2-3 minutes;

[0080] 7) Centrifuge at 12,000 × g for 15 minutes at 4°C;

[0081] 8) After centrifugation, the solution in the tube is clearly separated into three layers. Carefully pipette approximately 600 μL of the upper aqueous phase into a new centrifuge tube, taking care not to absorb the protein in the middle layer. Add 1.5 times the amount of anhydrous ethanol (900 μL) of the upper aqueous phase and mix thoroughly by pipetting.

[0082] 9) Transfer the sample to a purification column with a collection tube, transferring up to 700 μL of sample at a time. Cap tightly and centrifuge at 8000 × g for 15 seconds at room temperature. Discard the filtrate in the collection tube.

[0083] 10) Repeat step 9 until all the sample is added to the purification column;

[0084] 11) Add 700 μL of Buff RWT to the centrifuge column, cap tightly, and centrifuge at 8000 × g for 15 seconds at room temperature. Discard the filtrate in the collection tube.

[0085] 12) Add 500 μL of Buff RPE to the centrifuge column, cap tightly, and centrifuge at 8000 × g for 15 seconds at room temperature. Discard the filtrate in the collection tube.

[0086] 13) Add 500 μL of 80% anhydrous ethanol, cap tightly, and centrifuge at 8000 × g for 2 minutes at room temperature. Discard the collection tube and the filtrate.

[0087] 14) Replace the collection tube with a new one, open the purification cap, and centrifuge at high speed (about 15,000 rpm) at room temperature for 5 minutes to dry the film on the purification column. Discard the filtrate and collection tube.

[0088] 15) Replace the tube with a 1.5 ml recovery tube and carefully add 30 μL of RNase-free water to the center of the purification column membrane. Let it stand at room temperature for 1 minute. Centrifuge at high speed (about 15,000 rpm) for 1 minute to elute the RNA into the recovery tube.

[0089] 3. Reverse transcription of plasma total RNA

[0090] 1) Prepare 2× reverse transcription mixture (Table 2, all components are from the High capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Cat. No. 4374967) and keep on ice until use;

[0091] Table 2 shows the reverse transcription reaction system

[0092]

[0093] 2) Prepare the reverse transcription mixture and add it to each of the 8-tube strips. Add 15 μL of RNA sample to each tube to make a 30 μL reaction system. Mix by gently pipetting, cap tightly, and centrifuge briefly.

[0094] 3) Place the tube in a 96-well PCR instrument and set the reaction conditions as follows (Table 3):

[0095] Table 3 shows the reverse transcription PCR conditions.

[0096]

[0097] Example 1: circ_0035796 is highly expressed in the plasma and lung cancer tissues of lung cancer patients

[0098] 1. Discovery of circular RNA circ_0035796

[0099] Using the second-generation high-throughput sequencing technology, deep sequencing of the whole transcriptome in the plasma of patients with non-small cell lung cancer (lung adenocarcinoma and lung squamous cell carcinoma) and benign lung nodules revealed that circular RNA circ_0035796 (hsa_circ_0035796) was the circRNA with the highest up-regulated fold ( Figure 1 The nucleotide sequence of circular RNA circ_0035796 is from position 21 to 1163 of SEQ ID NO: 1.

[0100] 2. Expression of circular RNA circ_0035796 in plasma and lung cancer tissues of patients with non-small cell lung cancer

[0101] 1. Primer design Primers were designed based on the gene sequences of circ_0035796 and GAPDH for real-time fluorescence quantitative PCR reaction. The specific primer names and sequences are shown in Table 4:

[0102] Table 4 shows the primer sequence information used in the PCR reaction

[0103]

[0104] 2. qRT-PCR reaction system and conditions

[0105] Extract RNA from the plasma to be tested, reverse transcribe to obtain cDNA as a template, and perform qRT-PCR using the following system and procedure:

