Application of CPSF7 as a therapeutic and prognostic target for ovarian cancer

Abstract

The application discloses application of CPSF7 as an ovarian cancer treatment and prognosis target, and relates to the technical field of biological medicine.The application research finds that overexpression of CPSF7 is related to poor prognosis of ovarian cancer, indicating that CPSF7 can be used as a prognosis index and potential treatment target of ovarian cancer.Further research shows that CPSF7 promotes malignant progression of ovarian cancer by promoting proliferation, migration and invasion of ovarian cancer cells, and inhibition of CPSF7 expression can significantly inhibit proliferation, migration and invasion of ovarian cancer cells.The application also finds that UBE2K is one of key downstream targets of CPSF7 in the ovarian cancer cell-mediated carcinogenic process, and inhibition of UBE2K expression can weaken the overexpression of CPSF7 induced enhancement effect of ovarian cancer cell proliferation, migration and invasion.The application provides a new target for treatment and prognosis prediction of ovarian cancer, and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of CPSF7 as a target for the treatment and prognosis of ovarian cancer. Background Technology

[0002] Ovarian cancer is the leading cause of death among gynecological malignancies, with high-grade serous ovarian cancer (HGSOC) accounting for the largest proportion and exhibiting the most severe malignancy. Due to its insidious onset, 70% of patients are diagnosed at an advanced stage, and the cancer is prone to drug resistance and recurrence after treatment, resulting in a 5-year survival rate of only about 50%. Therefore, elucidating the mechanisms of ovarian cancer development, developing early diagnostic biomarkers, and employing precise targeted therapy strategies are urgent clinical challenges that need to be addressed.

[0003] Variable polyadenylation (APA) is a key mechanism by which precursor mRNA regulates gene expression by selecting different polyadenylation sites (PAS) in the 3′ untranslated region (3′ UTR), affecting mRNA stability, translation efficiency, and interactions with regulatory factors. APA is synergistically regulated by complexes such as cleavage and polyadenylation-specific factor (CPSF) and cleavage-stimulating factor (CSTF). Among them, the CFIm complex (containing CPSF5 / CFIm25, CPSF6 / CFIm68, and CPSF7 / CFIm59 subunits) regulates PAS selection by recognizing the upstream UGUA motif of PAS.

[0004] Cleavage and polyadenylation specific factor 7 (CPSF7), also known as CFIm59, is a large subunit of a cleavage factor involved in pre-mRNA cleavage and polyadenylation.

[0005] Aberrant APAs are ubiquitous in tumors and can participate in tumor progression by altering the 3′ UTR conformation of oncogenes / tumor suppressor genes. For example, CPSF5 inhibits bladder cancer progression by regulating APAs in ANXA2 and LIMK2; CPSF6 promotes cancer cell proliferation through phase separation; and CPSF7 promotes metastasis in hepatocellular carcinoma by regulating the WWP2 / PTEN / AKT pathway. Currently, the expression characteristics, function, and APA regulatory network of CPSF7 in ovarian cancer have not been systematically elucidated. Summary of the Invention

[0006] The purpose of this invention is to provide the application of CPSF7 as a therapeutic and prognostic target for ovarian cancer, thereby addressing the problems existing in the prior art. This invention has found that overexpression of CPSF7 is associated with poor prognosis in ovarian cancer, promoting malignant progression by promoting the proliferation, migration, and invasion of ovarian cancer cells. This invention confirms that CPSF7 can serve as a prognostic indicator and potential therapeutic target for ovarian cancer.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] This invention provides the application of a reagent that reduces CPSF7 expression levels in the preparation of drugs for treating ovarian cancer.

[0009] Furthermore, the reagent is siRNA;

[0010] The nucleotide sequence of the siRNA is shown in SEQ ID NO.2.

[0011] Furthermore, the reagent is siRNA;

[0012] The nucleotide sequence of the siRNA is shown in SEQ ID NO.3.

[0013] The present invention also provides a drug for treating ovarian cancer, wherein the active ingredient includes a reagent that reduces CPSF7 expression levels;

[0014] The reagent is siRNA with a nucleotide sequence as shown in SEQ ID NO.2 or SEQ ID NO.3.

[0015] This invention also provides the use of reagents that reduce UBE2K expression levels in the preparation of drugs for treating ovarian cancer.

[0016] Furthermore, the reagent is siRNA;

[0017] The nucleotide sequence of the siRNA is shown in SEQ ID NO.4.

