Circular RNA combined as a molecular marker for lung adenocarcinoma

Abstract

The application discloses application of circular RNA combination as a molecular marker of lung adenocarcinoma, and the circular RNA combination is composed of circLZIC and circCEP350, and has special correlation with lung adenocarcinoma. By detecting the expression of circLZIC and circCEP350 of a subject, whether the subject has lung adenocarcinoma or whether the subject has the risk of lung adenocarcinoma can be judged, so that the application can be applied to screening of lung adenocarcinoma drugs. In addition, the combination of circLZIC and circCEP350 can also be used as an effective target for screening of lung adenocarcinoma diagnosis and treatment, and can be made into diagnostic kits, test papers, chips and other diagnostic tools for early screening and diagnosis of lung adenocarcinoma. Furthermore, the application proves that the combination of circLZIC and circCEP350 can inhibit the biological process of lung adenocarcinoma proliferation, and therefore considers that the combination of circLZIC and circCEP350 is a drug target for treating lung adenocarcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and in particular relates to the application of a circular RNA combination as a molecular marker for lung adenocarcinoma. Background Technology

[0002] Lung adenocarcinoma is the most common subtype of non-small cell lung cancer, accounting for approximately 40%. Despite the rapid development of emerging treatments such as targeted therapy and immunotherapy in recent years, the clinical prognosis for lung cancer patients remains poor. The 5-year survival rate for most patients diagnosed with metastatic advanced NSCLC is still low, at only about 17%. The main reason is the lack of effective methods for early screening and diagnosis.

[0003] Currently accepted low-dose computed tomography (LDCT) screening for lung cancer has drawbacks such as a high false-positive rate and radiation damage. Various serum tumor markers commonly used clinically, such as carcinoembryonic antigen (CEA), serum sialic acid (SA), squamous cell carcinoma antigen (SCC), and cytokeratin fragment 19, all have low sensitivity and specificity.

[0004] Circ RNAs (circRNAs) are a class of covalently closed circular non-coding RNAs characterized by their wide distribution, diverse types, and multiple regulatory functions. In recent years, liquid biopsy technology has been widely used for early disease screening due to its minimally invasive nature and the ability to repeatedly collect samples. Due to their good stability, circRNAs in body fluids have become candidate biomarkers for tumor diagnosis. Song et al. found that hsa_circ_0137439 was significantly upregulated in the urine of bladder cancer patients, promoting tumor proliferation and migration through the hsa_ciec_0137439 / miR-142-5p / mtdh axis, and was associated with tumor stage, lymph node metastasis, and muscle invasion, potentially serving as a biomarker for bladder cancer diagnosis and prognostic assessment. Zhao et al. also detected high expression of hsa_circ_0001874 and hsa_circ_0001971 in the saliva of oral cancer patients. The area under the ROC curve for their combined application was 0.922. Postoperative expression levels of both circRNAs in saliva were significantly lower than preoperative levels, suggesting their potential as biomarkers for oral squamous cell carcinoma diagnosis. hsa_circRNA_0056616 was significantly upregulated in plasma, with an AUC of 0.812, and its expression level was significantly correlated with tumor size and lymph node metastasis in lung cancer patients, suggesting that hsa_circRNA_0056616 has potential application value as a diagnostic biomarker for lung cancer. However, due to the low abundance of circRNA in plasma, false negative results may occur when using traditional qRT-PCR to detect plasma circRNA. RT-ddPCR, due to its high sensitivity and ability to achieve absolute quantification, is a better choice for detecting plasma circRNA. Guo et al. used RT-ddPCR to analyze the expression of circRNA in the plasma of gastric cancer patients and healthy individuals, and found that the expression of hsa_circ_0001017 and hsa_circ_0061276 was significantly reduced in the plasma of gastric cancer patients. The combined diagnostic efficacy of the two circRNAs was evaluated, with an AUC of 0.966, which can serve as an effective diagnostic biomarker for gastric cancer. However, there are currently no reports on the use of RT-ddPCR detection of peripheral blood circRNA for early diagnosis in lung adenocarcinoma. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the prior art, the primary objective of this invention is to provide an application of circular RNA as a molecular marker for lung adenocarcinoma.

[0006] Another object of the present invention is to provide the application of the above-mentioned circular RNA as a molecular marker for lung adenocarcinoma in the screening of lung adenocarcinoma drugs.

[0007] Another object of the present invention is to provide the application of the above-mentioned circular RNA combination in the preparation of antitumor drugs.

[0008] This invention is achieved by using a circular RNA synthesis in the preparation of tools for the diagnosis or prognosis of lung adenocarcinoma, the circular RNA synthesis consisting of circ LZIC and circ CEP350.

[0009] The relevant information of two circ RNA molecules was analyzed using the UCSC and CircBase databases: circ LZIC (hsa_circ_0000014) is derived from the LZIC gene on chromosome 1, with the sequence chr1:9,991,948-9,994,918, consisting of exons 5, 6, and 7 of the LZIC gene; circ CEP350 (hsa_circ_0003942) is derived from the CEP350 gene on chromosome 1, with the sequence chr1:179,972,308-179,975,702, consisting of exons 7 and 8 of the CEP350 gene.