[0106] According to the primer synthesis instructions, add an appropriate amount of RNase-free water to prepare a primer solution with a concentration of 10 μM. TM For the SYBR qPCR Mix Without ROX kit, prepare the reaction system according to the instructions as follows (Table 5):

[0107] Table 5 shows the qRT-PCR reaction system

[0108]

[0109] The prepared qRT-PCR reaction system shown in Table 5 was added to a 96-well PCR plate. 18 μL of the qRT-PCR reaction system shown in Table 5 was added to each reaction well. 2 μL of cDNA was then added to make a 20 μL reaction system. The plate was placed in a real-time fluorescence quantitative PCR instrument for reaction. The PCR reaction conditions were as follows (Table 6):

[0110] Table 6 shows the qRT-PCR reaction conditions.

[0111]

[0112] Each sample was assayed in triplicate, with GAPDH used as the internal reference gene. After the qPCR reaction, the average Ct value of the three replicate wells for each target gene was used as the final result. The relative expression level of each target gene was calculated using the formula 2^-ΔCt, where ΔCt = Ct(target gene) - Ct(reference gene).

[0113] qRT-PCR was used to detect the expression of circ_0035796 in the plasma of 48 patients with non-small cell lung adenocarcinoma (referred to as lung adenocarcinoma patients in the figure), 17 patients with non-small cell lung squamous cell carcinoma (referred to as lung squamous cell carcinoma patients in the figure), and 17 patients with benign lung nodules (referred to as normal patients in the figure).

[0114] The results are as follows Figure 2 As shown in the results, this circRNA was highly expressed in the plasma of patients with non-small cell lung cancer compared with normal patients (P<0.01).

[0115] The respective ROC curves were drawn in GraphPad Prim 6 according to the relative expression levels of circ_0035796.

[0116] The results are as follows Figure 3 As shown in the figure, the possibility of the circRNA as a diagnostic molecular marker for non-small cell lung cancer was evaluated by the AUC value (area under the curve). The larger the AUC value, the higher the sensitivity and specificity of the gene as a diagnostic molecular marker for non-small cell lung cancer. It can be seen that the AUC value of the circRNA is 0.7790 (P=0.0014<0.01), indicating that the circRNA can be used as a diagnostic molecular marker for non-small cell lung cancer, and has high sensitivity and specificity for the diagnosis of non-small cell lung cancer.

[0117] The relative expression of circ_0035796 in lung cancer tissue and adjacent tissues from 10 patients with non-small cell lung cancer (8 with non-small cell lung adenocarcinoma and 2 with non-small cell lung adenocarcinoma) was determined using qRT-PCR. The template was cDNA reverse-transcribed from RNA from the tissues to be tested.

[0118] The results are as follows Figure 4 As shown in the results, the relative expression level of circ_0035796 in lung cancer tissues was significantly higher than that in adjacent adjacent tissues (P<0.05).

[0119] Therefore, circ_0035796 can be used as a molecular marker for the diagnosis or screening of non-small cell lung cancer. Specifically, the expression level of circ_0035796 in the blood or tumor tissue of the patient can be detected to determine or screen whether the patient has lung cancer.

[0120] If the expression level of circ_0035796 in the blood of the patient to be tested is higher than the expression level of circ_0035796 in the blood of the non-cancer patient, the patient to be tested is or is a candidate for non-small cell lung cancer;

[0121] If the expression level of circ_0035796 in the tumor tissue to be tested is higher than that in the adjacent adjacent tissue, the tumor tissue to be tested is derived from or is a candidate for being derived from a patient with non-small cell lung cancer.

[0122] The patient to be tested or the tumor to be tested is a patient suspected of having non-small cell lung cancer.

[0123] Example 2: Application of circ_0035796 in the treatment of lung cancer

[0124] 1. Construction of circ_0035796 overexpression plasmids or cells and knockdown plasmids or cells

[0125] 1. Construction of circ_0035796 overexpression plasmid and knockdown plasmid

[0126] Overexpression vector pLC5-circ_0035796: The DNA molecule containing the linear sequence of circ_0035796 (SEQ ID NO: 1) was used to replace the DNA molecule between the EcoRI and BamHI restriction sites of the circRNA overexpression vector pLC5-ciR (Guangzhou Gisai Biotechnology Co., Ltd.).