[0018] Furthermore, the reagent is siRNA;

[0019] The nucleotide sequence of the siRNA is shown in SEQ ID NO.5.

[0020] The present invention also provides a medicament for treating ovarian cancer, wherein the active ingredient includes a reagent that reduces UBE2K expression levels;

[0021] The reagent is siRNA with a nucleotide sequence as shown in SEQ ID NO.4 or SEQ ID NO.5.

[0022] This invention also provides the application of reagents for detecting CPSF7 expression levels in the preparation of products for predicting the prognosis of ovarian cancer.

[0023] Furthermore, the product is a reagent kit.

[0024] The present invention discloses the following technical effects:

[0025] This invention found that CPSF7 overexpression is associated with poor prognosis in ovarian cancer, suggesting it may serve as a prognostic indicator and potential therapeutic target for ovarian cancer. Further research showed that CPSF7 promotes the malignant progression of ovarian cancer by enhancing its proliferation, migration, and invasion, while inhibiting CPSF7 expression significantly suppresses these processes. This invention also discovered that UBE2K is one of the key downstream targets of CPSF7 in the ovarian cancer cell-mediated carcinogenesis process; inhibiting UBE2K expression weakens the enhanced proliferation, migration, and invasion effects of CPSF7 overexpression induced by ovarian cancer cell proliferation. This invention provides a novel target for the treatment and prognostic prediction of ovarian cancer, possessing significant clinical application value. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 The figures show the results of CPSF7 expression detection in ovarian cancer and its association with poor prognosis in patients; where A is a representative image of CPSF7 immunohistochemical staining in ovarian cancer tissue and normal fallopian tube tissue; B is a graph of overall survival analysis based on immunohistochemical staining; C is a graph of progression-free survival analysis based on immunohistochemical staining; in B and C, the low expression group (n=83) and the high expression group (n=83) are respectively.

[0028] Figure 2 Figure 1 shows the qPCR results of CPSF7 expression levels in SKOV3, HEY, and OVCAR8 cells.

[0029] Figure 3 The image shows the results of Western blot analysis of CPSF7 expression levels in SKOV3, HEY, and OVCAR8 cells.

[0030] Figure 4 The figure shows the results of the MTT assay used to evaluate the effect of CPSF7 silencing on the proliferation of SKOV3, HEY and OVCAR8 ovarian cancer cell lines.

[0031] Figure 5 The figure shows the results of evaluating the effect of CPSF7 knockdown on the colony-forming ability of SKOV3, HEY and OVCAR8 cells using a colony formation assay.

[0032] Figure 6Figure 1 shows the results of EdU incorporation experiments on the changes in the proportion of cells synthesizing DNA in SKOV3, HEY and OVCAR8 cells after CPSF7 silencing.

[0033] Figure 7 The figure shows the effect of CPSF7 knockdown on the metastatic potential of SKOV3 and HEY cells using Transwell migration and invasion assays.

[0034] Figure 8 This is a schematic diagram of representative nude mouse subcutaneous xenografts derived from the PLKO.1-shCPSF7 group and the PLKO.1 control group (n=5 mice in each group);

[0035] Figure 9 Statistical graphs of tumor weight (A) and tumor volume (B) in the PLKO.1-shCPSF7 group and the PLKO.1 control group (n=5 mice in each group);

[0036] Figure 10 Heatmap of differentially expressed transcripts in OVCAR8 cells after CPSF7 knockdown;

[0037] Figure 11 A gene GO function analysis diagram of 291 differentially expressed polyadenylation sites-related genes using the DAVID database;

[0038] Figure 12 Venn diagram of differentially expressed APA transcripts and differentially expressed genes after CPSF7 silencing;

[0039] Figure 13 A heatmap showing the expression patterns of the selected genes (UBE2K, CSDE1, AIMP1, AMIGO2, SLC25A24, and FUBP3) in the fallopian tube (n=5), normal ovary (n=88), and ovarian cancer tissue (n=426) based on the TCGA-GTEx database.

[0040] Figure 14 The analysis plot (A) shows the differential expression of UBE2K in fallopian tubes (n=5), normal ovaries (n=88), and ovarian cancer tissues (n=426) using TCGA-GTEx data, and the correlation analysis plot (B) shows the correlation between CPSF7 and UBE2K expression in ovarian cancer samples (n=426).

