Lung adenocarcinoma diagnostic marker based on m6A methylation modified circular RNA and application of lung adenocarcinoma diagnostic marker

Abstract

The invention relates to the technical field of biology, in particular to a lung adenocarcinoma diagnosis marker based on m6A methylation modified circular RNA and application of the lung adenocarcinoma diagnosis marker. The invention discloses a circular RNA (Ribonucleic Acid) hsacirc0079039 combined with m6A recognition protein HNRNPC (Hepatoxin Nuclear Receptor Protein) and application of the circular RNA hsacirc0079039 in methylation modification of the circular RNA m6A. The hsacirc0079039 subjected to methylation modification of the m6A can be used as a biomarker for diagnosing lung adenocarcinoma. The invention has important value for screening and early diagnosis of lung adenocarcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a lung adenocarcinoma diagnostic marker based on m 6 A methylation-modified circular RNA and its application in the diagnosis of lung adenocarcinoma. Background Art

[0002] The incidence and mortality of lung cancer have been increasing year by year, and it has currently become one of the main causes of cancer-related deaths. In China, in terms of the ranking of the number of new cases, lung cancer also ranks first among malignant tumors. As the most common histological subtype of non-small cell lung cancer, lung adenocarcinoma accounts for 40% of the incidence of lung cancer. 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 to be one of the important causes of carcinogenesis. Among them, N6-methyladenosine (m 6 A) is the most abundant RNA modification found in eukaryotes so far. It is estimated that more than 7,000 coding RNAs and 3,000 non-coding RNAs in the human body have m 6 A modification. m 6 A 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. The overall abundance of m 6 A and the expression levels of its regulatory factors are closely related to the malignant biological activities such as the occurrence, proliferation, invasion and metastasis of malignant tumors.

[0004] In recent years, more and more scholars have begun to study the effect of m 6 A modification on tumor non-coding RNAs. Researchers have identified some specific m 6 A-modified circRNAs in the tissues and blood samples of early-stage lung adenocarcinoma patients. These abnormal circRNAs are more likely to promote the malignant biological processes of tumor cells and regulate the progression and outcome of tumors by acting as miRNA sponges or binding to proteins. At the same time, circRNAs have the characteristics of high stability, high specificity and wide expression. m 6 A-modified circRNAs can play an important role in the diagnosis and screening of lung adenocarcinoma. However, the current detection methods for such RNAs are not yet standardized, and existing studies still have problems such as low sensitivity and specificity. In order to overcome the deficiencies of detection means and detection efficiency, the purpose of the present invention is to provide a diagnostic marker with high diagnostic efficacy based on m 6A modified hsa_circ_0079039 as a screening and diagnostic kit for biomarker molecules, aiming to improve the screening and diagnostic efficiency of lung adenocarcinoma. Summary of the Invention

[0005] Object of the Invention: The technical problem to be solved by the present invention is to provide a lung adenocarcinoma diagnostic biomarker based on m 6 A methylation-modified circular RNA and its application in view of the deficiencies of the prior art.

[0006] To solve the above technical problems, the present invention discloses a lung adenocarcinoma diagnostic biomarker based on m 6 A methylation-modified circular RNA and its application. The specific technical solutions are as follows:

[0007] The present invention provides the use of a reagent for detecting the m 6 A methylation modification level of circular RNA in the preparation of a lung adenocarcinoma diagnostic kit, 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 a reagent for detecting the m 6 A methylation modification level of circular RNA in a sample, and the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No.1.

[0009] Among them, the lung adenocarcinoma diagnostic kit includes a reagent for detecting the m 6 A methylation modification level of the 3'UTR region of the circular RNA located in exon 7 of the sample.

[0010] Among them, the lung adenocarcinoma diagnostic kit detects the m 6 A methylation modification level of circular RNA in a sample by MeRIP-qRTPCR technology.

[0011] Among them, the reagent for detecting the m 6 A methylation modification level of circular RNA in a sample includes any one or two primer pairs in the following (1) to (2):

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

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

[0014] Among them, the reagent for detecting the m 6The reagent for detecting the mA methylation modification level includes a primer pair for amplifying a reference gene, and the reference gene is GAPDH. The nucleotide sequences of the primer pair for amplifying the reference gene are shown in SEQ ID No. 6-7.

[0015] Among them, the sample includes 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, and the primer pair is composed of the nucleotide sequences shown in SEQ ID No. 4 and SEQ ID No. 5.

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

[0018] In the fourth aspect, the present invention provides the application of a reagent for inhibiting the m 6 A methylation modification level of circular RNA in the preparation of drugs for treating lung adenocarcinoma. The circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No. 1.

[0019] In addition, the present invention finds through research that heterogeneous nuclear ribonucleoprotein HNRNPC binds to and regulates the m 6 A methylation modification level of circular RNA hsa_circ_0079039 in lung adenocarcinoma. HNRNPC is an RNA m 6 A recognition protein. The present invention also provides a primer pair for detecting the binding of the m 6 A modification site of hsa_circ_0079039 to HNRNPC based on MeRIP-WB. The primer pair includes a sense strand primer shown in SEQ ID No. 12 and an antisense 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 the application of a reagent for detecting the m 6 A methylation modification level of the biomarker RNA in the screening and early diagnosis of lung adenocarcinoma. The technical problem to be solved is not limited to the described technical theme, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0022] Technical effects and advantages of the present invention:

[0023] 1. The present invention collected 6 cases of lung adenocarcinoma tumors and normal tissues, and detected them by RNA methylation immunoprecipitation sequencing (MeRIP-seq / m 6 A-seq) technology. The sequencing results showed that compared with normal tissues, there were 177 differentially expressed circRNAs in tumors, among which 148 were significantly up-regulated and 29 were significantly down-regulated. Compared with normal tissues, m 6 A-methylated hsa_circ_0079039 was up-regulated in tumor tissues. The m 6 A methylation modification peak was located in the 3’UTR region of exon 7, and the conserved motifs were GGACC and GGACA.