[0127] The nucleotide sequence of the DNA molecule containing the circ_0035796 linear sequence is sequence 1, in which positions 3-8 of sequence 1 are EcoRI restriction sites, positions 9-18 are forward cyclization mediating sequences, positions 19-20 are AG acceptors, positions 1164-1165 are GT donors, positions 1166-1175 are reverse cyclization mediating sequences, and positions 1176-1181 are BamHI restriction sites.

[0128] The silencing expression vector pLKO.1-circ_0035796 is a vector obtained by replacing the DNA molecule between the AgeI and EcoRI restriction sites of the interference plasmid pLKO.1 (Addgene) with a DNA molecule that interferes with the expression of circ_0035796 (sequence 4, sequence GCATCACTATCAGATATCA (circular RNA junction, sequence 1 positions 1151-1163 and 21-26).

[0129] 2. Construction of circ_0035796 overexpression and knockdown cells

[0130] The above-mentioned overexpression vector pLC5-circ_0035796, empty vector pLC5-ciR, silent expression vector pLKO.1-circ_0035796 and empty vector pLKO.1 were transfected into lung cancer cells NCI-H3255 (Shanghai Qincheng Biotechnology Co., Ltd., QC473), respectively, to obtain pLC5-circ_0035796-transfected cells, pLC5-ciR-transfected cells, pLKO.1-circ_0035796-transfected cells and pLKO.1-transfected cells.

[0131] RNA was extracted from cells transfected with pLC5-circ_0035796, cells transfected with pLC5-ciR, cells transfected with pLKO.1-circ_0035796, and cells transfected with pLKO.1, and reverse transcribed according to the above method to obtain cDNA as a template for the above-mentioned RT-PCR amplification.

[0132] The expression results of pLKO.1-circ_0035796 cells (denoted as knockdown in the figure) and pLKO.1 cells (denoted as control in the figure) are shown in the figure. Figure 5 As shown in the right figure, it can be seen that compared with the pLKO.1 transfected cells, the expression level of circ_0035796 in the pLKO.1-circ_0035796 transfected cells was significantly downregulated (P<0.01), indicating that cells with silenced circ_0035796 expression were obtained.

[0133] The expression levels of pLC5-circ_0035796 transfected cells (denoted as overexpression in the figure) and pLC5-ciR transfected cells (denoted as control in the figure) are as follows Figure 5 As shown in the left figure, it can be seen that compared with the empty vector, the expression level of circ_0035796 in the pLC5-circ_0035796 transfected cells was upregulated (P<0.01), indicating that cells overexpressing circ_0035796 were obtained.

[0134] 2. circ_0035796 is associated with the proliferation ability of non-small cell lung cancer cells

[0135] The proliferation ability of lung cancer cells was detected by CCK-8 assay and EdU assay.

[0136] 1. CCK-8 assay to detect cell proliferation

[0137] The pLC5-circ_0035796-transfected cells, pLC5-ciR-transfected cells, pLKO.1-circ_0035796-transfected cells, and pLKO.1-transfected cells obtained above were counted using a Bio-Rad cell counting chamber and prepared into a cell suspension (density of approximately 2×10 4 / mL), 100 μL per well was inoculated into a 96-well plate, with three replicates per group. After the cells adhered, 10 μL of CCK-8 solution (Cell Counting Kit-8, APExBIO Catalog No. K1018) was added to each well. The cells were incubated in a 37°C cell culture incubator for 120 min. The optical density (OD) (450 nm) was then measured using a microplate reader. The optical density of each group of cells was then measured using the same method after culturing for 24, 48, 72, and 96 h. The values ​​were recorded and growth curves were plotted.

