Inhibitors targeting the ccdc137 gene and their use in the preparation of medicaments for the diagnosis and treatment of acute myeloid leukemia

Abstract

The present application relates to the biomedical technology field, and is an inhibitor targeting CCDC137 gene and application thereof in preparation of a drug for diagnosing and treating acute myeloid leukemia, wherein the inhibitor targeting CCDC137 gene is shRNA. The present application discloses, for the first time, that CCDC137 gene is a new therapeutic target and a prognostic biomarker for acute myeloid leukemia. Experiments prove that inhibition of CCDC137 gene can significantly reduce AML cell proliferation, prolong the survival of an animal model, and reduce tumor load. Based on this finding, the present application provides a therapeutic strategy for treating AML by inhibiting the expression of CCDC137 gene, provides a new therapeutic strategy for AML patients, and reduces the recurrence cost through targeted therapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to an inhibitor targeting the CCDC137 gene and its application in the preparation of a drug for treating acute myeloid leukemia. It also includes a pharmaceutical composition and the application of primers for amplifying the CCDC137 gene in the preparation of diagnostic reagents or kits for acute myeloid leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is not only the second most common type of leukemia in adults and children, but also the 15th most common cancer and the 11th leading cause of cancer-related death worldwide. Each year, it causes 437,033 new cases and 309,006 deaths. The disease is characterized by the uncontrolled proliferation and differentiation arrest of clonal myeloid progenitor cells, ultimately leading to bone marrow failure. Despite advances in chemotherapy and targeted therapy, the overall prognosis for AML patients remains poor, with a five-year survival rate of only 27%. This poor prognosis is closely related to the high heterogeneity of the disease, driven by complex cytogenetic abnormalities and relapsing gene mutations. The median age of diagnosis for AML is 67 years, and the incidence increases significantly with age. Elderly patients often cannot tolerate intensive chemotherapy, and the relapse rate can be as high as 80%. These factors pose significant challenges to the clinical management of AML. While targeted therapies targeting molecular targets such as FLT3, IDH1 / 2, and BCL2 have changed the treatment landscape, acquired resistance and disease relapse remain significant obstacles to improving long-term survival. Therefore, deepening our understanding of the pathogenesis of AML and identifying new prognostic biomarkers and therapeutic targets are of urgent clinical significance for developing more effective treatment strategies.

[0003] The pathogenesis of AML involves multi-level regulatory dysregulation. Driver mutations in epigenetic regulators are present in over 70% of AML cases, indicating that epigenetic dysregulation is one of the core mechanisms of the disease. Simultaneously, aberrant activation of key signaling pathways, such as PI3K / AKT and RAS / RAF / MEK / ERK (MAPK), plays a central role in regulating leukemia cell proliferation, survival, metabolic reprogramming, and chemotherapy resistance. In the search for key regulators of these processes, the coiled-coil domain (CCDC) family has received considerable attention. This family comprises approximately 180 members, whose encoded proteins are widely involved in key biological processes such as cell signal transduction, transcriptional regulation, and cell division. Notably, several members of this family have been shown to play important roles in tumor growth, metastasis, stem cell characterization, and treatment resistance by regulating core signaling pathways such as PI3K / AKT, c-Myc, and ERK.

[0004] As a member of the CCDC family, CCDC137 has received considerable attention in the field of solid tumor research in recent years. Pan-cancer analysis shows that it is significantly overexpressed in various solid tumors, including hepatocellular carcinoma, lung adenocarcinoma, and colorectal cancer, and is associated with poor prognosis. Functional studies have shown that CCDC137 is associated with tumor stem cell characteristics and may activate the AKT signaling pathway. For example, in colorectal cancer, CCDC137 has been identified as a super-enhancer-associated oncogene, promoting liver metastasis by enhancing cancer cell proliferation and stemness. Many members of the CCDC family (such as CCDC6 and CCDC88A) have been shown to function as molecular "scaffolds" due to the protein-protein interaction properties inherent in their coil-coil domains. These proteins can recruit a variety of signaling molecules, thereby effectively integrating and regulating downstream networks. Similarly, RNA-binding proteins (RBPs), such as AKAPs and SPOPs, can also function as "platforms," ​​coupling post-transcriptional regulation with post-translational modifications to achieve precise control over gene expression. However, the expression pattern, biological function, and potential molecular mechanisms of CCDC137 in AML remain unknown. Against this backdrop, this study is the first to combine bioinformatics analysis and experimental verification to explore the function and mechanism of CCDC137 in AML, providing a new perspective for understanding the pathogenesis of AML.

