Use of circ_0009061 expression enhancer in preparation of drug for treating ovarian cancer

Abstract

The application relates to application of a circ_0009061 expression enhancer in preparation of a drug for treating ovarian cancer. The circ_0009061 expression enhancer is a circ_0009061 overexpression lentivirus, can specifically up-regulate expression of circ_0009061, and the nucleotide sequence of the circ_0009061 gene is shown in SEQ ID NO. 12. It is found in the application that circ_0009061 is specifically lowly expressed in human ovarian cancer tissues and cell lines, and high expression of the circ_0009061 is positively correlated with higher overall survival rate and disease-free survival rate of patients. A nude mouse subcutaneous xenotransplant tumor model and an abdominal cavity metastasis model also prove that overexpression of the circ_0009061 can significantly inhibit growth and abdominal cavity metastasis of ovarian cancer tumors. Therefore, the application proposes that the circ_0009061 can be used as a potential drug target for targeted treatment of ovarian cancer, and provides important experimental basis and application guidance for treatment of ovarian cancer.

Description

Technical Field

[0001] This invention relates to the field of circular RNA and protein technology, and in particular to the application of a circ_0009061 expression enhancer in the preparation of drugs for treating ovarian cancer. Background Technology

[0002] Ovarian cancer is a heterogeneous tumor originating from epithelial cells, germ cells, stroma, and fallopian tubes. In recent years, the incidence of ovarian cancer has been rising annually, and the incidence is trending towards younger ages. Furthermore, the ovaries are located deep in the pelvic cavity, and early symptoms are often subtle, which is a significant reason for its high mortality rate. Due to the lack of specific symptoms, early screening methods, and the highly invasive nature of ovarian cancer in the peritoneal cavity and its tendency to metastasize, approximately 70% of ovarian cancer patients are already in advanced stages (stage III or IV) at initial diagnosis, resulting in poor treatment outcomes and prognosis.

[0003] A growing body of reports indicates that circRNAs are important regulators in various types of cancer. Currently, the functional role of circRNAs in ovarian cancer, acting as miRNA sponges to regulate the expression of miRNA target genes, is extensively studied. The dependence of cancer cells on glycolysis has been proposed as a method for tumor localization and diagnosis, and as a therapeutic target for tumor suppression, showing great clinical potential. circRNAs play a crucial role in glucose metabolism in ovarian cancer, regulating the expression of glucose metabolism-related proteins such as glucose transporters and hexokinases, promoting glycolysis in cancer cells, increasing glucose uptake and ATP production, and influencing energy supply and biosynthesis to support tumor growth and invasion.

[0004] For example, Chinese patent CN112921086A discloses a circular RNA circ-CRIM1 (hsa_circ_0002346) as a diagnostic biomarker for ovarian cancer. PCR amplification was performed using specific primers designed across splice sites (junctions), and the amplified products were sequenced using Sanger sequencing to verify their end-to-end circular structure. Simultaneously, high expression of circ-CRIM1 in ovarian cancer tissues was verified using qRT-PCR. However, this approach uses circ-CRIM1 as a ceRNA sponge to adsorb miRNAs, promoting ovarian cancer progression. It is an oncogene involving a miRNA sponge mechanism (ceRNA), and high expression of circ-CRIM1 suggests a poor prognosis.

[0005] For example, Chinese patent CN111617249A discloses the application of hsa_circ_0007444 in the preparation of drugs for treating ovarian cancer. By preparing a has_circ_0007444 overexpression plasmid vector, and overexpressing lentivirus and specific siRNA, its function in ovarian cancer cells was verified. It was found that overexpression of has_circ_0007444 can significantly inhibit the proliferation, invasion, and migration of ovarian cancer, and promote its apoptosis. Knockdown of has_circ_0007444 can significantly promote the proliferation, invasion, and migration of ovarian cancer, and inhibit its apoptosis. Simultaneously, has_circ_0007444 overexpression can inhibit the growth and lung metastasis of ovarian cancer in vivo, making it a very important target for ovarian cancer treatment. However, this approach involves a ceRNA mechanism (sponge-adsorbed miRNA).

[0006] Crotonylation, one of the newly discovered protein post-translational modifications (PTMs) in 2011, has been found in both histones and non-histones and has been found to be involved in a variety of diseases and biological processes, such as neuropsychiatric disorders, carcinogenesis, spermatogenesis, tissue damage and inflammation. This modification mainly occurs at lysine sites.

[0007] MCCC1 and MCCC2 are two human nuclear-encoding genes that synthesize two protein subunits of the key mitochondrial enzyme 3-methylcrotonyl-CoA carboxylase (MCC). Research on the role of MCCC1 / MCCC2 in tumors is scarce. However, existing studies have demonstrated that the enzymatic activity of MCC can influence the crotonylation modification of the protein.

[0008] Epithelial ovarian cancer is characterized by low incidence and high mortality. One of its main causes is the widespread implantation, dissemination, and distant metastasis of ovarian cancer cells within the peritoneal cavity. Because ovarian tumors are located deep in the pelvic cavity, over 75% of ovarian cancer patients have already experienced extensive metastasis at diagnosis, which is a significant reason for its high mortality rate. Furthermore, ovarian cancer metastasis shows a marked tendency towards the greater omentum and mesentery, which may be closely related to the high-lipid environment of the greater omentum and mesentery.

[0009] Therefore, exploring the mechanisms of peritoneal seeding metastasis in ovarian cancer and finding new combination therapy strategies are of great significance for improving the survival rate of ovarian cancer patients. Summary of the Invention

[0010] The purpose of this invention is to overcome the problems of high peritoneal invasiveness and implantation metastasis in epithelial ovarian cancer, and to provide the application of circ_0009061 expression enhancer in the preparation of drugs for treating ovarian cancer.

[0011] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides the application of a circ_0009061 expression enhancer in the preparation of a drug for treating ovarian cancer, wherein the nucleotide sequence of the circ_0009061 gene is shown in SEQ ID NO.12.

[0012] Preferably, the circ_0009061 expression enhancer is a circ_0009061 overexpression lentivirus.

[0013] Furthermore, the circ_0009061 overexpression lentivirus was prepared by co-transfecting cells with a lentivirus overexpression vector and a lentivirus packaging plasmid.

[0014] Furthermore, the lentiviral overexpression vector is formed by inserting the circ_0009061 gene with the nucleotide sequence shown in SEQ ID NO.12 into the empty lentiviral vector pLenti ciR6024; the packaging plasmids are pMD2.G and psPAX2; and the cells are HEK-293T cells.

[0015] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0016] Furthermore, the application of circ_0009061 expression enhancer in the preparation of drugs that inhibit the expression of MCCC1 and MCCC2 proteins to suppress the growth of ovarian cancer tumors.

[0017] Furthermore, the application of circ_0009061 expression enhancer in the preparation of drugs that inhibit cell proliferation and migration of HEY and SKOV3 cells.

[0018] Furthermore, the application of circ_0009061 expression enhancer in the preparation of drugs that inhibit aerobic glycolysis, glycolytic ATP and lactate production in HEY and SKOV3 cells.

[0019] Furthermore, the application of circ_0009061 expression enhancer in the preparation of drugs that inhibit glucose uptake in HEY and SKOV3 cells.

[0020] Furthermore, the application of the circ_0009061 expression enhancer in the preparation of a drug that inhibits the growth of subcutaneous xenograft tumors in nude mice.

[0021] Furthermore, the application of the circ_0009061 expression enhancer in the preparation of drugs that inhibit the growth of peritoneal metastatic tumors and the formation of metastatic nodules in nude mice.

[0022] Furthermore, the circ_0009061 expression enhancer achieves its anti-ovarian cancer effect through the following molecular mechanism: 1) It binds directly to the amino acid region of MCCC1 protein from amino acid position 100 to 151; 2) It binds directly to the amino acid region of MCCC2 protein from amino acid position 338 to 389; 3) It promotes the K48-ubiquitination degradation of MCCC1 and MCCC2 proteins mediated by the E3 ubiquitin ligase TRIM21; 4) It drives the increase of histone H3K36cr crotonylation modification level; 5) It inhibits the transcription and expression of key glycolytic enzymes HK2 and PKM2.

[0023] In a second aspect, the present invention provides a pharmaceutical composition for treating ovarian cancer, comprising a circ_0009061 expression enhancer and a pharmaceutically acceptable vector, the nucleotide sequence of the circ_0009061 gene being shown in SEQ ID NO.12.

[0024] Preferably, the circ_0009061 expression enhancer is a circ_0009061 overexpression lentivirus.

[0025] Thirdly, the present invention provides the application of a reagent for detecting circ_0009061 gene expression in the preparation of a kit for detecting ovarian cancer.