[0138] The results are as follows Figure 6 As shown, the right figure is the silence group and the left figure is the overexpression group. It can be seen that in the CCK-8 experiment, after 96 hours of culture, the optical density value at 450nm of the overexpression group was significantly increased compared with the pLC5-ciR transfected cells (denoted as control in the figure) (P < 0.01, Figure 6 Left panel); In the silenced group, compared with cells transfected with pLKO.1 (denoted as control in the figure), cells transfected with pLKO.1-circ_0035796 (denoted as knockdown in the figure) had significantly lower optical density at 450 nm (P < 0.01, Figure 6 right).

[0139] 2. EdU experiment

[0140] EdU labeling was performed using the BeyoClick Edu-594 Cell Proliferation Assay Kit (Beyotime, Cat. No. C0078L) in pLC5-circ_0035796-, pLC5-ciR-, pLKO.1-circ_0035796-, and pLKO.1-transfected cells. Cells were fixed with 4% paraformaldehyde for 15 minutes, washed twice with PBS for 5 minutes each, and permeabilized with 1% Triton-X100 for 10 minutes at room temperature. The permeabilization buffer was removed and the cells were washed twice with PBS for 5 minutes each. 1 mL of staining solution was prepared with the following formula: 860 μL Click Reaction Buffer, 40 μL CuSO₄, 2 μL Azide 594, and 100 μL Click Additive Solution. Cells were stained with this freshly prepared staining solution for 30 minutes in the dark and then washed twice with PBS for 5 minutes each. The cells were stained for DNA using mounting medium with DAPI (Abcam, catalog number ab104139) in the dark, and then photographed under a fluorescence microscope.

[0141] The results are as follows Figure 7 As shown, the left group is the overexpression group, and the right group is the knockout group. Five visual fields were collected respectively. In the overexpression group, the positive cell rate of cells transfected with pLC5-circ_0035796 (denoted as overexpression in the figure) was 51.64%, and the positive cell rate of cells transfected with pLC5-ciR (denoted as control in the figure) was 28.15%. The number of positive cells increased significantly, and the cell proliferation activity increased significantly (P<0.01, Figure 7 Left panel); in the knockout group, the positive cell rate in the pLKO.1-circ_0035796 transfected cells (denoted as knockdown in the figure) was 8.93%, while the positive cell rate in the pLKO.1 transfected cell group (denoted as control in the figure) was 32.39%, and the number of positive cells was significantly reduced, and the cell proliferation activity was significantly reduced (P < 0.01, Figure 7 right).

[0142] The above results indicate that silencing the expression of circ_0035796 can inhibit the proliferation of non-small cell lung cancer cells.

[0143] circ_0035796 is associated with the migration and invasion ability of non-small cell lung cancer cells

[0144] The migration and invasion abilities of non-small cell lung cancer cells were detected by cell scratch assay and Transwell chamber migration and invasion assay.

[0145] 1. Cell scratch test

[0146] The cells were seeded onto a streaked 6-well cell culture plate the day before transfection, with the cell density ideally reaching 70% to 80% on the next day. For transfection experiments, pLC5-ciR and the overexpression vector pLC5-circ_0035796 were transfected into NCI-H3255 (Shanghai Qincheng Biotechnology Co., Ltd., catalog number QC473) / BEAS-2B cells (Shanghai Gaining Biotechnology Co., Ltd., CM-H365), and pLKO.1 and the silencing expression vector pLKO.1-circ_0035796 were transfected into NCI-H3255 / A549 cells (ATCC, catalog number CCL-185).

[0147] After changing the medium within 4-6 hours of transfection, continue culturing the cells until they fill the wells. Use the pipette tip to scratch the lines, applying even pressure. Discard the culture medium and rinse the disrupted cells with PBS. Add DMEM supplemented with 1% fetal bovine serum. Immediately photograph the cells under a microscope, marking this time as 0 hours, and use a marker to record the observation position. After photographing, return the cells to the incubator and continue culturing. Photograph the cells at the same location every 12 hours. Repeat the experiment three times.

