Molecular markers for esophageal cancer and uses thereof

Abstract

The application provides an esophageal cancer molecular marker and use thereof, and belongs to the technical field of biological medicine. Specifically relates to the use of a reagent for detecting circular RNA in the preparation of an esophageal cancer sample screening kit, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and hsa_circ_0043278 has a nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence with at least 90% identity thereto. Through gene chip screening combined with qRT-PCR verification, it is found that the expression level of hsa_circ_0043278 in the esophageal cancer sample is significantly higher than that in the normal control sample adjacent to the cancer, and can be used as an effective molecular marker for distinguishing esophageal cancer samples from non-cancer samples.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology. Specifically, this application relates to molecular markers for esophageal cancer and their uses, particularly the use of reagents for detecting circular RNA in the preparation of kits for screening esophageal cancer samples, the use of agents that downregulate the expression level of circular RNA in the preparation of drugs for treating esophageal cancer, kits, and drugs. Background Technology

[0002] Esophageal cancer is one of the most common malignant tumors of the digestive tract worldwide, ranking among the top cancers in both incidence and mortality. The disease is highly malignant, with insidious early symptoms, and most patients are diagnosed at an advanced stage. Traditional treatments, including surgery, radiotherapy, and chemotherapy, generally have poor efficacy, with a low five-year survival rate. Therefore, identifying highly sensitive and specific molecular biomarkers for early diagnosis and novel therapeutic targets is of significant clinical importance for improving the early detection rate and prognosis of esophageal cancer patients.

[0003] Currently, the clinical diagnosis of esophageal cancer mainly relies on serum tumor marker detection, imaging examinations, and endoscopic biopsy. Among these, serum tumor marker detection, as a non-invasive method, is widely used in clinical screening. However, existing serum tumor markers generally suffer from insufficient sensitivity and specificity, making it difficult to meet the clinical needs for accurate early diagnosis of esophageal cancer. Summary of the Invention

[0004] This application aims to solve at least one of the aforementioned related technical problems. In view of this, this application provides a molecular biomarker for esophageal cancer and its uses. Through gene chip screening combined with qRT-PCR verification, it was found that the expression level of hsa_circ_0043278 in esophageal cancer samples was significantly higher than that in adjacent normal control samples, and it can serve as an effective molecular biomarker for distinguishing esophageal cancer samples from non-cancer samples.

[0005] In a first aspect, this application proposes the use of a reagent for detecting circular RNA in the preparation of a kit for screening esophageal cancer samples, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with it.

[0006] This application's embodiments discovered and validated hsa_circ_0043278 as a novel molecular marker for esophageal cancer (especially esophageal squamous cell carcinoma), achieving non-invasive, highly sensitive, and highly specific early diagnosis and prognostic assessment. Compared with traditional serum protein markers, hsa_circ_0043278 in this application's embodiments exhibits higher expression stability and superior detection consistency, significantly improving the accuracy of esophageal cancer screening and auxiliary diagnosis. Simultaneously, the kit is easy to use and has good reproducibility, effectively assisting clinicians in the rapid screening and auxiliary diagnosis of esophageal cancer-related samples, providing reliable molecular evidence for accurate pathological subtyping and clinical decision-making in esophageal cancer.

[0007] Furthermore, this application also demonstrates for the first time that hsa_circ_0043278 has a significant pro-cancer effect. By downregulating the expression level of this circular RNA, the proliferation and migration ability of esophageal cancer cells can be significantly inhibited, indicating that it can not only serve as a diagnostic marker, but also as an important intervention target for molecular targeted therapy of esophageal cancer, thus providing a new, efficient, and low-cost strategy for the precise diagnosis and treatment of esophageal cancer.

[0008] It should be noted that the aforementioned nucleotide sequences with at least 90% identity include nucleotide sequences with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity.

[0009] It should be noted that the aforementioned functional fragments refer to nucleotide fragments that retain some of the biological or detection functions of hsa_circ_0043278. For example, these functional fragments may retain back-splice junction sequences, miRNA binding sites, protein binding regions, or functional regions involved in regulating the malignant phenotype of esophageal cancer cells for specific detection. These functions can be evaluated by detecting their impact on the proliferation, migration, and invasion abilities of esophageal cancer cells, or by detecting their specific expression ability in esophageal cancer samples.

[0010] In an exemplary preferred embodiment, the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO: 1.

[0011] In exemplary embodiments, the reagents include, but are not limited to, probes, primers, or transcriptome sequencing reagents that can specifically recognize hsa_circ_0043278. Based on the aforementioned reagents, techniques such as quantitative real-time PCR (qRT-PCR), digital PCR, RNA in situ hybridization, and RNA sequencing can be used to detect the expression level of hsa_circ_0043278 in esophageal cancer samples. The aforementioned detection methods can be selected from conventional detection methods in the art, and will not be elaborated further here.

[0012] For example, the primers or probes can be designed to target the unique backsplicing sites of hsa_circ_0043278, thereby achieving specific recognition of circular RNA and effectively avoiding interference from linear transcripts, thus improving the accuracy and specificity of detection.

[0013] In exemplary embodiments, the esophageal cancer-related biological samples include, but are not limited to, plasma, serum, whole blood, peripheral blood, tissue samples, biopsy tissue, surgically resected tissue, cell samples, exosome samples, saliva, or other bodily fluid samples derived from the subject. In a preferred embodiment, the biological sample is a plasma sample.

[0014] In an exemplary embodiment, the esophageal cancer includes esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma, and adenosquamous carcinoma, etc.

[0015] As those skilled in the art will know, esophageal squamous cell carcinoma is also known as esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is the most common histological subtype of esophageal cancer in China, accounting for over 90%. This disease is highly malignant, progresses rapidly, and often presents with subtle early symptoms, meaning most patients are already in the middle or late stages when they seek medical attention. Traditional treatments for esophageal squamous cell carcinoma mainly include surgery, radiotherapy, and chemotherapy; however, its overall five-year survival rate remains less than 20%.

[0016] Currently, the clinical diagnosis of esophageal cancer mainly includes: (1) Serum tumor marker detection: Commonly used serum markers include squamous cell carcinoma antigen (SCC-Ag), carcinoembryonic antigen (CEA), cytokeratin 19 fragment (CYFRA21-1), etc., but the sensitivity and specificity of these markers are not ideal. For example, the sensitivity of SCC-Ag in diagnosing esophageal squamous cell carcinoma is only 30%-60%, and false positives are easily observed in benign esophageal diseases; the diagnostic sensitivity of CEA is even lower (about 20%-40%), making it difficult to use as a single marker in clinical practice. (2) Imaging examinations: including upper gastrointestinal barium meal radiography, enhanced CT of the chest and abdomen, and PET-CT, etc. For the detection of early esophageal squamous cell carcinoma, the barium meal has a high rate of missed diagnosis, CT lacks characteristic manifestations, cannot effectively distinguish between inflammation and early cancer, and has problems of radiation exposure and high cost. (3) Endoscopic examinations: including electronic gastroscopy, endoscopic ultrasound (EUS), iodine staining endoscopy, etc. Although gastroscopy combined with biopsy is currently the gold standard for diagnosing esophageal cancer, this method is highly invasive, has poor patient compliance, is not suitable for large-scale population screening, and has limited ability to detect early submucosal lesions.