[0010] In the "screening, diagnosis or prognosis" used in this invention, screening refers to screening for the early occurrence of lung adenocarcinoma and predicting the lesion of lung adenocarcinoma; diagnosis refers to determining whether the subject has lung adenocarcinoma; and prognosis refers to determining whether the subject has the possibility of lung adenocarcinoma recurrence or determining whether the subject has already recurred.

[0011] Preferably, the kit, test strip chip, or high-throughput sequencing platform includes primers or probes for detecting circ LZIC and circCEP350.

[0012] Specifically, the chip includes a solid support and primers or probes fixed on the solid support for detecting circLZIC and circCEP350; the solid support can be made of various conventional materials in the chip field, including but not limited to nylon membranes, glass or silicon wafers modified with active groups (such as aldehydes, aminos, etc.), unmodified glass wafers, plastic sheets, etc.

[0013] The high-throughput sequencing platform includes primers or probes for detecting circ LZIC and circ CEP350;

[0014] The kit and test strips include primers or probes for detecting circ LZIC and circ CEP350.

[0015] More specifically, the primers for circ LZIC are:

[0016] circ LZIC F: 5'-GCAGGTGCTATACTCAGCCA-3';

[0017] circ LZIC R: 5'-TCCATCTCTGCTAACCTTGTCC-3';

[0018] The primers for circ CEP350 are:

[0019] circ CEP350 F: 5'-GCACAGTTATCAAGTACAGAATGC-3';

[0020] circ CEP350 R: 5'-CACACTTTCCCATCAGGTGTT-3';

[0021] The probe is modified with a fam group at 5' and a tamra group at 3' of the primer.

[0022] This invention further discloses the application of circular RNA synthesis as a molecular marker for lung adenocarcinoma in screening drugs for lung adenocarcinoma, wherein the circular RNA synthesis consists of circ LZIC and circ CEP350.

[0023] Having learned of the close correlation between circ LZIC and circ CEP350 and lung adenocarcinoma, substances that promote or inhibit the expression of circ LZIC and circ CEP350 can be screened based on this characteristic; thus, drugs truly useful for treating nodular lung adenocarcinoma can be found from these substances. Therefore, the present invention also provides a method for screening potential substances for treating lung adenocarcinoma, the method comprising: treating a nodular lung adenocarcinoma-related cell system with a candidate substance; if the candidate substance can promote or inhibit the expression or activity of circ LZIC and circ CEP350, then the candidate substance is a potential substance for treating nodular lung adenocarcinoma.

[0024] Specifically, the cell system can be a subcellular system, solution system, tissue system, organ system, or animal system (such as an animal model, preferably a non-human mammalian animal model, such as a mouse, rabbit, sheep, monkey, etc.); preferably, the obtained potential substances are subjected to further cell experiments and / or animal experiments to further select and identify substances that are truly useful for treating lung adenocarcinoma.

[0025] The present invention further discloses the application of circular RNA combination in the preparation of antitumor drugs, wherein the circular RNA combination consists of circ LZIC and circ CEP350, and the tumor is lung adenocarcinoma.

[0026] Preferably, the antitumor drug is a drug that inhibits the proliferation of lung adenocarcinoma cells.

[0027] Preferably, the antitumor drug comprises an effective dose of inhibitors of circ LZIC and circ CEP350; the inhibitor is a protein, oligonucleotide, small molecule compound, lentivirus and / or expression plasmid.

[0028] The inhibitor can inhibit the expression of circ LZIC and circ CEP350, or inhibit the activity of circ LZIC and circ CEP350, or inhibit the effective duration of action of circ LZIC and circ CEP350, or inhibit the stability of circ LZIC and circ CEP350.

[0029] Specifically, based on the prediction of downstream miRNAs and target genes of circ LZIC and circ CEP350, it was found that the molecular function (MF) of circ LZIC is mainly enriched in: extracellular matrix structural components, mRNA 3'-UTR binding, β-catenin binding, and platelet-derived growth factor binding; the biological processes (BP) of downstream target genes of circ LZIC are mainly enriched in: intercellular adhesion via plasma membrane adhesion molecules, adhesion of homogeneous cells via plasma membrane adhesion molecules, vesicle localization, and synaptic vesicle circulation; the cellular composition (CC) of circ LZIC is mainly composed of: collagen trimers, extracellular matrix components, and ribbon-like collagen fibers. The molecular functions (MF) of circ CEP350 are mainly enriched in: RNA polymerase II proximal promoter sequence-specific DNA binding, proximal promoter sequence-specific DNA binding, ubiquitin-like protein ligase binding, etc.; the downstream target gene biological processes (BP) of circ CEP350 are mainly enriched in: regulation of membrane permeability, positive regulation of mitochondrial membrane permeability, receptor recycling, and mitochondrial regulation during apoptosis; the cellular composition (CC) of circ CEP350 mainly consists of: extracellular vesicles, intercellular tight junctions, apical junction complexes, and axonal portions.

[0030] This invention further utilizes the DAVID website to perform KEGG analysis on the target circ RNA, downstream miRNAs, and target genes. It was found that the pathways involved by circ LZIC, with the highest number of enriched genes, were the Relaxin signaling pathway, dopaminergic synapses, and neurotrophic protein signaling pathways, etc.; while the main pathways involved by circ CEP350 were, in order, signaling pathways regulating pluripotency of stem cells, the Hippo signaling pathway, colorectal cancer, pancreatic cancer, and axonal guidance, etc.