[0041] Figure 15 Sashimi plot showing the effect of CPSF7 knockdown on UBE2K 3'-UTR length, plotted in IGV software using RNA-seq data;

[0042] Figure 16A schematic diagram of UBE2K transcripts (UBE2K-201 and UBE2K-202) based on the Ensembl database;

[0043] Figure 17 A statistical graph showing the expression levels of UBE2K-201 and UBE2K-202 transcripts in several ovarian cancer cell lines (n=47) from the CCLE database;

[0044] Figure 18 The expression profiles of UBE2K-201 and UBE2K-202 in ovarian cancer tissues (n=426) from the TCGA database;

[0045] Figure 19 A graph showing the correlation analysis of CPSF7 expression with UBE2K transcriptional variants UBE2K-201 (A) and UBE2K-202 (B) in TCGA ovarian cancer samples (n=426);

[0046] Figure 20 Figure 1 shows the qPCR results of transcript expression levels of UBE2K-201 (A) and UBE2K-202 (B) in SKOV3, HEY and OVCAR8 cells after CPSF7 knockdown.

[0047] Figure 21 Western blot results of UBE2K protein expression levels in SKOV3, HEY, and OVCAR8 cells after CPSF7 knockdown;

[0048] Figure 22 A schematic diagram illustrating the use of the RBP Binding Sites tool in the POSTAR3 database to identify RNA-binding proteins that interact with UBE2K mRNA.

[0049] Figure 23 The figure shows the degradation rate of UBE2K-201 mRNA in SKOV3, HEY and OVCAR8 cells after CPSF7 knockdown was treated with actinomycin D to inhibit transcription, and the results of qPCR detection.

[0050] Figure 24 The figure shows the results of qPCR verification of the knockdown efficiency of UBE2K in SKOV3, HEY and OVCAR8 cells;

[0051] Figure 25 The figure shows the results of Western blot analysis to verify the knockdown efficiency of UBE2K in SKOV3, HEY and OVCAR8 cells;

[0052] Figure 26The figure shows the results of the MTT assay used to evaluate the effect of UBE2K silencing on the proliferation of SKOV3, HEY and OVCAR8 ovarian cancer cells.

[0053] Figure 27 The figure shows the results of evaluating the effect of UBE2K knockdown on the colony formation ability of SKOV3, HEY and OVCAR8 cells using a colony formation assay.

[0054] Figure 28 This is a graph showing the changes in the proportion of DNA-synthesizing cells in SKOV3, HEY, and OVCAR8 cells after UBE2K knockdown, as demonstrated by an EdU incorporation experiment.

[0055] Figure 29 Figure 1. Transwell migration and invasion assay results showing the effect of UBE2K knockdown on the metastasis potential of SKOV3, HEY and OVCAR8 cells.

[0056] Figure 30 The figure shows the results of the MTT assay used to evaluate the effect of UBE2K-201 overexpression on HEY cell proliferation.

[0057] Figure 31 The figure shows the results of the EdU assay evaluating the effect of UBE2K-201 overexpression on the proliferation ability of HEY cells.

[0058] Figure 32 The figure shows the results of a clonogenic assay to verify the effect of UBE2K-201 overexpression on the cloning ability of HEY cells.

[0059] Figure 33 This is a diagram showing the effect of UBE2K-201 overexpression on the metastasis ability of HEY cells using Transwell migration and invasion assays.

[0060] Figure 34 These are representative images of subcutaneous xenograft tumors in nude mice. The samples were obtained from the PLKO.1-shUBE2K group and the PLKO.1 control group (n=5 mice in each group).

[0061] Figure 35 Comparison of tumor volume (A) and tumor weight (B) between the PLKO.1-shUBE2K group and the PLKO.1 control group (n=5 mice per group);

[0062] Figure 36The following figures illustrate experimental results to investigate whether UBE2K is a key downstream component of CPSF7 in promoting tumor growth in ovarian cancer. Figure A shows the results of the MTT assay evaluating the role of UBE2K in regulating CPSF7-mediated ovarian cancer cell proliferation; Figure B shows the results of the Transwell assay evaluating the role of UBE2K in regulating CPSF7-mediated metastatic phenotypes of ovarian cancer cells; Figure C shows the results of a nude mouse subcutaneous xenograft assay evaluating the role of UBE2K knockdown in CPSF7 overexpression-mediated tumor growth promotion; Figure D shows a statistical graph based on tumor volume measurements in Figure C; and Figure E shows a statistical graph based on tumor weight measurements in Figure C. Detailed Implementation

[0063] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0064] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0065] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0066] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0067] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0068] The experimental materials and their sources involved in the following examples are as follows:

[0069] HEY cells were donated by the laboratory of Zhaojian Liu at Shandong University; SKOV3 cells were purchased from the Cell Bank of the Chinese Academy of Sciences; and OVCAR8 cells were donated by the laboratory of Academician Ma Ding at Huazhong University of Science and Technology. Antibody information is as follows: CPSF7 antibody (PTG, catalog number 55195-1-AP), β-actin antibody (Sigma, catalog number A5441), and UBE2K antibody (PTG, catalog number 11834-3-AP).

