A method for diagnosing lung cancer in a subject, comprising detecting the presence or absence of a methylation modification of a circular RNA in a biological sample obtained from the subject 6 A lung adenocarcinoma diagnosis marker based on methylation modification of circular RNA and application thereof

Abstract

The application relates to the field of biotechnology, and particularly relates to a lung adenocarcinoma diagnosis biomarker based on m 6 A lung adenocarcinoma diagnosis biomarker based on methylation modification of circular RNA and application thereof 6 A lung adenocarcinoma diagnosis biomarker based on methylation modification of circular RNA and application thereof 6 A lung adenocarcinoma diagnosis biomarker based on methylation modification of circular RNA and application thereof 6 A lung adenocarcinoma diagnosis biomarker based on methylation modification of circular RNA and application thereof The hsa_circ_0079039 modified by methylation can be used as a biomarker for diagnosing lung adenocarcinoma. The application has important value for screening and early diagnosis of lung adenocarcinoma.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method based on m 6 Diagnostic biomarkers for lung adenocarcinoma based on methylated circular RNA and their application in the diagnosis of lung adenocarcinoma. Background Technology

[0002] The incidence and mortality rates of lung cancer are rising year by year, and it has become one of the leading causes of cancer-related deaths. In my country, lung cancer ranks first among malignant tumors in terms of the number of cases. Lung adenocarcinoma, the most common histological subtype of non-small cell lung cancer, accounts for 40% of lung cancer cases. The diagnosis rate of early-stage lung adenocarcinoma is not high, but the survival rate of stage I lung cancer can be as high as 85%. Therefore, early screening and diagnosis of lung adenocarcinoma are extremely important.

[0003] Abnormal epigenetic alterations are considered a significant cause of cancer. Among these, N6-methyladenosine (m...) is particularly important. 6 A) is the most abundant RNA modification discovered in eukaryotes to date. It is estimated that over 7000 coding RNAs and 3000 non-coding RNAs exist in the human body. 6 A modifies. m 6 RNA is widely present in mRNA, miRNA, lncRNA, and circRNA, and is closely related to the biological functions of various RNAs, such as splicing, degradation, and translation. 6 The overall abundance of A and the expression level of its regulatory factors are closely related to malignant biological activities such as occurrence, proliferation, invasion, and metastasis of malignant tumors.

[0004] In recent years, an increasing number of scholars have begun to study m 6 The impact of A modification on tumor non-coding RNA. Researchers identified some specific m in tissue and blood samples from patients with early-stage lung adenocarcinoma. 6 Modified circRNAs (A-type circRNAs) are more likely to promote malignant biological processes in tumor cells and regulate tumor progression and outcome by acting as miRNA sponges or binding to proteins. Simultaneously, circRNAs possess characteristics such as high stability, high specificity, and widespread expression. 6 A-modified circRNAs play an important role in the diagnosis and screening of lung adenocarcinoma; however, current detection methods for these RNAs are not standardized, and existing studies suffer from low sensitivity and specificity. To overcome these shortcomings in detection methods and efficiency, the present invention aims to provide a highly effective detection method based on m... 6A screening and diagnostic kit using A-modified hsa_circ_0079039 as a biomarker molecule was developed to improve the screening and diagnostic efficacy of lung adenocarcinoma. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the existing technology and provide a method based on m 6 Diagnostic biomarkers for lung adenocarcinoma based on methylated circular RNA and their applications.

[0006] To address the aforementioned technical problems, this invention discloses a method based on m 6 A diagnostic biomarker for lung adenocarcinoma based on methylated circular RNA and its application. The specific technical solution is as follows:

[0007] This invention provides a method for detecting circular RNA m 6 The application of reagents with varying levels of methylation modification in the preparation of diagnostic kits for lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1.

[0008] Secondly, the present invention provides a lung adenocarcinoma diagnostic kit, which includes an m-type detector for detecting circular RNA in a sample. 6 A reagent for methylation modification level, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1.

[0009] The lung adenocarcinoma diagnostic kit includes a detection method for detecting circular RNA located in the 3'UTR region of exon 7 in a sample. 6 A reagent with a methylation modification level.

[0010] The lung adenocarcinoma diagnostic kit uses MeRIP-qRTPCR technology to detect the m-bandinium of circular RNA in the sample. 6 A. Methylation modification level.

[0011] Among them, the m of the circular RNA in the detection sample 6 The reagents for A-level methylation modification include any one or two primer pairs from (1) to (2) below:

[0012] (1) Primer pairs consisting of the nucleotide sequences shown in SEQ ID No. 8 and SEQ ID No. 9;

[0013] (2) Primer pairs consisting of the nucleotide sequences shown in SEQ ID No. 10 and SEQ ID No. 11.

[0014] Among them, the m of the circular RNA in the detection sample 6The reagent for A-level methylation modification includes a primer pair for amplifying an internal reference gene, wherein the internal reference gene is GAPDH. The nucleotide sequence of the primer pair for amplifying the internal reference gene is shown in SEQ ID No. 6-7.

[0015] The samples mentioned include the patient's serum, plasma, or tumor tissue.

[0016] Preferably, the present invention also provides a primer pair for detecting the expression level of hsa_circ_0079039, the primer pair consisting of the nucleotide sequences shown in SEQ ID No. 4 and SEQ ID No. 5.

[0017] Thirdly, the present invention provides the application of circular RNA as a therapeutic target in the preparation of drugs for treating lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1.

[0018] Fourthly, the present invention provides m-type inhibitors for inhibiting circular RNA. 6 The application of a reagent with a methylation modification level in the preparation of a drug for treating lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1.

[0019] Furthermore, this study found that the heterogeneous nucleoribonucleoprotein HNRNPC binds to and regulates the m-molecule of the circular RNA hsa_circ_0079039 in lung adenocarcinoma. 6 A. Methylation modification level. HNRNPC is RNA m 6 In addition to the protein A recognition method, this invention also provides a method for detecting hsa_circ_0079039 based on MeRIP-WB. 6 A primer pair that binds to the modified site HNRNPC. The primer pair consists of the positive strand primer shown in SEQ ID No. 12 and the negative strand primer shown in SEQ ID No. 13.

[0020] Beneficial effects:

[0021] The technical problem to be solved by the present invention is to provide a biomarker, or a reagent for detecting the biomarker, or a m-type RNA of the biomarker. 6 Application of reagents with varying levels of methylation modification in lung adenocarcinoma screening and early diagnosis. The technical problems to be solved are not limited to the described technical topics; other technical topics not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0022] The technical effects and advantages of this invention are as follows:

[0023] 1. This invention collected 6 cases of lung adenocarcinoma tumors and normal tissues, and used RNA methylation immunoprecipitation sequencing (MeRIP-seq / m 6 Analytical sequencing (A-seq) using alpha-seq technology revealed 177 differentially regulated circRNAs in tumors compared to normal tissues, with 148 significantly upregulated and 29 significantly downregulated. Compared to normal tissues, m 6 A-methylated hsa_circ_0079039 was upregulated in tumor tissues. 6 The A methylation modification peak is located in the 3'UTR region of exon 7, with conserved motifs GGACC and GGACA.