[0148] The results are as follows Figure 8 As shown, the right figure is the knockout group, and the left figure is the overexpression group. It can be seen that in the cell scratch test, the relative migration rate of cells transfected with pLC5-ciR in the overexpression group (denoted as control in the figure) was 41%, and the relative migration rate of cells transfected with pLC5-circ_0035796 (denoted as overexpression in the figure) was 70.2%. Compared with cells transfected with pLC5-ciR, the relative migration rate of cells transfected with pLC5-circ_0035796 increased by 1.71 times (P<0.01, Figure 8 Left panel); in the knockout group, the relative migration rate of cells transfected with pLKO.1 (denoted as control in the figure) was 68.9%, and the relative migration rate of cells transfected with pLKO.1-circ_0035796 (denoted as knockdown in the figure) was 40.3%. Compared with cells transfected with pLKO.1, the relative migration rate of cells transfected with pLKO.1-circ_0035796 was reduced by 1.73 times (P < 0.01, Figure 8 ).

[0149] The above results indicate that silencing the expression of circ_0035796 can inhibit the migration of lung cancer cells.

[0150] 2. Transwell chamber migration assay and Matrigel invasion assay

[0151] A. Transwell chamber migration assay

[0152] pLC5-ciR and overexpression vector pLC5-circ_0035796 were transfected into NCI-H3255 / BEAS-2B cells, respectively, and pLKO.1 and silenced expression vector pLKO.1-circ_0035796 were transfected into NCI-H3255 / A549 cells, respectively.

[0153] 2.1 Matrigel plating

[0154] Dilute BD Matrigel 1:8 (depending on the amount of MMP produced by the cells) and coat the upper surface of the bottom membrane of the Transwell chamber. Incubate at 37°C for 2 hours to allow the Matrigel to polymerize. Hydrate the basement membrane before use.

[0155] 2.2 Preparation of cell suspension

[0156] Digest the cells, centrifuge and discard the culture medium after digestion (wash 1-2 times with PBS), and resuspend in serum-free medium. Adjust the cell density to 5×10 5 / ml.

[0157] 2.3 Cell inoculation

[0158] ① Take 100 μl of cell suspension and add it to the Transwell chamber.

[0159] ② Generally, 600 μl of culture medium containing 20% ​​serum is added to the lower chamber of the 24-well plate, and no cells are produced.

[0160] ③Cultivate cells: conventional culture for 10-48 hours

[0161] 2.4 Results Statistics

[0162] Remove the Transwell chamber, discard the culture medium in the well, wash twice with PBS, fix with 90% ethanol for 30 minutes, and air-dry the chamber appropriately.

[0163] Stain with 0.1% crystal violet for 30 min, gently wipe off the upper layer of non-migrated cells with a cotton swab, and wash three times with PBS.

[0164] The cells were observed in five fields of view under a 400x microscope and counted.

[0165] B. Matrigel invasion assay

[0166] 1) Preparation of invasion chambers: Matrigel Matrix basement membrane (Corning, Cat. No. 354234) solidifies quickly at room temperature. Therefore, remove it from -20°C and thaw at 4°C. Keep on ice throughout the entire process. Dilute it with serum-free medium (1:5-1:8), then spread 400 μL onto a Transwell membrane. Place a Transwell chamber (Transwell prmeable supports 6.5 mm insert, 24-well plate, Cat. No. 00721069) in a 6-well plate and incubate at 37°C for 3 h to allow it to solidify.

[0167] 2) Cell inoculation: Experimental cells (cells transfected with pLC5-circ_0035796, cells transfected with pLC5-ciR, cells transfected with pLKO.1-circ_0035796, and cells transfected with pLKO.1) were digested and centrifuged, and then resuspended in serum-free medium. 1 mL of tumor cell suspension was added to the chamber, and the cell count was 3×10 5 -6×10 5 (The appropriate cell number is selected based on the invasive ability of the tumor cells.) Add 3 mL of culture medium containing 1.5% fetal bovine serum to the outside of the chamber.