[0026] Fourthly, this invention provides an application of the circ_0009061 gene as a target in screening drugs for the treatment of ovarian cancer.

[0027] Fifthly, the present invention provides a method for screening drugs for treating ovarian cancer, comprising: applying a candidate drug to cells expressing circ_0009061, detecting changes in the expression level of circ_0009061, and selecting a candidate drug that increases the expression level of circ_0009061.

[0028] Compared with the prior art, the present invention has the following beneficial effects: This invention investigates the differential expression of circ_0009061 in normal human ovaries and ovarian cancer tissues and cell lines. It was found that circ_0009061 is specifically lowly expressed in human ovarian cancer tissues and cell lines, and its high expression is positively correlated with higher overall survival and disease-free survival rates. Further, it was confirmed that circ_0009061 can directly bind to MCCC1 / MCCC2 proteins, promoting their degradation via TRIM21-mediated K48-ubiquitination, thereby driving an increase in histone H3K36cr crotonylation modification levels, inhibiting the transcription and expression of key glycolytic enzymes HK2 and PKM2, and ultimately inhibiting ovarian cancer cell proliferation, migration, and aerobic glycolysis. Subcutaneous xenograft tumor models and peritoneal metastasis models in nude mice confirmed that overexpression of circ_0009061 can significantly inhibit ovarian cancer tumor growth and peritoneal metastasis. Therefore, this invention proposes that circ_0009061 can be used as a potential drug target for targeted therapy of ovarian cancer, and further provides the application of circ_0009061 expression enhancers in the preparation of drugs for treating ovarian cancer, providing important experimental evidence and application guidance for the treatment of ovarian cancer. Attached Figure Description

[0029] Figure 1 This is a schematic diagram illustrating the application and mechanism of action of circular RNA circ_0009061 in the preparation of drugs for treating ovarian cancer.

[0030] Figure 2 shows the screening and identification of ovarian cancer-associated circular RNA circ_0009061; Figure 2 includes Figure 2A , Figure 2B , Figure 2C , Figure 2D , Figure 2E , Figure 2F , Figure 2G , Figure 2H , Figure 2I , Figure 2J , Figure 2K , Figure 2LA shows the results of circRNA-Seq screening of circ_0009061 in normal ovaries, primary EOC lesions, EOC greater omental metastases, and EOC mesenteric metastases, where OV represents normal ovaries; EOC represents primary EOC lesions; GO represents EOC greater omental metastases; and ME represents EOC mesenteric metastases. B shows the Reactome analysis of mRNA-Seq, confirming that a large number of differentially expressed genes in normal ovaries and EOCs are related to tumor metabolism. C shows the expression of circ_0009061 in normal ovarian cells and multiple ovarian cancer cell lines. D shows the expression level of circ_0009061 in 71 normal ovarian tissues and 64 EOC tissues, where Normol represents normal ovarian tissue and Tumor represents EOC tissue. The diagram shows the overall survival rate of circ_0009061 in the FUSCC-cohort, E; the disease-free survival rate of circ_0009061 in the FUSCC-cohort, F; the Sanger sequencing verification diagram, confirming that the reverse splicing site of circ_0009061 is CAGT; H is a statistical diagram of the stability of circ_0009061 detected by qRT-PCR after cell treatment with actinomycin D (Act D); I is a WB diagram of the RNase R digestion experiment, proving that circ_0009061 is resistant to RNase R digestion; J is a schematic diagram of the distribution of circ_0009061 in HEY and SKOV3 cell lines during RNA-FISH assay; K and L are schematic diagrams of the subcellular localization of circ_0009061 in HEY and SKOV3 cell lines during nucleocytoplasmic separation assay; GAPDH is used as a cytoplasmic internal control, and U6 and Lamin B1 are used as nuclear internal controls.

[0031] Figure 3 shows the in vitro validation of circ_0009061's inhibition of ovarian cancer cell motility and aerobic glycolysis; Figure 3 includes Figures 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H, 3I, 3J, 3K, 3L, 3M, 3N, 3O, 3P, and 3Q; where A and B represent the circ_0009061 overexpression stable cell line (circ_0009061 stable-transfected HEY and SKOV3 cells) and the knockdown stable cell line (circ_0009061 stable-transfected cells) detected by qRT-PCR. Statistical graphs showing the expression of circ_0009061 in HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061-sh2; C and D show the proliferation of HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061 during CCK-8 assay; E and F show the proliferation of HEY and SKOV3 cells stably transfected with circ_shNC / circ_0009061-sh1 / circ_0009061-sh2 during CCK-8 assay. Statistical charts of colony formation; G and H represent the colony formation ability of HEY and SKOV3 cells stably transfected by circ_NC / circ_0009061 during the colony formation assay; I and J represent the colony formation ability of HEY and SKOV3 cell lines stably transfected by circ_shNC / circ_0009061-sh1 / circ_0009061-sh2 during the colony formation assay; K and L represent the colony formation ability of circ_NC / circ_0009061 and c during the Transwell assay. Schematic diagram of the migration ability of HEY and SKOV3 cells stably transfected with irc_shNC / circ_0009061-sh1 / circ_0009061-sh2; M is a statistical graph showing the inhibition of ATP and lactate production by glycolysis in HEY and SKOV3 cells stably transfected with circ_0009061 by overexpression; N is a schematic diagram showing the inhibition of glucose uptake by HEY and SKOV3 cells stably transfected with circ_0009061 by overexpression; O is a flowchart showing the glucose uptake ability of circ_0009061 overexpressed and control tumors under 18F-FDG PET-CT detection; P is a schematic diagram showing the glucose uptake ability of tumors under 18F-FDG PET-CT detection after bilateral injection of circ_0009061 overexpressed and control cells; Q is the glucose uptake ability of tumors under 18F-FDG PET-CT detection after unilateral injection of circ_0009061 overexpressed and control cells.

[0032] Figure 4 shows the binding of circ_0009061 to MCCC1 / MCCC2 proteins; Figure 4 includes Figures 4A, 4B, 4C, and 4D. Figure 4E Figures 4F, 4G, and 4H are shown. A represents the mass spectrometry detection of RNA pull-down after silver staining using circ_0009061-specific and control probes; B represents the Western blotting (WB) graph verifying the enrichment and binding of MCCC1 and MCCC2 proteins in the RNA-pull-down sample; C represents the WB graph verifying the binding of MCCC1 / MCCC2 protein antibodies to magnetic beads; D represents the statistical graph of MCCC1 / MCCC2 protein enrichment of circ_0009061 in HEY and SKOV3 cell lines during RIP-qRT-PCR; E represents a schematic diagram of the catRAPID-predicted binding sites of circ_0009061 and MCCC1 / MCCC2 proteins; FG represents the statistical graph of the enrichment degree of circ_0009061 by the plasmid-Flag fragment during qRT-PCR; and H represents the WB graph verifying the binding of the Flag protein antibody to magnetic beads and the successful transfection of the plasmid.

[0033] Figure 5 illustrates the biological function of the MCCC1 / circ_0009061 / MCCC2 complex in promoting H3K36cr crotonylation modification and inhibiting peritoneal metastasis of ovarian cancer; Figure 5 includes... Figure 5A Figures 5B, 5C, 5D, 5E, 5F, and 5G. Figure 5H Figure 5I, Figure 5J, Figure 5K, Figure 5L, Figure 5M, Figure 5N, Figure 5O Figure 5PFigures 5Q, 5R, 5S, 5T, 5U, 5V, 5W, and 5Y; where A is a schematic diagram of the co-localization of circ_0009061 (green), MCCC1 (pink), and MCCC2 (red) in the cytoplasm of EOC cells confirmed by IF and FISH; B is a schematic diagram of the binding of MCCC1 and MCCC2 at the protein level after PLA assay to detect the overexpression of circ_0009061; C is a Western blotting diagram of the changes in protein expression of MCCC1 and MCCC2 after overexpression of circ_0009061; D is a graph showing the changes in MCCC1 / MCCC2 protein levels after treatment of EOC cells stably transfected with circ_0009061 with MG-132, 3-MA, and chloroquine; E is a graph showing the changes in MCCC1 / MCCC2 protein levels after MG-132 treatment. Figure 1 shows the changes in MCCC1 / MCCC2 protein ubiquitination levels in circ_0009061 and control stable-transformed HEY and SKOV3 cell lines; Figure 2 shows the changes in ubiquitin K48 site in circ_0009061 overexpression and control stable-transformed cells after MG132 treatment; Figure 3 shows the changes in ubiquitin K63 site in circ_0009061 overexpression and control stable-transformed cells after MG132 treatment; Figure 4 shows the changes in ubiquitin K63 site in circ_0009061 and control stable-transformed cells after MG132 treatment. Image 1 shows the Co-IP combined silver staining mass spectrometry experiment of MCCC1 / MCCC2 after HEY and SKOV3 cell treatment; I is the intersection of Co-IP mass spectrometry results of MCCC1 / MCCC2, screening out the E3 ubiquitin ligase TRIM21; J is the schematic diagram of TRIM21 binding to MCCC1 / MCCC2 after MG132 treatment by Co-IP detection; K and L are the Co-IP detection results of MCCC1 / MCCC2 after MG132 treatment. Schematic diagram of MCCC1 / MCCC2 binding to TRIM21; M is a WB plot of crotonylation levels detected in circ_0009061 overexpression and control stable transgenic strains; N is a crotonylation modification proteomics plot, screening proteins with significantly different crotonylation modification levels in circ_0009061 overexpression and control stable transgenic strains; O is a KEGG enrichment analysis plot of crotonylation modification proteomics; P is a COG analysis plot of crotonylation modification proteomics; Q is a crotonylation modification proteomics detection plot. R represents the motif sequence analysis of the crotonylation modification site; S represents the results of histone crotonylation modification analysis and screening, identifying H3K36cr and H3K23cr; T represents the Western blot (WB) of H3K36cr and H3K23cr crotonylation modification levels in circ_0009061 overexpression and control stable transgenic strains; U represents the WB plot, confirming that simultaneous overexpression of MCCC1 / MCCC2 can reverse the promoting effect of circ_0009061 on H3K36cr.V is a Western blotting diagram showing the protein levels of PKM2 and HK2 in the circ_0009061 overexpression and control stable transgenic strains; W and Y are schematic diagrams showing the mRNA levels of PKM2 and HK2 in the circ_0009061 overexpression and control stable transgenic strains detected by qRT-PCR.