[0024] 2. The present invention infected PC-9 cells with lentiviral vectors overexpressing and knocking down HNRNPC respectively. qRT-PCR experiments showed that after knocking down HNRNPC, the expression level of hsa_circ_0079039 decreased significantly. After treatment with actinomycin D, the stability of hsa_circ_0079039 in the HNRNPC overexpression group increased significantly (P<0.05).

[0025] 3. The present invention used m 6 A methylation immunoprecipitation combined with real-time fluorescence quantitative PCR (MeRIP-qPCR) to verify the m 6 A methylation modification level. The results showed that in the control group and HNRNPC knockout group cells, the m 6 A methylation modifications of the 2 motifs located in the 3’UTR region of Exon7 of hsa_circ_0079039 could be detected. In HNRNPC knockout cells, the methylation modification level of hsa_circ_0079039 was significantly lower than that of the empty vector group; while in HNRNPC overexpression cells, the methylation modification level of hsa_circ_0079039 was significantly higher than that of the empty vector group (P<0.05).

[0026] 4. The present invention used RNA pull-down assay combined with Western blot (WB) assay to verify the binding site of hsa_circ_0079039 and HNRNPC. The results showed that in the control group cells, HNRNPC could bind to the m 6 A methylation modification site of hsa_circ_0079039. Compared with the control group cells, more HNRNPC bound to hsa_circ_0079039 in HNRNPC overexpression cells.

[0027] 5. The present invention used MeRIP-qPCR to detect 40 cases of lung adenocarcinoma tumors and normal tissues. Compared with normal tissues, m 6The expression level of m6A - modified hsa_circ_0079039 was significantly up - regulated in lung adenocarcinoma tumor tissues (P < 0.0001); the analysis results of the ROC curve showed that 6 the area under the curve of m6A - modified hsa_circ_0079039 for differentiating lung adenocarcinoma tumors from normal tissues was 0.894, the sensitivity was 80.0%, and the specificity was 92.5%.

[0028] 6. In the present invention, by knocking out / overexpressing HNRNPC / hsa_circ_0079039, it was verified that HNRNPC could up - regulate the m6A methylation modification level of hsa_circ_0079039. Through cell viability and animal experiments, it was verified that inhibiting the expression level of HNRNPC could down - regulate the m6A methylation modification level of hsa_circ_0079039 and reduce the viability of lung adenocarcinoma cells, thereby treating lung adenocarcinoma. 6 A methylation modification level. Through cell viability and animal experiments, it was verified that inhibiting the expression level of HNRNPC could down - regulate the m6A methylation modification level of hsa_circ_0079039. 6 A methylation modification level, reduce the viability of lung adenocarcinoma cells, thereby treating lung adenocarcinoma.

[0029] In summary, the present invention first found that the level of m6A - methylated hsa_circ_0079039 in the tissues of lung adenocarcinoma patients was significantly increased, and hsa_circ_0079039 could interact with the RNA m6A recognition protein HNRNPC. 6 A methylation modification level of hsa_circ_0079039 was significantly increased, and hsa_circ_0079039 could interact with the RNA m6A 6 recognition protein HNRNPC. 6 M6A - methylated hsa_circ_0079039 could be used as a screening and diagnostic target for early - stage lung adenocarcinoma.

[0030] Compared with the prior art, the present invention has the following advantages: the present invention first found that the level of m6A - modified hsa_circ_0079039 in the tissues of lung adenocarcinoma patients was significantly increased, and first found that hsa_circ_0079039 could interact with the RNA m6A 6 recognition protein HNRNPC. In addition, the current detection methods for the m6A methylation modification level of circular RNAs have not been standardized, and existing studies still have problems such as low sensitivity and specificity. In order to overcome the deficiencies of detection means and detection efficiency, the present invention provides a screening and diagnostic kit with high diagnostic efficiency using m6A - methylated hsa_circ_0079039 as a biomarker molecule, in order to improve the screening and diagnostic efficiency of lung adenocarcinoma. 6 recognition protein HNRNPC. In addition, the current detection methods for the m6A methylation modification level of circular RNAs have not been standardized, and existing studies still have problems such as low sensitivity and specificity. In order to overcome the deficiencies of detection means and detection efficiency, the present invention provides a screening and diagnostic kit with high diagnostic efficiency using m6A - methylated hsa_circ_0079039 as a biomarker molecule, in order to improve the screening and diagnostic efficiency of lung adenocarcinoma. 6 recognition protein HNRNPC. In addition, the current detection methods for the m6A methylation modification level of circular RNAs have not been standardized, and existing studies still have problems such as low sensitivity and specificity. In order to overcome the deficiencies of detection means and detection efficiency, the present invention provides a screening and diagnostic kit with high diagnostic efficiency using m6A - methylated hsa_circ_0079039 as a biomarker molecule, in order to improve the screening and diagnostic efficiency of lung adenocarcinoma. 6 recognition protein HNRNPC. In addition, the current detection methods for the m6A methylation modification level of circular RNAs have not been standardized, and existing studies still have problems such as low sensitivity and specificity. In order to overcome the deficiencies of detection means and detection efficiency, the present invention provides a screening and diagnostic kit with high diagnostic efficiency using m6A - methylated hsa_circ_0079039 as a biomarker molecule, in order to improve the screening and diagnostic efficiency of lung adenocarcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above - mentioned and / or other advantages of the present invention will become clearer.

[0032] Figure 1 To analyze the m 6 A methylation modification and differential expression of circRNAs in lung adenocarcinoma and normal tissues based on transcriptome data and epitranscriptome data. Figure 1 In it, A is the volcano plot of circRNAs with differential expression of m 6 A methylation modification in lung adenocarcinoma tumor tissues; red dots represent m 6 circRNAs with statistically significant m 6 A methylation modification, and light gray dots represent m Figure 1 In B, B is the expression level of the top 20 upregulated or downregulated circRNAs with the most significant fold change in m 6 A methylation modification.