[0024] 2. In this invention, PC-9 cells were infected with lentiviral vectors that overexpressed and knocked down HNRNPC, respectively. qRT-PCR experiments showed that knocking down HNRNPC significantly reduced the expression level of hsa_circ_0079039. Treatment with actinomycin D significantly increased the stability of hsa_circ_0079039 in the HNRNPC overexpression group (P<0.05).

[0025] 3. This invention uses m 6 A-methylation immunoprecipitation combined with real-time quantitative PCR (MeRIP-qPCR) to verify m 6 A. Methylation modification level. Results showed that both motifs of hsa_circ_0079039 located in the 3'UTR region of Exon7 were detectable in both the control group and the HNRNPC knockout group cells. 6 A. Methylation modification. In HNRNPC knockout cells, the methylation modification level of hsa_circ_0079039 was significantly lower than that in the empty vector group; while in HNRNPC overexpression cells, the methylation modification level of hsa_circ_0079039 was significantly higher than that in the empty vector group (P<0.05).

[0026] 4. This invention uses an RNA pull-down assay combined with Western blotting (WB) to verify the binding site of hsa_circ_0079039 to HNRNPC. The results showed that in the control group cells, HNRNPC could bind to the mα of hsa_circ_0079039. 6 Binding occurs at the A methylation modification site. Compared with control cells, HNRNPC-overexpressing cells have more HNRNPC binding to hsa_circ_0079039.

[0027] 5. This invention uses MeRIP-qPCR to detect lung adenocarcinoma tumors and normal tissues in 40 cases. Compared with normal tissues, m 6A-modified hsa_circ_0079039 significantly upregulated expression in lung adenocarcinoma tumor tissues (P<0.0001); ROC curve analysis showed that m 6 The area under the curve (AUC) of the modified hsa_circ_0079039 in distinguishing lung adenocarcinoma from normal tissue was 0.894, with a sensitivity of 80.0% and a specificity of 92.5%.

[0028] 6. This invention verifies that HNRNPC can upregulate the m-value of hsa_circ_0079039 by knocking out / overexpressing HNRNPC / hsa_circ_0079039. 6 A methylation modification level. Cell viability and animal experiments verified that inhibiting HNRNPC expression levels could downregulate the m-methylation level of hsa_circ_0079039. 6 A methylation modification level reduces the activity of lung adenocarcinoma cells, thereby treating lung adenocarcinoma.

[0029] In summary, this invention is the first to discover m in the tissue of patients with lung adenocarcinoma. 6 A-methylation significantly increased the level of hsa_circ_0079039, and hsa_circ_0079039 can bind to RNA m 6 A recognizes the interaction with the HNRNPC protein, m 6 A-methylated hsa_circ_0079039 can serve as a screening and diagnostic target for early-stage lung adenocarcinoma.

[0030] Compared with existing technologies, this invention has the following advantages: This invention is the first to discover m in the tissue of lung adenocarcinoma patients. 6 A-modified hsa_circ_0079039 levels were significantly increased, and this is the first time that hsa_circ_0079039 has been found to interact with RNA m 6 A recognizes the interaction with the HNRNPC protein. Additionally, current m-type RNA targeting circular RNA... 6 There is no standardized method for detecting A-methylation modification levels, and existing studies suffer from low sensitivity and specificity. To overcome these shortcomings in detection methods and efficiency, this invention provides a method with high diagnostic efficacy based on m... 6 A screening and diagnostic kit using methylated hsa_circ_0079039 as a biomarker was developed to improve the screening and diagnostic efficacy of lung adenocarcinoma. Attached Figure Description

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0032] Figure 1 To analyze mRNA m from lung adenocarcinoma and normal tissues based on transcriptomic and epitranscriptomic data 6 A. Methylation modification and differential expression of circRNAs. Figure 1 In this context, A represents m in lung adenocarcinoma tumor tissue. 6 Volcano plot of circRNAs showing differential expression due to A-methylation modification; red dots represent m 6 circRNAs with statistically significant differences in A methylation modification are represented by light gray dots. 6 A-methylation modifications did not show statistically significant differences in circRNAs. Figure 1 B in the equation is m 6 The expression levels of the 20 circRNAs with the most significant fold differences in methylation modification (up or down).

[0033] Figure 2 m in tumors and normal tissues 6 The KEGG enrichment pathway of circRNAs with varying levels of A-methylation modification. Figure 2 In this context, A represents m in the tumor tissue. 6 KEGG enrichment pathway of circRNAs with significantly upregulated A methylation levels; Figure 2 In this context, B represents m in the tumor tissue. 6 The KEGG enrichment pathway of circRNAs with significantly downregulated A methylation levels.

[0034] Figure 3 RNA m of hsa_circ_0079039 in tumors and normal tissues 6 A diagram showing the methylation modification peaks and sites. Figure 3 In this context, A represents the RNA m of hsa_circ_0079039 in both tumor and normal tissues. 6 Peak analysis of A-modified variants; Figure 3 In this context, B represents the RNA m of hsa_circ_0079039 in both tumor and normal tissues. 6 A schematic diagram of the site modified by A.

[0035] Figure 4 Comparison of the relative expression levels of hsa_circ_0079039 in different cell groups. Figure 4 In the figure, A represents the comparison of the level changes of hsa_circ_0079039 in the control group (ctrl), empty vector group (NC), and HNRNPC overexpression (OE-HN) cells in the qRT-PCR validation experiment. Figure 4In the figure, B represents the relative expression level of hsa_circ_0079039 over time in cells of the empty vector group and the HNRNPC overexpression group after treatment with actinomycin D. *p<0.05, **p<0.01, ***p<0.001, NS: no statistical difference.

[0036] Figure 5 For different cell groups, the m of hsa_circ_0079039 6 A. Methylation modification level. Figure 5 In this context, A represents the relative m of hsa_circ_0079039 detected using the hsa_circ_0079039-1 primer pair in knockout empty vector (shNC) and HNRNPC knockout (shHN) cells. 6 A methylation modification ratio (Relative m) 6 A Level); Figure 5 In the figure, B represents the detection of m-values ​​of hsa_circ_0079039 using the hsa_circ_0079039-1 primer pair in cells overexpressing the empty vector (OE-NC) and HNRNPC (OE-HN). 6 A. Methylation modification ratio; Figure 5 In the C group, HNRNPC knockout and overexpression cells are used to detect the m of hsa_circ_0079039-1 using the hsa_circ_0079039-1 primer. 6 A. Methylation enrichment factor (Relative Fold Enrichment); Figure 5 In the D group, HNRNPC knockout cells were used to detect the m of hsa_circ_0079039 using the hsa_circ_0079039-2 primer pair. 6 A. Methylation modification ratio; Figure 5 In the E group, HNRNPC overexpression cells were used to detect the m of hsa_circ_0079039 using the hsa_circ_0079039-2 primer pair. 6 A. Methylation modification ratio; Figure 5 In the figure, F represents the hsa_circ_0079039-2 primer pair used to detect the m of hsa_circ_0079039 in HNRNPC knockout and overexpression cells. 6 A. Enrichment factor of methylation modification; Figure 5 In the figure, G represents the effect of targeting m in HNRNPC-overexpressing cells. 6 WB map of the detection of hsa_circ_0079039 binding to HNRNPC using methylation primer pairs at methylation modification sites; Figure 5 In this context, H represents the effect of targeting m in the control group and HNRNPC overexpression group cells.6 WB comparison of methylation primer pairs at methylation modification sites for detecting the binding of hsa_circ_0079039 to HNRNPC. p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference.