[0005] Regarding patent application literature, there are currently reports of the application of the CCDC family in cancer treatment and other fields. For example, patent application literature with publication number CN117018199A discloses the application of CCDC154 gene inhibitors in the preparation of drugs for treating colorectal cancer or improving the prognosis of colorectal cancer. It was found through puncture or surgical resection specimens of human colorectal cancer patients that the overall survival of patients with low CCDC154 expression was significantly longer than that of patients with high CCDC154 expression. This indicates that CCDC154 can be used as a biomarker to predict the prognosis of colorectal cancer patients. Kits containing CCDC154 gene inhibitors can be prepared for the detection of colorectal cancer or the monitoring of colorectal cancer prognosis, providing new methods and tools for individualized assessment of colorectal cancer prognosis and the formulation of treatment strategies.

[0006] There are currently no reports on the application of inhibitors targeting the CCDC137 gene in the diagnosis and treatment of acute myeloid leukemia. Summary of the Invention

[0007] This invention provides an inhibitor targeting the CCDC137 gene and its application in the preparation of drugs for the diagnosis and treatment of acute myeloid leukemia. It is the first time that the application of the CCDC137 gene inhibitor in the diagnosis and treatment of acute myeloid leukemia has been disclosed, providing a new target and direction for the treatment of acute myeloid leukemia.

[0008] One of the technical solutions of the present invention is achieved through the following measures: an inhibitor targeting the CCDC137 gene, comprising NC-shRNA, shRNA#1 and shRNA#2.

[0009] The following are further optimizations and / or improvements to one of the above-mentioned technical solutions: Furthermore, the nucleotide sequence of the above-mentioned NC-shRNA is shown in SEQ ID NO.1.

[0010] Furthermore, the nucleotide sequence of the above-mentioned shRNA#1 is shown in SEQ ID NO.2.

[0011] Furthermore, the nucleotide sequence of the above-mentioned shRNA#2 is shown in SEQ ID NO.3.

[0012] The second technical solution of the present invention is achieved by the following measures: a pharmaceutical composition comprising an inhibitor targeting the CCDC137 gene as described in the first technical solution.

[0013] The third technical solution of the present invention is achieved through the following measures: the application of a primer for amplifying the CCDC137 gene in the preparation of diagnostic reagents or kits for acute myeloid leukemia, wherein the primer includes a forward primer and a reverse primer, the nucleotide sequence of the forward primer is shown in SEQ ID No. 4 of the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 5 of the sequence listing.

[0014] The fourth technical solution of the present invention is achieved through the following measures: the application of the inhibitor targeting the CCDC137 gene described in one of the technical solutions in the preparation of a drug for treating acute myeloid leukemia, wherein the inhibitor targeting the CCDC137 gene includes NC-shRNA, shRNA#1 and shRNA#2.

[0015] This invention discloses for the first time the use of the CCDC137 gene as a novel therapeutic target and prognostic biomarker for acute myeloid leukemia (AML). Experiments have demonstrated that inhibiting the CCDC137 gene significantly reduces AML cell proliferation, prolongs survival in animal models, and reduces tumor burden. Based on this discovery, this invention provides a therapeutic strategy for AML by inhibiting CCDC137 gene expression, offering a new treatment approach for AML patients and reducing the cost of relapse through targeted therapy. Attached Figure Description

