Application of CCDC78 gene

Abstract

The invention discloses application of a CCDC78 gene, relates to application of a gene, and provides application of the CCDC78 gene as a diagnosis marker, a prognosis evaluation marker and a treatment target of colon cancer in order to solve the problems that a CCDC78 mechanism is unknown and application of a single gene is deleted in the prior art. The expression inhibitor of the CCDC78 gene is used for preparing medicines for treating colon cancer. According to the application, by integrating multi-omics analysis and functional experiments, the dual value of the CCDC78 as an independent prognostic marker and a targeted therapy target is established, the mechanism that the CCDC78 possibly drives the progress of colon adenocarcinoma (COAD) by regulating a CDKN1A-CDK4 / E2F1 signal axis is disclosed, and the limitation that the CCDC78 mechanism is unknown and single gene application is deleted in the prior art is broken through. The method is applied to the technical field of biomedicine.

Description

Technical Field

[0001] This application relates to the application of the CCDC78 gene. Background Technology

[0002] Colorectal cancer (e.g., colon adenocarcinoma, COAD) is a major global public health problem. Its incidence has risen significantly in recent years, making it the third most common cancer worldwide, and its mortality rate ranks second among cancer-related deaths. Despite advancements in surgery, chemotherapy, targeted therapy, and immunotherapy, COAD patients still face poor prognoses, primarily due to tumor recurrence and metastasis. Statistics show that approximately 25% of patients have metastatic disease at diagnosis, with the liver being the most common site of distant metastasis. The five-year survival rate for patients with advanced metastatic COAD is only about 14%. This situation underscores the urgency of early diagnosis and intervention, as timely diagnosis can significantly improve patient outcomes. Molecular biomarkers are crucial for the early diagnosis and prognostic assessment of COAD; therefore, in-depth exploration of key regulatory molecules in the pathogenesis of COAD and the identification of specific biomarkers are of great significance for optimizing clinical treatment strategies.

[0003] The Coiled-Coil Domain-Containing (CCDC) family comprises approximately 180 genes, all characterized by highly conserved coiled-coil domains. CCDC family members are involved in various physiological processes, including signal transduction, transcriptional regulation, embryonic development, cell cycle regulation, angiogenesis, and ciliary movement. Recent research has revealed the important role of this family members in tumorigenesis and metastasis. CCDC78, located on human chromosome 16p13.3, encodes a protein composed of 438 amino acids. Previous reports have indicated that CCDC78 is an important component of centrioles and sarcoplasmic membranes, participating in multiciliated epithelial cell differentiation and skeletal muscle contraction. Furthermore, it is associated with diseases such as central nucleus myopathy and muscular dystrophy. However, CCDC78 is currently only considered a statistically associated molecule in multigene models, and its diagnostic or therapeutic potential as a single biomarker has not yet been explored. Summary of the Invention

[0004] The purpose of this application is to address the problems of unclear mechanisms of CCDC78 and lack of single-gene application in the existing technology, and to propose the application of the CCDC78 gene, specifically involving the coiled-coil domain protein 78 (CCDC78) gene, a prognostic marker for colorectal cancer, and its use in colorectal cancer assessment and drug development.

[0005] This application relates to the use of the CCDC78 gene as a diagnostic biomarker, prognostic marker, and therapeutic target for colorectal cancer.

[0006] This application discloses an inhibitor of the CCDC78 gene expression for the preparation of a drug for treating colon cancer.

[0007] The beneficial effects of this application are:

[0008] I. Analysis of the TCGACOAD dataset revealed high expression of CCDC78 in cancer patients. This result was validated using an external dataset. Furthermore, significant high expression of CCDC78 was detected at the cellular level in colon cancer cell lines, with an AUC of 0.97, indicating high diagnostic value. Simultaneously, pan-cancer analysis showed high expression of CCDC78 in 19 tumor types, suggesting its potential universality in cancer. Further research can explore its role and potential mechanisms in other cancer types.