[0034] Figure 6 shows the inhibition of circ_0009061 on ovarian cancer tumor growth and metastasis in nude mice; Figure 6 includes Figures 6A, 6B, and 6C. Figure 6D , Figure 6E Figure 6F, Figure 6G, Figure 6H Figure 6I A shows a photograph of the tumors in BALB / c nude mice after subcutaneous injection of HEY cells stably transfected with circ_NC / circ_0009061, taken at week 4; B shows the growth curve plotted by measuring tumor length and width every seven days; C shows the weight statistics of subcutaneous tumors in nude mice; D shows the expression of circ_0009061, Ki-67, MCCC1, MCCC2, and H3K36cr in subcutaneous tumors of nude mice detected by ISH and IHC; E shows weekly in vivo imaging images of HEY cells stably transfected with circ_NC / circ_0009061 injected intraperitoneally into BALB / c nude mice; F shows a photograph of the tumors removed from the peritoneum of nude mice at week 5; G and H show the number and weight statistics of metastatic nodules in the peritoneum of nude mice; I shows the expression of Ki-67, MCCC1, MCCC2, and H3K36cr in tumors within the peritoneum of nude mice detected by IHC. Detailed Implementation

[0035] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0036] This invention first screens and identifies circular RNA circ_0009061 as an important tumor suppressor regulatory molecule in ovarian cancer. The steps include: 1) Samples of normal ovarian tissue, primary epithelial ovarian cancer (EOC), EOC omental metastases, and EOC mesenteric metastases were collected. Total RNA was extracted and circRNA-Seq next-generation sequencing was performed. The sequencing results were analyzed, and circRNA molecules showing a gradient downregulation trend in normal ovarian tissue, primary EOC, EOC omental metastases, and EOC mesenteric metastases were screened using the screening criterion of "Fold Change > 3 or < 0.3, and P < 0.01" compared with the normal group. 2) The expression levels of the screened circRNAs in the normal ovarian epithelial cell line HOSEpiC and multiple epithelial ovarian cancer cell lines (HEY, SKOV3, OVCAR-8, A2780, etc.) were detected using qRT-PCR. At the same time, the expression differences were verified in 71 normal ovarian tissue samples and 64 EOC tissue samples. 3) The results of RNA-Seq analysis and qRT-PCR validation were summarized to identify circ_0009061, which was significantly downexpressed in both ovarian cancer tissues and cell lines, as the final research subject. Kaplan-Meier survival analysis was used to assess the correlation between circ_0009061 expression levels and overall survival and disease-free survival, confirming a positive correlation between high circ_0009061 expression and a favorable prognosis in ovarian cancer patients.

[0037] This invention also investigates the biological function of circ_0009061 in inhibiting the proliferation, migration, and aerobic glycolysis of ovarian cancer cells. The specific method is as follows: 1) Based on the expression levels of circ_0009061 in different ovarian cancer cell lines, ovarian cancer cell lines stably overexpressing or knocking down circ_0009061 were constructed. An overexpression plasmid for circ_0009061 was constructed using the lentiviral overexpression vector pLenticiR6024, and a shRNA plasmid targeting circ_0009061 (shRNA sequence: sh1 nucleotide sequence as shown in SEQ ID NO.4; sh2 nucleotide sequence as shown in SEQ ID NO.5) was constructed using the lentiviral knockdown vector pLKO.1. After lentiviral packaging, HEY and SKOV3 cells were infected, and stable cell lines were obtained through puromycin selection. 2) In vitro experiments using Cell Counting Kit-8, colony formation, and Transwell migration assays demonstrated that circ_0009061 has a significant inhibitory effect on the proliferation and migration of ovarian cancer cells. 3) In vitro and in vivo experiments, including the use of a glycolysis ATP and lactate production assay kit, a glucose uptake fluorescent probe (2-NBDG), and 18F-FDG PET-CT imaging of a BALB / c nude mouse subcutaneous tumor model, demonstrated that circ_0009061 has an important inhibitory effect on aerobic glycolysis in ovarian cancer cells.

[0038] This invention investigates the molecular mechanism by which circ_0009061 binds to MCCC1 / MCCC2 proteins and regulates their ubiquitination and degradation. The method is as follows: 1) A 5′ biotin-labeled circ_0009061-specific probe and a negative control probe were designed and synthesized. RNA pull-down assays were performed in HEY cell lysates to enrich the protein complex bound to circ_0009061. Differential protein bands were extracted after SDS-PAGE silver staining and identified by mass spectrometry. Western blotting was used to verify the binding of circ_0009061 to MCCC1 / MCCC2 proteins. 2) RNA immunoprecipitation assays were performed using MCCC1 or MCCC2 antibodies to capture the RNA-protein complex. qRT-PCR was used to detect the enrichment level of circ_0009061, thus verifying the intracellular binding of the two proteins. 3) Using the catRAPID database to predict binding regions, Flag tag expression plasmids for different truncated fragments of MCCC1 and MCCC2 proteins were constructed. After transfection into HEY cells, RIP experiments were performed. The enrichment level of circ_0009061 in each truncated fragment was detected by qRT-PCR, confirming that circ_0009061 binds to amino acid positions 100-151 of the MCCC1 protein and amino acid positions 338-389 of the MCCC2 protein. 4) Cells were treated with the proteasome inhibitor MG-132, the autophagy inhibitor 3-MA, and chloroquine, respectively. Changes in MCCC1 / MCCC2 protein levels were detected by Western blotting, confirming that the degradation pathway was proteasome-dependent. Ubiquitination levels were detected by immunoprecipitation combined with Western blotting, and K48 and K63 ubiquitin chain-specific antibodies were used to confirm that ubiquitination modification was K48 chain-dependent. 5) Mass spectrometry was used to identify the Co-IP products of MCCC1 / MCCC2, and Co-IP / WB verification was performed to screen and confirm that the E3 ubiquitin ligase TRIM21 mediates the above ubiquitination process.

[0039] This invention investigates the molecular mechanism by which the MCCC1 / circ_0009061 / MCCC2 complex drives histone H3K36cr crotonylation modification to inhibit the transcription of key glycolytic enzymes. The method is as follows: 1) Crotonylation modification proteomics was performed in HEY cells overexpressing circ_0009061 to analyze the subcellular localization of differentially expressed proteins and the enrichment of the KEGG pathway. Western blotting was performed using crotonylation pan-antibody, H3K36cr-specific antibody, H3K23cr-specific antibody, and H3K36me3-specific antibody to verify changes in H3K36cr levels. 2) MCCC1-CDS-3×Flag and MCCC2-CDS-3×Flag overexpression plasmids were simultaneously transiently transfected into HEY cells overexpressing circ_0009061. Western blotting was used to detect whether H3K36cr levels recovered, verifying that this modification is dependent on MCCC1 / MCCC2. 3) qRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of the key glycolytic enzymes HK2 and PKM2, demonstrating that increased H3K36cr crotonylation modification levels lead to inhibition of HK2 and PKM2 expression.