[0016] Figure 1 It is one of the technical routes for in vitro experiments to investigate the effects of CCDC137 on AML proliferation and apoptosis; Figure 2 This is the second technical route for in vitro experiments to investigate the effects of CCDC137 on AML proliferation and apoptosis; Figure 3 This is the technical route for in vivo experiments to investigate the effect of CCDC137 on AML proliferation; Figure 4 , Figure 5 , Figure 6 The CCDC137 gene is highly expressed in AML; (A) Expression level of CCDC137 in peripheral blood mononuclear cells of 23 patients with relapsed / refractory acute myeloid leukemia (RR); (B) Expression level of CCDC137 in peripheral blood mononuclear cells of 10 newly diagnosed acute myeloid leukemia (ND) patients; (C) Expression level of CCDC137 in peripheral blood mononuclear cells of 11 patients with complete remission acute myeloid leukemia (CR); (D) CCDC137 expression levels in healthy hematopoietic stem cell donors and AML patients; (E) CCDC137 expression levels in AML samples and normal samples; (F) Protein levels of CCDC137 in five AML cell lines; (G) mRNA levels of CCDC137 in five AML cell lines; (H) Protein expression levels of CCDC137 in five AML cell lines.

[0017] Figure 7 , Figure 8 , Figure 9 High expression of CCDC137 is a predictor of a poor prognosis for AML; (A) Sangerbox database analysis; Based on the Sangerbox database (sample sources: Cancer Genome Atlas (TCGA), TARGET database, GTEx database; sample size: N = 19,131; number of genes: 60,499), a Cox proportional hazards regression model was constructed using the survival analysis R package (version 3.2-7), and the statistical significance was evaluated by the log-rank test; (B) Prognostic value of CCDC137 in the TCGA-LAML cohort; using the maxstat R package (v0.7-25) and constrained by the 25%–75% quartile range, the optimal cutoff value of CCDC137 expression (ENSG00000185298) was determined to be 2.971. Kaplan-Meier survival curves were plotted using the survfit function. The difference between high and low expression groups was assessed by the log-rank test (p = 1.1 × 10⁻⁻⁴). 5 ); (C) Using the GEPIA2 platform, the prognostic analysis of CCDC137 expression in the TCGA-LAML dataset (n = 173) was evaluated with median expression level as the cutoff value (p = 0.0068). (D) Univariate and multivariate Cox regression analysis (Using the CAOMIP and UALCAN platforms to perform univariate and multivariate Cox regression analysis on AML patients in the TCGA-LAML dataset). (E) Relationship between CCDC137 expression level and AML pathological classification in the TCGA-LAML cohort; *p<0.05, *p<0.01, *p<0.001.

[0018] Figure 10 , Figure 11 , Figure 12 CCDC137 promotes the proliferation of AML cell lines in vitro; (A–C) CCDC137 was knocked down in three AML cell lines: Kasumi-1, THP-1, and MOLM-13, and the knockdown was validated at the protein (A) and mRNA (B, C) levels. (D–L) The proliferation capacity of AML cells after CCDC137 knockdown was assessed. Cell growth was assessed by measuring absorbance at 450 nm (D–F); proliferation capacity was quantified using EDU labeling (G–L); *p<0.05, *p<0.01, *p<0.001.

[0019] Figure 13 , Figure 14 Knockdown of CCDC137 induces G2 / M phase arrest in AML cells; (A, B) Flow cytometry analysis showed that knockdown of CCDC137 significantly inhibited the G2 / M phase of acute myeloid leukemia cells; (C) Western blot analysis showed an imbalance in the expression of key cell cycle-related proteins. *p<0.05, *p<0.01, *p<0.001.

[0020] Figure 15 , Figure 16 Knocking down CCDC137 promotes apoptosis in AML cells; (A, B) Flow cytometry analysis showed that knockdown of CCDC137 significantly promoted apoptosis in acute myeloid leukemia cells; (C) Western blot analysis showed an imbalance in the expression of key apoptosis-related proteins. *p<0.05, *p<0.01, *p<0.001.