[0017] The detection method described in this application is particularly suitable for esophageal squamous cell carcinoma. Compared with protein markers such as SCC-Ag and CEA commonly used in related technologies, the hsa_circ_0043278 detection kit provided in this application shows higher diagnostic efficacy in esophageal cancer screening. It can significantly improve the sensitivity and specificity of detection, effectively reduce the false positive rate and false negative rate, and provide important molecular evidence for clinical pathological diagnosis.

[0018] Furthermore, the detection scheme in this application embodiment is particularly suitable for liquid biopsy scenarios, requiring only the collection of peripheral blood or plasma samples from the subject to complete the detection. Compared with traditional tissue biopsy methods, it has advantages such as less trauma, higher patient compliance, and repeatable dynamic monitoring, making it more suitable for large-scale screening, early diagnosis, and postoperative recurrence monitoring of high-risk groups for esophageal cancer.

[0019] Secondly, this application proposes the use of an agent that downregulates the expression level of a circular RNA in the preparation of a drug for treating esophageal cancer, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with it.

[0020] This application's embodiments are the first to discover and confirm that hsa_circ_0043278 is abnormally highly expressed in esophageal cancer and plays an important regulatory role in the malignant biological behavior of esophageal cancer cells. By constructing an hsa_circ_0043278 knockdown model, the inventors found that after specifically downregulating hsa_circ_0043278 expression, the proliferation, DNA replication, and migration abilities of esophageal cancer cells were significantly reduced, indicating that hsa_circ_0043278 plays a pro-cancer role in the occurrence and development of esophageal cancer and can serve as an important molecular therapeutic target for esophageal cancer.

[0021] Specifically, the embodiments of this application, through CCK-8 assays, found that knocking down hsa_circ_0043278 significantly reduced the proliferation rate of Eca-109 and TE12 esophageal cancer cells. Further EdU incorporation assays revealed that silencing hsa_circ_0043278 significantly reduced cellular DNA synthesis capacity, suggesting its involvement in the regulation of esophageal cancer cell proliferation. Furthermore, scratch assays and Transwell migration assays further confirmed that downregulating hsa_circ_0043278 expression significantly inhibited the migration ability of esophageal cancer cells. These experimental results collectively demonstrate that hsa_circ_0043278 is not only a molecular marker associated with esophageal cancer but also an important functional circRNA involved in the malignant progression of esophageal cancer.

[0022] Based on the above findings, this application proposes targeted intervention for esophageal cancer by downregulating the expression level of hsa_circ_0043278. Compared with traditional treatments such as surgery, radiotherapy, and chemotherapy, the RNA-targeted therapy strategy in this application has advantages such as strong targeting, a clear mechanism of action, high programmability, and lower potential toxic side effects. By directly inhibiting key circular RNA molecules that drive the malignant progression of esophageal cancer, the proliferation and migration of tumor cells can be blocked at the source, thereby effectively inhibiting tumor progression.

[0023] It should be noted that the aforementioned nucleotide sequences with at least 90% identity include nucleotide sequences with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity.

[0024] It should be noted that the aforementioned functional fragments refer to nucleotide fragments that retain some of the biological or detection functions of hsa_circ_0043278. For example, these functional fragments may retain back-splice junction sequences, miRNA binding sites, protein binding regions, or functional regions involved in regulating the malignant phenotype of esophageal cancer cells for specific detection. These functions can be evaluated by detecting their impact on the proliferation, migration, and invasion abilities of esophageal cancer cells, or by detecting their specific expression ability in esophageal cancer samples.

[0025] In an exemplary preferred embodiment, the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO: 1.

[0026] In exemplary embodiments, the formulations that downregulate the expression level of circular RNA include, but are not limited to, specific siRNA, shRNA, antisense nucleic acid (ASO), interfering RNA, nucleic acid aptamers, CRISPR intervention systems, RNA degradation molecules, or small molecule inhibitors targeting hsa_circ_0043278.

[0027] In some embodiments, the siRNA can specifically recognize and degrade hsa_circ_0043278 through an RNA interference mechanism, thereby reducing its expression level in esophageal cancer cells. Preferably, the siRNA can be designed to target the back-splice junction specific to hsa_circ_0043278 to improve the recognition specificity of circular RNA and reduce non-specific interference with linear transcripts.

[0028] In some embodiments, the shRNA can be introduced into esophageal cancer cells via a viral vector, plasmid vector, or other expression system and continuously expressed within the cells, thereby achieving long-term stable silencing of hsa_circ_0043278.

[0029] In some embodiments, the antisense nucleic acid (ASO) can inhibit its biological function by inducing RNase H-mediated RNA degradation through base complementary pairing with hsa_circ_0043278, or by blocking its binding to downstream regulatory molecules.

[0030] In some embodiments, the small molecule inhibitor can inhibit the functional activity of hsa_circ_0043278 by inhibiting its generation, stability, subcellular localization, or interaction with functional proteins / miRNAs.

[0031] In an exemplary embodiment, the functional fragment of hsa_circ_0043278 includes a nucleotide fragment that retains its proliferative and migration-promoting functions, or a fragment that retains functional regions such as miRNA binding sites, protein binding sites, and backsplicing sites. The function of the functional fragment can be evaluated by detecting its effect on the proliferation, migration, and invasion abilities of esophageal cancer cells.

[0032] In exemplary embodiments, the formulation that downregulates circular RNA expression levels may further comprise a pharmaceutically acceptable delivery system to improve the in vivo stability, delivery efficiency, and tumor targeting of the nucleic acid drug. The delivery system includes, but is not limited to, lipid nanoparticles (LNPs), liposomes, cationic polymers, polyethylene glycol-modified carriers, peptide delivery systems, exosome delivery systems, or viral vectors.

[0033] In exemplary embodiments, the drug may be prepared as an injection, a lyophilized powder for injection, a lipid nanoparticle formulation, a sustained-release formulation, a targeted formulation, or other dosage forms suitable for clinical administration.

[0034] In exemplary embodiments, the drug can be used alone or in combination with chemotherapy drugs, radiotherapy, immunotherapy drugs, molecularly targeted drugs or other anti-tumor treatment regimens to further improve the treatment effect of esophageal cancer.

[0035] In an exemplary embodiment, the esophageal cancer includes esophageal squamous cell carcinoma (ESCC), esophageal adenocarcinoma, and adenosquamous carcinoma, etc.

[0036] In a preferred embodiment, the esophageal cancer is esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is the most common histological subtype of esophageal cancer in China, accounting for more than 90% of all esophageal cancer cases. It is characterized by high malignancy, strong invasiveness, easy recurrence and metastasis, and poor overall prognosis. This application's embodiments found that hsa_circ_0043278 is significantly highly expressed in esophageal squamous cell carcinoma, and its expression level is closely related to the malignant proliferation and migration ability of esophageal squamous cell carcinoma cells. Therefore, by downregulating the expression level of hsa_circ_0043278, the malignant biological behavior of esophageal squamous cell carcinoma cells can be effectively inhibited, providing a new precise molecular targeted therapy strategy for esophageal squamous cell carcinoma.