[0031] Therefore, based on the predicted and verified results, the targets of the inhibitors are not limited to circ LZIC and circCEP350 themselves, but also include the upstream and downstream of circ LZIC and circ CEP350, such as: the genomic sequences encoding circ LZIC and circCEP350, the target genes of circ LZIC and circ CEP350, and the proteins or genes that regulate circ LZIC and circ CEP350.

[0032] In terms of pharmaceutical properties, preferably, the inhibitor includes proteins, oligonucleotides, small molecule compounds, oligonucleotide expression vectors, lentiviruses, expression plasmids, and / or small interfering RNA.

[0033] In terms of drug form, preferably, the antitumor drug includes a pharmaceutically acceptable carrier; the carrier is a diluent, buffer, suspension, emulsion, granule, encapsulation agent, excipient, filler, binder, spray, transdermal absorbent, humectant, disintegrant, absorption enhancer, surfactant, colorant, flavoring agent or adsorbent.

[0034] The antitumor drug can be administered alone or in combination with other drugs that can treat lung adenocarcinoma; the subject can be a human or other mammal; more specifically, the subject is an organ, tissue, or cell. The antitumor drug can be administered ex vivo, specifically by introducing or transfecting a circular RNA expression vector into human autologous or xenogeneic cells (or foreign cells) in vitro, followed by in vitro cell amplification, and then reintroducing it into the human body. The antitumor drug can also be administered in vivo, specifically by directly introducing a circular RNA expression vector into the body; the expression vector can be viral or non-viral, or even naked DNA or RNA.

[0035] The term "effective dose" as used in this invention refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. The effective doses of circ LZIC and circ CEP350 may vary depending on the administration method and the severity of the disease to be treated. Preferably, the selection of the effective dose can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the circ LZIC and circ CEP350 inhibitors, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration.

[0036] The methods for analyzing circular RNA expression profiles in this invention include, but are not limited to, the following: one-step reverse transcription droplet digital polymerase chain reaction (RT-ddPCR), reverse transcription polymerase chain reaction (RT-PCT), real-time quantitative polymerase chain reaction (Real-time PCR), in situ hybridization, Northern blotting, RNase protection assay, Solexa sequencing technology, and bioarrays. In a specific embodiment of this invention, one-step reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) is used.

[0037] The term "circular RNA" used in this invention is interchangeable with "circular RNA" and "circ RNA".

[0038] The term "treatment of lung adenocarcinoma" as used in this invention includes improvement, relief, and cure of the disease.

[0039] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects:

[0040] (1) This invention first discovered that circ LZIC and circ CEP350, when used as a combined indicator, have a special correlation with lung adenocarcinoma. By detecting the expression of circ LZIC and circ CEP350 in the subject, it is possible to determine whether the subject has lung adenocarcinoma or whether the subject is at risk of having lung adenocarcinoma, which can be applied to the screening of lung adenocarcinoma drugs.

[0041] (2) The combination of circ LZIC and circ CEP350 of the present invention can also be used as an effective target for screening lung adenocarcinoma diagnosis and treatment. They can be made into diagnostic kits, test strips, chips and other diagnostic tools that can be used for early screening and diagnosis of lung adenocarcinoma.

[0042] (3) This invention demonstrates through prediction that the combination of circ LZIC and circ CEP350 can inhibit the biological processes of lung adenocarcinoma proliferation, thus suggesting that the combination of circ LZIC and circ CEP350 is a drug target for the treatment of lung adenocarcinoma. As a novel molecular biomarker for the diagnosis and treatment of lung adenocarcinoma, compared with traditional detection methods, circular diagnosis is more timely, more specific, and more sensitive, enabling early diagnosis of lung adenocarcinoma and thus reducing the mortality rate of lung adenocarcinoma. This molecular biomarker provides a theoretical basis for clinicians to develop personalized treatment plans and can be used for early screening, diagnosis, and treatment of gastrointestinal tumors in clinical practice, showing broad application prospects in clinical settings. Attached Figure Description

[0043] Figure 1 is a graph showing the results of differential expression profiling of plasma circRNA in lung adenocarcinoma patients according to the present invention; wherein... Figure 1A This is a distribution map of circRNAs on chromosomes; Figure 1B This is a distribution diagram of the sequence length of circ RNA; Figure 1C This is a heatmap of circ RNA; darker colors represent elevated expression. Figure 1D The volcano plot shows that dark dots represent upregulated circRNAs and light dots represent downregulated circRNAs.