[0037] Figure 6 The m of hsa_circ_0079039 in tumor tissue and normal tissue of lung adenocarcinoma patients 6 A. Methylation modification level and evaluation. Figure 6 In this context, A is hsa_circ_0079039 relative to m. 6 A methylation modification ratio (Relative m) 6 A Level); Figure 6 B in the equation is m of hsa_circ_0079039 6 A-methylation enrichment factor (Relative Fold Enrichment) Figure 6 In this context, C represents the relative m of hsa_circ_0079039 in tumor tissue. 6 The correlation between the A methylation modification ratio and HNRNPC expression level (qPCR value); Figure 6 In the middle D, the correlation between the expression levels of hsa_circ_0079039 and HNRNPC in tumor tissue is shown. Figure 6 E represents the receiver operating characteristic curve. p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference.

[0038] Figure 7 For HNRNPC, increase the m of hsa_circ_0079039. 6 A methylation modification level inhibits the activity of lung adenocarcinoma cells. Figure 7 In this context, A represents the relative m of each group of cells. 6 A methylation modification ratio (Relative m) 6 A Level); Figure 7 In this context, B represents the cell viability of each group of cells. Figure 7 In the diagram, C represents the cell colony formation experimental plate diagram for each group of cells; Figure 7 In this context, D represents the cell clone formation data for each group of cells; Figure 7 E in the figure represents the results of subcutaneous tumor formation experiments in nude mice treated with cells from each group; Figure 7 In the figure, F represents the subcutaneous tumor weight data of each group of cells in the subcutaneous tumor formation experiment in nude mice. Figure 7 G represents the relative m of subcutaneous tumor tissue from nude mice treated with cells in each group. 6A. Methylation modification ratio. p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference. Detailed Implementation

[0039] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0041] The sources of the key materials, reagents, and experimental techniques used in the following examples are as follows:

[0042] m 6 A MeRIP kit ( m 6 A MeRIP Kit (catalog number: GS-ET-006), RNA Pull down kit ( The RNA Pull down Kit (catalog number: GS-EG-010) was provided by Shanghai Yunxu Biotechnology Co., Ltd.

[0043] HNRNPC antibody (recombinant Anti-hnRNP C1 / C2 antibody [9G1], catalog number: ab314004), m 6 Antibody A (Anti-N6-methyladenosine (m6A) antibody [17-3-4-1], catalog number: ab208577), GAPDH antibody (Anti-GAPDH antibody [6C5]-Loading Control, catalog number: ab8245), IgG (Normal rabbit IgG, monoclonal antibody [EPR25A], catalog number: ab172730), HRP-goat anti-rabbit secondary antibody (HRP Goat Anti-Rabbit, catalog number: ab6721), and HRP-rabbit anti-mouse secondary antibody (HRP Goat Anti-Rabbit, catalog number: ab6728) were all provided by Shanghai Abcam Company.

[0044] 2× Protein Loading Buffer (Catalog No.: G2032), SDS-PAGE Gel Preparation Kit (Catalog No.: G2003), and Ultrasensitive ECL Chemiluminescence Kit (Catalog No.: G2020) were all provided by Wuhan Sewell Biotechnology Co., Ltd.

[0045] qRT-PCR assay kits: qRT-PCR kits ( The 2-Step RT-qPCR System (catalog number: A6010) was provided by Prometheus (Beijing) Biotechnology Co., Ltd.

[0046] Cell lysate (Pierce) TM IP lysis buffer, catalog number: 87787), TRIzol reagent (TRIzol TM Product code: 15596026CN; Biotinylation kit (Pierce) TM Biotin 3' end DNA labeling kit (catalog number: 89818) is manufactured by Thermo Scientific. TM Provided by the company.

[0047] Example 1: Recruitment of Lung Adenocarcinoma Patients and Collection of Clinical Samples

[0048] This retrospective analysis included data from 46 patients with lung adenocarcinoma who underwent surgery at Nanjing Drum Tower Hospital between January 2022 and January 2023. None of the patients had received radiofrequency ablation, chemotherapy, or radiotherapy prior to surgery. Postoperative pathological diagnosis was lung adenocarcinoma. Ninety-two frozen tumor tissue and normal tissue samples (distance >5 cm from the tumor) were collected. All patients signed informed consent forms to donate samples, and the donation was approved by the Ethics Committee of Nanjing Drum Tower Hospital (Ethics No.: 2023-485-01). Tumor and normal tissue sections were pathologically sectioned and stained with hematoxylin and eosin (HE). All sections were examined by two pathologists, and the samples were staged and classified according to the 2017 IASLC (International Association for the Study of Lung Cancer) 8th edition TNM staging criteria and the 2015 WHO lung adenocarcinoma classification criteria.

[0049] Example 2: Lung adenocarcinoma tissue m 6 A. circRNA sequencing and differential circRNA analysis

[0050] 1. Extraction of total RNA from frozen tissue

[0051] Frozen tumor and normal paired tissues from six recruited patients in Example 1 were selected, and total RNA was extracted from the frozen tissues of the six lung adenocarcinoma patients and the corresponding normal frozen tissues using the Trizol method. RNA concentration and purity were determined and assessed using a NanoDrop One (Thermo) ultra-micro UV-Vis spectrophotometer. The average A260 / A280 ratio was 1.8–2.0.

[0052] 2. RNA m 6 A-methylation sequencing

[0053] RNA m 6 A-methylation sequencing service was provided by Shanghai Yunxu Biotechnology Co., Ltd. (via m...) 6 A MeRIP kit for m 6 A MeRIP reaction, briefly described as follows: The extracted total RNA is randomly fragmented into fragments of approximately 200 nt. Using... m 6 A MeRIP Kit that combines magnetic beads with m 6 Antibody A was incubated at room temperature by rotation for 1 hour to allow the antibody to bind to the magnetic beads. The RNA fragment was then incubated with the antibody bound to the magnetic beads at 4° for 4 hours to allow the RNA to bind to the antibody. The bound complex was washed several times to further elute the RNA from the complex. An RNA sequencing library was constructed. The constructed library was quality controlled using an Agilent 2100 bioanalyzer, and then subjected to high-throughput sequencing on the sequencer.

[0054] 3. circRNAs analysis and screening criteria

[0055] RNA m 6 The A-methylation sequencing data analysis service was provided by Shanghai Yunxu Biotechnology Co., Ltd. The analysis workflow is briefly described as follows: After sequencing, image analysis, base identification, and quality control, raw reads are generated. First, Q30 is used for quality control. Then, cutadapt software (v1.9.3) is used to remove adapters and low-quality reads, obtaining clean reads. STAR software (v2.5.1b) is used to align high-quality reads to a reference genome, and DCC software (v0.4.4) is used for circular RNA detection and identification. The identified circular RNAs are then annotated using the circBase database and Circ2Traits.