[0037] Furthermore, due to the high natural stability, strong tissue specificity, and resistance to degradation of circRNA, the RNA-targeted therapy system constructed against hsa_circ_0043278 has good clinical application potential and can be used to treat esophageal cancer, especially mid-to-late stage esophageal squamous cell carcinoma, recurrent esophageal cancer, or patients who are not sensitive to traditional radiotherapy and chemotherapy.

[0038] Thirdly, this application proposes a kit for screening biological samples susceptible to or with esophageal cancer, the kit containing reagents suitable for detecting circular RNA, the circular RNA being hsa_circ_0043278 or a functional fragment thereof, the hsa_circ_0043278 having the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with it.

[0039] This application's embodiments, through gene chip screening and subsequent qRT-PCR verification, revealed that the expression level of hsa_circ_0043278 in esophageal cancer patients was significantly higher than that in healthy controls, and it also exhibited high expression abundance in esophageal cancer plasma samples. Therefore, the kit constructed in this application's embodiments can be used for the auxiliary diagnosis, early screening, prognostic assessment, recurrence monitoring, and risk assessment of high-risk groups of esophageal cancer.

[0040] Because circRNAs have a naturally closed circular structure and are not easily degraded by RNA exonucleases, hsa_circ_0043278 exhibits higher stability and a longer half-life compared to traditional linear RNA or protein biomarkers, making it more suitable for clinical liquid biopsy and molecular diagnostic applications. The kit in this application utilizes hsa_circ_0043278 as a molecular detection target, effectively improving the sensitivity and specificity of esophageal cancer screening.

[0041] It should be noted that the aforementioned nucleotide sequences with at least 90% identity include nucleotide sequences with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity.

[0042] It should be noted that the aforementioned functional fragments refer to nucleotide fragments that retain some of the biological or detection functions of hsa_circ_0043278. For example, these functional fragments may retain back-splice junction sequences, miRNA binding sites, protein binding regions, or functional regions involved in regulating the malignant phenotype of esophageal cancer cells for specific detection. These functions can be evaluated by detecting their impact on the proliferation, migration, and invasion abilities of esophageal cancer cells, or by detecting their specific expression ability in esophageal cancer samples.

[0043] In exemplary embodiments, the reagents in the kit suitable for detecting circular RNA include, but are not limited to, specific primers, probes, reverse transcription reagents, amplification enzymes, nucleic acid detection buffers, RNA extraction reagents, RNA stabilizers, fluorescent labeling reagents, sequencing reagents, or other reagents for detecting the expression level of hsa_circ_0043278.

[0044] In some embodiments, the reagent includes specific primers or probes designed for the hsa_circ_0043278 backsplicing site to achieve specific recognition of circular RNA. Because the unique backsplicing structure of circular RNA differs from that of linear RNA transcripts, detection systems designed for this region can effectively avoid interference from linear RNA amplification, thereby improving detection accuracy and specificity.

[0045] In some embodiments, the kit is suitable for quantitative real-time PCR (qRT-PCR), digital PCR (dPCR), RNA sequencing, RNA in situ hybridization (RNA-FISH), or other nucleic acid detection technology platforms.

[0046] In some embodiments, the kit may further include an internal control component for correcting RNA extraction levels and detection errors between samples. The internal control includes, but is not limited to, β-actin, GAPDH, U6, or other commonly used internal control molecules in the art.

[0047] In exemplary embodiments, the biological samples include, but are not limited to, plasma, serum, whole blood, peripheral blood, tissue samples, biopsy tissue, surgically excised tissue, cell samples, exosome samples, saliva, pleural or peritoneal fluid, or other biological samples derived from the subject.

[0048] In a preferred embodiment, the biological sample is a plasma sample. Because plasma collection is simple, minimally invasive, and easy to repeat, the kit described in this application is particularly suitable for liquid biopsy scenarios and can be used for large-scale screening, early auxiliary diagnosis, and postoperative dynamic monitoring of high-risk groups for esophageal cancer.

[0049] In some embodiments, when the expression level of hsa_circ_0043278 in a subject's sample is detected to be significantly higher than the normal reference level, it can be determined that the subject has a high risk of esophageal cancer or already has esophageal cancer lesions.

[0050] In an exemplary embodiment, the esophageal cancer includes esophageal squamous cell carcinoma, esophageal adenocarcinoma, and adenosquamous carcinoma, etc.

[0051] In a preferred embodiment, the esophageal cancer is esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is the most common type of esophageal cancer in China, accounting for more than 90% of all esophageal cancer cases. The inventors found that hsa_circ_0043278 is significantly overexpressed in patients with esophageal squamous cell carcinoma, and its expression level is closely related to the malignant progression of esophageal squamous cell carcinoma. Therefore, the kit described in this application is particularly suitable for the auxiliary screening and molecular diagnosis of esophageal squamous cell carcinoma.

[0052] Compared with traditional serum protein markers such as SCC-Ag, CEA, and CYFRA21-1, the hsa_circ_0043278 detection kit in this application exhibits higher stability, sensitivity, and specificity, effectively reducing false positive and false negative rates. Furthermore, compared to invasive methods such as endoscopic biopsy, the kit in this application only requires the collection of peripheral blood or plasma samples from the subject, making it more suitable for large-scale clinical screening and long-term dynamic follow-up of esophageal cancer.

[0053] Fourthly, this application proposes a medicament for the prevention and / or treatment of esophageal cancer, the medicament comprising an agent that downregulates the expression level of a circular RNA, the circular RNA being hsa_circ_0043278 or a functional fragment thereof, the hsa_circ_0043278 having the nucleotide sequence shown in SEQ ID NO: 1 or a nucleotide sequence having at least 90% identity with it.

[0054] This application's embodiments reveal for the first time that hsa_circ_0043278 is abnormally highly expressed in esophageal cancer, and its expression level is closely related to the malignant proliferation and migration ability of esophageal cancer cells. Further research showed that specifically downregulating the expression of hsa_circ_0043278 can significantly inhibit the proliferation, DNA replication, and migration abilities of esophageal cancer cells, thereby effectively inhibiting the malignant progression of esophageal cancer. Therefore, hsa_circ_0043278 in this application's embodiments can not only serve as a molecular marker for esophageal cancer but also as an important therapeutic target for esophageal cancer.

[0055] Based on the above findings, this application proposes to construct an anti-esophageal cancer drug targeting hsa_circ_0043278. By inhibiting the expression or function of this circular RNA, precise intervention in esophageal cancer can be achieved. Compared with traditional radiotherapy, chemotherapy, or surgery, the RNA-targeted therapy strategy in this application has advantages such as strong targeting, clear mechanism, specific regulation of carcinogenic molecules, and lower potential toxic side effects, and can block the malignant biological behavior of esophageal cancer at the molecular level.

[0056] It should be noted that the aforementioned nucleotide sequences with at least 90% identity include nucleotide sequences with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.9% identity.

[0057] It should be noted that the aforementioned functional fragments refer to nucleotide fragments that retain some of the biological or detection functions of hsa_circ_0043278. For example, these functional fragments may retain backsplicing sites, miRNA binding sites, protein binding regions, or functional regions involved in regulating the malignant phenotype of esophageal cancer cells for specific detection. These functions can be evaluated by detecting their impact on the proliferation, migration, and invasion abilities of esophageal cancer cells, or by detecting their specific expression ability in esophageal cancer samples.