[0168] 3) Cell culture: Routine culture for 24-48 hours (depending on the invasive ability of tumor cells).

[0169] 4) Fixation and staining: Remove the Transwell chamber, wash once with PBS, wipe off the cells on the top of the microporous membrane with a cotton swab, fix and stain with 1% crystal violet-formaldehyde solution for 20-30 minutes, aspirate the staining solution, and then slowly wash away the residual staining solution with tap water and air dry.

[0170] 5) Microscopic examination: Take photos under an inverted microscope, count 5 fields of view for each sample, and perform statistical analysis after counting.

[0171] The results are as follows Figure 9 As shown, the upper figure is the overexpression group and the lower figure is the knockout group. It can be seen that in the Transwell chamber migration experiment, in the overexpression group, cells transfected with pLC5-circ_0035796 (denoted as overexpression in the figure) and cells transfected with pLC5-ciR (denoted as control in the figure) were photographed and counted at 10 hours. Compared with cells transfected with pLC5-ciR, the number of migrating cells transfected with pLC5-circ_0035796 increased significantly, increasing by 10.9 times (P<0.01, Figure 9); In the invasion assay, cells transfected with pLC5-circ_0035796 (denoted as overexpression in the figure) and cells transfected with pLC5-ciR (denoted as control in the figure) were photographed and counted at 16 hours. Compared with cells transfected with pLC5-ciR, the number of migrating cells in cells transfected with pLC5-circ_0035796 was significantly increased, increasing by 8 times (P<0.01, Figure 9 ); The above results show that overexpression of circ_0035796 can enhance the migration and invasion of lung cancer cells; in the Transwell chamber migration assay, in the knockout group, cells transfected with pLKO.1-circ_0035796 (denoted as knockdown in the figure) and cells transfected with pLKO.1 (denoted as control in the figure) were photographed and counted at 16 hours. Compared with cells transfected with pLKO.1, the number of migrating cells in the knockdown group was significantly reduced, which was reduced by 6 times (P<0.01, Figure 9 ), in the invasion assay, pictures were taken and counted at 22 hours. Compared with cells transfected with pLKO.1, the number of invasive cells in cells transfected with pLKO.1-circ_0035796 was significantly reduced, and the number of invasive cells in cells transfected with pLKO.1-circ_0035796 was reduced by 12.5 times (P < 0.01, Figure 9 ).

[0172] The above results indicate that silencing the expression of circ_0035796 can inhibit the migration or invasion of non-small cell lung cancer cells.

[0173] 4. cir_0035796 is associated with tumorigenesis and metastasis in vivo

[0174] To investigate the effect of circ_0035796 on tumor growth and metastasis in vivo, NCI-H3255 cells stably overexpressing circ_0035796 and their corresponding control group cells were inoculated subcutaneously into female nude mice, and a nude mouse xenograft tumor growth and metastasis model was successfully established.

[0175] The details are as follows:

[0176] 1) Conventional culture control cells (pLC5-ciR transfected cells and pLKO.1 transfected cells) or transfected cell lines carrying stable overexpression of circ-0035796 (pLC5-circ_0035796 transfected cells) and cell lines with knockdown of circ_0035796 expression (pLKO.1-circ_0035796 transfected cells) were expanded to a sufficient number and prepared for inoculation.

[0177] 2) Digest the cells with trypsin, collect them by centrifugation, count the cells, and resuspend them in PBS. The general inoculation volume is 100 μL per cell, which contains 4×10 6Place the cells to be injected on ice.

[0178] 3) The nude mice to be injected were briefly induced to coma with ether, and the cell suspension was injected subcutaneously in the armpit using a 1 mL syringe.