[0033] Figure 2 For circRNAs with different m 6 A methylation modification levels in tumor and normal tissues, the KEGG enrichment pathways. Figure 2 In A, A is the KEGG enrichment pathway of circRNAs with significantly upregulated m 6 A methylation modification levels in tumor tissues; Figure 2 In B, B is the KEGG enrichment pathway of circRNAs with significantly downregulated m 6 A methylation modification levels in tumor tissues.

[0034] Figure 3 For the RNA m 6 A methylation modification peak and site display diagram of hsa_circ_0079039 in tumor and normal tissues. Figure 3 In A, A is the differential peak analysis of RNA m 6 A modification of hsa_circ_0079039 in tumor and normal tissues; Figure 3 In B, B is the site schematic diagram of RNA m 6 A modification of hsa_circ_0079039 in tumor and normal tissues.

[0035] Figure 4 For the comparison of relative expression levels of hsa_circ_0079039 in different cell groups. Figure 4 In A, A is the comparison diagram of the level changes in the qRT-PCR verification experiment of hsa_circ_0079039 in the control group (ctrl), empty vector group (NC), and HNRNPC overexpression (OE-HN) cells. Figure 4B in it is the comparison of the relative expression levels 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 is the m 6 A methylation modification level of hsa_circ_0079039 in different cell groups. Figure 5 A in it is the relative m 6 A methylation modification ratio (Relative m 6 A Level) of hsa_circ_0079039 detected by using the hsa_circ_0079039-1 primer pair in knockout group empty vector (shNC) and HNRNPC knockout (shHN) cells; Figure 5 B in it is the m 6 A methylation modification ratio of hsa_circ_0079039 detected by using the hsa_circ_0079039-1 primer pair in overexpression group empty vector (OE-NC) and HNRNPC overexpression (OE-HN) cells; Figure 5 C in it is the m 6 A methylation modification enrichment fold (Relative FoldEnrichment) of hsa_circ_0079039 detected by using the hsa_circ_0079039-1 primer in HNRNPC knockout group and overexpression group cells; Figure 5 D in it is the m 6 A methylation modification ratio of hsa_circ_0079039 detected by using the hsa_circ_0079039-2 primer pair in HNRNPC knockout group cells; Figure 5 E in it is the m 6 A methylation modification ratio of hsa_circ_0079039 detected by using the hsa_circ_0079039-2 primer pair in HNRNPC overexpression group cells; Figure 5 F in it is the m 6 A methylation modification enrichment fold detected by using the hsa_circ_0079039-2 primer pair of hsa_circ_0079039 in HNRNPC knockout group and overexpression group cells; Figure 5 G in it is in HNRNPC overexpressing cells, WB graph of the binding of hsa_circ_0079039 to HNRNPC detected by using methylation primer pairs targeting m 6 A methylation modification sites; Figure 5 H in it is in control group and HNRNPC overexpression group cells, using methylation primer pairs targeting m6 Western blot comparison graph of the methylation primer pair at the A methylation modification site 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 m of hsa_circ_0079039 in tumor tissues (Tumor) and normal tissues (Normal) of lung adenocarcinoma patients 6 A methylation modification level and evaluation. Figure 6 A in it is the relative m of hsa_circ_0079039 6 A methylation modification ratio (Relative m 6 A Level); Figure 6 B in it is the m of hsa_circ_0079039 6 A methylation modification enrichment fold (Relative Fold Enrichment), Figure 6 C in it is the relative m of hsa_circ_0079039 in tumor tissues 6 A correlation between the methylation modification ratio of hsa_circ_0079039 and the expression level of HNRNPC (qPCR value); Figure 6 D in it is the correlation between the expression level of hsa_circ_0079039 and the expression level of HNRNPC in tumor tissues; Figure 6 E in it is the receiver operating characteristic curve. p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference.

[0038] Figure 7 HNRNPC inhibits the activity of lung adenocarcinoma cells by upregulating the m 6 A methylation modification level of hsa_circ_0079039. Figure 7 A in it is the relative m of each group of cells 6 A methylation modification ratio (Relative m 6 A Level); Figure 7 B in it is the cell viability of each group of cells; Figure 7 C in it is the plate graph of the cell colony formation experiment of each group of cells; Figure 7 D in it is the cell colony formation data of each group of cells; Figure 7 E in it is the result of the subcutaneous tumor formation experiment in nude mice treated with each group of cells; Figure 7 F in it is the subcutaneous tumor weight data of the subcutaneous tumor formation experiment in nude mice treated with each group of cells; Figure 7 G in it is the relative m of the subcutaneous tumor tissues of nude mice treated with each group of cells 6A methylation modification ratio. p < 0.05, **p < 0.01, ***p < 0.001, NS: no statistical difference. Detailed implementation manners

[0039] The present invention will be further described in detail below in conjunction with the specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0040] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0041] The sources of the important materials, reagents and test techniques used in the following embodiments are as follows:

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

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

[0044] 2× protein loading buffer (product number: G2032), SDS-PAGE gel preparation kit (product number: G2003), ultra-sensitive ECL chemiluminescence kit (product number: G2020) are all provided by Wuhan Sevier Biotechnology Co., Ltd.

[0045] Reagents kits required for qRT-PCR experiment: The qRT-PCR kit ( 2-Step RT-qPCR System, product number: A6010) was provided by Promega (Beijing) Biotechnology Co., Ltd.;

[0046] Cell lysis buffer (Pierce TM IP lysis buffer, product number: 87787), TRIzol reagent (TRIzol TM , product number: 15596026CN), Biotinylation kit (Pierce TM 3'-End DNA Biotinylation Kit, product number: 89818) were all provided by Thermo Scientific TM Company.