[0040] This invention studies the biological function of circ_0009061 in inhibiting the in vivo growth of ovarian cancer. The method is as follows: 1) Construction of a BALB / c nude mouse subcutaneous xenograft tumor model: HEY cells stably overexpressing circ_0009061 and control cells were cultured at 5×10⁻⁶ cells per cell line. 6 1) A dose per mouse was injected subcutaneously into the right axilla of nude mice. The long and short diameters of the tumor were measured weekly, and the tumor volume was calculated. After 4 weeks, the nude mice were sacrificed, the tumor was dissected, weighed, and photographed. 2) Construction of BALB / c nude mouse peritoneal metastasis model: HEY cells stably overexpressing circ_0009061 and carrying luciferase labeling and control cells were injected at 5×10⁻⁶ cells per mouse. 6 Administer a dose per animal via intraperitoneal injection. Weekly in vivo small animal imaging is performed to observe peritoneal metastasis. Nude mice are sacrificed after 5 weeks, and the number and weight of metastatic nodules in the peritoneal cavity are recorded via dissection. 3) Subcutaneous tumor and peritoneal metastatic tumor tissues are collected for immunohistochemical detection (Ki67, MCCC1, MCCC2, H3K36cr) and in situ hybridization detection (circ_0009061) to verify the in vivo mechanism.

[0041] Unless otherwise specified, the experimental conditions or methods in the following examples are generally performed under standard conditions—that is, the conditions described in "Molecular Cloning" (3rd ed.) or as recommended by the manufacturer.

[0042] The sequences used in this invention are shown in Table 1. Table 1 Sequence

[0043] Example 1: Screening and identification of ovarian cancer-associated circular RNA circ_0009061 1) Sample collection and sequencing Normal ovarian tissue (4 cases) and paired primary epithelial ovarian cancer (EOC) lesions (4 cases), EOC greater omental metastases (4 cases), and EOC mesenteric metastases (4 cases) were collected. Total RNA was extracted using the TRIzol method, treated with RNase R, and then subjected to circRNA-Seq next-generation sequencing. The sequencing results were analyzed. Differentially expressed circRNAs were screened based on the screening criteria of "fold change (FoldChange) > 3 or < 0.3 between the EOC group (primary epithelial ovarian cancer lesion, EOC greater omental metastases, and EOC mesenteric metastases) and the normal group (normal ovarian tissue)" (P < 0.01). The results are as follows: Figure 2A As shown, circ_0009061 (i.e. hsa_circ_0009061) was found to show a gradient downregulation trend in normal ovarian tissue, primary EOC lesion, EOC greater omental metastasis lesion, and EOC mesenteric metastasis lesion.

[0044] Transcriptome sequencing was performed on four normal ovarian tissues and four primary epithelial ovarian cancer (EOC) lesions using RNA-seq. Raw sequencing data were filtered for quality control and aligned to the human reference genome (hg38) using STAR. Gene expression was quantified using featureCounts. Differentially expressed genes were screened using DESeq2 with |log2FoldChange| > 1 and FDR < 0.05. The list of differentially expressed genes was uploaded to the Reactome database (https: / / reactome.org) for pathway enrichment analysis. The hypergeometric distribution test or GSEA algorithm provided by Reactome was used to evaluate the significance of differentially expressed gene enrichment in each pathway against the Reactome pathway database. The analysis results are presented as a bubble chart showing the enriched pathways. Figure 2B As shown, Reactome analysis based on mRNA-Seq revealed that a large number of differentially expressed genes in normal ovarian and ovarian cancer tissues are related to tumor metabolism.

[0045] 2) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to verify the low expression of circ_0009061 in EOC cell lines and tissues. Primers F (nucleotide sequence shown in SEQ ID NO.1) and R (nucleotide sequence shown in SEQ ID NO.2) specifically designed for the circ_0009061 cross-splicing site. Total RNA was extracted from the normal ovarian epithelial cell line HOSEpiC and epithelial ovarian cancer cell lines HEY, SKOV3, OVCAR-8, A2780, OVCA-420, OVCA-433, and OVCA-429 [all cells were obtained from the American Type Culture Collection (ATCC)] using the TRIzol method, and then reverse transcribed and detected by qRT-PCR.

[0046] The results are as follows Figure 2C As shown, compared with the normal ovarian epithelial cell line HOSEpiC, circ_0009061 showed a significant trend of low expression in various epithelial ovarian cancer cell lines (HEY, SKOV3, OVCAR-8, A2780, OVCA-420, OVCA-433, OVCA-429).

[0047] Meanwhile, total RNA was extracted and reverse transcribed from 71 selected normal ovarian tissue samples and 64 primary epithelial ovarian cancer lesion tissue samples. Confirmation was performed by qRT-PCR, and the results are as follows: Figure 2D As shown, compared with normal ovarian tissue, circ_0009061 showed a significantly low expression trend in the primary epithelial ovarian cancer tumor tissue sample.

[0048] The results of circRNA-Seq analysis and qRT-PCR validation were summarized to identify circ_0009061, which was significantly downexpressed in both primary epithelial ovarian cancer lesions and epithelial ovarian cancer cell lines, as the final study target. Kaplan-Meier survival analysis was used to assess the correlation between circ_0009061 expression levels and overall survival and disease-free survival in the FUSCC-cohort study. The results are as follows: Figure 2E As shown in Figures F and F, high expression of circ_0009061 was positively correlated with a good prognosis in ovarian cancer patients.

[0049] 3) Verify the circular property of circ_0009061. The PCR products [i.e., cDNA (obtained by reverse transcription of total cellular RNA) and gDNA (as a negative control)] of the circ_0009061 crosslinker site were simultaneously amplified using both polymerization and divergence primers. The nucleotide sequence of polymerization primer F is shown in SEQ ID NO.13; the nucleotide sequence of polymerization primer R is shown in SEQ ID NO.14; the nucleotide sequence of divergence primer F is shown in SEQ ID NO.15; and the nucleotide sequence of divergence primer R is shown in SEQ ID NO.16] were then Sanger sequenced and compared with the reference sequence of hsa_circ_0009061 in the circBase database. The results are as follows: Figure 2G As shown, it exhibits a CAGT splicing site with the 5′ and 3′ ends joined end-to-end. HEY cells were treated with actinomycin D (MedChemExpress, HY-17559) (2 μg / mL) and incubated at 37 ℃ in a 5% CO2 incubator. RNA was extracted after 0, 6, 12, 18, and 24 hours of treatment for qRT-PCR detection. The results are shown below. Figure 2H As shown, it was found that the level of maternal gene KDM1A gradually decreased with increasing treatment time, while circ_0009061 remained in a stable state.

[0050] RNase R digestion of circ_0009061 verification experiment: RNase R- group: Total RNA was extracted from ovarian cancer cell lines Hey and SKOV3 and amplified using polymerization primers (nucleotide sequences as shown in SEQ ID NO.13 and 14) and divergent primers (nucleotide sequences as shown in SEQ ID NO.15 and 16), respectively. RNase R+ group: Total RNA was extracted from ovarian cancer cell lines Hey and SKOV3, and the total RNA was treated with RNase R (EpiCentre, Inc.) (6 U / μg RNA) at 37 ℃ for 90 minutes. Amplification was performed using polymerization primers and divergence primers, respectively. gDNA group: Genomic DNA was extracted from ovarian cancer cell lines Hey and SKOV3 and amplified using polymerization primers and divergence primers, respectively; Analyze the PCR results, and the results are as follows: Figure 2IAs shown, agarose gel electrophoresis revealed that in the SKOV3 ovarian cancer cell line, both the divergent primer and the convergent primer showed bands in RNase R(-), indicating the simultaneous presence of linear maternal mRNA and circ_0009061 in the sample. In RNase R(+), the divergent primer showed a band, while the convergent primer showed no band, indicating that circ_0009061 is resistant to RNase R digestion, and the stability of its linear maternal gene is disrupted after RNase R treatment. Furthermore, the divergent primer could amplify circ_0009061 from cDNA but not from gDNA, indicating that circ_0009061 is resistant to RNase R digestion. In the ovarian cancer cell line Hey, both the divergent primer and the convergent primer showed bands in RNase R(-), indicating the simultaneous presence of linear maternal mRNA and circ_0009061 in the sample; both the divergent primer and the convergent primer showed bands in RNase R(+), indicating that circ_0009061 has a circular structure, and the divergent primer can amplify circ_0009061 from cDNA but not from gDNA, indicating that circ_0009061 is resistant to RNase R digestion.