[0056] According to RNA m 6 A. Screening for target circRNAs based on methylation sequencing results: (1) fold change (FC) greater than 1 between normal tissue and lung adenocarcinoma tissue, p-value less than 0.05; (2) circRNAs with length between 200bp and 1000bp; (3) circRNAs from lung adenocarcinoma m 6 A. circRNAs with a methylation peak intensity greater than 500. Comprehensive analysis revealed differentially expressed circRNAs in the cohort of lung adenocarcinoma patients, and KEGG pathway enrichment analysis was performed on the source genes of these differentially modified circRNAs.

[0057] 4. Results

[0058] Sequencing results showed that, compared with normal tissue, the tumor group had m 6Among the differentially regulated circRNAs, 177 were selected with p < 0.05 and FC ≥ 1. Of these, 148 were significantly upregulated and 29 were significantly downregulated. Figure 1 (A) Due to the large number of differentially expressed circRNAs, the 20 most significant mRNAs were selected by fold change. 6 AcircRNAs were used to create a heatmap. Figure 1 B). The upregulated differentially expressed circRNAs were enriched by KEGG primarily in ubiquitin-mediated proteolytic pathways and tumor-associated molecular pathways (B). Figure 2 In the A group, the KEGG enrichment pathways of downregulated differentially regulated circRNAs are mainly concentrated in the peroxidation and glucose metabolism pathways. Figure 2 (B) Compared with normal tissue, tumor tissue showed a significant difference. The hsa_circ_0079039 modification peak was located in the 3'UTR region of exon 7. Figure 3 In the diagram, A), the conserved motifs are GGACC and GGACA, and a schematic diagram is shown based on their positions. Figure 3 (B) and designed primers for MeRIP-qPCR.

[0059] Example 3: HNRNPC modulates the expression level of hsa_circ_0079039

[0060] 1. Cell Culture and Construction

[0061] 1.1 Cell Acquisition and Culture

[0062] Human lung cancer cells PC-9 were cultured in DMEM medium (containing 10% v / v fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin) in a cell culture incubator at 37°C and 5% CO2. Cells in good condition were digested, resuspended, and seeded into 6-well plates, then incubated overnight at 37°C.

[0063] 1.2 Construction of overexpression cells

[0064] A lentiviral vector (pLVX-CMV) overexpressing heterogeneous nucleoribonucleoprotein (HNRNPC, encoded by a gene with the nucleotide sequence as shown in SEQ ID No. 16) was constructed by Shanghai Jikai Gene Medical Technology Co., Ltd. The required volumes of empty vector (empty viral vector) and HNRNPC virus were calculated based on an infection MOI of 30, with a total volume of approximately 1 mL. Polybrene, a gene transfection enhancer, was added to a final concentration of 5 μg / mL to obtain the viral solution. The original culture medium in 6-well plates was replaced with the viral solution, and the plates were incubated overnight at 37°C. After 24 hours, the viral solution was aspirated, and fresh DMEM medium containing puromycin (1 μg / mL) was added, and the plates were incubated at 37°C. The medium was replaced with fresh DMEM medium containing puromycin (1 μg / mL) every 2-3 days, and cell status was observed daily until all cells in the empty vector group died. The HNRNPC overexpressing cells were then passaged, and the expression levels of the target gene in each cellular component were detected by real-time quantitative PCR (qRT-PCR). Meanwhile, cells that were not transfected with any vector were used as a control group (ctrl).

[0065] 1.3 Actinomycin D treatment

[0066] Collect cells and transfer them to 15 mL centrifuge tubes. Centrifuge at 1500 rpm for 5 min at 4 °C. Add fresh DMEM medium, gently mix, count cells, and adjust the volume to 2 × 10⁶ cells / mL. 6 Cells were seeded at 2.5 mL per well in 6-well plates. Cells were treated with 10 μg / mL actinomycin D, and cells were collected after 0, 2, and 4 hours of treatment for subsequent operations. 2. qRT-PCR was used to detect the expression level of hsa_circ_0079039.

[0067] 2.1 Total RNA Extraction

[0068] Total RNA was extracted from HNRNPC overexpressing cells (OE-HN), control cells (ctrl), and empty vector group cells (NC) using the TRIzol method. RNA concentration and purity were determined and assessed using a NanoDrop One ultra-micro UV-Vis spectrophotometer (Thermo). The mean A260 / A280 ratio was 1.8–2.0.

[0069] 2.2 cDNA Synthesis

[0070] (1) GAPDH was selected as the internal reference gene. Primers were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primer sequences are shown in Table 1:

[0071] Table 1. List of qRT-PCR primers

[0072]

[0073] (2) Add the RNA template and primer mixture to a new reaction tube according to Table 2. Place the reaction tube in a 70°C temperature-controlled module and incubate for 5 min. Immediately cool in an ice bath for at least 5 min. Centrifuge each reaction tube for 10 seconds on a microcentrifuge to allow the contents to settle to the bottom and maintain the original volume. Tightly cap the reaction tube and incubate on ice.

[0074] Table 2 RNA template and primer mixing system

[0075] Components Amount added per reaction RNA template 0.8μg Random Primers 1μL Oligo(dT) 15 Primer 1μL Nuclease-Free Water XμL Total volume 10μL

[0076] (3) Prepare the RT-MiX mixture on ice according to Table 3. Add each component to a 1.5 mL sterile microcentrifuge reaction tube, gently vortex to mix, and place on ice.

[0077] Table 3 Reverse transcription RT-MiX mixture system

[0078]

[0079] (4) Add 10 μL of the reverse transcription reaction mixture from step (3) to the reaction tube from step (2), and bring the volume to a final volume of 20 μL. Incubate at 25°C for 5 min, then at 25°C for 60 min, and finally at 70°C for 15 min. Store the obtained cDNA on ice or at -20°C.

[0080] 2.3 qRT-PCR

[0081] (1) Prepare the reaction mixture according to Table 4 (without adding cDNA template).

[0082] Table 4 qRT-PCR amplification system

[0083]

[0084] (2) Sample addition

[0085] Add the cDNA template-free mixture to each well of the Roche 384 PCR plate, and add the synthesized cDNA template (or template-free control water) to the corresponding wells. Seal with Sealing Film and briefly centrifuge to mix. Place the prepared PCR plate on ice before setting the PCR program. Perform triple replicates for each sample.

[0086] (3) qRT-PCR amplification program settings: 95℃, 2min; thermal cycling: 95℃, 15s; 60℃, 1min (40 cycles); melting curve: 95℃, 15s; 60℃, 1min, 95℃, 15s.

[0087] 3. Statistical methods

[0088] All statistical analyses were performed using R software (version 4.0.2), SPSS software (version 19.0), and GraphPad Prism (version 8.4.1). A 2-1 index was used. -ΔΔCT The relative expression levels of HNRNPC and hsa_circ_0079039 in qRT-PCR were calculated, and the data are expressed as mean ± standard deviation. The Shapiro-Wilk test was used to check the normality of the data. For normally distributed data, independent samples t-tests or one-way ANOVA were used to analyze differences between groups. For non-normally distributed data, the Mann-Whitney U test was used to analyze differences between groups.