[0009] II. KM curve analysis revealed that high CCDC78 expression in COAD patients was associated with poorer overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI). This indicates that high CCDC78 expression generally predicts a poor prognosis for COAD patients, and CCDC78 is a potential prognostic biomarker for COAD patients.

[0010] III. In vitro functional validation results showed that knockdown of CCDC78 expression in colorectal cancer cells (HT29, HCT116) slowed cell proliferation, reduced colony formation rate, and inhibited migration and invasion, indicating that CCDC78 is a potential oncogene and a potential new target for COAD treatment. Furthermore, bioinformatics analysis, combined with in vitro siRNA loss-of-function experiments, suggested that CCDC78 drives cell cycle progression by inhibiting CDKN1A expression and / or maintaining CDK4-E2F1 axis activity, thereby influencing tumorigenesis and development. Attached Figure Description

[0011] Figure 1 The expression levels of CCDC78 mRNA varied among 33 types of tumor and normal tissues.

[0012] Figure 2 The expression level of CCDC78 mRNA differs between COAD tumor tissues and normal tissues;

[0013] Figure 3 To verify the expression and distribution of CCDC78 mRNA and protein in colorectal cancer cell lines (HT29, HCT116 and Caco-2) in in vitro cell experiments;

[0014] Figure 4 ROC curve for CCDC78 expression level in COAD diagnosis;

[0015] Figure 5 The correlation between CCDC78 expression levels and various clinical characteristics;

[0016] Figure 6 Survival curve analysis to show the impact of CCDC78 expression level on overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) in COAD;

[0017] Figure 7 Differential gene and functional enrichment analysis for CCDC78 high-expression vs. low-expression groups;

[0018] Figure 8 The effect of siRNA knocking down CCDC78 expression;

[0019] Figure 9 To verify the effect of CCDC78 knockdown expression on the proliferation, migration and invasion of colorectal cancer cells in in vitro cell experiments;

[0020] Figure 10 Preliminary exploration of the mechanism of action of CCDC78. Detailed Implementation

[0021] The technical solution of this application is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0022] This application provides an application of the CCDC78 gene as a diagnostic marker, prognostic marker, or therapeutic target for colorectal cancer.

[0023] In some implementations, the CCDC78 gene is used to prepare kits for the diagnosis and / or prognosis of colorectal cancer.

[0024] In one example, the kit can be used for detection using CCDC78 ribonucleic acid (RNA).

[0025] In yet another example, the kit can use the CCDC78 protein as a detection target.

[0026] In some implementations, inhibitors of the CCDC78 gene expression are used to prepare drugs for treating colon cancer.

[0027] In one example, the use of an inhibitor of CCDC78 gene expression in the preparation of a drug that blocks G1 / S phase transition in colon cancer by inhibiting the activity of the CDK4 / 6-cyclin D complex.

[0028] In yet another example, the use of an inhibitor of CCDC78 gene expression in the development of a drug that blocks G1 / S phase transition in colon cancer by inhibiting CDK4-E2F1.

[0029] In some embodiments, the expression inhibitor of the CCDC78 gene is siCCDC78-2, wherein the sense strand of siCCDC78-2 is shown in SEQ ID NO.3, and the antisense strand of siCCDC78-2 is shown in SEQ ID NO.4.

[0030] In some other embodiments, the expression inhibitor of the CCDC78 gene is siCCDC78-2, wherein the sense strand of siCCDC78-2 is shown in SEQ ID NO.3, and the antisense strand of siCCDC78-2 is shown in SEQ ID NO.4.

[0031] In this application, colon cancer may include other subtypes such as colonic adenocarcinoma (COAD), mucinous adenocarcinoma, and signet ring cell carcinoma. To further illustrate this application, COAD in colon cancer will be used as an example and described in detail below.

[0032] Specific Implementation Method 1: This implementation method applies the CCDC78 gene as a diagnostic marker for colon cancer.