[0058] In exemplary embodiments, the agents that downregulate the expression level of circular RNA include, but are not limited to, siRNA, shRNA, antisense nucleic acid (ASO), double-stranded RNA, RNA interference molecules, CRISPR intervention systems, nucleic acid aptamers, small molecule inhibitors, or other agents that can reduce the expression level of hsa_circ_0043278 or inhibit its biological function.

[0059] In some embodiments, the siRNA can specifically bind to hsa_circ_0043278 and induce its degradation through an RNA interference mechanism, thereby reducing its expression level in esophageal cancer cells.

[0060] In some embodiments, the siRNA is preferably designed to target the backsplicing site of hsa_circ_0043278 to enhance specific recognition of circular RNA and reduce nonspecific effects on the corresponding linear RNA transcript.

[0061] In some embodiments, the shRNA can be introduced into esophageal cancer cells via a viral vector or expression vector and continuously expressed within the cells, thereby achieving long-term stable silencing of hsa_circ_0043278.

[0062] In some embodiments, the antisense nucleic acid (ASO) can induce RNase H-mediated RNA degradation by base complementary pairing with hsa_circ_0043278, or block its interaction with downstream miRNAs, RNA-binding proteins or other regulatory molecules, thereby inhibiting its oncogenic function.

[0063] In some embodiments, the small molecule inhibitor can inhibit the biological activity of hsa_circ_0043278 by inhibiting its generation, stability, subcellular localization, or functional interactions.

[0064] In an exemplary embodiment, the functional fragment of hsa_circ_0043278 includes nucleotide fragments that retain their functions of promoting proliferation and migration, or fragments that retain functional regions such as their back splicing sites, miRNA binding sites, or protein binding sites.

[0065] In an exemplary embodiment, the drug may be prepared as an injectable formulation.

[0066] Because RNA drugs typically suffer from low in vivo stability and limited oral absorption efficiency, the drugs in this application are preferably administered via injection to improve the bioavailability and tumor delivery efficiency of the nucleic acid drugs. The injection methods include, but are not limited to, intravenous injection, local injection, intratumoral injection, subcutaneous injection, or intraperitoneal injection.

[0067] In some embodiments, the injectable may be a solution-type injectable, a lyophilized powder injectable, a lipid nanoparticle injectable, a sustained-release injectable, or other injectable dosage forms suitable for clinical administration.

[0068] In an exemplary embodiment, the drug may further comprise pharmaceutically acceptable excipients or carriers. These pharmaceutically acceptable excipients or carriers include, but are not limited to, buffers, stabilizers, preservatives, osmotic pressure regulators, emulsifiers, solubilizers, excipients, sustained-release materials, or delivery carriers.

[0069] In some embodiments, the delivery carrier includes, but is not limited to, lipid nanoparticles (LNPs), liposomes, cationic liposomes, polymer nanoparticles, polyethylene glycol-modified carriers, peptide delivery systems, exosome delivery systems, or viral vectors.

[0070] In some embodiments, the excipients in the drug can improve the stability of the nucleic acid preparation, its circulation time in vivo, its cellular uptake efficiency, or its ability to accumulate in tumor tissue, thereby further improving the therapeutic effect of the drug.

[0071] In exemplary embodiments, the drug can be used alone or in combination with chemotherapy drugs, radiotherapy, immunotherapy, targeted therapy or other anti-tumor treatment regimens to further improve the treatment effect of esophageal cancer.

[0072] In an exemplary embodiment, the esophageal cancer includes esophageal squamous cell carcinoma, esophageal adenocarcinoma, and adenosquamous carcinoma, etc.

[0073] In a preferred embodiment, the esophageal cancer is esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is the most common subtype of esophageal cancer in China, characterized by high invasiveness, easy recurrence and metastasis, and a generally poor prognosis. This application's embodiments found that hsa_circ_0043278 is significantly overexpressed in esophageal squamous cell carcinoma and participates in regulating the malignant proliferation and migration of esophageal cancer cells. Therefore, by downregulating the expression level of hsa_circ_0043278, the malignant biological behavior of esophageal squamous cell carcinoma can be effectively inhibited, providing a new precise molecular targeted therapy strategy for esophageal squamous cell carcinoma.

[0074] Furthermore, due to the stable expression, strong tissue specificity, and resistance to degradation of circRNA, the RNA-targeted therapy system based on hsa_circ_0043278 has good clinical application prospects, especially suitable for patients with advanced, recurrent, or esophageal cancer who are not sensitive to traditional radiotherapy and chemotherapy.

[0075] Fifthly, this application proposes a method for screening esophageal cancer biological samples. According to an embodiment of this application, the method includes: 1) determining the expression level of a circular RNA in the biological sample, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO: 1; 2) comparing the expression level of the circular RNA obtained in step 1) with a predetermined threshold to screen esophageal cancer biological samples.

[0076] This application's embodiments, through gene chip screening and subsequent qRT-PCR verification, revealed that hsa_circ_0043278 is significantly highly expressed in esophageal cancer patients, and its expression level is closely related to the occurrence and development of esophageal cancer. Therefore, by detecting the expression level of hsa_circ_0043278 in subject samples and comparing it with normal reference levels or predetermined thresholds, esophageal cancer samples can be effectively distinguished from non-cancer samples, enabling auxiliary screening, early diagnosis, risk assessment, and prognostic analysis of esophageal cancer.

[0077] In an exemplary embodiment, the method for determining the expression level of circular RNA in step 1) includes, but is not limited to, quantitative real-time PCR (qRT-PCR), digital PCR (dPCR), RNA sequencing (RNA-seq), microarray detection, RNA in situ hybridization (RNA-FISH), Northern blot, or other nucleic acid detection techniques suitable for detecting circRNA expression levels.

[0078] In some embodiments, the detection method employs specific primers or probes designed for the hsa_circ_0043278 backsplicing site to improve the detection specificity for circular RNA and reduce detection interference caused by the corresponding linear RNA transcripts.

[0079] In some embodiments, step 1) further includes processing steps such as RNA extraction, reverse transcription, and nucleic acid amplification of the biological sample.

[0080] In some embodiments, the RNA extraction may employ the Trizol method, centrifugation column method, magnetic bead method, or other conventional RNA extraction methods in the art.

[0081] In some embodiments, the biological samples include, but are not limited to, plasma, serum, whole blood, peripheral blood, tissue samples, biopsy tissue, surgically excised tissue, cell samples, exosome samples, saliva, pleural or peritoneal fluid, or other biological samples derived from the subject.

[0082] In a preferred embodiment, the biological sample is a plasma sample. Because plasma sample collection is simple, minimally invasive, and easy to repeat, the method in this embodiment is particularly suitable for liquid biopsy scenarios and can be used for large-scale screening of high-risk groups for esophageal cancer, postoperative recurrence monitoring, and dynamic follow-up.

[0083] In some other preferred embodiments, the biological sample is an esophageal tissue sample. Since hsa_circ_0043278 is significantly highly expressed in esophageal cancer tissue, the method in this embodiment is also applicable to molecular detection of pathological tissue samples, and can assist clinicians in pathological diagnosis and molecular subtyping of esophageal cancer tissue.

[0084] In an exemplary embodiment, the predetermined threshold in step 2) includes the mean expression level of hsa_circ_0043278 in the normal control group, the standard deviation range, the optimal cutoff value of the ROC curve, the empirical statistical threshold, or other reference values ​​that can distinguish esophageal cancer samples from non-cancer samples.