[0089] 4. Results

[0090] (1) qRT-PCR experiments showed that, compared with the control group (ctrl) and empty vector group (NC) cells, the expression level of hsa_circ_0079039 was significantly increased after overexpression of HNRNPC (OE-HN group). Figure 4 A in the middle;

[0091] (2) After treatment with actinomycin D, the stability of hsa_circ_0079039 in the HNRNPC overexpression group was significantly increased compared with the empty vector group (NC) (P<0.05). Figure 4 (B in the middle).

[0092] Example 4: HNRNPC regulation of hsa_circ_0079039's m 6 A methylation modification level

[0093] 1. Construction of knockout and overexpression cells

[0094] The HNRNPC overexpression lentiviral vector (same as in Example 3) and the HNRNPC knockout lentiviral vector (pLKO.1 vector) were constructed by Shanghai Jikai Gene Medical Technology Co., Ltd. The construction methods for overexpression or knockout cells are the same as those in Sections 1.1-1.2 of Example 3.

[0095] 2. MeRIP assay for methylated RNA

[0096] 2.1 Total RNA Extraction

[0097] The method is the same as in Section 2.1 of Example 3.

[0098] 2.2 RNA fragmentation

[0099] (1) Nuclease-free water was used to adjust the total RNA concentration of each group of cells in Example 2.1 to 1 μg / μL.

[0100] (2) Add 18 μL (~18 μg) of total RNA to each PCR tube. Add 2 μL of 10×Fragmentation Buffer to each tube, vortex to mix and centrifuge briefly;

[0101] (3) Place the PCR tubes in a PCR instrument preheated to 70°C and incubate for 6 min; add 2 μL of Stop Buffer to each tube, vortex to mix and briefly centrifuge, then place the tubes on ice. Repeat this step 2-3 times until all RNA fragments are formed.

[0102] (4) Combine the reaction solutions of the same sample and transfer them to a new 1.5 mL EP tube. Adjust the total volume to 270 μL with Nuclease-free water. Add 30 μL PC Buffer, 1 μL PC Enhancer, and 750 μL anhydrous ethanol. Mix gently and incubate at -80°C overnight for precipitation.

[0103] (5) Transfer the overnight precipitated sample to a refrigerated centrifuge and centrifuge at 4°C and 15,000g for 25 min. Aspirate the supernatant and keep the RNA precipitate stable. Gently and slowly add 1 mL of 75% ethanol. Centrifuge at 4°C and 15,000g for 15 min.

[0104] (6) Aspirate the supernatant to stabilize the RNA precipitate. Open the tube cap and allow the sample tube to dry at room temperature for 2-5 minutes. Add 50 μL of Nuclease-free water to fully dissolve the RNA precipitate and place on ice for later use.

[0105] (7) RNA fragment size and concentration were detected using the Qsep100 Automatic Nucleic Acid and Protein Analysis System (Bioptic). The RNA fragment size was ~200 nt.

[0106] (8) Take 1-3 μg of fragmented RNA as the Input group and store it at -80℃ for later use. The remaining fragmented RNA will be used for subsequent immunoprecipitation experiments as the IP group.

[0107] 2.3 Preparation of Immunoprecipitation Magnetic Beads

[0108] (1) Prepare a new 1.5 mL EP tube for each reaction and add 25 μL of resuspended PGM magnetic beads into the reaction tube.

[0109] (2) Wash the PGM beads with 1×IP buffer. Add 200 μL of 1×IP buffer to each tube; place the reaction tube on a magnetic rack until the solution is clear; aspirate the supernatant without stirring the beads. Repeat this step once.

[0110] (3) Add 50 μL of 1×IP buffer to each tube and mix gently with a pipette.

[0111] (4) IP group: Add 2μL m to each tube 6 Antibody A. IgG group: Add 2 μL of IgG antibody to each tube as a control. Incubate on a shaker at room temperature for 1 hour. Briefly centrifuge and collect the solution to the bottom of the tube. Place the reaction tube on a magnetic rack until the solution becomes clear. Discard the supernatant, without stirring the magnetic beads.

[0112] (5) Add 200 μL of 1×IP buffer to each centrifuge tube to wash the magnetic beads. Place the reaction tube on a magnetic rack until the solution is clear. Discard the supernatant without stirring the magnetic beads. Remove the centrifuge tube, cap it, and place it on ice for later use.

[0113] 2.4 Immunoprecipitation

[0114] (1) Prepare the MeRIP reaction solution according to Table 5:

[0115] Table 5 MeRIP reaction solution system

[0116] reagents volume Fragmented RNA XμL Nuclease-free Water (200-X)μL 5×IP buffer 50μL Total 250μL

[0117] (2) Add 250 μL of the above mixture to the magnetic beads prepared in step 2.3. Gently pipette several times to mix and completely resuspend the magnetic beads. Incubate at 4°C with rotation for 1 hour. Briefly centrifuge to collect the solution to the bottom of the tube. Place the reaction tube on a magnetic rack until the solution becomes clear. Discard the supernatant without stirring the magnetic beads.

[0118] (3) Add 200 μL of 1×IP buffer to each tube to wash the magnetic beads, and place the reaction tube on a magnetic rack until the solution is clear; discard the supernatant without stirring the magnetic beads. Repeat this step once.

[0119] (4) Add 200 μL of 1×LB buffer to each tube to wash the magnetic beads, and place the reaction tube on a magnetic rack until the solution is clear; discard the supernatant without stirring the magnetic beads. Repeat this step once.

[0120] (5) Add 200 μL of 1×HS buffer to each tube to wash the magnetic beads, and place the reaction tube on a magnetic rack until the solution is clear; discard the supernatant without stirring the magnetic beads. Repeat this step once.

[0121] 2.5 RNA purification

[0122] (1) Add 30 μL of RLT Buffer to each tube to resuspend the prepared magnetic beads and incubate at room temperature for 1 min. Place the centrifuge tube on a magnetic rack until the solution is clear. Transfer the supernatant to a new 1.5 mL centrifuge tube and place on ice for later use.

[0123] (2) For each reaction, prepare a new 1.5 mL EP tube and transfer 20 μL of resuspended MS magnetic beads into the new tube and place it on a magnetic rack until the solution is clear. Discard the supernatant without stirring the MS magnetic beads.

[0124] (3) Add 100 μL of RLT Buffer to each tube to completely resuspend the MS beads. Place the reaction tube on a magnetic rack until the solution is clear. Discard the supernatant without stirring the MS beads.

[0125] (4) Add 30 μL of RLT Buffer to each tube to resuspend the MS magnetic beads and add it to the supernatant of step (1) in 3.4.

[0126] (5) Add 60 μL of anhydrous ethanol to each tube, mix gently, and incubate at room temperature for 1 min. Place the centrifuge tubes on a magnetic rack until the solution becomes clear. Discard the supernatant, without stirring the MS magnetic beads.

[0127] (6) Add 200 μL of 75% ethanol to each tube to clean the MS magnetic beads. Place the centrifuge tube on a magnetic rack until the solution becomes clear. Discard the supernatant without stirring the magnetic beads. Repeat this step to clean the beads again.