[0033] Bioinformatics analysis was used to differentially analyze the expression of CCDC78 in pan-cancer tissues based on The Cancer Genome Atlas (TCGA) data. The results showed that the mRNA expression level of CCDC78 differed between human tumors and normal tissues, and was significantly elevated in 16 types of tumors (such as breast cancer (BRCA), cholangiocarcinoma (CHOL), colorectal adenocarcinoma (COAD), and hepatocellular carcinoma (LIHC)). Particularly in COAD, CCDC78 expression was significantly upregulated. Furthermore, analysis of the external dataset GSE39582 and RT-qPCR of clinical samples validated these results, confirming that the CCDC78 mRNA level in tumor tissues was significantly higher than that in adjacent normal tissues.

[0034] Receiver Operating Characteristic (ROC) curve analysis showed that CCDC78 had high diagnostic accuracy (AUC value of 0.970, 95% confidence interval: 0.955-0.986), indicating that it can effectively distinguish cancerous tissue from normal tissue. Its high diagnostic efficacy suggests that CCDC78 is a potential biomarker for COAD.

[0035] Specific Implementation Method Two: This implementation method applies the CCDC78 gene as a prognostic biomarker for colorectal cancer.

[0036] Clinicopathological parameters of 478 patients with colorectal adenocarcinoma (COAD) were obtained from the TCGA database, and the correlation between CCDC78 expression levels and these characteristics was analyzed using the Wilcoxon rank-sum test. The results showed that CCDC78 expression differed significantly across different clinical characteristics, particularly in lymph node metastasis stage (N1 / N2), distant metastasis stage (M1), and pathological stage (III / IV). However, high CCDC78 expression was not associated with T stage, age, or sex. These analyses indicate that high CCDC78 expression is closely related to specific adverse prognostic pathological features.

[0037] Survival analysis using Cox regression showed that patients with high CCDC78 expression had significantly shorter overall survival (OS) (HR = 1.84, p < 0.001). Further improvements in disease-specific survival (DSS) (HR = 2.31, p < 0.001) and progression-free interval (PFI) (HR = 1.47, p < 0.001) indicate a significantly increased prognostic risk in cancer patients with high CCDC78 expression. These results demonstrate that high CCDC78 expression is an independent prognostic risk factor for worsened survival in colorectal cancer patients and is significantly positively correlated with the risk of tumor progression.

[0038] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method Two in that the prognostic assessment evaluates overall survival, disease-specific survival, and progression-free survival. Everything else is the same as in Specific Implementation Method Two.

[0039] Specific Implementation Method 4: This implementation method applies the CCDC78 gene as a therapeutic target for colon cancer.

[0040] The expression level of CCDC78 was detected in three COAD cell lines (HT29, Caco2, and HCT116). RT-qPCR results showed that CCDC78 expression was significantly increased in HT29 and HCT116 cells. Immunofluorescence staining showed that CCDC78 was mainly located in the cytoplasm of COAD cells. Figure 3C). Functional experiments were conducted using HT29 cells. A siRNA (small interfering RNA) targeting CCDC78 was designed to knock down its expression. Results showed that both the mRNA and protein levels of CCDC78 were significantly decreased in both HT29 and HCT116 cells. CCK-8 assays demonstrated that CCDC78 knockdown significantly inhibited the viability of both HT29 and HCT116 cells. CCDC78 knockdown induced a significant upregulation of the key cell cycle regulators CDKN1A / p21 (4.8-fold and 2.2-fold, respectively, p<0.001). This molecule blocks the G1 / S phase transition by inhibiting the activity of the CDK4 / 6-cyclin D complex, accompanied by a downregulation of CDK4 expression (60.6% and 57.5%, respectively). Further analysis revealed that CCDC78 may exert its function by regulating the major transcription factor E2F1, which enters the S phase. Knockdown of CCDC78 significantly inhibited E2F1 expression (82.1% and 57.8%, respectively), suggesting that it promotes G1 / S phase transition through E2F1-mediated transcriptional activation. Furthermore, colony formation assays showed that CCDC78 knockdown inhibited cell proliferation. Transwell migration and wound healing assays demonstrated that knockdown significantly weakened cell migration ability. These results indicate that CCDC78 is a key oncogene driving COAD proliferation and migration. Reducing CCDC78 expression significantly inhibits the growth of colon cancer cells, suggesting that CCDC78 could serve as a therapeutic target for colon cancer.