[0051] 4) Subcellular localization of circ_0009061 RNA fluorescence in situ hybridization (RNA-FISH) was performed in HEY and SKOV3 cells using a circ_0009061 specific probe (its nucleotide sequence is shown in SEQ ID NO.3). The results are as follows: Figure 2J As shown, circ_0009061 is mainly distributed in the cytoplasm. Further, the nucleus and cytoplasm were separated using a nuclear-cytoplasmic separation kit (Thermo Fisher Scientific, PARIS™ Kit). Nuclear proteins Lamin B1 and GAPDH were selected as positive controls for Western blotting. In subsequent qRT-PCR detection, GAPDH was used as a cytoplasmic internal control, and U6 and Lamin B1 as nuclear internal controls. The results showed the expression level of circ_0009061 in the cytoplasm, as shown in the figure. Figure 2K As shown in Figures 1 and 2, circ_0009061 is mainly located in the cytoplasm.

[0052] In summary, qRT-PCR analysis revealed that, using the normal ovarian epithelial cell line HOSEpiC as a control, circ_0009061 showed a significantly low expression trend in various epithelial ovarian cancer cell lines (HEY, SKOV3, OVCAR-8, A2780, OVCA-420, OVCA-433). Figure 2C Simultaneously, samples of 71 normal ovarian tissues and 64 epithelial ovarian cancer (EOC) tissues were collected, and RNA was extracted and reverse transcribed. qRT-PCR analysis confirmed that circ_0009061 showed a significantly lower expression trend in epithelial ovarian cancer samples compared to normal ovarian tissues. Figure 2D To further validate the results of qRT-PCR, a specific probe for circ_0009061 was designed. The distribution and expression abundance of circ_0009061 in HEY and SKOV3 cell lines were detected by fluorescence in situ hybridization (FISH). The results showed that circ_0009061 was mainly located in the cytoplasm.

[0053] Example 2: circ_0009061 inhibits the proliferation, migration, and aerobic glycolysis of ovarian cancer cells. 1) Construction of stable cell lines Overexpression of circ_0009061: The circ_0009061 overexpression plasmid was constructed using the lentiviral overexpression vector pLenti ciR6024 (Lingke). The plasmid, along with the helper packaging plasmids pMD2.G and psPAX2 (Lingke), was cultured with cells in a 5% CO2, 37°C incubator for 4-6 hours. HEK-293T cells were transfected, and the supernatant was collected. HEY and SKOV3 cells were infected with the viral supernatant, and puromycin (Solepro, IP1280) was added to the culture dish to a final concentration of 2 μg / mL. Stable overexpression cell lines (i.e., HEY and SKOV3 cells stably transfected with circ_0009061) were obtained after 24 hours of selection.

[0054] Knockdown of circ_0009061: A shRNA plasmid targeting circ_0009061 was constructed using the lentiviral knockdown vector pLKO.1 (Lingke). (shRNA sequence: sh1 nucleotide sequence as shown in SEQ ID NO.4; sh2 nucleotide sequence as shown in SEQ ID NO.5). The above plasmid was co-cultured with the helper packaging plasmids pMD2.G and psPAX2 (Lingke) in a 5% CO2, 37°C incubator for 4-6 h. HEK-293T cells were then transfected, and the supernatant was collected to obtain viral supernatant. HEY and SKOV3 cells were infected with the viral supernatant. Puromycin (Solepro, IP1280) was added to the dish to a final concentration of 2 μg / mL, and the cells were screened for 24 h to obtain cell lines with knockdown of circ_0009061 (i.e., HEY and SKOV3 cells stably transfected with circ_0009061-sh1 / circ_0009061-sh2).

[0055] The circ_0009061 overexpression plasmid and shRNA plasmid were constructed by Linke (Shanghai) Biotechnology Co., Ltd. and identified by sequencing.

[0056] In addition, HEK-293T cells were co-transfected with the empty lentivirus overexpression vector pLenti ciR6024 and the helper packaging plasmids pMD2.G and psPAX2, with the remaining procedures being the same as described above, to obtain the control cell line circ-NC (HEY / SKOV3); HEK-293T cells were co-transfected with the empty lentivirus knockdown vector pLKO.1 and the helper packaging plasmids pMD2.G and psPAX2, with the remaining procedures being the same as described above, to obtain the control cell line circ-shNC (HEY / SKOV3).

[0057] Total RNA was extracted from HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061 and circ_shNC / circ_0009061-sh1 / circ_0009061-sh2 and qRT-PCR was used to verify the overexpression and knockdown efficiency. The results are as follows: Figures 3A and 3B As shown, compared with control cells (circ-NC / circ-shNC), the protein level of circ_0009061 was significantly increased in cells overexpressing circ_0009061 (Figure 3A); the protein expression level of circ_0009061 was significantly downregulated in cells with knockdown of circ_0009061 (circ_0009061_sh1, circ_0009061_sh2) (Figure 3B).

[0058] 2) The effect of circ_0009061 on cell proliferation was detected using the CCK-8 assay. HEY and SKOV3 cells stably overexpressing circ_0009061, along with control cells, were seeded at 1500 cells / well in 96-well plates. CCK-8 working solution was added at 0, 24, 48, 72, and 96 hours (100 μL of CCK-8 working solution was prepared per well under light-protected conditions, consisting of 95 μL of basal culture medium and 5 μL of CCK-8 stock solution mixed thoroughly). The 6-well plates were wrapped with aluminum foil and incubated at 37°C for 2 hours. The absorbance was measured at 450 nm.

[0059] The results are as follows Figures 3C, D, E, F As shown, with control cells (circ-NC / circ-shNC) as a reference, overexpression of circ_0009061 significantly inhibited the proliferation of HEY and SKOV3 cells (Figure 3C, D); while knockdown of circ_0009061 promoted cell proliferation (Figure 3E, F).

[0060] 3) Detect the effect of circ_0009061 on cell colony formation ability. Logarithmically growing cells (HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061; HEY and SKOV3 cells stably transfected with circ_shNC / circ_0009061-sh1 / circ_0009061-sh2) were collected. An appropriate amount of pre-warmed trypsin was added according to the size of the culture dish. The dish was gently shaken to ensure the trypsin covered the entire bottom, and the cells were incubated at 37°C for 1-2 minutes to digest the cells. After centrifugation, the cells were resuspended in DMEM basal medium to prepare a single-cell suspension. 500-600 cells were seeded into 6-well cell culture plates and cultured overnight at 37°C with 5% CO2. After 10-14 days, the cells were fixed with methanol, stained with crystal violet, and the number of cell clones was counted to determine the effect of circ_0009061 overexpression and knockdown on cell colony formation ability.

[0061] The results are as follows Figure 3G, H, I, J As shown, with control cells (circ-NC / circ-shNC) as a reference, overexpression of circ_0009061 significantly inhibited the proliferation of HEY and SKOV3 cells (Figure 3G, H); while knockdown of circ_0009061 promoted cell proliferation (Figure 3I, J).

[0062] 4) The effect of circ_0009061 on cell migration was detected using the Transwell assay. Collect logarithmically growing cells (HEY and SKOV3 cells stably transfected by circ_NC / circ_0009061; HEY and SKOV3 cells stably transfected by circ_shNC / circ_0009061-sh1 / circ_0009061-sh2), add preheated trypsin according to the size of the culture dish, digest for 1-2 min to prepare a single-cell suspension and count the cells. Add 10,000-20,000 cells / well and 150 μL of cell suspension / well to a 24-well plate, with three replicates per well. Add 200 μL of basal culture medium (DMEM) to each well. Incubate at 37°C for 48 hours before detection. Before detection, aspirate the old culture medium, wash with PBS, fix with anhydrous ethanol, stain with ammonium oxalate crystal violet for 30 min, wash with PBS, wipe away excess dye with a cotton swab, and photograph and record the experimental results.

[0063] The results are as follows Figure 3K, L As shown, overexpression of circ_0009061 significantly inhibited the migration ability of HEY and SKOV3 cells (Fig. 3K), while knockdown of circ_0009061 promoted cell migration (Fig. 3L).

[0064] 5) Detection of the effect of circ_0009061 on cellular aerobic glycolysis Stable transgenic cells (HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061) were seeded at 2000 cells per well in 24-well plates. Oxidative phosphorylation was inhibited by treating the cells with DMEM basal medium containing 1 mmol / L Oligomycin (DOJINDO, Glycolysis / OXPHOS Assay Kit) at 37°C for 6 hours. Lactate content was measured in the supernatant, and ATP production was simultaneously detected using chemiluminescence immunoassay. The results, shown in Figure 3M, indicated that the lactate and ATP production in the circ_0009061 overexpression group (HEY and SKOV3 cells stably transfected with circ_0009061) were significantly lower than those in the control group (HEY and SKOV3 cells stably transfected with circ_NC).