[0085] In some embodiments, when the expression level of hsa_circ_0043278 in the biological sample to be tested is higher than the predetermined threshold, the biological sample can be determined to be a positive sample for esophageal cancer, or it can be indicated that the subject has a high risk of esophageal cancer.

[0086] In some embodiments, when the expression level of hsa_circ_0043278 in the biological sample to be tested is lower than the predetermined threshold, the biological sample can be determined to be a non-esophageal cancer sample, or it can be indicated that the subject does not have a significant risk of esophageal cancer.

[0087] In an exemplary embodiment, the esophageal cancer includes esophageal squamous cell carcinoma, esophageal adenocarcinoma, and adenosquamous carcinoma, etc.

[0088] In a preferred embodiment, the esophageal cancer is esophageal squamous cell carcinoma. Esophageal squamous cell carcinoma is the most common histological subtype of esophageal cancer in China, characterized by inconspicuous early symptoms, high malignancy, and poor prognosis. This application's embodiments found that hsa_circ_0043278 is significantly highly expressed in esophageal squamous cell carcinoma; therefore, the method in this application's embodiments is particularly suitable for the auxiliary screening and early diagnosis of esophageal squamous cell carcinoma. Compared with traditional serum protein markers such as SCC-Ag, CEA, and CYFRA21-1, the detection method in this application's embodiments has higher sensitivity and specificity, effectively reducing false positive and false negative rates. Furthermore, compared to invasive detection methods such as endoscopic biopsy, the method in this application's embodiments can achieve non-invasive detection by detecting circRNA in the peripheral blood or plasma of the subject, making it more suitable for large-scale clinical screening and long-term dynamic monitoring of esophageal cancer. In addition, because circRNA has a closed-circular structure and is not easily degraded by RNA exonucleases, hsa_circ_0043278 has good theoretical stability and is more suitable as a molecular marker in liquid biopsy. Attached Figure Description

[0089] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0090] Figure 1 This diagram illustrates the results of differential expression analysis of circular RNA between the experimental group of esophageal squamous cell carcinoma patients and the healthy control group provided in this application embodiment. In this diagram, A represents the hierarchical clustering analysis results of differentially expressed circular RNA between the experimental group (T) of esophageal squamous cell carcinoma patients and the healthy control group (C); B represents the chromosome distribution results of differentially expressed circRNAs between the experimental group (T) of esophageal squamous cell carcinoma patients and the healthy control group (C); and C represents the results of significantly differentially expressed circular RNAs obtained based on the selection of log2-fold change > 2 and P < 0.05.

[0091] Figure 2The diagram illustrates the results of circ_0043278 expression verification and transfection efficiency detection provided in the embodiments of this application. A shows the results of qRT-PCR detection of circ_0043278 expression levels in esophageal cancer tissue and adjacent normal tissue; B shows the results of qRT-PCR verification of the knockdown efficiency of three siRNAs (si-circ#1, si-circ#2, and si-circ#3) targeting circ_0043278 in Eca-109 cells; C shows the results of qRT-PCR verification of the knockdown efficiency of three siRNAs targeting circ_0043278 in TE12 cells; D shows the results of qRT-PCR detection of the transfection efficiency of the circ_0043278 overexpression plasmid in Eca-109 cells; and E shows the results of qRT-PCR detection of the transfection efficiency of the circ_0043278 overexpression plasmid in TE12 cells.

[0092] Figure 3 This is a schematic diagram of the subcellular localization analysis results of circ_0043278 provided in the embodiments of this application. In this diagram, A shows the results of subcellular distribution analysis of circ_0043278 in Eca-109 and TE12 cells by combining nucleocytoplasmic separation experiment with qRT-PCR, with U6 as the nucleus control and β-actin as the cytoplasm control; B shows the results of spatial localization detection of circ_0043278 in Eca-109 and TE12 cells by FISH experiment, where the fluorescence signal of circ_0043278 is green, and the nucleus is blue after DAPI counterstaining.

[0093] Figure 4 This diagram illustrates the detection results of the effect of circ_0043278 on the proliferation ability of esophageal cancer cells provided in the embodiments of this application. In this diagram, A shows the results of the CCK-8 assay in Eca-109 cells to detect the cell proliferation ability of the si-NC group and the si-circ_0043278 group; B shows the results of the CCK-8 assay in TE12 cells to detect the cell proliferation ability of the si-NC group and the si-circ_0043278 group; C shows the results of the CCK-8 assay in Eca-109 cells to detect the cell proliferation ability of the circ_0043278 overexpression group and the empty vector control group; and D shows the results of the CCK-8 assay in TE12 cells to detect the cell proliferation ability of the circ_0043278 overexpression group and the empty vector control group.

[0094] Figure 5This is a schematic diagram of the detection results of the effect of circ_0043278 on the DNA synthesis ability of esophageal cancer cells provided in the embodiments of this application. In this diagram, A is a schematic diagram of the EdU experiment results of Eca-109 and TE12 cells after circ_0043278 knockdown or overexpression. EdU-positive proliferating cells are shown in red, and the cell nuclei are shown in blue after DAPI counterstaining. B is the quantitative analysis result of the proportion of EdU-positive cells in Eca-109 and TE12 cells.

[0095] Figure 6 This is a schematic diagram showing the detection results of the effect of circ_0043278 on the migration ability of esophageal cancer cells provided in the embodiments of this application. In this diagram, A shows the scratch experiment results of Eca-109 and TE12 cells after being transfected with si-NC, si-circ_0043278, empty vector or circ_0043278 overexpression plasmid, respectively, at 0 hours and 48 hours after scratching; B shows the quantitative analysis results of the relative scratch healing area of ​​Eca-109 and TE12 cells. Detailed Implementation

[0096] The embodiments of this application are described in detail below, with examples of these embodiments shown in the accompanying drawings. Throughout, the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application.

[0097] In the process of describing this application, the terms used herein have been explained and clarified. These explanations and clarifications are merely for the purpose of facilitating the understanding of the solution and should not be regarded as a limitation on the solution protected by this application.

[0098] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0099] In this application, "molecular marker" is synonymous with "biomarker," which refers to cellular / biochemical or molecular alterations that can be detected in biological media, including various body fluids, tissues, cells, etc., and in this context, plasma.

[0100] In this invention, for comparing two or more nucleotide sequences, the percentage of "sequence identity" between a first and a second sequence can be calculated by dividing the number of nucleotides in the first sequence that are identical to those at the corresponding positions by the number of nucleotides in the second sequence. The percentage is calculated by subtracting the total number of nucleotides in the first sequence from the number of nucleotides in the second sequence and then multiplying by 100%, where each deletion, insertion, substitution, or addition of a nucleotide in the second sequence—relative to the first sequence—is considered a difference at a single nucleotide (position).

[0101] Alternatively, standard settings can be used to calculate the degree of sequence identity between two or more nucleotide sequences using known computer algorithms for sequence alignment, such as NCBI Blastv2.0.

[0102] Other techniques, computer algorithms, and settings used to determine the degree of sequence identity include, for example, those in WO 04 / 037999, EP 0 967 284, EP 1 085 089, WO 00 / 55318, WO 00 / 78972, WO 98 / 49185, and GB2357768-A.