[0047] Example 1 Recruitment of patients with lung adenocarcinoma and collection of clinical samples

[0048] The data of 46 patients with lung adenocarcinoma who underwent surgery in Nanjing Drum Tower Hospital from January 2022 to January 2023 were retrospectively analyzed. None of the patients received radiofrequency ablation, chemotherapy, or radiotherapy before surgery. The postoperative pathological diagnosis was lung adenocarcinoma. 92 samples of frozen tumor tissue and normal tissue (distance from the tumor > 5 cm) were collected from the patients. All patients signed the informed consent form to agree to donate samples, which were reviewed and approved by the Ethics Committee of Nanjing Drum Tower Hospital (Ethics number: 2023-485-01). The tumor tissue and normal tissue were subjected to pathological sectioning and HE staining, and all sections were examined by 2 pathologists. The samples were staged and typed according to the 8th edition TNM staging standard of IASLC (International Association for the Study of Lung Cancer) in 2017 and the lung adenocarcinoma typing standard of WHO in 2015.

[0049] Example 2 m 6 A circRNA sequencing and analysis of differential circRNAs

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

[0051] Six pairs of frozen tumor and normal tissues from the 6 recruited patients in Example 1 were selected, and the total RNA of 6 frozen lung adenocarcinoma tissues and the corresponding normal frozen tissues was extracted from them respectively using the Trizol method. The concentration of RNA was measured and the purity was evaluated using a NanoDrop One (Thermo) ultra-micro ultraviolet-visible spectrophotometer. The average value of A260 / A280 was 1.8 - 2.0.

[0052] 2. RNA m 6 A methylation sequencing

[0053] RNA m 6 The m 6 A MeRIP sequencing service was provided by Shanghai Yunxu Biotechnology Co., Ltd. The m 6 A MeRIP reaction was carried out using the m m 6 A MeRIP Kit. The procedure is briefly described as follows: The extracted total RNA was randomly fragmented into fragments of about 200 nt. Using the 6 m

[0054] 3. Analysis and screening criteria for circRNAs

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

[0056] According to the results of RNA m 6 A methylated sequencing, the target circRNAs were screened: (1) The fold change (FC) in the expression between normal tissues and lung adenocarcinoma tissues was greater than 1, and the p-value was less than 0.05; (2) circRNAs with lengths between 200 bp and 1000 bp; (3) circRNAs with m 6 A methylation peak intensity greater than 500 in lung adenocarcinoma. Through comprehensive analysis, the differentially expressed circRNAs in lung adenocarcinoma patients in the cohort were found, and KEGG pathway enrichment analysis was performed on the source genes of the differentially modified circRNAs.

[0057] 4. Results

[0058] The sequencing results showed that compared with normal tissues, there were m 6Among the differential circRNAs, 177 with p < 0.05 and FC ≥ 1 were screened out. Among them, 148 were significantly up-regulated and 29 were significantly down-regulated( Figure 1 A in 6 . Due to the excessive number of differential circRNAs, the top 20 most significant m Figure 1 A circRNAs were screened by fold change and a heat map was made( Figure 2 B in Figure 2 . The KEGG enrichment pathways of up-regulated differential circRNAs were mainly concentrated in the ubiquitin-mediated proteolysis pathway and tumor-related molecular pathways( Figure 3 A in Figure 3 . The KEGG enrichment pathways of down-regulated differential circRNAs were mainly concentrated in the peroxidation and sugar metabolism pathways(

[0059] Example 3 HNRNPC regulates 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. The cells in good condition were digested and resuspended, and then seeded into a 6-well plate and cultured overnight in a 37 °C incubator

[0063] 1.2 Construction of overexpressing cells

[0064] The lentiviral vector (pLVX-CMV) overexpressing heterogeneous nuclear ribonucleoprotein (HNRNPC, encoded by a gene with a nucleotide sequence such as SEQ ID No. 16) was constructed by Shanghai GeneChem Co., Ltd. The required volume of the empty vector (the empty vector group is the empty viral vector) and the HNRNPC virus was calculated according to the infection multiplicity of infection (MOI) = 30, with a total volume of approximately 1 mL. Then, the gene transfection enhancer Polybrene was added at a final concentration of 5 μg / mL to obtain the virus solution. The original culture medium in the 6-well plate was replaced with the virus solution, and the cells were cultured overnight in a 37 °C incubator; after 24 h, the virus solution was aspirated, and fresh DMEM medium containing puromycin (1 μg / mL) was added, and the cells were cultured in a 37 °C incubator. The fresh DMEM medium containing puromycin (1 μg / mL) was changed every 2 - 3 days, and the cell status was observed daily until all the cells in the empty vector group died. Then, the cells in the HNRNPC overexpression group were continuously passaged, and the expression levels of the target gene in each cell component were detected by real-time quantitative fluorescence PCR (qRT-PCR). At the same time, cells transfected with no vector were used as the control group (ctrl).

[0065] 1.3 Treatment with actinomycin D

[0066] Collect the cells and transfer them to a 15 mL centrifuge tube, centrifuge at 4 °C and 1500 rpm for 5 min. Add fresh DMEM medium, gently resuspend and mix well, count the cells and adjust the cell density to 2×10 6 / mL, and seed the cells in a 6-well plate, 2.5 mL per well. Treat the cells with 10 μg / mL actinomycin D, and collect the cells after treatment for 0, 2, and 4 h respectively for subsequent operations. 2. Detection of the expression level of hsa_circ_0079039 by qRT-PCR

[0067] 2.1 Total RNA extraction

[0068] Extract the total RNA of HNRNPC overexpressing cells (OE-HN), control group cells (ctrl), and empty vector group cells (NC) by the TRIzol method. Use a NanoDrop One ultra-micro ultraviolet-visible spectrophotometer (Thermo) to measure the RNA concentration and evaluate the purity. The average value of A260 / A280 is 1.8 - 2.0.