[0065] Glucose uptake experiment: Stable cells (HEY and SKOV3 cells stably transfected by circ_NC / circ_0009061) were seeded at a 70% confluence density in 24-well plates and incubated with the glucose fluorescent analog 2-NBDG at 37°C in a 5% CO2 incubator for 15 minutes. The cells were then observed and quantified under a fluorescence microscope.

[0066] The results, as shown in Figure 3N, indicate that the fluorescence intensity of the circ_0009061 overexpression group (HEY and SKOV3 cells stably transfected with circ_0009061) was significantly lower than that of the control group (HEY and SKOV3 cells stably transfected with circ_NC).

[0067] Constructing a subcutaneous xenograft tumor model in nude mice: HEY cells stably transfected with circ_0009061 and circ_NC (HEY cells stably overexpressing circ_0009061 and control cells) were expanded and cultured. An appropriate amount of preheated trypsin was added according to the size of the culture dish. The dish was gently shaken to ensure the trypsin covered the entire bottom, and the cell culture dish was placed in a 37°C incubator for 1-2 minutes to digest the cells. After digestion, the cells were resuspended in PBS, and the cell density was adjusted to 5 × 10⁶ cells / mL. 7 Cells / mL. 100 μL of cell suspension (i.e., 5 × 10⁶ cells / mL) was subcutaneously injected into the right axilla of 6-8 week old female BALB / c nude mice. 6 (cells / each).

[0068] 18F-FDG PET-CT imaging was performed, and the results are as follows: Figure 3O, P, Q As shown, the tumors overexpressing circ_0009061 showed a significant decrease in glucose uptake.

[0069] In summary, CCK-8 and colony formation assays revealed that, using control cells (circ-NC / circ-shNC) as a reference, overexpression of circ_0009061 significantly inhibited the proliferation of HEY and SKOV3 cells, while knockdown of circ_0009061 promoted cell proliferation. Transwell assays showed that overexpression of circ_0009061 significantly inhibited the migration of HEY and SKOV3 cells, while knockdown of circ_0009061 promoted cell migration. To further verify the effect of circ_0009061 on aerobic glycolysis in ovarian cancer cells, cells were treated with oligomycin-containing medium to inhibit oxidative phosphorylation, and the production of ATP and lactate from glycolysis was measured. The results showed that the ATP and lactate production in the circ_0009061 overexpression group was significantly lower than that in the control group. Simultaneously, the glucose uptake capacity of cells was detected by incubation with the glucose fluorescent analog 2-NBDG, and the results showed that the fluorescence intensity in the circ_0009061 overexpression group was significantly lower than that in the control group. In addition, a subcutaneous xenograft tumor model was constructed in nude mice and 18F-FDG PET-CT imaging was performed. The results showed that the glucose uptake capacity of the tumor was significantly reduced in the group overexpressing circ_0009061.

[0070] Example 3: Binding of circ_0009061 to MCCC1 / MCCC2 proteins and localization of the binding region 1) RNA pull-down combined with mass spectrometry identification A 5′ biotin-tagged circ_0009061-specific probe (nucleotide sequence as shown in SEQ ID NO. 6) and a negative control probe (nucleotide sequence as shown in SEQ ID NO. 7) were designed and synthesized.

[0071] Using the Pierce™ Magnetic RNA-Protein Pull-Down Kit from Thermo Fisher Scientific, HEY and SKOV3 cell lysates were collected. The probes were mixed with streptavidin magnetic beads and incubated at room temperature for 5-6 hours, followed by overnight (12-16 hours) incubation with HEY and SKOV3 cell lysates at 4°C. Eluted bound proteins were subjected to SDS-PAGE electrophoresis and silver staining. Differential protein bands near 71 kDa and 61 kDa were excised for mass spectrometry identification and Western blotting verification.

[0072] The results are shown in Figures 4A and 4B. The circ_0009061 specific probe can enrich MCCC1 and MCCC2 proteins.

[0073] 2) RIP Reverse Verification Antibody-magnetic bead complexes were formed by incubating MCCC1 antibody (14861-1-AP) or MCCC2 antibody (12117-1-AP) (Proteintech®) with Protein A / G magnetic beads (B23202, Bimake) at 4°C overnight. These complexes were then incubated with HEY and SKOV3 cell lysates at 4°C overnight (12-16 hours). After washing, the co-precipitated RNA was extracted, validated by Western blotting, and the enrichment level of circ_0009061 was detected by qRT-PCR.

[0074] The results are shown in Figures 4C and 4D. The enrichment of circ_0009061 in the MCCC1 antibody group and the MCCC2 antibody group was significantly higher than that in the IgG control group (P<0.001).

[0075] 3) Combined with regional positioning The binding regions of circ_0009061 with MCCC1 and MCCC2 proteins were predicted using the catRAPID database.

[0076] The results are as follows Figure 4EAs shown, truncated fragments of MCCC1 protein (100-151aa, 125-176aa, 176-227aa, 375-426aa) and truncated fragments of MCCC2 protein (88-139aa, 238-289aa, 338-389aa, 426-477aa) were selected.

[0077] Flag-tagged expression plasmids for truncated fragments of MCCC1 protein (100-151aa, 125-176aa, 176-227aa, 375-426aa) and MCCC2 protein (88-139aa, 238-289aa, 338-389aa, 426-477aa) were constructed using pCDNA3.1 plasmid. The DNA sequences of these truncated fragments were synthesized by Sangon Biotech (Shanghai) Co., Ltd. After transfecting HEY cells for 48 hours, RIP experiments were performed using the Flag antibody (DYKDDDDK, 20543-1-AP, Proteintech). The enrichment level of circ_0009061 in each truncated fragment was detected by qRT-PCR and verified by Western blotting.

[0078] The results are as follows Figures 4F, G, H As shown, circ_0009061 binds to amino acid regions 100-151 of the MCCC1 protein and amino acid regions 338-389 of the MCCC2 protein (Figure 4F, G). In addition, Western blotting verified the binding of the Flag protein antibody to the magnetic beads and the successful transfection of the plasmid (Figure 4H).

[0079] In summary, RNA pull-down combined with mass spectrometry identification revealed that the circ_0009061 specific probe could enrich MCCC1 and MCCC2 proteins (Figure 4A, B). Reverse verification using RIP experiments confirmed that the enrichment level of circ_0009061 in the MCCC1 and MCCC2 antibody groups was significantly higher than that in the IgG control group (P<0.001) (Figure 4C, D). Using the catRAPID database to predict binding regions, flag-tagged expression plasmids of truncated fragments of MCCC1 and MCCC2 proteins were constructed for RIP experiments. The results showed that circ_0009061 bound to amino acid positions 100-151 of the MCCC1 protein and amino acid positions 338-389 of the MCCC2 protein (…). Figures 4F, G, H ).

[0080] Example 4: circ_0009061-mediated TRIM21-ubiquitination degradation of MCCC1 / MCCC2 1) Protein level and dimer detection In HEY and SKOV3 cells stably transfected with circ_0009061, the protein levels of MCCC1 and MCCC2 were detected by Western Blot. The results are shown in Figure 5C, which show that overexpression of circ_0009061 significantly reduced the protein levels of both.

[0081] Immunofluorescence and RNA-FISH co-localization assays confirmed that circ_0009061 co-localized with MCCC1 and MCCC2 in the cytoplasm. Figure 5A The formation of MCCC1 / MCCC2 dimers was detected by the adjacent linkage assay (PLA), showing that overexpression of circ_0009061 reduced the formation of MCCC1 / MCCC2 dimers (Figure 5B).

[0082] 2) Identification of degradation pathways HEY and SKOV3 cells overexpressing circ_0009061 were cultured for 8 hours at 37°C in a 5% CO2 incubator using the proteasome inhibitor MG-132 (20 μM) (Maclean, M832899), the autophagy inhibitor 3-MA (Maclean, M833793) (20 μM), and chloroquine (Maclean, C843545) (20 μM).

[0083] The results are shown in Figure 5D. The results indicate that only MG-132 treatment can significantly reverse the reduction in MCCC1 / MCCC2 protein levels caused by overexpression of circ_0009061.