[0044] Figure 2 shows the screening results of the target circRNA in this invention, wherein, Figure 2A Differences in the copy number of upregulated circRNA in the plasma of lung adenocarcinoma patients Figure 2B For the analysis of the origin and maternal genes of circ LZIC, Figure 2C Analysis of the origin and maternal genes of circ CEP350;

[0045] Figure 3 shows the results of RT-ddPCR verification of the differential copy number of target circRNA in plasma according to this invention. Figure 3A The difference in circ LZIC copy number in plasma. Figure 3B The difference in circ CEP350 copy number in plasma. Figure 3C To detect the difference in plasma circ LZIC copy number using RT-ddPCR, Figure 3D To detect the difference in plasma circ CEP350 copy number using RT-ddPCR, Figure 3E Changes in circ LZIC copy number in plasma before and after surgery. Figure 3F Changes in circ CEP350 copy number in plasma before and after surgery;

[0046] Figure 4 is a ROC curve graph evaluating the diagnostic efficacy of the target circRNA as a diagnostic tool for lung adenocarcinoma; among which, Figure 4A ROC curves for circLZIC and circCEP350 in diagnosing lung adenocarcinoma. Figure 4B ROC curves for early diagnosis of lung adenocarcinoma using circ LZIC and circ CEP350;

[0047] Figure 5 shows the predicted downstream miRNAs and target genes of the target circRNA; among them... Figure 5A This is a network interaction diagram of circLZI with miRNA and downstream target genes. Figure 5B Interaction diagram of circ CEP350 with miRNA and downstream target gene network;

[0048] Figure 6 shows the analysis results of the downstream target genes GO and KEGG pathway of the target circRNA, in which... Figure 6A The results of GO function analysis of downstream target genes of CircLZIC. Figure 6B The results of GO function analysis of downstream target genes of circ CEP350 are shown. Figure 6C The results of KEGG analysis of downstream target genes of Circ LZIC are as follows. Figure 6D The results of KEGG analysis of downstream target genes of circ CEP350 are shown. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0050] This invention employs RNA seq technology to screen differentially expressed circRNAs in the plasma of patients with early-stage lung adenocarcinoma as target circRNAs. RT-ddPCR is used to detect the copy number of circRNAs in the plasma of healthy individuals, patients with early-stage lung adenocarcinoma, and patients with advanced-stage lung adenocarcinoma. ROC curves are plotted, and the diagnostic value of combined detection is analyzed, laying an experimental basis for finding accurate molecular markers for the early diagnosis of lung adenocarcinoma.

[0051] I. Methods

[0052] (I) RT-dd PCR amplification of circ RNA

[0053] 1. Research Subjects

[0054] All lung cancer cases in this invention were obtained from patients diagnosed with lung adenocarcinoma at the Department of Respiratory and Critical Care Medicine and Department of Thoracic Surgery of the First Affiliated Hospital of Bengbu Medical College (hereinafter referred to as the First Affiliated Hospital) between September 2018 and March 2021 (with authorization from the patients and their families, and approval from the hospital's ethics committee). No treatment (including chemotherapy, radiotherapy, targeted therapy, and immunotherapy) was administered before sample collection. A healthy control group of 25 healthy individuals who underwent physical examinations at the hospital during the same period was also included. All studies were approved by the hospital's ethics committee. Clinical data: TNM staging was performed on lung adenocarcinoma patients whose plasma samples were collected according to the latest international guidelines. A total of 50 lung adenocarcinoma patients' plasma samples were collected, with the oldest patient being 82 years old, the youngest 35 years old, and the average age being 61.1 years. Among them, 32 patients had tumor lesions ≥3cm in diameter, 22 had tumor lesions <3cm in diameter, and 22 were classified as N2-N3. Further TNM staging determined the lesions to be stage Ia-IIb (25 cases) and stage IIIa-IV (25 cases).

[0055] 2. Extraction of total RNA from plasma

[0056] (1) Take out the plasma stored at -80℃, thaw it, transfer 300μL of plasma to a 2mL enzyme-free EP tube, add 700μL of TRIzol-LS reagent to the EP tube, and let it stand for 5min.

[0057] (2) Add 200 μL of chloroform, shake manually for 15 seconds, and let stand at room temperature for 5 minutes;

[0058] (3) Transfer the EP tube to a centrifuge and centrifuge at 12000r for 15 min at 4℃. The solution will separate into layers. Slowly aspirate 400 μL of the upper layer and transfer it to a 1.5 mL enzyme-free EP tube. Add isopropanol to each tube, mix well, and let stand at 4℃ for 10 min.

[0059] (4) Centrifuge at 4℃ for 10 min, remove the supernatant, add 1 mL of anhydrous ethanol and mix well so that the precipitate in the EP tube can float in the liquid.

[0060] (5) Centrifuge at 7500r for 5 min at 4℃, discard the ethanol, and then place at room temperature for 8 min;

[0061] (6) Add 15 μL of DEPC water to dissolve the RNA precipitate, mix well, and place on ice for later use;

[0062] (7) Extract 2 μL of RNA solution and place it in a spectrophotometer to detect RNA concentration and purity.

[0063] (8) The extracted total RNA should be stored at -80℃ for a long time.

[0064] 3. Plasma total RNA quality detection

[0065] (1) Determination of total RNA purity and concentration

[0066] Before using the UV spectrophotometer, zero the instrument and aspirate 1 μL of RNA sample onto the surface of the measurement base for detection.

[0067] (2) Total RNA integrity assay

[0068] RNA integrity was detected by 1% denaturing agarose gel electrophoresis. The gel was then photographed under ultraviolet light to observe the brightness and width of the bands and to analyze whether the RNA had degraded.