[0069] 2.2 cDNA synthesis

[0070] (1) Select GAPDH as the internal reference gene, and the primers were synthesized by Shanghai Sangon Biotech Co., Ltd. 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 temperature control module at 70 °C and incubate for 5 min. Immediately cool on ice for at least 5 min. Centrifuge each reaction tube in a microcentrifuge for 10 seconds to sediment the contents and maintain the original volume. Tightly cap the reaction tube and incubate on ice.

[0074] Table 2 RNA template and primer mixing system

[0075] Component Amount Added per Reaction RNA Template 0.8 μg Random Primers 1 μL Oligo(dT)15 Primer 1 μL Nuclease-Free Water X μL Total Volume 10 μL

[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 in step (2) and make up to a final volume of 20 μL. Incubate at 25 °C for 5 min, incubate at 25 °C for 60 min, and incubate at 70 °C for 15 min. Place the resulting cDNA on ice for later use or store at -20 °C.

[0080] 2.3 qRT-PCR

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

[0082] Table 4 qRT-PCR amplification system

[0083]

[0084] (2) Loading

[0085] Add the mixture without cDNA template to each well corresponding on the Roche fluorescence quantitative 384-PCR plate. Add the synthesized cDNA template (or template-free control reaction water) to the corresponding wells of the reaction plate. Stick on the Sealing Film and briefly centrifuge to mix. Place the prepared PCR plate on ice before setting the PCR program. Set 3 replicates for each sample.

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

[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). The 2 -ΔΔCT -method was used to calculate the relative expression levels of HNRNPC and hsa_circ_0079039 in qRT-PCR, and the data were expressed as mean ± standard deviation. The Shapiro-Wilk test was used to examine the normality of the data. For data conforming to a normal distribution, independent sample t-tests or one-way analysis of variance (ANOVA) were used to analyze the differences between groups. For data not conforming to a normal distribution, the rank sum test (Mann-Whitney U test) was used to analyze the differences between groups.

[0089] 4. Results

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

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

[0092] Example 4 Regulation of the m 6 A methylation modification level of hsa_circ_0079039 by HNRNPC

[0093] 1. Construction of knockout and overexpression cells

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

[0095] 2. Methylated RNA immunoprecipitation (MeRIP) experiment

[0096] 2.1 Total RNA extraction

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

[0098] 2.2 RNA fragmentation

[0099] (1)Adjust the total RNA concentration of each group of cells in Example 2.1 to 1 μg / μL with nuclease-free water.

[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 and mix well, 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 and mix well, and centrifuge briefly. Place the tubes on ice. Repeat this step 2 - 3 times until all RNA is fragmented.

[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 of PC Buffer, 1 μL of PC Enhancer, and 750 μL of absolute ethanol, mix gently, and precipitate overnight at -80 °C.

[0103] (5)Transfer the samples precipitated overnight to a refrigerated centrifuge and centrifuge at 4 °C and 15,000 g 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 15,000 g for 15 min.

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

[0105] (7)Use the Qsep100 Automatic Nucleic Acid and Protein Analysis System (Bioptic) to detect the RNA fragment size and concentration. The RNA fragmentation size is ~200 nt.

[0106] (8)Take out 1 - 3 μg of fragmented RNA as the Input group and store it at -80 °C for later use. The remaining fragmented RNA is 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 to 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 stand until the solution becomes clear; aspirate the supernatant without disturbing the beads. Repeat this step once.

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

[0111] (4) IP group: Add 2 μL of m 6 A antibody. IgG group: Add 2 μL of IgG antibody to each tube as a control. Incubate with rotation on a shaker at room temperature for 1 hour. Centrifuge briefly to collect the solution at the bottom of the tube. Place the reaction tube on a magnetic stand until the solution becomes clear. Aspirate the supernatant without disturbing the beads.

[0112] (5) Wash the beads with 200 μL of 1×IP buffer in each tube. Place the reaction tube on a magnetic stand until the solution becomes clear; aspirate the supernatant without disturbing the beads. Remove the centrifuge tube and cover it, then 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 Composition of MeRIP reaction solution

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

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

[0118] (3) Wash the beads with 200 μL of 1×IP buffer in each tube. Place the reaction tube on a magnetic stand until the solution becomes clear; aspirate the supernatant without disturbing the beads. Repeat this step once.

[0119] (4) Wash the beads with 200 μL of 1×LB buffer in each tube. Place the reaction tube on a magnetic stand until the solution becomes clear; aspirate the supernatant without disturbing the beads. Repeat this step once.

[0120] (5) Wash the beads with 200 μL of 1×HS buffer in each tube. Place the reaction tube on a magnetic stand until the solution becomes clear; aspirate the supernatant without disturbing the 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 the magnetic stand until the solution becomes clear. Transfer the supernatant to a new 1.5 mL centrifuge tube and place it on ice for later use.

[0123] (2) For each reaction, prepare a new 1.5 mL EP tube, transfer 20 μL of resuspended MS magnetic beads to the new tube, and place it on the magnetic stand until the solution becomes clear. Aspirate and discard the supernatant without disturbing the MS magnetic beads.

[0124] (3) Add 100 μL of RLT Buffer to each tube to completely resuspend the MS magnetic beads. Place the reaction tube on the magnetic stand until the solution becomes clear. Aspirate and discard the supernatant without disturbing the MS magnetic beads.

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

[0126] (5) Add 60 μL of absolute ethanol to each tube, gently mix, and incubate at room temperature for 1 min. Place the centrifuge tube on the magnetic stand until the solution becomes clear. Aspirate and discard the supernatant without disturbing the MS magnetic beads.