[0070] Example 1: Analysis of CPSF7 Expression and Clinical Significance in Ovarian Cancer Tissue

[0071] 1. Experimental Methods

[0072] Tissue samples were collected from 166 patients with serous ovarian cancer at Qilu Hospital of Shandong University. All included patients were diagnosed with serous ovarian cancer at Qilu Hospital of Shandong University and underwent initial surgical treatment. Patients who received neoadjuvant chemotherapy or had other cancers were excluded from this study.

[0073] Tissue microarrays were fabricated, and the expression level of CPCF7 protein was detected by immunohistochemistry (kit purchased from Zhongshan Jinqiao, catalog number PV-9000), with normal fallopian tube tissue samples as controls. The specific steps are as follows:

[0074] (1) Sample pretreatment: The tissue microarray was dewaxed with xylene and hydrated with gradient ethanol;

[0075] (2) Antigen retrieval: Antigen retrieval was performed using EDTA antigen retrieval solution;

[0076] (3) Blocking and blocking: Incubate with peroxidase inhibitor (reagent A) for 10 minutes to inhibit endogenous peroxidase activity, and then block with serum (reagent B) for 30 minutes;

[0077] (4) Antibody incubation: Add CPSF7 antibody (1:50 dilution) and incubate overnight at 4°C; the next day, add biotin-labeled secondary antibody (reagent C) and incubate at room temperature for 30 minutes, then react with streptomycin-biotin-peroxidase (reagent D) at room temperature for 20 minutes.

[0078] (5) Color development and counterstaining: DAB solution for color development, mature hematoxylin for counterstaining for 5 minutes, 1% (v / v) hydrochloric acid ethanol for color separation for 3 seconds, and running water for blueing for 15 minutes;

[0079] (6) Mounting and observation: After dehydration with graded ethanol and clearing with xylene for 5 minutes, the slide is mounted with neutral resin and photographed under a microscope in bright field.

[0080] Based on the immunohistochemical results, patients were divided into a high-CPSF7 expression group and a low-CPSF7 expression group, and prognostic analysis was performed in conjunction with clinical survival data.

[0081] 2. Experimental Results

[0082] Immunohistochemistry (IHC) was used to detect ovarian cancer tissue samples. The results showed that CPSF7 protein expression in cancer tissue was significantly higher than in normal fallopian tube tissue. Figure 1 (A). Notably, high expression of CPSF7 is significantly associated with poor clinical prognosis in ovarian cancer patients ( ). Figure 1 (Chinese BC).

[0083] The above results suggest that CPSF7 overexpression is associated with poor prognosis, indicating that it may serve as a prognostic indicator and potential therapeutic target for ovarian cancer.

[0084] Example 2: Effects of CPSF7 on the biological function of ovarian cancer cells

[0085] 1. Experimental Methods

[0086] 1.1 siRNA-mediated CPSF7 gene silencing

[0087] siRNA sequences targeting CPSF7 were designed (si-CPSF7#1: CAGUGGCCUGCGUAAUAGAdTdT, SEQ ID NO.2; si-CPSF7#2: GAUAUCAUGAAGCGAAACAdTdT, SEQ ID NO.3), and si-NC (UUCUCCGAACGUGUCACGUdTdT, SEQ ID NO.1) was used as a negative control. Gene silencing was performed in SKOV3, HEY, and OVCAR8 cells, and the steps are as follows:

[0088] 1) Cell seeding: After digesting and centrifuging the logarithmic growth phase cells, count them and seed them in 6cm culture dishes, and incubate overnight;

[0089] 2) Preparation of transfection system: Dissolve 10 μL of siRNA (si-NC, si-CPSF7#1 or si-CPSF7#2) in 500 μL of Opti-MEM medium, and dissolve 10 μL of Lipo-2000 in 500 μL of Opti-MEM medium. After standing for 5 minutes, mix the two solutions and incubate at room temperature for 25 minutes.