[0103] In this application, the parts of the name "hsa_circ_0043278" represent the following: hsa, representing Homosapiens (scientific human), i.e., of human origin; circ, representing circular RNA; and 0043278, a unique sequence number assigned by the database. It should be noted that the length of "hsa_circ_0043278 or its functional fragment" as described in this application is not particularly limited; it can be a complete gene, a functional fragment of a gene, or, moreover, any relevant nucleic acid fragment of any length can be selected depending on different experimental purposes.

[0104] In this application, "circular RNA (circRNA)" refers to a class of non-coding RNA molecules with a covalently closed circular structure. Due to the absence of a 5' cap and a 3' poly(A) tail, circRNA is less susceptible to degradation by exonucleases, exhibiting higher stability, a longer half-life, and superior stability in body fluid assays compared to linear RNA. Based on these structural characteristics, circRNA is particularly suitable as a disease molecular biomarker in liquid biopsies, possessing significant application value in early tumor screening, auxiliary diagnosis, prognostic assessment, and efficacy monitoring.

[0105] In this application, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly in humans.

[0106] In this application, "administration" refers to the process of introducing a formulation or drug for regulating the expression level of hsa_circ_0043278 into a subject's body or acting on the subject's target tissues / cells through a suitable route of administration, so that the formulation or drug reaches an effective concentration in the body, thereby achieving regulation of hsa_circ_0043278 expression or function. The administration includes, but is not limited to, systemic administration or local administration, wherein systemic administration includes intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, etc.; local administration includes local injection into tumor sites, targeted tissue delivery, or direct administration to the lesion site, etc. In some embodiments, the administration is preferably by injection to improve the stability and targeted delivery efficiency of nucleic acid formulations (such as siRNA, shRNA, or ASO, etc.) in vivo. In some embodiments, the administration can be achieved using a delivery carrier, including lipid nanoparticles (LNP), liposomes, polymer nanoparticles, or exosomes, to improve the drug's accumulation capacity in target tissues.

[0107] In this application, "treatment" refers to the process of improving, alleviating, delaying, or reversing esophageal cancer-related pathological states by regulating the expression level or biological function of hsa_circ_0043278. The treatment includes, but is not limited to: inhibiting the abnormal proliferation of esophageal cancer cells; inhibiting the migration and invasion capabilities of esophageal cancer cells; blocking or delaying tumor progression; reducing tumor burden; improving patient survival or quality of life; and reducing the risk of recurrence or metastasis. In the embodiments of this application, the treatment is mainly achieved by downregulating the expression level of hsa_circ_0043278, which, as a pro-cancer circular RNA, significantly reduces the malignant biological behavior of esophageal cancer cells when its expression is inhibited. In some embodiments, the treatment is suitable for preventive intervention, early intervention, mid-to-late stage treatment, or adjuvant therapy for recurrent esophageal cancer, especially for patients with esophageal squamous cell carcinoma. In some embodiments, the treatment can be implemented alone or in combination with surgical treatment, radiotherapy, chemotherapy, immunotherapy, or other targeted therapies to improve the overall treatment effect.

[0108] In this application, the term "downregulation" is used to describe the change in the expression level of the circular RNA (hsa_circ_0043278), which refers to the process or state in which the expression level of the circular RNA is reduced relative to the control level through exogenous intervention.

[0109] For example, the “downregulation” includes reducing or significantly reducing the expression level of hsa_circ_0043278, where “significantly reduced” means that its expression level drops below a preset threshold level compared to a control sample that has not received any intervention (e.g., a healthy individual sample or an untreated esophageal cancer cell / tissue sample).

[0110] In some embodiments, the control sample may be normal esophageal tissue, plasma samples from healthy subjects, or cell samples that have not undergone intervention with siRNA, shRNA, or antisense nucleic acids.

[0111] In some implementations, the “downregulation” is achieved through RNA interference (RNAi), antisense nucleic acid (ASO), gene editing systems, or other means that can reduce the expression or stability of the target circular RNA.

[0112] In some preferred embodiments, the “downregulation” is manifested as a decrease in the relative expression level of hsa_circ_0043278, for example, a decrease to less than 50%, less than 30%, or lower than the expression level of the control group. However, this application is not limited to the specific reduction range, as long as it can functionally inhibit the proliferation or migration ability of esophageal cancer cells.

[0113] In the context of this application, whether "downregulation" has occurred is typically determined by comparison with a preset threshold, which includes, but is not limited to, the expression level in normal control tissues, the expression level in the plasma of healthy individuals, or a statistically determined reference range (such as mean ± standard deviation or cut-off value determined by ROC analysis).

[0114] The sequences involved in this application are shown in Table 1.

[0115] Table 1

[0116] The embodiments of this application will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0117] Example 1: Screening of Molecular Markers To screen for circular RNA molecular markers associated with the development and progression of esophageal squamous cell carcinoma, this embodiment constructs a differential expression profile of esophageal squamous cell carcinoma circRNAs using gene chip technology. Specifically, this includes: From March to October 2021, 31 patients with esophageal squamous cell carcinoma and 35 healthy controls were recruited from Ningbo University Affiliated Li Huili Hospital, and plasma samples were collected for subsequent analysis. Recruitment criteria were as follows: Cohort A consisted of patients with histologically confirmed esophageal squamous cell carcinoma (ESCC) (age ≥19 years) who had not previously received radiotherapy, chemotherapy, or targeted therapy. Cohort B consisted of patients with untreated non-malignant esophageal diseases. Exclusion criteria included: (i) other malignancies; (ii) prior radiotherapy, chemotherapy, or biotherapy; and (iii) serious comorbidities. This study adhered to the ethical standards and operating procedures approved by the Li Huili Hospital Ethics Review Committee, and written informed consent was obtained from all participants.

[0118] This embodiment analyzed circular RNA microarray data from samples of three patients with esophageal squamous cell carcinoma and three healthy controls. Total RNA in each sample was quantified using a NanoDrop ND-1000 ultra-micro spectrophotometer. Sample preparation and microarray hybridization experiments were strictly performed according to Arraystar's standard experimental procedures. A brief description of the experiments is as follows: Total RNA was enzymatically digested using Epicentre's RNase R enzyme to remove linear RNA and enrich circular RNA. The enriched circular RNA was then amplified using a random primer method combined with the Arraystar Super RNA labeling kit and transcribed into fluorescently labeled cRNA. The labeled cRNA was hybridized to an Arraystar human circular RNA microarray V2 (8×15K). After microarray elution, the microarray signal was scanned using an Agilent G2505C microarray scanner. The acquired microarray images were analyzed using Agilent feature extraction software (version 11.0.1.1). Quantile normalization and subsequent data processing were performed using the limma package in R. Differentially expressed circular RNAs with statistically significant differences between groups were screened using volcano plots, and differentially expressed circular RNAs between pairs of samples were determined by fold change. Hierarchical cluster analysis was also performed to visually demonstrate the differences in circular RNA expression characteristics among samples.