[0127] (6) Add 200 μL of 75% ethanol to each tube to wash the MS magnetic beads. Place the centrifuge tube on the magnetic stand until the solution becomes clear; aspirate and discard the supernatant without disturbing the magnetic beads. Repeat this step for another wash.

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

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

[0130] 3. Experimental procedures for qRT-PCR

[0131] (1) cDNA synthesis

[0132] Design corresponding primers according to two m 6 A modification sites. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. The primer sequences are shown in Table 6. Other methods are the same as those in Section 2.2 of Example 3:

[0133] Table 6 qRT-PCR primers for m 6 A modification sites

[0134]

[0135] (2) qRT-PCR

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

[0137] (3) Calculation of methylation modification level

[0138] The methylation modification ratio (Relative m 6 A Level) and enrichment fold (Relative Fold Enrichment) of each transcript were calculated using the following formula, where DF and IF are the dilution factors of the m 6 A antibody and IgG antibody, respectively:

[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 Preparation of hsa_circ_0079039m 6 A modification sequence probe

[0144] According to the hsa_circ_0079039m 6 A modification sequence, sense and antisense strand probes were designed as shown in Table 7, and the probes were synthesized by Shanghai Yunxu Biotechnology Co., Ltd.

[0145] Table 7 m 6 A modification sequence sense and antisense strand probe sequences

[0146] Name Primer Sequence (5’-3’ end) Sequence Number Methylated Sense Strand Probe AGAGCTTGAGGACCGTCttggctttcacggtcgc SEQ ID No.12 Methylated Antisense Strand Probe gcgaccgtgaaagccaaGACGGACCTCAAGCTCT SEQ ID No.13

[0147] 4.2 Labeling of probes

[0148] (1) Thaw all components in the kit except terminal deoxynucleotidyl transferase (TdT) and place them on ice. Keep TdT at -20 °C until use. Immediately 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 components in the order listed in Table 8 to prepare a labeling reaction for the control system. Prepare the sample labeling reaction in the same manner, substituting the unlabeled control oligonucleotide with a 5 pmol probe with a 3'-OH terminus.

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

[0151] Table 8 Biotin Labeling Reaction Mixture System

[0152] Component Amount Added per Reaction 5X TdT Reaction Buffer 10 μL Biotin-11-UTP (5 μM) 5 μL Diluted TdT (1.5 U / μL) 5 μL Unlabeled Control Oligo (1 μM) or Probe 5 μL Ultra-Pure Water X μL Total Volume 50 μL

[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, then centrifuge at high speed in a microcentrifuge for 1 - 2 min to separate the layers. Remove and save the upper layer (aqueous phase).

[0155] 4.3 Cell Lysis

[0156] (1) Collect the cell suspensions of HNRNPC overexpressing cells and control group cells (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 pellet 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 pellet the cells again.

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

[0159] (4) Incubate on ice for 5 min, mixing regularly during this period to ensure effective lysis.

[0160] (5) Centrifuge at 13,000 × g for 10 min at 4 °C 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) 10 μL of the protein lysate can be taken as the input group, and the remaining protein lysate is divided into two equal parts, one for the positive probe RNA Pull-down reaction and one for the negative probe RNA Pull-down reaction.

[0162] 4.4 RNA Pull-down

[0163] The RNA Pull-down experiment was conducted according to the kit instructions, and the specific steps are as follows:

[0164] (1) Take 3 μL of the biotin-labeled probe at 100 μM, add 47 μL of 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 and mix well, and rotate and mix at room temperature for 1 h;

[0166] Table 9 Probe-Protein Binding Reaction Master Mix System

[0167] Component Amount Added per Reaction RNase Inhibitor 5 μL Protease Inhibitor 2.5 μL 10×Binding Buffer 50 μL Biotin-Labeled Probe 50 μL Cell Lysate 192.5 μL Nuclease-Free Water 200 μL Total Volume 500 μL

[0168] (3) Add 100 μL of the master mix to the magnetic beads in step (1), and mix well by pipetting or gently vortexing. Incubate at 4 °C for 60 min, and keep stirring or rotating to ensure thorough mixing;

[0169] (4) Prepare new 1.5 mL EP tubes according to the number of reactions, and label the EP tubes; briefly oscillate and mix the SA Beads, and take 50 μL each and add them to each prepared EP tube; place the EP tubes on the magnetic rack until the solution becomes clear (about 2 min); aspirate the supernatant without disturbing the magnetic beads, 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 and mix well; incubate with rotation at room temperature for 2 h. Briefly centrifuge, place the EP tube on the magnetic rack until the solution becomes clear (about 2 min); aspirate the supernatant without disturbing the magnetic beads; wash the SA Beads with 500 μL of 1×Binding Buffer pre-cooled on ice: place the EP tube on the magnetic rack until the solution becomes clear (about 2 min); aspirate the supernatant without disturbing 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 that have completed the above operations, and gently pipette and mix well; incubate at 95 °C for 5 - 10 min. Place the EP tube on the magnetic rack until the solution becomes clear (about 2 min), aspirate the supernatant into a new EP tube without disturbing the magnetic beads; the supernatant is used for subsequent experiments.

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

[0175] Use an SDS - PAGE gel preparation kit to prepare 10 mL of 16% separating gel. After mixing evenly, pour the gel immediately until it reaches 2 - 3 mm below the lower edge of the comb. Let it stand at room temperature for 45 min until the gel is completely polymerized. Prepare 5 mL of 4% stacking gel, mix evenly and pour it immediately until it reaches the top, then vertically insert the comb. Let it stand at room temperature for 20 min until the gel polymerizes, then pull out the comb and place it in the electrophoresis tank.