[0021] Figure 17 , Figure 18 In a xenograft mouse model, knocking down CCDC137 inhibited the proliferation of human refractory acute myeloid leukemia cells; (A) mRNA and protein expression levels of CCDC137 in lentivirus-transduced MOLM-13 cells in the control and experimental groups; (B) Representative bioluminescence images of MOLM-13 leukemia cells in vivo from mice. Mice were injected via tail vein with 200 μL of PBS (containing 8 × 10⁻⁶ ppm). 4 (Number of transduced cells). Images were acquired every six days (n = 5 per group); (C) Quantitative analysis of bioluminescence intensity; data were analyzed using two-way ANOVA (factors: treatment group and time), followed by Sidak multiple comparison test; (D) Changes in mouse body weight; data were analyzed using two-way ANOVA (factors: treatment group and time), followed by Sidak multiple comparison test; (E) Survival curves. Statistical significance was determined using the log-rank (Mantel-Cox) test; (F) Gross morphology and length of the spleen in the control and experimental groups; (G) Representative images of hematoxylin-eosin (H&E) staining and Ki-67 immunohistochemical (IHC) staining in spleen sections from a xenograft model; scale bar: 50 μm (H&E and Ki-67 images) and 100 μm (Ki-67 image); in H&E staining, blue / purple represents cell nuclei, and pink represents cytoplasm. In Ki-67 IHC staining, brown cell nuclei represent Ki-67 positive cells; *p<0.05, *p<0.01, *p<0.001. Detailed Implementation

[0022] The present invention is not limited to the following embodiments, and the specific implementation can be determined according to the technical solution of the present invention and the actual situation.

[0023] In this invention, CCDC137 refers to the CCDC137 gene. Kasumi-1: AML1-ETO* AML-M2 cells; THP-1: AML-M5 cells that can be induced into macrophages by PMA; MOLM-13: FLT3-ITD* AML-M5 cells.

[0024] The present invention will be further described below with reference to embodiments: Example 1: An inhibitor targeting the CCDC137 gene, comprising NC-shRNA, shRNA#1, and shRNA#2.

[0025] CCDC137 gene related information: HGNC:33451; NCBI Gene:339230; Ensembl:ENSG00000185298 OMIM®: 614271UniProtKB / Swiss-Prot: Q6PK04.

[0026] Example 2: As an optimization of the above example, the nucleotide sequence of the NC-shRNA is: 5´-ACTACCGTTGTTATAGGTGT-3´, as shown in SEQ ID NO.1.

[0027] Example 3: As an optimization of the above example, the nucleotide sequence of shRNA#1 is: 5´-TCCGAGACACGGTGAAGTTTG-3´, as shown in SEQ ID NO.2.

[0028] Example 4: As an optimization of the above example, the nucleotide sequence of shRNA#2 is: 5´-TTTCCGGCTCCGGGAGATTAT-3´, as shown in SEQ ID NO.3.

[0029] Example 5: A pharmaceutical composition comprising the inhibitor targeting the CCDC137 gene described in the above examples.

[0030] Example 6: Application of the primers for amplifying the CCDC137 gene in the preparation of diagnostic reagents or kits for acute myeloid leukemia, wherein the primers include forward primers and reverse primers. Forward primer: 5´-TGGAGATGGCGGGAGCTG-3´, as shown in SEQ ID No. 4 of the sequence listing; Reverse primer: 5´-TCACTTTCTTCTTCTCTTTGCTGC-3´, as shown in SEQ ID No. 5 of the sequence listing.

[0031] Example 7: Application of the inhibitor targeting the CCDC137 gene in the preparation of a drug for treating acute myeloid leukemia, wherein the inhibitor targeting the CCDC137 gene includes NC-shRNA, shRNA#1 and shRNA#2.

[0032] The following are relevant experimental studies on the diagnosis and treatment of acute myeloid leukemia using inhibitors targeting the CCDC137 gene: The relevant technical approach in this experimental study is as follows: Figures 1 to 3 As shown.

[0033] Materials and Methods 1. Sources of healthy donors and patient samples The samples were obtained from the First Affiliated Hospital of University of Science and Technology of China. After obtaining the informed consent of healthy hematopoietic stem cell donors (HSCT) and patients with acute myeloid leukemia (AML), 3 mL to 5 mL of bone marrow or peripheral blood samples were collected. Mononuclear cells were isolated by density gradient centrifugation, and proteins were extracted for subsequent experiments. This study was approved by the Ethics Committee of the First Affiliated Hospital of University of Science and Technology of China (Approval number: 2025KY伦审第160号).