[0041] The sequence of the CCDC78 gene described in the above specific embodiments is shown in SEQ ID NO.5.

[0042] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the CCDC78 gene is used to prepare a kit for the diagnosis and prognosis of colorectal cancer. Everything else is the same as in Specific Implementation Methods One to Four.

[0043] This kit uses CCDC78 RNA detection and / or CCDC78 protein as the detection target. The detection primers are: upstream primer 5'-AATGTTGTGCTACGAGCCAAG-3', downstream primer 5'-CTGGGGTCAGACTCCACTG-3', used for specific amplification of the CCDC78 gene. The internal control used is β-actin, with upstream primer 5'-CTGAACCCCAAGGCCAAC-3' and downstream primer 5'-TTCATGAGGTAGTCAGTCAGGT-3'. CCDC78 RNA detection is performed using real-time quantitative PCR. In addition to the above specific primers, TRizol RNA extraction reagent (TRizol, isopropanol, chloroform, RNase-free water), reverse transcription reagent (reverse transcription primers, reverse transcriptase, RNA inhibitors, etc.), and real-time quantitative PCR reagent (SYBR method) are required. CCDC78 protein detection is performed using CCDC78 antibody to stain tumor sections from COAD patients using immunohistochemistry (IHC).

[0044] Specific Implementation Method Six: This implementation method differs from Specific Implementation Method Five in that the kit uses CCDC78 RNA for detection. Everything else is the same as in Specific Implementation Method Five.

[0045] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Method Six in that the kit uses the CCDC78 protein as the detection target. Everything else is the same as in Specific Implementation Method Six.

[0046] Specific implementation method eight: In this implementation method, the expression inhibitor of the CCDC78 gene is used to prepare a drug for treating colon cancer.

[0047] Specific Implementation Method Nine: This implementation method differs from Specific Implementation Method Eight in that it involves the application of an inhibitor of the CCDC78 gene expression in the preparation of a drug that blocks the G1 / S phase transition of colon cancer by inhibiting the activity of the CDK4 / 6-cyclin D complex. Everything else is the same as in Specific Implementation Method Seven.

[0048] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Eight in that it involves the application of an inhibitor of the CCDC78 gene expression in the preparation of a drug that blocks the G1 / S phase transition in colon cancer by inhibiting CDK4-E2F1. Everything else is the same as in Specific Implementation Method Eight.

[0049] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Eight in that the CCDC78 gene expression inhibitor is siCCDC78-1, wherein the sense strand of siCCDC78-1 is shown in SEQ ID NO.1, and the antisense strand of siCCDC78-1 is shown in SEQ ID NO.2. Everything else is the same as in Specific Implementation Method Eight.

[0050] Specific Implementation Method Ten: This implementation method differs from Specific Implementation Method Eight in that the CCDC78 gene expression inhibitor is siCCDC78-2, wherein the sense strand of siCCDC78-2 is shown in SEQ ID NO.3, and the antisense strand of siCCDC78-2 is shown in SEQ ID NO.4. Everything else is the same as in Specific Implementation Method Eight.

[0051] The beneficial effects of this application are verified using the following embodiments:

[0052] Example 1

[0053] Bioinformatics methods were used to detect the differences in CCDC78 mRNA expression levels between 33 types of tumor (pan-cancer) tissues and normal tissues. mRNA expression profiles and corresponding clinical information for various cancers were downloaded from the TCGA database. Clinical information and TPM (transcripts per million) expression values ​​were extracted using R language. Data were processed using the Log2(value+1) method, and the Wilcoxon rank sumtest method was used to detect the differences in CCDC78 expression levels between tumor and normal tissues.