[0176] (3) Electrophoresis

[0177] Install the gel into the electrophoresis tank, and add 1×TGS (14.4 g glycine, 3.03 g Tris - base, and 1 g SDS are fully dissolved in 1 L of deionized water) into the electrophoresis tank. After loading the sample, set the voltage to 80 V for electrophoresis. When the Marker protein shows obvious stratification, adjust the electrophoresis instrument parameters to 120 V and continue electrophoresis for about 1 h.

[0178] (4) Protein transfer and immunodetection

[0179] Use the semi - dry transfer method for membrane transfer. Place the PVDF membrane in an appropriate amount of transfer buffer (14.4 g glycine, 2.9 g Tris - base, 200 mL methanol are fully dissolved in deionized water and then made up to 1 L). Take out the separating gel, place it on the activated PVDF membrane, and transfer it to the filter paper soaked with the transfer buffer, then place another layer of filter paper soaked with the transfer buffer on it. Use a semi - dry transfer instrument for membrane transfer, set the corresponding voltage (15 - 20 V) according to the number of gels, and the time is 40 min.

[0180] After membrane transfer, wash the membrane 3 times with 1×TBST buffer and block it with 5% skim milk for 1 h. Incubate overnight at 4℃ with the antibody against HNRNPC, then wash the membrane 3 times with 1×TBST buffer. Use a suitable HRP - labeled secondary antibody. Incubate at room temperature for 1 hour and wash the membrane 3 times with 1×TBST buffer. Use an ELC chemiluminescent kit to detect the protein signal, and the detection instrument is the chemiluminescent gel imaging system of Bio - Rad.

[0181] 5. Results

[0182] (1) Use the hsa_circ_0079039 - 1 and hsa_circ_0079039 - 2 primers to detect the m 6 A methylation modification level of each group of cells. The results show that, as Figure 5 shown in A, C, D, and F below, the m 6 A methylation modification level of hsa_circ_0079039 in the HNRNPC knockout group of cells (shHN) is significantly lower than that in the empty vector control group (shNC); asFigure 5 As shown in B, C, E, and F in 6 the methylation modification level of hsa_circ_0079039 in the HNRNPC overexpression group cells (OE-HN) was significantly higher than that in the empty vector control group (OE-NC).

[0183] (2) In HNRNPC overexpressing cells (OE-HN), HNRNPC was able to bind to the m 6 A site ([[]] Figure 5 G in Figure 5 ). Compared with the cells in the empty vector group (OE-NC), more HNRNPC bound to hsa_circ_0079039 in the HNRNPC overexpressing cells ( Figure 5 H in 6 ). These results revealed the mechanism by which HNRNPC regulates the m 6 A methylation modification level of hsa_circ_0079039.

[0184] Example 5 Evaluation of the diagnostic value of the m 6 A methylation modification level of hsa_circ_0079039

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

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

[0187] 2. Evaluation of the diagnostic value of the m 6 A methylation modification level of hsa_circ_0079039 for lung adenocarcinoma

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

[0189] 3. Results

[0190] (1) Compared with the normal control tissue (Normal), the expression level of m 6 A-modified hsa_circ_0079039 was significantly upregulated in the tumor tissues of lung adenocarcinoma (P < 0.001) ( Figure 6 A inFigure 6 in B);

[0191] (2) The m 6 A methylation modification level of hsa_circ_0079039 in lung adenocarcinoma tumor tissues 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 in C, Figure 6 in D).

[0192] (3) The analysis results of the ROC curve showed that the area under the curve (AUC) of m 6 A - modified hsa_circ_0079039 for distinguishing lung adenocarcinoma tumors from normal tissues was 0.894, the sensitivity (SE) was 80.0%, and the specificity (SP) was 92.5% ( Figure 6 in E).

[0193] Example 6 HNRNPC up - regulates the m 6 A methylation modification level of hsa_circ_0079039 to inhibit the activity of lung adenocarcinoma cells

[0194] 1. Construction of knockout and over - expression cells

[0195] The interfering RNA of hsa_circ_0079039 (the interfering RNA includes a sense - strand probe and an antisense - strand probe, the sequences are shown in Table 10, and the 3' end of the interfering RNA is overhanging with dTdT) and the over - expression lentiviral vector of hsa_circ_0079039 (pLVX - CMV) were synthesized or constructed by Shanghai GeneChem Co., Ltd. The constructed cell groups include:

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

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

[0198] HNRNPC knockout group cells (shHN), the corresponding empty - vector group (shNC),

[0199] HNRNPC over - expression group cells (OE - HN), the 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 construction method of the cells is the same as that 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’ end) Sequence Number Sense Strand Probe GUACAUCUUGCGUUUCCTC SEQ ID No.14 Antisense Strand Probe GAGGAAACGCAAGAUGUAC SEQ ID No.15

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

[0208] 2. MeRIP and qRT-PCR

[0209] Using the primer pair has_circ_0079039-1 in Table 6, MeRIP and qRT-PCR experiments were performed on each group of cells, and the steps were the same as in Example 4. The results showed that in HNRNPC knockout cells and hsa_circ_0079039 interfering cells, the m 6 A methylation modification level of hsa_circ_0079039 was significantly lower than that of the corresponding empty vector group. In HNRNPC overexpressing cells and hsa_circ_0079039 overexpressing cells, the m 6 A methylation modification level of hsa_circ_0079039 was significantly higher than that of the corresponding empty vector group. Overexpressing HNRNPC could increase the m 6 A methylation modification level of the hsa_circ_0079039 interference group, while knocking out HNRNPC could decrease the m 6 A methylation modification level of the hsa_circ_0079039 overexpression group ( Figure 7 A in). These results indicate that HNRNPC can up-regulate the m 6 A methylation modification level of hsa_circ_0079039.