[0034] 2 Bioinformatics analysis The gene expression data and clinical information of AML were downloaded from the GSE114868 dataset of the Gene Expression Omnibus (GEO) database (https: / / www.ncbi.nlm.nih.gov / geo / ). AML patients and normal bone marrow samples lacking gene expression data or clinical information were excluded. The mRNA expression levels of CCDC137 in AML and normal bone marrow samples were analyzed using R software, and the samples were divided into high- and low-expression groups based on the median expression value of CCDC137 mRNA in AML bone marrow samples. The expression of the CCDC137 gene in different cancer types was analyzed through the TNMplot database, and its pan-cancer expression and association with the overall survival (OS) of AML patients were evaluated using the Sangerbox database. The GEPIA database was used to analyze the effect of CCDC137 expression on the OS of AML patients (with the median expression level as the cut-off value). The CAOMIP database was used for Cox proportional hazards regression model and analysis of prognostic-related risk factors. The significance criterion for whether a variable was an independent prognostic factor was p < 0.05 after multiple comparison correction, and variables included in the multivariable model were screened based on p < 0.05. The UALCAN database was used to analyze the expression of CCDC137 in different AML subtypes. Database links are shown in Table 1.

[0035] 3 Cell culture and transfection Five AML cell lines (NB4, MV-4-11, THP-1, Kasumi-1, and MOLM-13) were purchased from the public research platform of the First Affiliated Hospital of University of Science and Technology of China. All cell lines were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) under standard conditions of 37°C and 5% CO2. Lentiviruses for interfering with or overexpressing CCDC137 were purchased from Beijing Tsingke Biotechnology Co., Ltd., and the cells were transfected according to the manufacturer's instructions. After 72 hours, the cells were screened with 5 μM puromycin, and the culture medium was changed every 48 hours to obtain cells with stable low or high expression of CCDC137.

[0036] 4 Real-time quantitative PCR Total RNA was extracted from cells and tissues using TRIzol reagent, and cDNA was synthesized by reverse transcription using the PrimeScript™ RT kit. Amplification was performed using real-time quantitative PCR with TB Green Premix Ex Taq II, and the relative mRNA expression level was calculated using the 2−ΔΔCt method with β-actin as an internal control.

[0037] 5 CCK-8 test Cells were spaced at 1×10⁶ cells per well. 4 The culture medium was seeded at a density of 100 μL per well in 96-well plates and incubated for 0, 24, 48, and 72 hours. After each stage, 10 μL of CCK-8 reagent was added to each well and incubated in the dark for 2 hours. The absorbance at 450 nm was then measured using a microplate reader.

[0038] 6 EdU detection Cells were loaded at 1.0 × 10 5 Nuclei were seeded at a density of 1 / mL in poly-L-lysine-coated confocal culture dishes. EdU was added to the culture medium to a final concentration of 10 μmol / L, and the mixture was incubated for 3 hours. The culture medium was discarded, and the nuclei were fixed with immunocytochemical fixative at room temperature for 15 minutes, followed by washing three times with immunocytochemical blocking solution. After permeation at room temperature for 10-15 minutes, the nuclei were washed again. A click reaction mixture was added, and the mixture was reacted in the dark for 30 minutes. After washing, the nuclei were stained with a 1:1000 diluted Hoechst 33342 solution for 10 minutes. The nuclei were then imaged and analyzed under a fluorescence microscope at 200x magnification using both the Azide 594 and Hoechst 33342 channels.

[0039] 7. Flow cytometry analysis Cell suspensions were washed with PBS, centrifuged to collect cells, and fixed with 75% ethanol at 4°C for 24 hours. Cells were stained with PI in the dark for 30 minutes, and cell cycle distribution was quantitatively analyzed by flow cytometry.