[0119] Differentially expressed circular RNAs that were significantly upregulated and significantly downregulated were screened, and the results are as follows: Figure 1As shown in A-1C. Specifically, a total of 11,381 circular RNAs were identified; after analysis using strict screening criteria (log2-fold change > 2 and P < 0.05), 1,012 circular RNAs showed significant differential expression between the esophageal squamous cell carcinoma experimental group (T) and the healthy control group (C), and their sources were mainly distributed on chromosomes 1-7 and the X chromosome (chr1, chr2, chr3, chr4, chr5, chr6, chr7 and chrX).

[0120] Furthermore, the top 10 circRNAs with the most significant differential expression were screened from the 1012 significantly differentially expressed circRNAs, and the results are shown in Table 2. The results indicate that there is a significantly abnormal circRNA expression profile in the plasma samples of esophageal squamous cell carcinoma patients. Among them, hsa_circ_0043278 showed a significantly high expression trend, with a log2fold change value reaching 34.5999, which was statistically significant (P=0.0086), significantly higher than most other differentially expressed molecules. These results suggest that hsa_circ_0043278 is strongly correlated with the occurrence and development of esophageal squamous cell carcinoma and can serve as a potential molecular marker candidate molecule for subsequent diagnostic and functional studies.

[0121] Table 2

[0122] Note: FC stands for fold change.

[0123] Example 2: Validation of hsa_circ_0043278 expression in plasma and tissue samples To verify the expression characteristics of candidate circRNAs in clinical samples, this embodiment detected the expression level of hsa_circ_0043278 in esophageal cancer tissue and plasma samples, and further constructed circ_0043278 knockdown and overexpression cell models to verify its expression characteristics in esophageal cancer and the feasibility of subsequent functional studies. Details are as follows: 1. RNA extraction and qRT-PCR detection from clinical samples esophageal tissue from The tissue was removed from the 80°C ultra-low temperature freezer, and 50 mg of the tissue was thoroughly ground. Total RNA was then extracted using TRIzol reagent from Invitrogen (USA) following standard experimental methods.

[0124] Take 2-4 mL of peripheral blood sample and centrifuge at 3000 rpm for 15 min; after stratification, separate the plasma components and store them at -80 ℃ for subsequent testing. Total RNA was extracted from the plasma sample using the Invitrogen TRIzol LS reagent according to standard experimental methods.

[0125] After RNA extraction, cDNA was synthesized via reverse transcription. Gene expression levels were detected using an ABI 7500 real-time quantitative PCR instrument from Applied Biosystems, USA, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from GenePharma, China, used as an internal control gene. Each experiment was performed in triplicate, using 2... - The relative expression levels of circular RNA were analyzed using the ^ΔΔCT method. The primer sequences used in this experiment are detailed in Table 3.

[0126] Table 3

[0127] 2. Construction of cell models for circ_0043278 knockdown and overexpression ECA109 or TE12 cells were cultured in RPMI 1640 DMEM medium containing 10% fetal bovine serum (FBS), 50 U / ml streptomycin, and 50 U / ml penicillin, respectively. Overexpression and silencing of hsa_circ_0043278 were performed in the esophageal cancer cell line. A recombinant plasmid upregulating hsa_circ_0043278 was constructed and ligated to the target gene fragment of pcd5 by double enzyme digestion. The overexpression and silencing efficiencies of hsa_circ_0043278 in both cell lines were determined using qRT-PCR. The overexpression vector of hsa_circ_0043278, si-hsa_circ_0043278, and negative controls were provided by GenePharma (Shanghai, China). The RNA oligonucleotide sequences are listed in Table 1. The siRNA and overexpression plasmid of hsa_circ_0043278 were transfected into ECA109 or TE12 cells using Lipofectamine 2000 (Invitrogen) for 48 hours. Transfection efficiency was determined by qRT-PCR.

[0128] 3. Subcellular localization analysis of circ_0043278 One × 10^7 cells were resuspended in hypotonic buffer (25 mM Tris-HCl, pH 7.4, 1 mM MgCl2, 5 mM KCl, 0.5% NP-40) and incubated on ice for 5 min. The cells were then centrifuged at 5000 g for 5 min, and the supernatant was collected as the cytoplasmic fraction. The pellet was washed twice with hypotonic buffer and then resuspended in nuclear buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF). After incubation on ice for 20 min, the cells were centrifuged to collect the nuclear fraction. Actin, circ_0043278, and U6 were used as controls for the cytoplasmic or nuclear fraction. RNA levels were detected using a Ribo™ fluorescence in situ hybridization kit (RiboBio) according to the manufacturer's protocol. Hybridization was performed using Cy3-labeled probes complementary to the backsplicing sites of U6 or circ_0043278, and the signals were detected using a confocal laser scanning microscope (Zeiss LSM510).

[0129] In this embodiment, the results of circ_0043278 expression verification and transfection efficiency detection are as follows: Figure 2 As shown, qRT-PCR detection revealed that the expression level of circ_0043278 was significantly increased in esophageal cancer tissues compared to adjacent normal tissues. Figure 2 The expression A in the figure indicates that circ_0043278 is abnormally highly expressed in esophageal cancer, suggesting that it may be involved in the development and progression of esophageal cancer. In Eca-109 and TE12 cells, three siRNAs targeting circ_0043278 effectively reduced the expression level of circ_0043278, with some siRNAs showing more significant knockdown efficiency, indicating the successful construction of a circ_0043278 low-expression cell model. Figure 2 (BC in the text). After transfection with the circ_0043278 overexpression vector, the expression level of circ_0043278 in Eca-109 and TE12 cells was significantly increased, indicating that the circ_0043278 overexpression cell model was successfully constructed. Figure 2 (DE in the above). The above knockdown and overexpression models can be used for subsequent functional experimental studies.

[0130] Furthermore, to explore the potential mechanism of action of circ_0043278, its subcellular localization was analyzed, and the results are as follows: Figure 3 As shown, the results of the nucleocytoplasmic separation experiment indicate that ( Figure 3In Eca-109 and TE12 cells, the expression abundance of circ_0043278 was significantly higher in the cytoplasmic component than in the nuclear component, indicating that circ_0043278 is mainly localized in the cytoplasm. FISH experiments further confirmed that the fluorescence signal of circ_0043278 was mainly distributed in the cytoplasmic region, while the signal in the nucleus was relatively weak. Figure 3 (B in the above results) indicates that circ_0043278 is a circRNA mainly located in the cytoplasm, suggesting that it may participate in esophageal cancer-related biological processes through post-transcriptional regulatory mechanisms.

[0131] Example 3: Functional validation of hsa_circ_0043278 in esophageal cancer cells To verify the effect of hsa_circ_0043278 on the proliferation of esophageal cancer cells, this embodiment constructed circ_0043278 knockdown and overexpression models in Eca-109 and TE12 cells, respectively. The effects on the proliferation and DNA replication ability of esophageal cancer cells were then detected using CCK-8 and EdU incorporation assays. Details are as follows: 1. CCK-8 cell proliferation experiment Following existing experimental protocols, cell proliferation dynamics were detected using the CCK-8 colorimetric assay kit from NCM (China). Transfected cells were seeded into 96-well NEST (China) plates, with 5000 cells per well. Cells were cultured continuously for 5 days, and cell growth status was assessed every 24 hours.

[0132] Following the instructions from Dojindo Molecular Technologies, 10 μL of CCK-8 working solution was added to each well, and the mixture was incubated under standard culture conditions (37 °C, 5% CO2) for 2 h. The absorbance at 450 nm was then measured using a microplate reader.