[0054] Figure 1 The study showed differences in CCDC78 mRNA expression levels among 33 types of tumor tissues and normal tissues. Figure 1 (A, B) Based on the TCGA database, the expression levels of CCDC78 mRNA in tumors and adjacent normal tissues (unpaired and paired samples) in pan-cancer tissues were compared. Data are expressed as mean ± standard deviation; ***p<0.001, **p<0.01, *p<0.05, ns indicate no significant difference. The results show that the expression of CCDC78 in 16 types of tumors (including COAD) is significantly higher than that in normal tissues. Figure 1 A). Furthermore, in the analysis of paired samples, CCDC78 was significantly more highly expressed in 13 types of tumors (including COAD) than in adjacent normal tissues. This indicates that CCDC78 is highly expressed in a variety of tumors, including COAD, suggesting that it plays an important role in the development and progression of various tumors.

[0055] Example 2

[0056] We used a similar approach to pan-cancer data processing to process CCDC78 in the TCGA-COAD dataset (containing 41 normal samples and 480 COAD samples) and the GSE39582 dataset (containing 19 normal samples and 566 COAD samples) to verify the difference in CCDC78 expression levels between COAD patients and normal tissue samples.

[0057] In addition, 10 tissue samples (5 normal tissues and 5 COAD tissues) were collected from patients who underwent colon cancer resection at Ningbo University Affiliated Li Huili Hospital. Total RNA was extracted from the tissues, and cDNA was obtained by reverse transcription. RT-qPCR was used to detect the difference in CCDC78 mRNA expression levels between patient and normal tissues. The collection of all clinical samples was strictly carried out in accordance with the ethical guidelines established by the hospital's ethics committee to ensure that the rights and welfare of patients were protected throughout the research process.

[0058] Figure 2 This study shows the difference in CCDC78 mRNA expression levels between COAD tumor tissues and normal tissues. (A, B) Based on the TCGA database, the mRNA expression levels in COAD tumor tissues and normal tissues (unpaired and paired samples) were compared. (C) GEO database analysis was used to compare the CCDC78 mRNA expression levels in COAD tumor tissues and normal tissues. (D) qRT-PCR was used to detect the CCDC78 mRNA expression levels in 5 pairs (tumor vs. adjacent normal) COAD samples. Data are expressed as mean ± standard deviation; ***p<0.001, **p<0.01. The results show that in the TCGA database, regardless of the mixed sample ( Figure 2 A) or paired samples ( Figure 2 B) CCDC78 mRNA expression was significantly higher in COAD tumor tissues than in normal tissues. This was consistent with the results of analysis using an external dataset from GEO (GSE39582). Figure 2 C). Furthermore, data from fresh tissue samples collected from clinical patients also showed that CCDC78 was significantly highly expressed in COAD tumor samples compared to adjacent normal tissue. Figure 2 D). This indicates that CCDC78 is indeed significantly highly expressed in COAD patients compared to adjacent normal tissues.

[0059] Example 3

[0060] The expression of CCDC78 in three colorectal cancer cell lines (HT29, HCT116, and Caco-2) was detected by real-time immunofluorescence (RT-qPCR), Western blot (WB), and immunofluorescence assay (IFA), with primary cells derived from adjacent normal tissue as controls.

[0061] In the RT-qPCR experiment, TRizol was used to lyse cells, RNA was extracted, and 1 μg of reverse transcriptase was used to convert the RNA into cDNA. The CCDC78 mRNA level was then detected by RT-qPCR. Human β-actin was used as an internal control. Results were obtained using 2... -ΔΔCT Calculation by method.

[0062] Protein expression levels were detected by lysing cells on ice for 30 min with RIPA lysis buffer containing 1 mM benzyl sulfonyl fluoride (PMSF), and protein concentration was determined using the BCA method. The loading volume was adjusted to maintain consistency based on concentration, and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After electrophoresis, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 h, followed by overnight incubation at 4°C with CCDC78 antibody (1:2000 dilution). After washing three times with TBST buffer, the membrane was incubated at room temperature with horseradish peroxidase (HRP)-labeled secondary antibody for 1 h. Finally, enhanced chemiluminescence (ECL) substrate was used to detect the protein signal, and β-actin was used as an internal control to standardize the loading volume.