[0040] 8. Flow cytometry detection of apoptosis To detect apoptosis, 1×10⁶ cells were collected. 5 Cells were centrifuged at 300×g for 3 minutes to collect the cell pellet, resuspended in pre-chilled PBS, and washed twice. Using the APExBIO apoptosis detection kit, 1× binding buffer was prepared by mixing 10× binding buffer with ddH2O at a 1:10 ratio, and the cells were resuspended in 500 μL of 1× binding buffer. Then, 5 μL of Annexin V-APC and 5 μL of propidium iodide (PI) were added, and the cells were incubated at room temperature in the dark for 15 minutes. Finally, the cells were quickly loaded onto the flow cytometer for quantitative analysis of the apoptotic cell population.

[0041] 9. Protein Blotting Total protein was extracted from cells using RIPA lysis buffer containing a phosphatase inhibitor, and the concentration was determined by the BCA method. After separation by 10% SDS-PAGE, the proteins were transferred to a PVDF membrane, blocked with 5% skim milk for 2 hours, incubated with primary antibody at 4°C for 12 hours, and then incubated with secondary antibody at room temperature for 2 hours. After ECL staining, the relative protein expression levels were calculated by analyzing the grayscale values ​​of the bands using ImageLab software.

[0042] 10. Animal experiments Six-week-old NOD / NSG mice were purchased from Shanghai Nanmo Biotechnology Co., Ltd. The experimental protocol was approved by the Experimental Animal Ethics Committee of Shihezi University School of Medicine (Approval No.: A2024-352). Mice were randomly grouped and injected with 8 × 10⁸ mg / L of 8 × 10⁸ mg / L via the tail vein. 5 Cells were photographed in vivo every 6 days to obtain in vivo fluorescence images.

[0043] 11 Immunohistochemical staining After dewaxing and hydration of paraffin sections, antigen was repaired using EDTA buffer, and endogenous peroxidase activity was blocked with 3% H2O2. The sections were blocked for 30 minutes, incubated with primary antibody overnight at 4°C, and then incubated with secondary antibody for 30 minutes at 37°C. After hematoxylin counterstaining, dehydration, and mounting, the sections were observed.

[0044] 12 Hematoxylin-eosin staining After dewaxing and hydration of paraffin sections, the nuclei were stained with hematoxylin for 10 minutes, differentiated with hydrochloric acid and ethanol, and the cytoplasm was stained with eosin. After dehydration, clearing, and mounting, the sections were examined under a microscope.

[0045] 13 Statistical Analysis All data were derived from at least three independent biological replicates. Data were analyzed using GraphPad Prism 8.0. Normality was tested using the Shapiro–Wilk test, and homogeneity of variance was tested using the Levene test. For parametric tests, t-tests were used for comparisons between two groups, one-way ANOVA for comparisons among multiple groups, and two-way ANOVA for two-way designs. Non-parametric data were analyzed using the Mann–Whitney U test (for two groups) or the Kruskal–Wallis test (for multiple groups). Gene expression correlations were analyzed using Spearman rank correlation analysis. p<0.05, *p<0.01, and **p<0.001 were considered statistically significant.

[0046] II. Results and Analysis 1. Upregulation of CCDC137 expression in AML patients To investigate the expression of CCDC137 in acute myeloid leukemia (AML) and its clinical relevance, from January 2025 to September 2025, a total of 23 patients with relapsed / refractory AML (RR), 10 newly diagnosed AML (ND), and 11 patients with complete remission AML (CR) were included in this study (see [link to study]). Figures 4 to 6Clinical results (A, B, C) have shown that CCDC137 is highly expressed in AML compared to healthy hematopoietic stem cell donors (see A, B, C). Figure 6 Analysis of the GSE114868 dataset (GPL17586 platform) further confirms that CCDC137 is upregulated in AML (see D); Figure 6 The GSE114868 database showed that the expression level of CCDC137 in AML samples (n = 194) was significantly higher than that in normal samples (n = 20) (|log2 FC|>1, p<0.05). To investigate the role of CCDC137 in AML, we compared the expression levels of CCDC137 in peripheral blood mononuclear cells (PBMCs) from healthy donors and newly diagnosed AML patients. The protein level of CCDC137 in the five AML cell lines was significantly higher than that in healthy donor PBMCs (n = 3) (see [link to relevant database]). Figure 6 The results of qRT-PCR and Western blot analysis showed that the expression levels of CCDC137 mRNA and protein in newly diagnosed AML patients were significantly higher than those in healthy donors (see F). Figure 6 To screen suitable cell models for subsequent functional studies, we compared the mRNA and protein expression levels of CCDC137 in healthy donor PBMCs and five AML cell lines (NB4, MV-4-11, THP-1, Kasumi-1, and MOLM-13). The mRNA level of CCDC137 in the five AML cell lines was significantly higher than that in healthy donor PBMCs (n = 3); *p<0.05, *p<0.01, *p<0.001. Kasumi-1, THP-1, and MOLM-13 cell lines showed high expression (see [link to study]). Figure 6 H).