[0128] (7) Dry at room temperature for 5 min. Resuspend completely in 11.25 μL of nuclease-free water and incubate at room temperature for 2 min.

[0129] (8) Transfer 10 μL of supernatant (eluted RNA) to a new centrifuge tube. The RNA sample can be used immediately for subsequent experiments or stored at -80°C for later use.

[0130] 3. qRT-PCR experimental procedure

[0131] (1) cDNA synthesis

[0132] Based on the two m 6 Primers were designed for the modified site A and synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 6. Other methods are the same as in Section 2.2 of Example 3.

[0133] Table 6 m 6 A modified site qRT-PCR primers

[0134]

[0135] (2) qRT-PCR

[0136] The methylation modification level of hsa_circ_0079039 was detected by qRT-PCR, using the same method as in Section 2.3 of Example 3.

[0137] (3) Calculation of methylation modification level

[0138] The methylation modification ratio of each transcript is calculated using the following formula (Relative m). 6 A Level) and Relative Fold Enrichment, where DF and IF are m 6 Dilution factors for antibody A and antibody IgG:

[0139] A = Merip - m6a / Input (%) = 2 (Ct Input-Ct IP) *DF*100

[0140] B = Merip-IgG / Input (%) = 2 (Ct Input-Ct IP) *IF*100

[0141] Relative Fold Enrichment=A / B

[0142] 4. RNA pull-down experiment

[0143] 4.1 hsa_circ_0079039m 6 Preparation of modified sequence probes

[0144] According to hsa_circ_0079039m 6 A modified sequence was used to design sense and antisense probes, as shown in Table 7. The probes were synthesized by Shanghai Yunxu Biotechnology Co., Ltd.

[0145] Table 7 m 6 A modified sequence for sense and antisense strand probe sequences.

[0146] name Primer sequence (5'-3' ends) Serial Number Methylation sense strand probe AGAGCTTGAGGACCGTCttggctttcacggtcgc SEQ ID No. 12 Methylated antisense probe gcgaccgtgaaagccaaGACGGACCTCAAGCTCT SEQ ID No. 13

[0147] 4.2 Labeled Probe

[0148] (1) Thaw all components of the kit except for terminal deoxynucleotidyl transferase (TdT) and place them on ice. Store TdT at -20°C until use. Just before use, dilute a portion of the TdT stock solution to a working concentration of 1.5 U / μL with TdT reaction buffer.

[0149] (2) Add the components in the order listed in Table 8 to prepare the labeling reaction for the control system. Prepare the sample labeling reaction in the same way, replacing the unlabeled control oligonucleotide with a 5 pmol 3'-OH-terminated probe.

[0150] (3) Incubate the reaction at 37°C for 30 min.

[0151] Table 8 Biotin-labeled reaction mixture system

[0152] Components Amount added per reaction 5X TdT Reaction Buffer 10μL Biotin-11-UTP (5μM) 5μL Diluted TdT (1.5 U / μL) 5μL Unlabeled Control Oligo (1μM) or probe 5μL Ultrapure water XμL Total volume 50μL

[0153] (4) Add 2.5 μL of 0.2 M EDTA to each reaction to terminate the reaction.

[0154] (5) Add 50 μL of chloroform:isoamyl alcohol (24:1 v / v) to each reaction to extract TdT. Vortex briefly to mix, then centrifuge at high speed for 1-2 min in a microcentrifuge to separate the solutions. Remove and store the upper layer (aqueous phase).

[0155] 4.3 Cell lysis

[0156] (1) Collect the cell suspensions of HNRNPC overexpressing cells and control group cells (as constructed by the method described in Example 3) into 1.5 mL centrifuge tubes;

[0157] (2) Centrifuge at 1,000×g for 5 min to precipitate the cells, and carefully discard the supernatant. Resuspend the cell pellet in ice-cold PBS and centrifuge at 1,000×g for 5 min to precipitate the cells again.

[0158] (3) Add ice-cold Pierce IP lysis buffer to the cell pellet at a ratio of 500 μL buffer per 50 mg wet weight cell pellet (buffer: wet weight cells = 10:1, volume μL / weight mg).

[0159] (4) Incubate on ice for 5 minutes, mixing regularly during the process to ensure effective lysis.

[0160] (5) Centrifuge at 13,000×g for 10 min at 4℃ to remove cell debris. Collect the supernatant: Transfer the supernatant to a clean, nuclease-free tube for protein concentration determination and subsequent analysis.

[0161] (6) Take 10 μL of protein lysis product as input group, and divide the remaining protein lysis product into two parts, one part for positive probe RNA pull-down reaction and the other part for negative probe RNA pull-down reaction.

[0162] 4.4 RNA Pull-down

[0163] The RNA pull-down experiment was performed according to the kit instructions, and included the following steps:

[0164] (1) Take 3 μL of 100 μM biotin-labeled probe, add 47 μL RNA Structure Buffer, heat at 95 °C for 2 min, immediately place on ice, let stand for 3 min, and then let stand at room temperature for 30 min.

[0165] (2) Prepare the incubation buffer according to Table 9. After preparation, gently pipette to mix and rotate at room temperature for 1 hour.

[0166] Table 9. Master Mixture System for Probe-Protein Binding Reaction

[0167] Components Amount added per reaction RNase Inhibitor 5μL Protease Inhibitor 2.5μL 10×Binding Buffer 50μL Biotin-labeled probes 50μL Cell lysis products 192.5μL Nuclease-Free Water 200μL Total volume 500μL

[0168] (3) Add 100 μL of the main mixture to the magnetic beads from step (1) and mix thoroughly by pipetting or gentle vortexing. Incubate at 4°C for 60 min, stirring or rotating to ensure thorough mixing;

[0169] (4) Prepare new 1.5 mL EP tubes according to the reaction number, and mark the EP tubes; briefly shake to mix the SA Beads, and take 50 μL into each prepared EP tube; place the EP tubes on a magnetic rack and wait for the solution to clarify (about 2 min); aspirate the supernatant, do not stir the magnetic beads, and remove the EP tubes from the magnetic rack; wash the SA Beads with 1× Binding Buffer. Repeat the washing step once.

[0170] (5) Add the prepared probe-protein complex to the prepared magnetic beads and gently pipette to mix. Incubate at room temperature for 2 hours. Briefly centrifuge and place the EP tube on a magnetic rack to allow the solution to clarify (approximately 2 minutes). Discard the supernatant without stirring the magnetic beads. Wash the SA beads with 500 μL of ice-cold 1× Binding Buffer. Place the EP tube on a magnetic rack to allow the solution to clarify (approximately 2 minutes). Discard the supernatant without stirring the magnetic beads. Repeat the washing step five times.

[0171] 4.5 WB detection

[0172] (1) Pretreatment before loading

[0173] Add 30 μL of 2×SDS-PAGE Loading Buffer to the magnetic beads after the above operations, and gently pipette to mix. Incubate at 95°C for 5-10 min. Place the EP tube on a magnetic rack and wait for the solution to clarify (about 2 min). Transfer the supernatant to a new EP tube, without disturbing the magnetic beads. The supernatant will be used for subsequent experiments.