[0210] 3. Detection of cell viability by CCK-8 method

[0211] Cells in the logarithmic growth phase of each group were seeded into 96-well plates (about 500 cells per well), and blank groups (cell-free medium), empty vector groups, and experimental groups were set up, with 3 replicates in each group. After pre-incubating in a 37 °C incubator for 24 hours for cell attachment, 10 μL of CCK-8 reagent was directly added to each well (avoiding air bubbles), and incubated in the dark for 2 hours. After the solution turned orange-yellow, the absorbance at 450 nm (OD 450 value) was measured with an enzyme-linked immunosorbent assay reader; the cell survival rate (%) was calculated using the formula = (OD of the experimental group 450 − OD of the blank group 450 ) / (OD of the control group 450 − OD of the blank group 450 ) × 100% to calculate the activity of each group. The results showed that the activities of HNRNPC knockout cells and hsa_circ_0079039 interfering cells were significantly lower than those of their respective empty vector groups, and the activities of HNRNPC and hsa_circ_0079039 overexpressing cells were significantly higher than those of their respective empty vector groups. Overexpression of HNRNPC could increase the cell activity of the hsa_circ_0079039 interfering group, while knockout of HNRNPC could decrease the cell activity of the hsa_circ_0079039 interfering group ( Figure 7 B in). These results indicate that HNRNPC can increase the activity of lung adenocarcinoma cells by upregulating the m 6 A methylation modification level of hsa_circ_0079039.

[0212] 4. Colony formation assay

[0213] Cells in the logarithmic growth phase of each group were collected, resuspended and counted. DMEM medium containing 10% v / v fetal bovine serum was added, and the cells were seeded in 6-well plates, with 500 cells added to each well, and the medium was added to 2 mL, and G418 (400 μg / mL) was maintained at half the amount. The size of cell colonies could be observed under a microscope every day for 3 days, and the 6-well plates were taken out after 10 days. Fixed with paraformaldehyde, stained with crystal violet at room temperature in the dark for 15 min, washed twice with PBS, blotted with absorbent paper to remove excess moisture, and air-dried. The results were scanned with a scanner and saved. The results showed that the colony formation ability of hsa_circ_0079039 interfering cells was significantly lower on average than that of the control group (PC-9 cells cultured normally without any treatment), and the colony formation ability of hsa_circ_0079039 overexpressing cells was significantly higher on average than that of the empty vector group. Overexpression of HNRNPC could increase the colony formation ability of the hsa_circ_0079039 interfering group, while knockout of HNRNPC could decrease the colony formation ability of the hsa_circ_0079039 overexpressing group ( Figure 7in C and D). These results indicate that HNRNPC can promote the clonogenic ability of lung adenocarcinoma cells by upregulating the m 6 A methylation modification level of hsa_circ_0079039.

[0214] 5. Establishment of subcutaneous xenograft tumor model

[0215] Male BALB / c nude mice (6 - 8 weeks old, 18 - 22 g) were selected. Cells from each group were subcutaneously injected into the inner side of the left axilla of each nude mouse at a dose of 10 7 per mouse, and 5 nude mice were injected with each type of cell. After 25 days, the nude mice were sacrificed, and the tumor tissues were dissected and weighed. The results showed that compared with the control group (injected with normally cultured PC-9 cells without any treatment), the size and weight of subcutaneous tumors in the hsa_circ_0079039 overexpression group were significantly increased. Knockdown of HNRNPC could inhibit the tumor growth in the overexpression group (OE-circRNA) ( Figure 7 in E and F), and at the same time, it also downregulated the m 6 A methylation modification level in the OE-circRNA group ( Figure 7 in G). These results indicate that inhibiting HNRNPC can reduce the m 6 A methylation modification level of hsa_circ_0079039, thereby inhibiting tumor growth.

[0216] The present invention provides an idea and method for a lung adenocarcinoma diagnostic marker based on m 6 A-methylated modified circular RNA and its application. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. Application of a reagent for detecting the m 6 A methylation modification level of circular RNA in the preparation of a diagnostic kit for lung adenocarcinoma, wherein, The circular RNA described above is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No.

1.

2. A lung adenocarcinoma diagnostic kit, characterized in that, It includes a reagent for detecting the m 6 A methylation modification level of circular RNA, and the circular RNA is hsa_circ_0079039, and its nucleotide sequence is shown in SEQ ID No.

1.

3. The lung adenocarcinoma diagnosis kit according to claim 2, wherein The described lung adenocarcinoma diagnostic kit includes reagents for detecting the m 6 A methylation modification level of the 3'UTR region of the circular RNA located in exon 7 in the sample.

4. The lung adenocarcinoma diagnosis kit according to claim 2, characterized in that, The described lung adenocarcinoma diagnostic kit detects the m 6 A methylation modification level of circular RNA in a sample by MeRIP-qRTPCR technology.

5. The lung adenocarcinoma diagnosis kit according to claim 2, characterized in that, The reagent for detecting the m 6 A methylation modification level of circular RNA in a test sample comprises any one or two primer pairs in the following (1) to (2): (1) A primer pair consisting of the nucleotide sequences shown in SEQ ID No.8 and SEQ ID No.9; (2) A primer pair consisting of the nucleotide sequences shown in SEQ ID No.10 and SEQ ID No.

11.

6. The lung adenocarcinoma diagnosis kit according to claim 2, wherein The reagent for detecting the m 6 A methylation modification level of circular RNA in the test sample comprises a primer pair for amplifying a reference gene, and the reference gene is GAPDH.

7. The lung adenocarcinoma diagnostic kit according to claim 6, characterized in that, The nucleotide sequence of the primer pair for amplifying the internal reference gene is shown in SEQ ID No.6-7.

8. The lung adenocarcinoma diagnosis kit according to claim 2, characterized in that, The sample described above includes the serum, plasma or tumor tissue of the patient.

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

1.

10. Application of reagent for inhibiting m 6 methylation modification level of circular RNA in preparing medicine for treating lung adenocarcinoma, wherein the circular RNA is hsa_circ_0079039 and its nucleotide sequence is shown as SEQ ID No.1.

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