[0069] 4. RNA library construction and sequencing

[0070] (1) After the sample quality inspection is qualified, take 10-20 ng of RNA and digest it with RNase R to remove linear RNA. Then, use the Clontech SMARTer Stranded Total RNA seq kit V2 pico input kit to construct a library. First, fragment the RNA and add a 5' end conversion template. Then, reverse transcribe to synthesize the first strand of cDNA. Add an indexed adapter through the first round of PCR. Then, purify the ligation product. Then, remove the ribosomal cDNA with Zap R and R-probes. Select the appropriate cycle number to amplify the library. After completion, it can be purified and recovered with magnetic beads. The result is the circRNA-Seq library of the present invention.

[0071] (2) After the library is initially prepared, it can be quantified using Qubit3.0, then the target library range can be detected using Agilenyzer, and then the effective concentration of the library can be accurately quantified using qPCR technology to verify the quality of the library.

[0072] (3) After the library passes the inspection, the PE150 mode is used for sequencing according to the effective concentration and required pooling.

[0073] 5. Reverse transcription of total plasma RNA

[0074] According to the instructions of the Takara MicroRNA Reverse Transcription Kit (Japan), total RNA was reverse transcribed into cDNA. Random primers were used for reverse transcription of total RNA in this experiment (Tables 1 and 2).

[0075] Table 1 Reverse transcription system

[0076]

[0077] Table 2 Reverse transcription reaction conditions

[0078]

[0079] Reverse transcription was performed under the above reaction conditions, and the resulting cDNA could be stored at -20°C for long-term use.

[0080] 6. RT-ddPCR detection of circRNA copy number in plasma

[0081] The reaction system was prepared according to the operating instructions of the RT-dd PCR instrument, and the reaction was carried out using the QX200 instrument.

[0082] (1) Design primers and probes

[0083] The primers were centrifuged in an EP tube for 1 min, then 10 times the recommended volume of DEPC water was added to bring the primer concentration to 10 μmol / μL. The primers were then stored at -20°C. The circ RNA has a circular structure, and the primers were designed and synthesized by Nanjing Qingke Biotechnology Co., Ltd. The probe was prepared by modifying the 5' end of the primer with a fam group and the 3' end with a tamra group. The primer sequences for the target circ RNA are shown in Table 3 below.

[0084] Table 3 Target circRNA primer sequences

[0085]

[0086]

[0087] (2) Preparation of the reaction system

[0088] Configure the reaction system according to the table in Table 4 below. During operation, the pipette tip should be close to the well wall and near the bottom of the well. The well of the PCR plate should correspond to the well of the pipette tip box. The sample should be added to the bottom of the PCR plate.

[0089] Table 4 Reaction System

[0090]

[0091] Add 20 μL of PCR reaction mixture to each droplet generating cartridge (DG8). Then, add 70 μL of droplet generating oil (DG Oil) to each well of the DG8 cartridge and place it in a droplet generator to generate droplets. Transfer the droplets to a 96-well PCR plate (it is recommended to carefully and slowly aspirate using a multi-channel pipette with a 200 μL tip, and then slowly inject the droplets into the corresponding wells, keeping the pipette tip close to the well wall and near the bottom to avoid damaging the droplets). Cover with aluminum foil and seal using a preheated PX1 heat sealer. Perform the PCR reaction on the PCR instrument. The RT-ddPCR reaction conditions are shown in Table 5 below.

[0092] Table 5 RT-ddPCR reaction conditions

[0093]

[0094] After the reaction, the 96-well plate was transferred to a reader, and the number of droplets was calculated using QuantaSoft software. The absolute quantification method used in RT-dd PCR involves the software directly calculating the number of positive droplets in the reaction system, resulting in relatively accurate results.

[0095] (II) Analysis of circRNA-miRNA-mRNA interaction network

[0096] Using the TargetScan and miRTarBase databases, the miRNAs that the target circ RNA may bind to were predicted. Then, mRNAs with tight binding sites were selected from the databases for ceRNA network analysis, and a circRNA-miRNA-mRNA network diagram was drawn using bioinformatics software.

[0097] (III) Functional enrichment analysis of target circRNA and downstream target genes

[0098] To further understand the potential biological functions of the target circRNA, DAVID technology was used to perform GO (Gene Ontology) functional enrichment and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis on the target circRNA and its downstream target genes. This helps the present invention to understand the biological processes and functions of the target circRNA in vivo, as well as the cell signaling pathways it participates in.

[0099] (IV) Statistical Analysis SPSS 22.0 and GraphPad Prism software were used for statistical processing. The t-test was used to analyze differentially expressed circRNAs in plasma, followed by hierarchical cluster analysis. Independent samples t-tests were used to compare the differences between the experimental and control groups. Paired t-tests were used to compare the copy number differences of target circRNAs in the plasma of patients before and after surgery. The chi-square test was used to analyze the relationship between target circRNAs in the plasma of lung adenocarcinoma patients and clinicopathological indicators. Receiver operating characteristic (ROC) curves were plotted using GraphPad software, and the area under the ROC curve (AUC) was calculated to assess the diagnostic efficacy of circRNAs. All tests were two-tailed, and a p-value < 0.05 was considered statistically significant.