[0047] 2. Upregulation of CCDC137 is associated with poor prognosis in AML. To assess the prognostic value of CCDC137 in AML, Sangerbox database analysis showed that CCDC137 was associated with poor prognosis in various cancers, suggesting its potential as a prognostic biomarker. Further analysis using the Sangerbox and GEPIA2 databases revealed that AML patients with high CCDC137 expression had significantly lower survival rates. To further explore the impact of CCDC137 on AML prognosis, we performed univariate and multivariate Cox regression analyses using the CAOMIP database to assess the association between CCDC137 expression and clinical variables. The results showed that CCDC137 expression was an independent predictor of overall survival. Furthermore, FAB subtyping analysis of AML using the UALCAN database revealed that the M3 subtype, with its favorable prognosis, had a cure rate of 80%, significantly better than other subtypes (see [link to UALCAN database]). Figures 7 to 9 (A, B, C, D, E).

[0048] 3. CCDC137 promotes AML cell proliferation Bioinformatics analysis showed that CCDC137 was significantly upregulated in AML, and overexpression was closely associated with poor prognosis. Western blotting and qRT-PCR further confirmed high expression of CCDC137 in three AML cell lines: Kasumi-1, THP-1, and MOLM-13 (see...). Figure 10 Based on the above findings, we propose the scientific hypothesis that CCDC137 may promote cell proliferation by accelerating cell cycle progression. We used lentiviral shRNA to knock down the expression of CCDC137 in Kasumi-1, THP-1, and MOLM-13 cell lines, and verified the knockdown efficiency by Western blotting and qRT-PCR. Subsequent experiments all used the validated knockdown model. The CCK-8 assay showed that inhibiting CCDC137 expression significantly reduced the proliferation capacity of AML cells. The EdU assay further confirmed that, compared with the control group, the proliferation activity of Kasumi-1, THP-1, and MOLM-13 cells in the CCDC137 knockdown group was significantly weakened (see A, B, and C). Figure 10 , Figure 11 , Figure 12 (of D, E, F, G, H, I, J, K, L).

[0049] 4. Knockdown of CCDC137 induces G2 / M phase arrest in AML cells To elucidate the role of CCDC137 in cell cycle regulation, we analyzed flow cytometry data stained with PI. The results showed that the proportion of cells in the G2 / M phase was significantly increased in CCDC137 knockdown Kasumi-1, THP-1, and MOLM-13 cells. Western blot analysis of the expression of key cell cycle regulatory proteins revealed that CDK1 protein expression was significantly reduced in all three cell lines, inducing G2 / M phase arrest in AML cells (see...). Figure 13 , Figure 14 (A, B, C).

[0050] 5. Knockdown of CCDC137 promotes apoptosis in AML cells. To investigate the regulatory mechanisms of apoptosis, pro-apoptotic proteins, which release apoptosis-related factors by increasing mitochondrial membrane permeability and thus initiating the apoptosis program, were selected to protect cells. Flow cytometry analysis, based on Annexin V and PI staining, revealed that compared to the Vector group, inhibition of CCDC137 increased the apoptosis rate in Kasumi-1, THP-1, and MOLM-13 cell lines, and significantly increased the expression of related apoptotic proteins. This indicates that inhibition of CCDC137 promotes apoptosis in AML cell lines, suggesting that CCDC137 may be a poor prognostic factor for AML (see [link to relevant documentation]). Figure 15 , Figure 16 (A and B).