[0090] 3) Transfection and culture: Discard the original cell culture medium, add the mixed transfection solution and 1 mL of Opti-MEM medium, incubate for 6 hours, then replace with normal culture medium;

[0091] 4) Sample collection: RNA was extracted 24 hours after transfection and protein was extracted 48 hours later. The CPSF7 silencing efficiency was verified by qRT-PCR and Western blot.

[0092] 1.2 Cell Function Experiments

[0093] Proliferation capacity assay: Control group and CPSF7 silenced cells were seeded at 1×10³ cells / well in 96-well plates. 20 μL of MTT solution was added at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h and 144 h, respectively. After incubation for 4 hours, the culture medium was discarded, and 100 μL of LDMSO was added to dissolve and crystallize the cells. The absorbance at 490 nm was measured.

[0094] Clonogenic assay: Detecting the effect of CPSF7 silencing on cell clonogenic ability;

[0095] EdU incorporation assay: Detecting changes in cell proportions during DNA synthesis.

[0096] 1.3 Transwell migration and invasion experiments

[0097] Using a Transwell chamber with a pore size of 0.8 μm, the control group and CPSF7 silenced cells were resuspended in serum-free culture medium and seeded in the upper chamber. After an appropriate culture time, the upper layer of unpermeable cells was wiped off with a cotton swab, and the lower layer of cells were fixed with methanol for 15 minutes and stained with 0.5% crystal violet for 20 minutes. Five fields of view were randomly selected under a microscope to count the permeable cells.

[0098] 1.4 Tumor formation experiment in nude mice

[0099] shRNAs were designed based on the si-CPSF7#1 and si-CPSF7#2 sequences, cloned into the PLKO.1 vector, and transfected into HEY cells to construct a stable CPSF7-silenced cell line (PLKO.1-shCPSF7 group) and a control cell line (PLKO.1-control group). The two cell groups (5 × 10⁻⁶ cells / year) were then divided into two groups. 6 (One per mouse) was injected into the armpit of nude mice. The tumor volume was measured every 2 days. After 2 weeks, the nude mice were sacrificed and the tumor was removed and weighed.

[0100] 2. Experimental Results

[0101] To evaluate the functional role of CPSF7, this invention employs specific siRNA to downregulate CPSF7 in ovarian cancer cell lines SKOV3, HEY, and OVCAR8. Figures 2-3 ).

[0102] Cell proliferation experiments showed that CPSF7 deficiency significantly inhibited the proliferation of ovarian cancer cells. Figure 4 ).

[0103] Clonogenic experiments also showed that knocking down CPSF7 significantly reduced clonogenic ability. Figure 5 ).

[0104] EdU incorporation experiments showed that CPSF7 knockdown reduced the proportion of cells in the DNA replication phase. Figure 6 ).

[0105] Furthermore, Transwell migration and invasion assays showed that the loss of CPSF7 expression inhibited the migration and invasion abilities of ovarian cancer cells. Figure 7 ).

[0106] In vivo, the tumor-promoting effect of CPSF7 was further verified using a subcutaneous xenograft mouse model: compared with the control group tumors, the tumor volume and weight of cells treated with CPSF7-shRNA were significantly reduced. Figures 8-9 ).

[0107] The above results indicate that CPSF7 promotes the malignant progression of ovarian cancer by promoting the proliferation, migration, and invasion of ovarian cancer cells.

[0108] Example 3: CPSF7 affects ovarian cancer cell function by regulating the expression of ubiquitin-binding enzyme E2K (UBE2K).

[0109] 1. Experimental Methods

[0110] CPSF7 was knocked down in SKOV3, HEY, and OVCAR8 cells (using the same method as in Example 2), and the UBE2K protein level was detected by Western blot.

[0111] 2. Experimental Results

[0112] To investigate the molecular mechanism by which CPSF7 drives ovarian cancer progression, this invention treated OVCAR8 cells with CPSF7 using either siRNA or control siRNA, extracted total RNA, and performed RNA-seq analysis. Differential expression analysis used |log2FoldChange|≥1 and Padj<0.05 as the screening threshold, identifying 526 significantly differentially expressed transcripts, of which 199 were upregulated and 327 were downregulated. The results are presented in heatmap format. Figure 10 ).