[0100] II. Results

[0101] (I) Differential expression profile analysis of plasma circRNA in patients with lung adenocarcinoma

[0102] This invention employed high-throughput sequencing technology to analyze the plasma of 5 patients with early-stage lung adenocarcinoma and 5 healthy individuals, ultimately detecting a total of 9310 circ RNAs. These were then screened according to differential expression levels (FC≥2, P<0.05), revealing 9 upregulated circ RNAs and 62 downregulated circ RNAs. The circ RNA sequence lengths were then analyzed. Figure 1A) and circ RNA distribution on chromosomes ( Figure 1B The results showed that the differentially expressed circRNAs ranged in length from 100 to 1200 base pairs (nt), but most circRNAs were between 100 and 500 nt in length; and circRNAs were scattered across genes on most chromosomes, including chromosomes 1, 5, 7, 8, 14, and 19. Further hierarchical cluster analysis of the differentially expressed circRNAs was performed, and heatmaps, scatter plots, and volcano plots were generated to present the results more intuitively. Figure 1C , Figure 1D ).

[0103] (II) Screening and analysis of target circRNAs

[0104] To identify potential biomarkers for the diagnosis of lung adenocarcinoma, this invention used RT-ddPCR to detect the copy number of nine upregulated circRNAs in the plasma of lung adenocarcinoma patients. The results showed that the copy numbers of circ LZIC (FC = 3.84, P < 0.05) and circCEP350 (FC = 2.55, P < 0.05) were elevated in the plasma of lung adenocarcinoma patients. Figure 2A After reviewing the literature, it was found that these two circRNAs had not been reported in lung cancer, so this invention selected these two circRNA molecules as the research objects.

[0105] The UCSC and CircBase databases were used to analyze the relevant information of two circRNA molecules: circ LZIC (hsa_circ_0000014), derived from the LZIC gene on chromosome 1, with the sequence chr1:9,991,948-9,994,918, consisting of exons 5, 6, and 7 of the LZIC gene; and circ CEP350 (hsa_circ_0003942), derived from the CEP350 gene on chromosome 1, with the sequence chr1:179,972,308-179,975,702, consisting of exons 7 and 8 of the CEP350 gene. Simultaneously, this invention utilized the UCSC database to analyze the maternal genes of the target circRNAs. Figure 2B , Figure 2C ).

[0106] (III) Validation of differential copy number of target circRNA in plasma using RT-ddPCR

[0107] This invention validated the results using RT-ddPCR on plasma from 25 healthy individuals, 25 patients with early-stage lung adenocarcinoma, and 25 patients with advanced-stage lung adenocarcinoma collected clinically. The results showed that the copy numbers of circ LZIC and circ CEP350 were elevated in the plasma of lung adenocarcinoma patients, with a highly statistically significant difference (P < 0.001), consistent with sequencing results. Figure 3A , Figure 3B , Figure 3C , 3D ).

[0108] This invention then explores whether the target circRNA can serve as a biomarker for assessing surgical efficacy. Plasma analysis was performed on three lung adenocarcinoma patients before and after surgery, revealing a significant decrease in the copy numbers of circ LZIC and circ CEP350 in the plasma after surgery. Figure 3E , Figure 3F ).

[0109] (iv) Analyzing the relationship between target circRNA and clinicopathological parameters

[0110] Based on the copy number of the target circRNA in plasma, this invention divides lung adenocarcinoma patients into a high-expression group and a low-expression group. Combining clinicopathological data of lung adenocarcinoma patients, this invention found that the copy number level of circ LZIC in the plasma of lung adenocarcinoma patients was not significantly correlated with gender (p=0.729), age (p=0.196), tumor size (p=0.584), and presence of lymph node metastasis (p=0.138), but was significantly correlated with TNM stage, as shown in Table 6 below.

[0111] Table 6. Correlation analysis of LZIC copy number in plasma with clinicopathological parameters.

[0112]

[0113] The plasma copy number level of circ CEP350 was not significantly correlated with sex (p = 0.427), age (p = 0.243), or presence of lymph node metastasis (p = 0.074), but was significantly correlated with TNM stage and tumor size (p < 0.05), as shown in Table 7 below.

[0114] Table 7. Correlation analysis of circ CEP350 copy number in plasma with clinicopathological parameters

[0115]

[0116]

[0117] Observing the above statistical results, this invention found that the copy number levels of circ LZIC and circ CEP350 in plasma are affected by TNM stage, suggesting that the difference in copy number between these two in plasma may have value for early diagnosis.

[0118] (V) ROC curve evaluation of the diagnostic efficacy of target circRNA in lung adenocarcinoma

[0119] This invention constructed ROC curves to evaluate the efficacy of plasma circ LZIC and circ CEP350 as diagnostic biomarkers for lung adenocarcinoma. The results showed that the AUC value of circ LZIC was 0.782, with a sensitivity and specificity of 52% and 84%, respectively; the AUC value of circ CEP350 was 0.764, with a sensitivity and specificity of 56% and 88%, respectively, demonstrating high diagnostic value. Logistic regression analysis was then performed to detect the efficacy of the two markers combined as diagnostic biomarkers. It was found that the combined use of circ LZIC and circ CEP350 resulted in an AUC as high as 0.863, with a sensitivity and specificity of 72% and 92%, respectively, exhibiting extremely high diagnostic value and significant clinical implications. Figure 4A ).