[0174] (2) Preparation of polyacrylamide adhesive (SDS-PAGE)

[0175] Prepare 10 mL of 16% separating gel using an SDS-PAGE gel preparation kit. After mixing, immediately pour the gel to 2–3 mm below the comb. Let it stand at room temperature for 45 min to allow complete polymerization. Prepare 5 mL of 4% stacking gel. After mixing, immediately pour the gel to the top and vertically insert the comb. Let it stand at room temperature for 20 min to allow polymerization. Remove the comb and place the gel in the electrophoresis tank.

[0176] (3) Electrophoresis

[0177] Install the gel into the electrophoresis tank and add 1×TGS (14.4g glycine, 3.03g Tris-base, and 1g SDS dissolved in 1L deionized water). After loading the sample, set the electrophoresis apparatus to 80V. When the marker proteins show clear stratification, adjust the electrophoresis parameters to 120V and continue electrophoresis for about 1 hour.

[0178] (4) Transfer protein and immunoassay

[0179] A semi-dry transfer method was used for membrane transfer. The PVDF membrane was placed in an appropriate amount of transfer buffer (14.4 g glycine, 2.9 g Tris-base, and 200 mL methanol were fully dissolved in deionized water and then brought to a final volume of 1 L). The separating gel was removed and placed on the activated PVDF membrane, then transferred to filter paper soaked in transfer buffer, and another layer of filter paper soaked in transfer buffer was placed on top. The membrane was transferred using a semi-dry transfer apparatus, with the voltage (15-20 V) set according to the amount of gel, and the transfer time was 40 min.

[0180] After transfer, the membrane was washed three times with 1×TBST buffer and blocked with 5% skim milk for 1 hour. It was then incubated overnight at 4°C with HNRNPC antibody, followed by three washes with 1×TBST buffer. A suitable HRP-labeled secondary antibody was applied. The membrane was incubated at room temperature for 1 hour, followed by three washes with 1×TBST buffer. Protein signals were detected using an ELC luminescence assay kit on a Bio-Rad chemiluminescent gel imaging system.

[0181] 5. Results

[0182] (1) The m of cells in each group was detected using primers hsa_circ_0079039-1 and hsa_circ_0079039-2, respectively. 6 A methylation modification level, the results showed, such as Figure 5 As shown in A, C, D, and F, the m of hsa_circ_0079039 in HNRNPC knockout cells (shHN) 6 The levels of A-methylation modification were significantly lower than those of the empty vector control group (shNC); such as Figure 5 As shown in B, C, E, and F, the m of hsa_circ_0079039 in HNRNPC overexpression group cells (OE-HN) 6 The levels of A-methylation modification were significantly higher than those of the empty vector control group (OE-NC).

[0183] (2) In HNRNPC-overexpressing cells (OE-HN), HNRNPC can bind to the m of hsa_circ_0079039. 6 Binding occurs at site A ( Figure 5 The G in the text). Compared with empty vector group (OE-NC) cells, HNRNPC-overexpressing cells had more HNRNPC binding to hsa_circ_0079039 (in G). Figure 5 (H in the text). These results reveal that HNRNPC regulates the m of hsa_circ_0079039. 6 Mechanism of A-methylation modification level.

[0184] Example 5 hsa_circ_0079039's m 6 Assessment of the diagnostic value of A-methylation modification levels in lung adenocarcinoma

[0185] 1. m in patients with lung adenocarcinoma hsa_circ_0079039 6 A methylation modification level

[0186] Using the primer pair has_circ_0079039-1 in Table 6, MeRIP and qRT-PCR experiments were performed on the tumors and corresponding normal lung tissues (Normal) of another 40 patients in Example 1, following the same steps as in Example 4.

[0187] 2. m of hsa_circ_0079039 6 Assessment of the diagnostic value of A-methylation modification level in lung adenocarcinoma

[0188] The receiver operating characteristic (ROC) curve can be used to evaluate the effectiveness of one or more indicators in classifying or diagnosing tumor tissue and normal tissue in patients with lung adenocarcinoma, and to calculate the area under the curve (AUC), specificity (SP), and sensitivity (SE).

[0189] 3. Results

[0190] (1) Compared with normal control tissue, m 6 A-modified hsa_circ_0079039 showed significantly upregulated expression levels in lung adenocarcinoma tumor tissues (P<0.001). Figure 6 A in Figure 6 (B) in the middle;

[0191] (2) m of hsa_circ_0079039 in lung adenocarcinoma tumor tissue 6 A methylation modification level was significantly correlated with HNRNPC (R = 0.5298, p = 0.0004), and the expression level of hsa_circ_0079039 was significantly correlated with HNRNPC (R = 0.4143, p = 0.0079). Figure 6 C in Figure 6 (D in the middle).

[0192] (3) The analysis results of the ROC curve show that m 6 The area under the curve (AUC) of the modified hsa_circ_0079039 in differentiating lung adenocarcinoma from normal tissue was 0.894, with a sensitivity (SE) of 80.0% and a specificity (SP) of 92.5%. Figure 6 (E in the text)

[0193] Example 6: HNRNPC increases the m of hsa_circ_0079039 6 A methylation modification level inhibits lung adenocarcinoma cell activity

[0194] 1. Construction of knockout and overexpression cells

[0195] The hsa_circ_0079039 interfering RNA (which includes a sense probe and an antisense probe, the sequences of which are shown in Table 10, and the 3' end of the interfering RNA has a dTdT dT dextrin) and the hsa_circ_0079039 overexpression lentiviral vector (pLVX-CMV) were synthesized or constructed by Shanghai Jikai Gene Medical Technology Co., Ltd. The cell groups constructed included:

[0196] hsa_circ_0079039 interference group cells (si-circRNA), corresponding empty vector group (si-NC-circRNA)

[0197] hsa_circ_0079039 overexpression group cells (OE-circRNA), corresponding empty vector group (OE-NC-circRNA)

[0198] HNRNPC knockout group cells (shHN) and corresponding empty vector group (shNC)

[0199] HNRNPC overexpression group cells (OE-HN), corresponding empty vector group (OE-NC)

[0200] hsa_circ_0079039 interference + HNRNPC knockout group cells (si-circRNA + shHN)

[0201] hsa_circ_0079039 interference + HNRNPC overexpression group cells (si-circRNA + OE-HN)

[0202] hsa_circ_0079039 overexpression + HNRNPC knockout group cells (OE-circRNA+shHN)

[0203] hsa_circ_0079039 overexpression + HNRNPC overexpression group cells (OE-circRNA+OE-HN).

[0204] The cell construction method is the same as that described in sections 1.1-1.2 of Example 3.

[0205] Table 10. HSA_circ_0079039 interfering RNA (si-circRNA) sequence

[0206] name Primer sequence (5'-3' ends) Serial Number Justice Chain Probe GUACAUCUUGCGUUUCCTC SEQ ID No. 14 antisense probe GAGGAAACGCAAGAUGUAC SEQ ID No. 15

[0207] Note: In the sequence list, U is represented by T.