[0084] 3) Ubiquitination level detection Following MG-132 treatment, immunoprecipitation was performed using either MCCC1 or MCCC2 antibodies: HEY and SKOV3 cells overexpressing circ_0009061 after MG-132 treatment were collected, washed twice with pre-chilled PBS, and lysed in IP lysis buffer (RIPA) containing a protease inhibitor (PMSF) for 30 minutes on ice with shaking every 10 minutes. The cells were centrifuged at 12000×g for 15 minutes at 4°C, and the supernatant was collected. Protein concentration was determined using a BCA kit (Beyotime). 500 μg of protein lysis buffer was added, along with 2 μg of MCCC1 antibody (Proteintech, 14861-1-AP) or MCCC2 antibody (Proteintech, 12117-1-AP). A negative control group was also included, containing an equal volume of normal rabbit IgG. The cells were incubated overnight (12-16 hours) at 4°C using a rotary incubator. The following day, 20 μL of Protein A / G magnetic beads (B23202, Bimake) (pre-equilibrated with lysis buffer) were added to each sample, and the mixture was incubated at 4°C for 2 hours. After incubation, the magnetic beads were washed three times with pre-cooled IP wash buffer (PBS containing 0.1% NP-40), centrifuged at 3000×g for 3 minutes each time at 4°C, and the supernatant was discarded. 30 μL of 2×SDS-PAGE loading buffer was added to the washed magnetic beads, and the mixture was boiled at 100°C for 10 minutes to denature and elute the proteins. After centrifugation, the supernatant was discarded, and the precipitates were analyzed by Western blotting using K48-Ubiquitin antibody (8081S, Cell Signaling Technology) and K63-Ubiquitin antibody (5621S, Cell Signaling Technology), respectively.

[0085] The results are shown in Figures 5E, F, and G. Overexpression of circ_0009061 significantly increased the ubiquitination levels of MCCC1 and MCCC2, with the K48 chain being the dominant type.

[0086] 4) Identification with E3 ligase Cells were collected and lysed. The protein supernatant was incubated overnight at 4°C with either MCCC1 antibody (Proteintech, 14861-1-AP) or MCCC2 antibody (Proteintech, 12117-1-AP). Protein A / G magnetic beads (B23202, Bimake) were added for capture. After washing, the cells were eluted, and the resulting eluent was the Co-IP product. The Co-IP products of MCCC1 / MCCC2 were identified by silver staining combined with mass spectrometry. The results are as follows: Figure 5H As shown, the E3 ubiquitin ligase TRIM21 was screened from the immunoprecipitation complex of MCCC1 and MCCC2.

[0087] Combined with co-immunoprecipitation (Co-IP) / WB verification, the E3 ubiquitin ligase TRIM21 was screened and confirmed to mediate the above ubiquitination process, and the binding of MCCC1 / MCCC2 to TRIM21 was enhanced after overexpression of circ_0009061. Figure 5I, J, K, L M).

[0088] In summary, Western blot analysis revealed that overexpression of circ_0009061 significantly reduced the protein levels of MCCC1 and MCCC2 (Figure 5C), while knockdown of circ_0009061 increased their protein levels. Immunofluorescence and RNA FISH co-localization experiments confirmed that circ_0009061 co-localized with MCCC1 and MCCC2 in the cytoplasm. Figure 5A The adjacent ligation assay (PLA) showed that overexpression of circ_0009061 reduced MCCC1 / MCCC2 dimer formation (Fig. 5B). Treatment of cells with the proteasome inhibitor MG-132, the autophagy inhibitor 3-MA, and chloroquine, respectively, showed that only MG-132 could restore MCCC1 / MCCC2 protein levels, indicating that the degradation pathway was proteasome-dependent. Immunoprecipitation combined with Western blotting showed that overexpression of circ_0009061 significantly increased the ubiquitination levels of MCCC1 and MCCC2, with the K48 chain being the predominant form (Fig. 5E, F, G). Mass spectrometry identification and Co-IP / WB verification confirmed that the E3 ubiquitin ligase TRIM21 mediated the above ubiquitination process, and that overexpression of circ_0009061 enhanced the binding of MCCC1 / MCCC2 to TRIM21. Figure 5H 、I、J、K、L、M).

[0089] Example 5: circ_0009061-driven H3K36cr crotonylation modification inhibits HK2 / PKM2 transcription 1) Crotonylation modification proteomics detection Crotonylation modification proteomics was performed on HEY cells overexpressing circ_0009061 and their control cells (circ_NC) to detect KEGG pathway enrichment and COG analysis.

[0090] The results are shown in Figures 5N, O, and P. Overexpression of circ_0009061 significantly increased the overall crotonylation level in cells. KEGG enrichment analysis showed that the differentially expressed protein was closely associated with aerobic glycolysis. COG analysis showed that circ_0009061 regulates functional proteins related to cell proliferation, migration, and glucose metabolism through crotonylation modification.

[0091] Western blotting was performed using anti-crotonyllysine (PTM-502, PTM BIO) and the results are shown in Figure 5M. The results showed that, compared with the control group (circ_NC), the overall crotonylation modification level of EOC cell line proteins was significantly increased after overexpression of circ_0009061.

[0092] Normal ovarian epithelial cell line HOSEpiC and ovarian cancer cell lines HEY, SKOV3, and A2780 were collected. Total protein was extracted and then digested with trypsin. Crotonylated peptides were immunoaffinity-enriched using a highly specific anti-crotonylated lysine antibody (PTM Biolabs). Peptide separation and analysis were performed using LC-MS / MS (liquid chromatography-tandem mass spectrometry). Mass spectrometry parameters were set as follows: primary mass spectrometry scan range 100-1700 m / z, and secondary mass spectrometry in data-dependent acquisition mode. Mass spectrometry data were processed using the MaxQuant search engine and compared with the human UniProt database, with a false discovery rate (FDR) set to <1%. The experimental results are shown in Figure 5Q. Crotonylation modification proteomics successfully identified crotonylated peptides in ovarian cancer cells before and after overexpression of circ_0009061, and differentially expressed crotonylated proteins were screened. Subsequently, motif sequence analysis was performed on all crotonylated peptides identified in Figure 5Q using motifs-x software. Six to ten amino acid residues were extracted upstream and downstream of the crotonylation modification site, and the frequency distribution of amino acids surrounding the lysine residue (Kcr) at the modification site was analyzed. The results are shown in Figure 5R. Motif sequence analysis revealed conserved sequence features around the crotonylation modification site and identified characteristic motif sequences (such as specific amino acid residues enriched around Kcr), indicating that crotonylation modification exhibits site-specific sequence bias. Based on the omics data from Figure 5Q, subcellular localization annotation and functional enrichment analysis were performed on the identified crotonylated proteins using gene ontology (GO) analysis, screening for differentially modified proteins located in the cell nucleus. Of these, 25.08% of the differentially modified proteins were subcellularly located in the cell nucleus. Based on the motif sequence analysis results in Figure 5R, we focused on the changes in crotonylation modification at various lysine sites on histone H3, and screened out the two sites with the most significant differences: H3K36cr and H3K23cr. The results are shown in Figure 5S, indicating that circ_0009061-mediated crotonylation modification mainly affects the histone H3K36 site.

[0093] 2) H3K36cr site verification The following antibodies were used: H3K36cr specific antibody anti-crotonyl-histone H3 (Lys36) (PTM Biolabs, PTM-536), H3K23cr specific antibody anti-crotonyl-histone H3 (Lys23) (PTM-519, PTM BIO), H3K36me3 specific antibody anti-tri-methyl-histone H3 (Lys36) (PTM-625RM, PTM BIO), H3K36ac specific antibody anti-crotonyl-histone H3 (Lys36) (PTM Biolabs, PTM-117), H3K36me1 specific antibody anti-crotonyl-histone H3 (Lys36) (PTM Biolabs, PTM-623), H3K36me2 specific antibody anti-crotonyl-histone H3 (Lys36) (PTM Biolabs, PTM-624), and H3 specific antibody anti-histone. Western blotting was performed on HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061 using H3 (PTM Biolabs, PTM-6613). The results, shown in Figure 5T, indicate that overexpression of circ_0009061 significantly increased H3K36cr levels and significantly decreased H3K36me3 levels, while H3K36ac, H3K36me1, H3K36me2, H3K23cr, and total H3 protein levels showed no significant changes.

[0094] 3) Recovery Experiment HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061 (HEY cells and SKOV3 cells overexpressing circ_0009061) were simultaneously transfected with MCCC1-CDS-3×Flag and MCCC2-CDS-3×Flag overexpression plasmids using lipo3000. Cells were seeded in 6-well plates (4×10⁶ cells / wells) 24 hours before transfection. 5Cell confluency was 60%-70% at transfection. Two centrifuge tubes were used to prepare plasmid mixtures (125 μL Opti-MEM + 2.5 μg MCCC1-CDS-3×Flag + 2.5 μg MCCC2-CDS-3×Flag + 10 μL P3000) and transfection reagent mixtures (125 μL Opti-MEM + 7.5 μL Lipo3000), respectively. After mixing, the mixtures were incubated at room temperature for 15 minutes. 250 μL of the complex was added to the cells, and the cells were incubated at 37°C for 4-6 hours. The medium was changed, and the cells were cultured for another 48 hours before harvesting for analysis. Western blotting was then performed. The results are shown in Figure 5. Simultaneous overexpression of MCCC1 / MCCC2 reversed the increase in H3K36cr and decrease in H3K36me3 levels induced by circ_0009061 overexpression.