[0113] Given the known role of CPSF7 in precursor mRNA cleavage and polyadenylation, this invention further investigated the effect of CPSF7 knockdown on variable polyadenylation (APA) events in ovarian cancer cells. Using APAtrap software with a threshold of ≥0.2% percentage difference and Padj <0.05, a total of 291 significantly different APA events were detected, of which 93 were accompanied by 3′-UTR prolongation and 198 by 3′-UTR shortening. GO enrichment analysis of genes exhibiting APA changes suggested their association with transcriptional regulation, inflammatory responses, embryonic development, blood-brain barrier transport, insulin stimulation response, cell proliferation, and the PI3K / AKT signaling pathway. Figure 11 ).

[0114] To screen for important APA targets regulated by CPSF7, this invention intersected 291 APA-affected genes with 526 differentially expressed transcripts, yielding six candidate genes: UBE2K (ENST00000261427), CSDE1 (ENST00000369530), AIMP1 (ENST00000394701), AMIGO2 (ENST00000550413), SLC25A24 (ENST00000648874), and FUBP3 (ENST00000650723). Figure 12 Expression analysis based on TCGA-GTEx data showed that UBE2K expression in ovarian cancer tissues was significantly higher than that in normal ovarian and fallopian tube tissues. Figure 13 , Figure 14 (A). Furthermore, in ovarian cancer samples, CPSF7 and UBE2K expression were positively correlated (correlation coefficient = 0.4556). Figure 14 (B) suggests that UBE2K may be one of the key downstream targets of CPSF7.

[0115] To verify the regulation of UBE2K APA by CPSF7, this invention visualized RNA-seq reads in IGV using Sashimi diagrams. The results showed that CPSF7 knockdown significantly shortened the 3′-UTR of UBE2K in OVCAR8 cells. Figure 15 This is consistent with previous reports that the CFIm complex promotes distal polyadenylation site use or inhibits proximal site selection. Further analysis of UBE2K transcript variants revealed that, despite having a longer 3′-UTR, UBE2K-201 (ENST00000261427) remained the dominant isoform in ovarian cancer cells; CPSF7 knockdown led to decreased UBE2K-201 expression, while UBE2K-202 (ENST00000438068) expression increased. According to Ensembl annotations, UBE2K-201 is the longest transcript, possessing a long 3′-UTR and encoding a functional full-length UBE2K protein (200 amino acids), while UBE2K-202 has a shorter 3′-UTR and does not encode a functional protein (…). Figure 16 ).

[0116] In CCLE ovarian cancer cell lines, the expression level of UBE2K-201 is generally higher than that of UBE2K-202 ( Figure 17 ); TCGA ovarian cancer tissue data also showed that most samples were predominantly UBE2K-201, with only a small number of samples showing high levels of UBE2K-202 expression. Figure 18Correlation analysis showed that CPSF7 had a stronger correlation with UBE2K-201 (r=0.5476), while its correlation with UBE2K-202 was weaker (r=0.2331). Figure 19 The study emphasizes the important role of CPSF7 in regulating the expression of UBE2K APA and its isoforms.

[0117] To experimentally verify the above results, this invention used isoform-specific primers for qPCR. The results showed that in the SKOV3, HEY, and OVCAR8 cell lines, knocking down CPSF7 significantly reduced UBE2K-201 levels, while slightly increasing UBE2K-202 levels. Figure 20 Consistent with altered transcriptional levels, Western blot results showed decreased UBE2K protein expression in all three cell lines after CPSF7 knockdown. Figure 21 ).

[0118] To elucidate the molecular mechanism by which CPSF7 regulates UBE2K, this invention searched the POSTAR3 database to identify RNA-binding proteins associated with UBE2K mRNA. The results showed that CPSF7 is a potential binding factor; further CLIP-seq data analysis revealed 13 CPSF7 binding sites in the UBE2K-201-specific 3′-UTR region. Figure 22 (and Table 1). This suggests that CPSF7 is more likely to bind to the distal polyadenylation site (PAS) in UBE2K-201 to promote the utilization of the distal poly(A) site, thereby maintaining the expression of the long 3′-UTR isoform.

[0119] Table 1. Binding sites of CPSF7 in the UBE2K-201 transcript-specific 3′-UTR fragment.