[0120] Next, this invention further evaluated the diagnostic efficacy of circ LZIC and circ CEP350 as early diagnostic markers for lung adenocarcinoma using ROC curves. The results showed that the AUC value of circ LZIC was 0.786, with a sensitivity and specificity of 72% and 64%, respectively; while the AUC value of circ CEP350 was 0.546, with a sensitivity and specificity of 64% and 56%, respectively. However, after combined analysis of circ LZIC and circ CEP350, the diagnostic efficacy was significantly improved, with an AUC value of 0.803 and a sensitivity and specificity of 60% and 92%, respectively. Figure 4B ).

[0121] (vi) Prediction of downstream miRNAs and target genes of target circRNA

[0122] The most frequently studied route for regulating downstream target genes is through sponge-like binding of circRNAs to miRNAs. Therefore, this invention, through integrated analysis of databases such as circBase and UCSC, discovered that circLZIC and circCEP350 have binding sites with many miRNAs. Several miRNAs with strong binding affinity to circLZIC and circCEP350 were screened, including miR-6504-5p, miR-1267, miR-130b-5p, miR-193b-5p, and miR-3160-5p. The top five miRNAs with the most binding sites to circCEP350 were: miR-125a-5p, miR-125b-5p, miR-4324, miR-4682, and miR-4496. Then, bioinformatics software was used to construct network interaction diagrams (5A and 5B).

[0123] (vii) Analysis of the downstream target genes GO and KEGG pathway of the target circRNA

[0124] This invention analyzes all circ LZIC and circ CEP350 molecules in databases such as miRbase and TangetMiner, screening for highly correlated downstream target genes. GO functional analysis of these downstream target genes is then performed using the DAVID website. The results show that circ LZIC molecular function (MF) is mainly enriched in: extracellular matrix structural components, mRNA 3'-UTR binding, β-catenin binding, and platelet-derived growth factor binding; circ LZIC downstream target gene biological processes (BP) are mainly enriched in: intercellular adhesion via plasma membrane adhesion molecules, homocellular adhesion via plasma membrane adhesion molecules, vesicle localization, and synaptic vesicle circulation; circ LZIC cellular composition (CC) mainly consists of: collagen trimers, extracellular matrix components, and ribbon-like collagen fibers. Figure 6A The molecular functions (MF) of circ CEP350 are mainly enriched in: RNA polymerase II proximal promoter sequence-specific DNA binding, proximal promoter sequence-specific DNA binding, ubiquitin-like protein ligase binding, etc.; the downstream target gene biological processes (BP) of circ CEP350 are mainly enriched in: regulation of membrane permeability, positive regulation of mitochondrial membrane permeability, receptor recycling, and mitochondrial regulation during apoptosis; the cellular composition (CC) of circ CEP350 mainly consists of: extracellular vesicles, intercellular tight junctions, apical junction complexes, and axonal portions. Figure 6B ).

[0125] This invention further utilizes the DAVID website to perform KEGG analysis on the target circRNA, downstream miRNAs, and target genes, revealing that the pathways involved in circ LZIC, with the highest number of enriched genes being the Relaxin signaling pathway, dopaminergic synapses, and neurotrophic protein signaling pathways, etc. Figure 6C The main pathways involved by circ CEP350 are, in order, signaling pathways regulating pluripotency of stem cells, the Hippo signaling pathway, colorectal cancer, pancreatic cancer, and axonal guidance. Figure 6D ).

[0126] III. Conclusion

[0127] In this invention, plasma analysis of lung adenocarcinoma patients and healthy individuals using RNA Seq technology identified 71 differentially expressed circRNAs, of which 9 were upregulated and 62 were downregulated. RT-ddPCR validation of the 9 upregulated circRNAs revealed significantly higher copy numbers of circ LZIC and circ CEP350 in the plasma of lung adenocarcinoma patients compared to healthy individuals, consistent with sequencing results. To verify the expression of the target circRNAs in lung adenocarcinoma tissue, four sets of sequencing data (GSE101586, GSE101684, GSE112214, and GSE158695) were downloaded from the GEO database. Bioinformatics analysis showed that circ LZIC and circ CEP350 expression was higher in lung adenocarcinoma tissue than in adjacent normal tissue, but the difference was not statistically significant (P>0.05). The analysis of this invention suggests that this result may be related to the fact that some circRNAs in plasma originate from exosomes secreted by tumor cells, while others are free circRNAs, resulting in different expression trends of target circRNAs in plasma and tissues.

[0128] Lung adenocarcinoma patients were divided into high-expression and low-expression groups based on plasma copy numbers of circ LZIC and circ CEP350. Further analysis of the relationship between these two circ RNAs and clinicopathological parameters revealed that plasma circ LZIC copy number was not correlated with gender, age, tumor diameter, or lymph node metastasis, but was significantly correlated with TNM stage. Conversely, plasma circ CEP350 copy number was not correlated with gender, age, or lymph node metastasis, but was significantly correlated with tumor diameter and TNM stage (p < 0.05). The close relationship between the differences in circ LZIC and circ CEP350 copy numbers and TNM stage suggests that they may be ideal biomarkers for the early diagnosis of lung adenocarcinoma.