[0208] 2. MeRIP and qRT-PCR

[0209] MeRIP and qRT-PCR experiments were performed on cells from each group using the primer pair has_circ_0079039-1 listed in Table 6, following the same procedures as in Example 4. The results showed that in HNRNPC knockout cells and hsa_circ_0079039 interference cells, the m of hsa_circ_0079039 was... 6 The methylation modification level of A was significantly lower than that of the corresponding empty vector group. In HNRNPC-overexpressing cells and hsa_circ_0079039-overexpressing cells, the methylation level of hsa_circ_0079039 was significantly lower. 6 The methylation modification level of A was significantly higher than that of the corresponding empty vector group. Overexpression of HNRNPC could increase the m in the hsa_circ_0079039 interference group. 6 A methylation modification level, while knocking out HNRNPC can reduce m in the hsa_circ_0079039 overexpression group 6 A methylation modification level ( Figure 7 (A in the text). These results indicate that HNRNPC can upregulate the m of hsa_circ_0079039. 6 A. Methylation modification level.

[0210] 3. CCK-8 assay for cell viability

[0211] Cells in the logarithmic growth phase were seeded into 96-well plates (approximately 500 cells per well), with three replicates for each group: a control group (cell-free culture medium), an empty vector group, and an experimental group. After pre-culturing at 37°C for 24 hours, cells adhered to the plates. 10 μL of CCK-8 reagent was added directly to each well (avoiding air bubbles), and the plates were incubated in the dark for 2 hours. Once the solution turned orange-yellow, the absorbance (OD) at 450 nm was measured using a microplate reader. 450 (value); Cell viability (%) is calculated using the formula: (experimental group OD) = (experimental group OD) / ... 450 - Blank group OD 450 ) / (control group OD 450 - Blank group OD 450 The activity of each group was calculated at 100% × 100%. The results showed that the activity of HNRNPC knockout cells and hsa_circ_0079039 interference cells was significantly lower than that of their respective empty vector groups, while the activity of HNRNPC and hsa_circ_0079039 overexpression cells was significantly higher than that of their respective empty vector groups. Overexpression of HNRNPC increased the cell activity of the hsa_circ_0079039 interference group, while knockout of HNRNPC decreased the cell activity of the hsa_circ_0079039 interference group. Figure 7 (B in the text). These results demonstrate that HNRNPC can achieve this by increasing the m of hsa_circ_0079039. 6 A methylation modification level increases the activity of lung adenocarcinoma cells.

[0212] 4. Cloning experiment

[0213] Cells in the logarithmic growth phase were collected, resuspended, and counted. DMEM medium containing 10% v / v fetal bovine serum was added, and cells were seeded in 6-well plates, with 500 cells per well. Medium was added to a final volume of 2 mL, and G418 (400 μg / mL) was added at half the recommended volume for maintenance. Cell colony size was observed daily under a microscope after 3 days. The 6-well plates were removed after 10 days. Cells were fixed with paraformaldehyde, stained with crystal violet at room temperature in the dark for 15 min, washed twice with PBS, blotted dry with absorbent paper, and air-dried. Results were scanned and saved. The results showed that the colony-forming ability of hsa_circ_0079039-interfering cells was significantly lower than that of the control group (normally cultured PC-9 cells without any treatment), while the colony-forming ability of hsa_circ_0079039-overexpressing cells was significantly higher than that of the empty vector group. Overexpression of HNRNPC enhanced the clonogenic ability of the hsa_circ_0079039 interfering group, while knockout of HNRNPC reduced the clonogenic ability of the hsa_circ_0079039 overexpressing group. Figure 7(C and D in the text). These results demonstrate that HNRNPC can achieve this by upregulating the m of hsa_circ_0079039. 6 Levels of A-methylation modification promote the cloning ability of lung adenocarcinoma cells.

[0214] 5. Establishment of a subcutaneous xenograft model in nude mice

[0215] Male BALB / c nude mice (6-8 weeks old, 18-22g) were selected, and cells from each group were divided into 10 groups per mouse. 7 Subcutaneous injections were administered to the medial left axilla of nude mice, with five mice injected with each cell type. The mice were sacrificed 25 days later, and tumor tissue was dissected and weighed. Results showed that compared to the control group (injected with untreated, normally cultured PC-9 cells), the size and weight of subcutaneous tumors in the hsa_circ_0079039 overexpression group were significantly increased. Knockdown of HNRNPC inhibited tumor growth in the overexpression group (OE-circRNA). Figure 7 In addition to E and F), it also downregulated m in the OE-circRNA group. 6 A methylation modification level ( Figure 7 (G in the middle). These results indicate that inhibiting HNRNPC can reduce the m of hsa_circ_0079039. 6 A methylation modification level, thereby inhibiting tumor growth.

[0216] This invention provides a method based on m 6 This invention relates to the concept and methods of using methylated circular RNA as a diagnostic biomarker for lung adenocarcinoma and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly described in this embodiment can be implemented using existing technologies.

Claims

1. Detection of m of circular RNA 6 The application of reagents with varying levels of methylation modification in the preparation of diagnostic kits for lung adenocarcinoma, among which... The circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No.

1.

2. A diagnostic kit for lung adenocarcinoma, characterized in that, It includes the detection of circular RNA in samples. 6 A reagent for methylation modification level, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1; the lung adenocarcinoma diagnostic kit detects the m of the circular RNA in the sample using MeRIP-qRTPCR technology. 6 A. Methylation modification level; The detection of circular RNA in the sample m 6 Reagents for A-level methylation modification include m 6 Antibody A; The detection of circular RNA in the sample m 6 The reagents for A-level methylation modification include any one or two primer pairs from (1) to (2) below: (1) A primer pair consisting of the nucleotide sequences shown in SEQ ID No. 8 and SEQ ID No. 9; (2) Primer pair consisting of the nucleotide sequences shown in SEQ ID No. 10 and SEQ ID No.

11.

3. The lung adenocarcinoma diagnostic kit according to claim 2, characterized in that, The lung adenocarcinoma diagnostic kit includes a method for detecting m-banded circular RNA located in the 3'UTR region of exon 7 in a sample. 6 A reagent with a methylation modification level.

4. The lung adenocarcinoma diagnostic kit according to claim 2, characterized in that, The detection of circular RNA in the sample m 6 The reagents for A-level methylation modification include primer pairs for amplifying the internal reference gene, which is GAPDH.

5. The lung adenocarcinoma diagnostic kit according to claim 4, characterized in that, The nucleotide sequences of the primer pairs for amplifying the internal reference gene are shown in SEQ ID No. 6~7.

6. The lung adenocarcinoma diagnostic kit according to claim 2, characterized in that, The samples mentioned include the patient's serum, plasma, or tumor tissue.

7. Application of circular RNA as a therapeutic target in the preparation of drugs for treating lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No.

1.

8. Inhibition of m of circular RNA 6 The application of a reagent for A-level methylation modification in the preparation of drugs for treating lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1; the m-type inhibitor of the circular RNA... 6 The reagent for A-level methylation modification is interfering RNA, which includes a sense strand probe and an antisense strand probe, the nucleotide sequences of which are shown in SEQ ID No. 14~15, respectively.

Images