[0133] 2. EdU cell proliferation detection experiment Logarithmic growth phase cells were seeded in culture plates and cultured at 37 ℃ in a 5% CO2 incubator. Once the cells adhered and met the preset treatment conditions, an appropriate amount of 5-ethynyl-2'-deoxyuridine (EdU) working solution was added to the cell culture medium, and incubation was continued at a suitable temperature for an appropriate duration to allow EdU to be fully incorporated into the newly generated DNA of the proliferating cells. After incubation, the culture medium was discarded, and the cells were washed with phosphate-buffered saline (PBS), fixed at room temperature with fixative, permeabilized with permeabilization solution, and then incubated with Click reaction solution in the dark to complete the EdU fluorescent labeling reaction. Subsequently, the cells were stained with nuclear dyes, washed with PBS to remove residual dye, and observed and acquired using a fluorescence microscope. Multiple fields of view were randomly selected to count the number of EdU-positive proliferating cells and the total number of cells, and the cell proliferation rate was calculated. Multiple replicates were set for each group, and the experiment was repeated three times independently to ensure the reliability of the results.

[0134] In this embodiment, the detection results of circ_0043278 on the proliferative capacity of esophageal cancer cells are as follows: Figure 4 As shown in the figure. CCK-8 assay revealed that in Eca-109 and TE12 cells, compared to the si-NC control group, silencing circ_0043278 significantly reduced cell proliferation rate, and cell growth was significantly inhibited within a 96-hour detection period. Figure 4 In contrast, overexpression of circ_0043278 significantly enhanced the proliferation capacity and cell growth rate of Eca-109 and TE12 cells (AB); Figure 4 The results indicate that circ_0043278 can promote the proliferation of esophageal cancer cells and has a pro-cancer effect in esophageal cancer.

[0135] Furthermore, to verify the effect of circ_0043278 on the DNA replication ability of esophageal cancer cells, an EdU incorporation assay was used for detection, and the results are as follows: Figure 5 As shown in the figure. Representative EdU experimental results of Eca-109 and TE12 cells after circ_0043278 knockdown or overexpression are shown in the figure. Figure 5 As shown in A, EdU-positive proliferating cells are displayed in red, and the cell nuclei are displayed in blue after being counterstained with DAPI.

[0136] Quantitative analysis results of the proportion of EdU-positive cells are as follows: Figure 5As shown in B in the figure. Compared with the si-NC control group, the proportion of EdU-positive cells in Eca-109 and TE12 cells was significantly reduced after transfection with circ_0043278 siRNA; conversely, the proportion of EdU-positive cells in both types of esophageal cancer cells was significantly increased after overexpression of circ_0043278. These results further demonstrate that circ_0043278 can enhance the DNA synthesis capacity and proliferation activity of esophageal cancer cells, thereby promoting the growth of esophageal cancer cells.

[0137] Example 4: Effect of hsa_circ_0043278 on the migration ability of esophageal cancer cells To further evaluate the role of hsa_circ_0043278 in esophageal cancer progression, this embodiment constructed circ_0043278 knockdown and overexpression models in Eca-109 and TE12 cells, respectively, and examined its effect on the migration ability of esophageal cancer cells using scratch assays and Transwell migration assays. Details are as follows: 1. Scratch test Esophageal cancer cell models with upregulated and downregulated expression of circ_0043278 were seeded in six-well culture plates and cultured until the cell monolayer confluence reached 90%. Using a sterile 200 μL pipette tip (Biosharp, China), linear scratches were uniformly made on the confluent cell layer, ensuring consistent scratch width across all groups using a calibrated measuring tool. Initial images of the scratches were captured using a phase-contrast microscope at 0 h. After scratch preparation, the culture medium was replaced with serum-free medium, and the cells were continuously observed for 48 h, with images taken at predetermined time points to record scratch healing progress. Cell migration ability was quantitatively analyzed by calculating the scratch closure rate at each time point.

[0138] 2. Transwell migration experiment Cell migration was measured using a 24-well Transwell chamber (Corning). Transfected cells were collected and resuspended in serum-free medium. 200 μL of cells were seeded into the upper chamber, and 500 μL of medium containing 20% ​​fetal bovine serum was added to the lower chamber. The cells were incubated for 24 hours. Finally, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. All experiments were performed in triplicate.

[0139] In this embodiment, the effect of circ_0043278 on the migration ability of esophageal cancer cells is as follows: Figure 6 As shown. The scratch test results are as follows: Figure 6As shown in A in the figure. Compared with the si-NC control group, knocking down circ_0043278 significantly widened the scratch area of ​​Eca-109 and TE12 cells 48 hours after scratching, and the scratch healing rate was significantly slowed down; conversely, compared with the empty vector control group, overexpression of circ_0043278 significantly increased the scratch healing area of ​​cells, indicating enhanced cell migration ability.

[0140] Furthermore, the quantitative analysis results of the relative scratch healing area are as follows: Figure 6 As shown in B in the figure, knockdown of circ_0043278 significantly decreased the scratch healing rate of both Eca-109 and TE12 cells; while overexpression of circ_0043278 significantly increased the scratch healing rate. These results indicate that circ_0043278 can promote the migration of esophageal cancer cells.

[0141] Furthermore, to further verify the effect of circ_0043278 on the migration ability of esophageal cancer cells, this embodiment further employed a Transwell migration assay. The results showed that, compared with the si-NC control group, silencing circ_0043278 significantly reduced the number of Eca-109 and TE12 cells migrating across the Transwell membrane; conversely, compared with the empty vector control group, overexpression of circ_0043278 significantly increased the number of cells that crossed the membrane.

[0142] The above results are consistent with the scratch assay results, further demonstrating that circ_0043278 can enhance the migration ability of esophageal cancer cells, suggesting that it plays an important role in promoting the invasion and metastasis of esophageal cancer.

[0143] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0144] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0145] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of a reagent for detecting circular RNA in the preparation of a kit for screening esophageal cancer samples, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that, The reagents include at least one of probes, primers, or transcriptome sequencing reagents.

3. The use according to claim 1 or 2, characterized in that, The esophageal cancer mentioned is esophageal squamous cell carcinoma.

4. Use of a formulation that downregulates the expression level of a circular RNA in the preparation of a drug for treating esophageal cancer, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO:

1.

5. The use according to claim 4, characterized in that, The formulation is a specific siRNA, shRNA, antisense nucleic acid, or small molecule inhibitor targeting hsa_circ_0043278.

6. The use according to claim 4 or 5, characterized in that, The esophageal cancer mentioned is esophageal squamous cell carcinoma.

7. A reagent kit, characterized in that, The kit is used to screen biological samples that are susceptible to or have esophageal cancer. The kit contains reagents suitable for detecting circular RNA, which is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO:

1.

8. A medicament for the prevention and / or treatment of esophageal cancer, characterized in that, The drug comprises an agent that downregulates the expression level of a circular RNA, wherein the circular RNA is hsa_circ_0043278 or a functional fragment thereof, and the hsa_circ_0043278 has the nucleotide sequence shown in SEQ ID NO:

1.

9. The medicament according to claim 8, characterized in that, The drug is an injectable form.

10. The medicament according to claim 8 or 9, characterized in that, The drug also contains pharmaceutically acceptable excipients or carriers.

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