[0129] This invention analyzed these two circRNAs using the circBase and UCSC databases, finding that circ LZIC originates from chromosome 1, consists of three exons, and its maternal gene is the LZIC gene. The leucine zipper and the ICAT domain (containing the leucine zipper and CTNNBIP1 domain, LZIC) make it a member of the WNT signaling family, primarily functioning through the expression of the LZIC protein. The LZIC protein is composed of 190 amino acids (21 kDa) and can inhibit normal cell mitosis by interacting with β-catenin. Recent studies have shown that decreased LZIC expression significantly promotes the progression of renal cell carcinoma cells, and low LZIC expression is clearly correlated with poor prognosis. circCEP350 originates from chromosome 1, consists of two exons, and its maternal gene is the CEP350 gene. Centrosomal protein 350 (CEP350), a member of the centrosomal protein family, is an active component of the centromere and plays a crucial role in centromere biogenesis and cell cycle control. In melanoma, CEP350 promotes DNA methylation in small cell lung cancer through the centrosomal mechanism, thereby reducing the response of small cell lung cancer to Aurora kinase inhibitors. Therefore, circ LZIC and circ CEP350 are closely related to the occurrence and development of malignant tumors and may serve as potential biomarkers for malignant tumors.

[0130] To verify the diagnostic efficacy of circ LZIC and circ CEP350 as diagnostic markers for lung adenocarcinoma, this invention performed ROC curve analysis on these two circ RNAs. The results showed that circ LZIC, as a diagnostic marker for lung adenocarcinoma, had an AUC of 0.782, with a sensitivity of 52% and a specificity of 84%; while circ CEP350, as a diagnostic marker for lung adenocarcinoma, had an AUC of 0.764, with a sensitivity of 56% and a specificity of 88%. When the two markers were combined, the AUC reached as high as 0.863, with a sensitivity of 72% and a specificity of 92%. When ROC curve analysis was used to analyze the efficacy of target circRNA as a diagnostic biomarker for early lung adenocarcinoma, circ LZIC had an AUC of 0.786, with a sensitivity and specificity of 72% and 64%, respectively; while circ CEP350 had an AUC of 0.546, with a sensitivity and specificity of 64% and 56%, respectively. Combining circ LZIC and circ CEP350 significantly improved diagnostic efficacy, with an AUC of 0.803, and a sensitivity and specificity of 60% and 92%, respectively. This indicates that combined detection of circ LZIC and circ CEP350 may be an ideal biomarker for the early diagnosis of lung adenocarcinoma.

[0131] To further explore the mechanisms by which circ LZIC and circ CEP350 function in tumors, this invention analyzed miRNAs that target and bind to circ LZIC and circ CEP350 using databases such as UCSC. The results showed that both circ LZIC and circ CEP350 have numerous miRNA binding sites. This invention also performed GO analysis on circ LZIC, circ CEP350, their closely bound miRNAs, and downstream target genes using databases. The analysis revealed that the biological processes (BP) of circ LZIC downstream target genes are mainly enriched in: intercellular adhesion via plasma membrane adhesion molecules, adhesion of homocellular cells via plasma membrane adhesion molecules, vesicle localization, and synaptic vesicle circulation; while the biological processes (BP) of circ CEP350 downstream target genes are mainly enriched in: regulation of membrane permeability, positive regulation of mitochondrial membrane permeability, receptor reuptake, and mitochondrial regulation during apoptosis.

[0132] KEGG pathway analysis of the target circRNA, downstream miRNAs, and target genes revealed that the most significant enrichment of downstream target genes of circLZIC was in the Relaxin signaling pathway. Relaxin (RLX) is an insulin-like polypeptide hormone initially reported to have pregnancy-related functions, such as cervical softening during childbirth. However, subsequent studies have shown that RLX has anti-fibrotic functions in tumors and non-reproductive tissues (such as skin, lungs, and other tissues). RLX mainly inhibits the accumulation of extracellular matrix (ECM) in solid tumors by suppressing collagen overexpression, thereby removing the physical barrier of dense ECM to drug penetration and diffusion. Beyer et al. demonstrated that hematopoietic stem cell (HSC)-mediated RLX expression effectively degraded ECM in Her2 / neu-positive breast tumors and improved the therapeutic efficacy of Her2 / neu-targeted therapeutic monoclonal antibody (Trastuzumab), indicating that RLX expression in tumors can overcome ECM-mediated drug resistance in solid tumors. The most significant downstream target gene enrichment of circ CEP350 is found in the Hippo signaling pathway, which has been previously studied to play a crucial role in the development and progression of malignant tumors and the regulation of stem cell function. The main effector molecules downstream of the Hippo signaling pathway are YAP / TAZ, which can promote the development of tumor stem cell characteristics and promote tumor growth and metastasis, such as in lung cancer and breast cancer. Therefore, combining GO and KEGG analyses, this invention identifies circ LZIC and circ CEP350 as being able to target miRNA molecules and act on downstream pathways, thereby playing a vital role in the development and progression of lung adenocarcinoma.

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a reagent for detecting the expression level of circular RNA in plasma in the preparation of tools for screening or diagnosing lung adenocarcinoma, characterized in that, The circular RNA is composed of hsa_circ_0000014 and hsa_circ_0003942, and its expression is downregulated in lung adenocarcinoma.

2. The application as described in claim 1, characterized in that, The tools include reagent kits, test strips, chips, or high-throughput sequencing platforms.

3. The application as described in claim 2, characterized in that, The kit, test strip, chip, or high-throughput sequencing platform includes primers or probes for detecting hsa_circ_0000014 and hsa_circ_0003942.

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