[0095] 4) HK2 / PKM2 expression detection In HEY and SKOV3 cells stably transfected with circ_NC / circ_0009061 (HEY and SKOV3 cells overexpressing circ_0009061), the protein levels of PKM2 and HK2 were detected by Western blotting, and the mRNA levels of HK2 and PKM2 were detected by qRT-PCR. The HK2 primer (upstream: nucleotide sequence as shown in SEQ ID NO.8, downstream: nucleotide sequence as shown in SEQ ID NO.9) and the PKM2 primer (upstream: nucleotide sequence as shown in SEQ ID NO.10, downstream: nucleotide sequence as shown in SEQ ID NO.11) were used.

[0096] The results are as follows Figure 5 V, W, Y As shown, overexpression of circ_0009061 significantly reduced the mRNA and protein levels of HK2 and PKM2.

[0097] In summary, crotonylation modification analysis revealed that overexpression of circ_0009061 significantly increased the overall crotonylation modification level in cells. KEGG enrichment analysis showed that the differentially expressed protein was closely associated with aerobic glycolysis (Figure 5N, O, P). Western blotting using an H3K36cr-specific antibody showed that overexpression of circ_0009061 significantly increased H3K36cr levels and significantly decreased H3K36me3 levels (Figure 5T). Reversal experiments showed that simultaneous overexpression of MCCC1 / MCCC2 could reverse the increase in H3K36cr and decrease in H3K36me3 levels induced by circ_0009061 overexpression (Figure 5U). qRT-PCR and Western blotting analysis of the expression of key glycolytic enzymes HK2 and PKM2 showed that overexpression of circ_0009061 significantly reduced both mRNA and protein levels of HK2 and PKM2. Figure 5 V, W, Y ).

[0098] Therefore, as Figure 1 As described above, circ_0009061 can directly bind to MCCC1 / MCCC2 proteins, promote their K48-ubiquitination degradation mediated by TRIM21, thereby driving an increase in histone H3K36cr crotonylation modification level, inhibiting the transcription and expression of key glycolytic enzymes HK2 and PKM2, and ultimately inhibiting the proliferation, migration and aerobic glycolysis of ovarian cancer cells.

[0099] Example 6: circ_0009061 inhibits subcutaneous tumor growth and peritoneal metastasis in nude mice 1) Nude mouse subcutaneous xenograft tumor model HEY cells stably transfected with circ_0009061 and circ_NC (HEY cells stably overexpressing circ_0009061 and control cells) were expanded and cultured. An appropriate amount of preheated trypsin was added according to the size of the culture dish. The dish was gently shaken to ensure the trypsin covered the entire bottom, and the cell culture dish was placed in a 37°C incubator for 1-2 minutes to digest the cells. After digestion, the cells were resuspended in PBS, and the cell density was adjusted to 5 × 10⁶ cells / mL. 7 Cells / mL. Five 6-8 week old female BALB / c nude mice were used as a subcutaneous injection of 100 μL of cell suspension (i.e., 5 × 10⁻⁶ cells / mL) into the right axilla. 6 (cells / tumor). Measure the long and short diameters of the tumor twice a week using calipers, and calculate using the formula V=π / 6×L×W. 2 Calculate the tumor volume. After 4 weeks, sacrifice the nude mice, remove the tumor, weigh it, and photograph it.

[0100] The results are as follows Figures 6A, B, and CAs shown, compared with the control group (circ_NC), the tumor volume of the circ_0009061 overexpression group decreased significantly from week 2 (P<0.01), and the average tumor volume decreased by about 65% and the average tumor weight decreased by about 58% by week 4 (P<0.001).

[0101] 2) Nude mouse peritoneal transfer model HEY cells stably overexpressing circ_0009061 and control cells (circ_NC) were adjusted to 5×10⁻⁶ cells. 7 Cells / mL, 100 μL of cell suspension (i.e., 5 × 10⁶ cells / mL) was injected intraperitoneally into each nude mouse. 6 (Cells / mouse). Fluorescein potassium salt (150 mg / kg) was injected intraperitoneally weekly, and the fluorescence signal was observed using a small animal in vivo imaging system 10-15 minutes later. Nude mice were sacrificed after 5 weeks, and the number of metastatic nodules in the peritoneal cavity was counted by dissection. The metastatic tumors were dissected and weighed.

[0102] The results are as follows Figure 6E As shown in Figures F, G, and H, the fluorescence signal intensity of the circ_0009061 overexpression group was significantly weaker than that of the control group. Figure 6E The number of peritoneal metastatic nodules decreased by approximately 72%, and the total weight of metastatic tumors decreased by approximately 68% (P<0.001). Figures 6F, G, H ).

[0103] 3) Immunohistochemistry (IHC) and in situ hybridization (ISH) detection Subcutaneous tumors and peritoneal metastases were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. IHC analysis was performed using Ki67 antibody (27309-1-AP, 1:4000), MCCC1 antibody (Proteintech, 14861-1-AP), MCCC2 antibody (Proteintech, 12117-1-AP), and H3K36cr antibody (PTM Biolabs, PTM-536). ISH analysis was performed using the circ_0009061 specific probe (nucleotide sequence shown in SEQ ID NO. 3).

[0104] IHC results ( Figure 6D I) showed that Ki67 expression was significantly reduced, MCCC1 / MCCC2 protein expression was significantly decreased, and H3K36cr expression was increased in the tumors of the circ_0009061 overexpression group; ISH ​​results ( Figure 6D This study validated the effective expression of circ_0009061 in the tumors of the overexpression group.

[0105] In summary, a subcutaneous xenograft tumor model was constructed in nude mice. HEY cells stably overexpressing circ_0009061 and control cells were cultured at a concentration of 5 × 10⁻⁶ cells / year. 6One mouse per mouse was injected subcutaneously into the right axilla of nude mice. Results showed that, compared to the control group, the tumor volume in the circ_0009061 overexpression group significantly decreased from week 2 (P<0.01), with an average tumor volume reduction of approximately 65% ​​and an average tumor weight reduction of approximately 58% by week 4 (P<0.001). Figures 6A, B, and C A nude mouse peritoneal transfer model was constructed by transferring HEY cells stably overexpressing circ_0009061 and carrying luciferase labeling at a rate of 5 × 10⁻⁶ cells per cell line. 6 Intraperitoneal injection was administered to each animal, and in vivo small animal imaging was performed weekly. Results showed that the fluorescence signal intensity in the circ_0009061 overexpression group was significantly weaker than that in the control group. Figure 6E The number of peritoneal metastatic nodules decreased by approximately 72%, and the total weight of metastatic tumors decreased by approximately 68% (P<0.001). Figures 6F, G, H Immunohistochemistry and in situ hybridization were performed on subcutaneous tumors and peritoneal metastases. The results showed that the Ki67 positivity rate was decreased, MCCC1 / MCCC2 protein expression was reduced, and H3K36cr expression was increased in the circ_0009061 overexpression group. Figure 6I ISH validated the effective expression of circ_0009061 in tumors of the overexpression group. Figure 6D ).

[0106] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of circ_0009061 expression enhancer in the preparation of drugs for treating ovarian cancer, characterized in that, The nucleotide sequence of the circ_0009061 gene is shown in SEQ ID NO.

12.

2. The application according to claim 1, characterized in that, The circ_0009061 expression enhancer is a lentivirus that overexpresses circ_0009061.

3. The application according to claim 2, characterized in that, The circ_0009061 overexpression lentivirus was prepared by co-transfecting cells with a lentivirus overexpression vector and a lentivirus packaging plasmid.

4. The application according to claim 3, characterized in that, The lentiviral overexpression vector is formed by inserting the circ_0009061 gene, whose nucleotide sequence is shown in SEQ ID NO.12, into the empty lentiviral vector pLenti ciR6024; The packaging plasmids are pMD2.G and psPAX2; The cells in question are HEK-293T cells.

5. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for treating ovarian cancer, characterized in that, Includes a circ_0009061 expression enhancer and a pharmaceutically acceptable vector, the nucleotide sequence of the circ_0009061 gene being shown in SEQ ID NO.

12.

7. The pharmaceutical composition according to claim 6, characterized in that, The circ_0009061 expression enhancer is a circ_0009061 overexpression lentivirus.

8. Application of reagents for detecting circ_0009061 gene expression in the preparation of kits for detecting ovarian cancer.

9. Application of the circ_0009061 gene as a target in screening drugs for the treatment of ovarian cancer.

10. A method for screening drugs for the treatment of ovarian cancer, characterized in that, include: Candidate drugs were applied to cells expressing circ_0009061, and changes in the expression level of circ_0009061 were detected. Candidate drugs that increased the expression level of circ_0009061 were selected.

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