[0179] 4) The nude mice were maintained for approximately one month, and tumor changes were observed regularly. Tumor volume was measured with a vernier caliper and recorded. Before tumor removal, in vivo imaging was performed. A 15 mg / ml luciferin substrate solution (Xenolight D-luciferin potassium salt, PerkinElmer, Catalog No. 12799) was prepared in PBS, filtered through a 0.2 μm sterilizing filter, and injected into the nude mice at a rate of 10 μL / g. Observation and imaging were performed using an imaging system (Living Image 4.5, PerkinElmer) 10-15 minutes after intraperitoneal injection.

[0180] 5) After removing the tumor, measure its weight and record the value for statistical analysis.

[0181] The results are as follows Figure 10 As shown in the figure, the overexpression group is mice injected with pLC5-circ_0035796 cells, the knockdown group is mice injected with pLKO.1-circ_0035796 cells, and the overexpression control group and knockdown control group are mice injected with pLC5-ciR cells and pLKO.1 cells, respectively. It can be seen that compared with the overexpression control group, the tumor volume and mass of the circ_0035796 overexpression group were significantly higher than those of the overexpression control group (P<0.01, Figure 10 ), the tumor volume and mass in the knockdown group were significantly lower than those in the knockdown control group (P<0.01, Figure 10 ).

[0182] The tumor growth curve showed that the tumor growth rate in the circ_0035796 overexpression group was significantly faster than that in the overexpression control group (P<0.01, Figure 10 ), while the tumor growth rate in the knockdown group was significantly slower than that in the knockdown control group (P<0.01, Figure 10 ).

[0183] The results of in vivo imaging of small animals (PE) showed that the fluorescence intensity of the circ_0035796 overexpression group was significantly enhanced compared with the control group (P<0.01, Figure 10 ), the fluorescence intensity of the knockdown group was significantly decreased compared with the control group (P<0.01, Figure 10 ).

[0184] The above results indicate that exogenous high expression of circ_0035796 significantly enhances tumor formation in non-small cell lung cancer cells in vivo, while low expression can significantly inhibit tumor formation in non-small cell lung cancer cells in vivo.

Claims

1. Use of a substance for detecting the expression of circular RNA circ_0035796 in the preparation of a product having any of the following functions: A1) Diagnosis or auxiliary diagnosis of non-small cell lung cancer; A2) Diagnosis or differentiation of benign pulmonary nodules from non-small cell lung cancer; A3) Screening or auxiliary screening for non-small cell lung cancer; A4) Diagnose or assist in diagnosing whether the patient is a non-small cell lung cancer patient; A5) Screening or auxiliary screening to determine whether the patient is a patient with non-small cell lung cancer; A6) Whether the sample to be tested is derived from non-small cell lung cancer tissue for diagnosis or auxiliary diagnosis; A7) Screening or auxiliary screening to determine whether the sample to be tested is derived from non-small cell lung cancer tissue; The nucleotide sequence of the circular RNA circ_0035796 is positions 21-1163 of sequence 1.

2. The use according to claim 1, characterized in that: The substance for detecting the expression of circular RNA circ_0035796 includes a probe that specifically binds to circ_0035796 or a primer that specifically amplifies circ_0035796.

3. Use of a substance that inhibits the expression of circular RNA circ_0035796 in the preparation of a product having any of the following functions: B1) Treatment of non-small cell lung cancer; B2) Inhibit the proliferation of non-small cell lung cancer cells; B3) Inhibits the migration of non-small cell lung cancer cells; B4) Inhibits the invasion of non-small cell lung cancer cells; B5) Inhibiting tumor formation in non-small cell lung cancer cells in vivo; the nucleotide sequence of the circular RNA circ_0035796 is positions 21-1163 of SEQ ID NO:

1.

4. The use according to claim 3, characterized in that: The substance that inhibits the expression of circular RNA circ_0035796 is a substance that interferes with or silences the expression of circ_0035796.

5. Use of circular RNA circ_0035796 in preparing an animal model or cell model for screening drugs for treating non-small cell lung cancer; the nucleotide sequence of the circular RNA circ_0035796 is positions 21-1163 of SEQ ID NO: 1.

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