[0063] In the IFA experiment, COAD cells with 70% confluence were seeded in confocal culture dishes and cultured for 24 h. After fixation with 4% paraformaldehyde, the cells were permeabilized with 0.5% Triton X-100 for 15 min. Blocking was performed, followed by overnight incubation at 4°C with rabbit-derived CCDC78 primary antibody (1:300 dilution, Proteintech). (Alexa data is not provided in the original text.) Cells were incubated with 546-labeled secondary antibody (Invitrogen) in the dark for 1 hour, followed by 5 minutes of Hoechst 33258 staining of the nuclei. Finally, fluorescence images were acquired using an Olympus BX51 fluorescence microscope.

[0064] Figure 3 This study demonstrates the expression and distribution of CCDC78 mRNA and protein in colorectal cancer cell lines (HT29, HCT116, and Caco-2) using in vitro cell experiments. (A) RT-qPCR was used to detect the mRNA expression level of CCDC78 in different cell lines. (B) Western blotting was used to detect the protein expression of CCDC78 in different cell lines. (C) Immunofluorescence was used to detect the localization of CCDC78 in COAD cells. Scale bar: 10 μm. Data are expressed as mean ± standard deviation; ***p<0.001, ns indicates no significant difference. The results showed that CCDC78 was significantly highly expressed in HT29 and HCT116 cell lines. Figure 3 AB), and IFA observation showed that its subcellular localization was mainly distributed in the cytoplasm ( ). Figure 3 C). Overall, CCDC78 is highly expressed in colon cancer cell lines.

[0065] Example 4

[0066] This implementation aims to evaluate the diagnostic value of CCDC78 expression level for COAD. Clinical information and CCDC78 expression data of COAD patients downloaded in Example 2 were used. ROC (Receiver Operating Characteristic) analysis was performed on the data using R (4.2.1) in conjunction with the pROC (1.18.0) R package, and the results were visualized using ggplot2 (3.4.4).

[0067] Figure 4 The ROC curve of CCDC78 expression level for the diagnosis of COAD is shown in the figure. As shown in the figure, the area under the curve (AUC) in the prediction results is 0.97, close to 1, CI: 0.955-0.986, indicating that the expression level of CCDC78 can effectively distinguish between COAD patients and non-patients, and has a good diagnostic effect in predicting the outcome.

[0068] Example 5

[0069] This study aims to evaluate the diagnostic value of CCDC78 expression levels for COAD. Using clinical information and CCDC78 expression levels of COAD patients downloaded in Example 2, the Wilcoxon rank sum test was employed to detect the correlation between CCDC78 expression levels and different clinical characteristics of COAD, namely TMN stage and pathological stage.

[0070] Figure 5 The correlation between CCDC78 expression levels and various clinical features is shown; (AC) association between CCDC78 expression and N stage (A), M stage (B), and pathological stage. Data are expressed as mean ± standard deviation; **p<0.01, *p<0.05.

[0071] The results showed that, compared with COAD patients without lymph node metastasis (N0 stage), CCDC78 was significantly upregulated in patients with positive lymph node metastasis (N1-N2 stage) (p<0.01). Figure 5 A). Compared with non-metastatic patients, CCDC78 expression was significantly increased in patients with tumor metastasis (p<0.05). Figure 5 B). Furthermore, its expression was significantly increased in patients with advanced tumors compared to those with earlier stages (p<0.01). Figure 5 C). The above results indicate that the higher the malignancy of COAD, the higher the expression level of CCDC78. The expression of CCDC78 is positively correlated with NM stage and overall severity, suggesting that it can be used as a marker for clinically assessing the severity of COAD.