[0051] 6. Knockdown of CCDC137 inhibits the proliferation of human refractory AML cells in a xenograft model. To validate the tumor-promoting function of CCDC137 in vivo, we used the MOLM-13-luciferase cell line and lentiviral particles expressing CCDC137-specific shRNA (shRNA-#2). Xenograft models were established by injecting cells into NOD / NSG mice via tail vein. Mice were randomly divided into two groups (n=5 per group), receiving either MOLM-13-luciferase cells carrying the control vector or the CCDC137 knockdown vector, respectively. In vivo imaging on days 6, 12, 18, and 24 post-injection showed that the tumor burden in the CCDC137 knockdown group was significantly lower than that in the control group. There was no significant change in body weight between the two groups, but the median survival time in the knockdown group was significantly prolonged. Dissection on day 39 post-surgery revealed a significant reduction in spleen length in the knockdown group. Histological analysis showed that the spleen structure in the control group was extensively destroyed due to infiltration of numerous abnormally proliferating cells, while infiltration was significantly reduced in the knockdown group; immunohistochemical results showed a significantly decreased Ki-67 proliferation index (see [link to relevant documentation]). Figure 17 , Figure 18 (A, B, C, D, E, F, G).

[0052] In summary, CCDC137 occupies a key oncogenic position in acute myeloid leukemia (AML), is significantly overexpressed in AML, and is closely associated with poor patient prognosis. Functional experiments show that CCDC137 drives AML progression by promoting cell proliferation and accelerating cell cycle progression. Furthermore, experiments have confirmed that inhibiting CCDC137 significantly reduces AML cell proliferation (CCK-8 absorbance decreases by 50%, EdU-positive cells decrease by 60%), prolongs survival in animal models (median survival increases by 40%), and reduces tumor burden (bioluminescence intensity decreases by 70%).

[0053] The above technical features constitute various embodiments of the present invention, which have strong adaptability and implementation effect. Unnecessary technical features can be added or removed according to actual needs to meet the needs of different situations.

Claims

1. An inhibitor targeting the CCDC137 gene, characterized in that, This includes NC-shRNA and / or shRNA#1 and / or shRNA#2.

2. The inhibitor targeting the CCDC137 gene according to claim 1, characterized in that, The nucleotide sequence of NC-shRNA is shown in SEQ ID NO.

1.

3. The inhibitor targeting the CCDC137 gene according to claim 1 or 2, characterized in that, The nucleotide sequence of shRNA#1 is shown in SEQ ID NO.

2.

4. The inhibitor targeting the CCDC137 gene according to claim 1, 2, or 3, characterized in that, The nucleotide sequence of shRNA#2 is shown in SEQ ID NO.

3.

5. A pharmaceutical composition, characterized in that, Including the inhibitors targeting the CCDC137 gene as described in any one of claims 1 to 4.

6. The application of a primer for amplifying the CCDC137 gene in the preparation of diagnostic reagents or kits for acute myeloid leukemia, characterized in that, The primers include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID No. 4 of the sequence listing, and the nucleotide sequence of the reverse primer is shown in SEQ ID No. 5 of the sequence listing.

7. The use of an inhibitor targeting the CCDC137 gene as described in any one of claims 1 to 4 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The inhibitors targeting the CCDC137 gene include NC-shRNA and / or shRNA#1 and / or shRNA#2.

8. The use of the inhibitor targeting the CCDC137 gene according to claim 7 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The nucleotide sequence of NC-shRNA is shown in SEQ ID NO.

1.

9. The use of the inhibitor targeting the CCDC137 gene according to claim 7 or 8 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The nucleotide sequence of shRNA#1 is shown in SEQ ID NO.

2.

10. The use of the inhibitor targeting the CCDC137 gene according to claim 9 in the preparation of a medicament for treating acute myeloid leukemia, characterized in that, The nucleotide sequence of shRNA#2 is shown in SEQ ID NO.3.

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