[0120]

[0121] Given the crucial role of the 3′-UTR in post-transcriptional regulation (such as mRNA stability, translation, and subcellular localization), this invention further investigated whether CPSF7 affects the stability of UBE2K mRNA. After CPSF7 knockdown, transcription was blocked in cells using actinomycin D, and the mRNA degradation rate was measured by qPCR. The results showed that reducing CPSF7 significantly reduced the mRNA stability of UBE2K-201. Figure 23 This suggests that CPSF7 plays a role in maintaining the stability of the UBE2K-201 transcript.

[0122] In summary, the results of this section indicate that CPSF7 affects the expression level of UBE2K by regulating the expression of its APA and isoforms. UBE2K is one of the key downstream targets of CPSF7 in the process of ovarian cancer cells mediating carcinogenesis.

[0123] Example 4: Effects of UBE2K on the biological function of ovarian cancer cells and rescue experiment

[0124] 1. Experimental Methods

[0125] 1.1 UBE2K gene silencing and its effects on cell function

[0126] siRNAs targeting UBE2K were designed (si-UBE2K#1: ACUCUCCGCACGGUAUUAUdTdT, SEQ ID NO.4; si-UBE2K#2: CCAGAAACAUACCCAUUUAdTdT, SEQ ID NO.5), with si-NC (SEQ ID NO.1) as a control. UBE2K was silenced in SKOV3, HEY, and OVCAR8 cells, and the silencing efficiency was verified by qPCR and Western blot. Cell proliferation (MTT), colony formation, EdU incorporation, and Transwell assays were then performed.

[0127] 1.2 Rescue Experiment Verifies the Downstream Effects of UBE2K

[0128] In HEY cells overexpressing CPSF7, UBE2K was simultaneously silenced, and phenotypic reversal was detected by cell proliferation and colony formation assays.

[0129] 1.3 Effects of UBE2K on CPSF7-regulated tumor growth in vivo

[0130] A nude mouse subcutaneous xenograft model was used. HEY cells overexpressing CPSF7 and CPSF7-overexpressing cells with UBE2K silenced were inoculated into the axilla of nude mice, and tumor growth was monitored.

[0131] 2. Experimental Results

[0132] To investigate the functional role of UBE2K, this invention employs specific siRNA knockdown of UBE2K in ovarian cancer cell lines SKOV3, HEY, and OVCAR8. Figures 24-25 Cell viability assays showed that loss of UBE2K significantly inhibited cell proliferation. Figure 26 Cloning experiments showed a significant decrease in its cloning ability. Figure 27 EdU incorporation experiments further confirmed that UBE2K knockdown reduced the proportion of cells in the active DNA replication phase. Figure 28Furthermore, Transwell migration and invasion assays showed that UBE2K deficiency significantly inhibited cell migration and invasion abilities. Figure 29 Conversely, to evaluate the role of the UBE2K-201 isoform, this invention constructed a UBE2K-201 overexpression plasmid (pcDNA3.1-UBE2K-201) and performed functional tests on ovarian cancer cells. Overexpression of UBE2K-201 significantly promoted cell proliferation, colony formation, migration, and invasion, supporting its oncogenic function in ovarian cancer progression. Figures 30-33 In an in vivo subcutaneous xenograft model, the tumor volume and weight in the UBE2K-deficient group were significantly lower than those in the control group, further confirming the key contribution of UBE2K to tumor growth. Figures 34-35 The above findings demonstrate that UBE2K promotes the malignant behavior of ovarian cancer cells.

[0133] To determine whether UBE2K mediates the pro-tumorigenic effect of CPSF7, this invention performed a recovery / inhibition experiment by transfecting UBE2K siRNA into ovarian cancer cells overexpressing CPSF7. The results showed that knockdown of UBE2K significantly attenuated the enhanced proliferation and migration / invasion effects induced by CPSF7 overexpression. Figure 36 In vivo subcutaneous transplantation experiments also confirmed that UBE2K knockdown significantly reduced tumor volume and weight increased due to CPSF7 overexpression. Figure 36 (CE). These results indicate that UBE2K is a key downstream effector molecule mediated by CPSF7 in the malignant progression of ovarian cancer.

[0134] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a reagent that reduces CPSF7 expression levels in the preparation of drugs for treating ovarian cancer, characterized in that, The reagent is siRNA; The nucleotide sequence of the siRNA is shown in SEQ ID NO.2 or SEQ ID NO.

3.

2. Application of reagents for detecting CPSF7 expression levels in the preparation of products for predicting the prognosis of ovarian cancer.

3. The application according to claim 2, characterized in that, The product in question is a reagent kit.

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