[0072] Example 6

[0073] Using the clinical information and CCDC78 expression levels of COAD patients downloaded in Example 2, patients were divided into high-expression and low-expression groups based on the median CCDC78 expression level. Differences in overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) between the two groups were analyzed. Cox regression was used, and the survival package was used for proportional hazards hypothesis testing and fitted survival regression. The results were analyzed using the survminer package, visualized using the ggplot2 package, and Kaplan-Meier (KM) survival curves were created.

[0074] Figure 6 Survival curve analysis showing the impact of CCDC78 expression levels on overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI) in COAD. (AC) Association between high and low CCDC78 expression levels and overall survival (OS), disease-specific survival (DSS), and progression-free survival (PFI). As shown in the figure, COAD patients with high CCDC78 expression had poorer overall survival (OS). Figure 6 A) Disease-specific survival (DSS) Figure 6 B) and progression-free survival (PFI) Figure 6 C) Related. This indicates that high expression of CCDC78 generally indicates a poor prognosis in COAD patients, and CCDC78 is a potential prognostic biomarker for COAD patients.

[0075] Example 7

[0076] The CCDC78 expression levels of COAD patients downloaded in Example 2 were analyzed using the DESeq2 package. Patients were divided into high and low expression groups based on the median CCDC78 expression value, and differentially expressed genes (DEGs) were analyzed between the two groups. The DEG selection criteria were p-value < 0.05 and |Log2 FC| > 1. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses (DEGs) and GSEA enrichment analyses (all molecules and their corresponding logFCs) were performed using the clusterProfiler package to identify biological processes and pathways related to CCDC78 expression.

[0077] Figure 7This shows the differentially expressed genes and functional enrichment analysis between the CCDC78 high-expression and low-expression groups. (A) Volcano plot of differentially expressed genes (DEGs) between the CCDC78 high-expression and low-expression groups. (BD) Results of hub gene screening, gene ontology (GO) functional annotation, KEGG pathway enrichment, and gene set enrichment analysis (GSEA) based on DEGs. The results show that there are a total of 328 DEGs compared with the CCDC78 high-expression group, of which 148 genes are upregulated and 180 genes are downregulated. Figure 7 A). PPI analysis of DEGs using the STRING database identified 16 hub genes, which are closely related to histones and their modifications. Figure 7 B). Further analysis using GO, KEGG, and gene set enrichment analysis (GSEA) revealed that DEGs were significantly enriched in biological processes such as protein-DNA complex assembly, nucleosome organization and assembly, DNA packaging complexes, nucleosomes, and protein heterodimerization activity. Figure 7 C); associated with disease pathways such as neutrophil extracellular trap formation ( Figure 7 C). Furthermore, gene set variation analysis (GSVA) ​​based on the Reactome pathway showed that the activities of cell cycle checkpoint, G1 phase of mitosis, G1 / S phase transition, DNA replication, and mitosis pathways were significantly positively correlated with the upregulation of CCDC78 expression. Figure 7 D). The above results analyzed the molecular characteristics and functional pathways of the CCDC78 high-expression group in COAD, revealing its possible mechanism of action in driving tumor progression by regulating cell cycle processes, and providing a theoretical basis for intervention therapy targeting CCDC78.

[0078] Example 8

[0079] Based on the CCDC78 gene, siRNA mediates post-transcriptional gene silencing (PTGS) by specifically binding to the CCDC78 mRNA. Its mechanism of action is as follows: The optimal length of siRNA is 21-23 nucleotides (nt). When recognized and bound by the RNA-induced silencing complex (RISC), the siRNA double strand unwinds. Subsequently, guided by the antisense strand of the siRNA, the RISC specifically recognizes and binds to the CCDC78 mRNA with a homologous sequence, cleaving it between the 10th and 11th bases downstream of its 5' end, leading to mRNA degradation. This blocks the translation of the CCDC78 protein, achieving post-transcriptional silencing of the target gene. The siRNA nucleotide sequence of the sense strand is: siCCDC78-1#5'-GACACUGAUAAUCACAGGAAA-3'; siCCDC78-2#5'-GCAAGUCACUUAAGCAUGGUU-3'. A GFP-targeting shRNA was used as a control; the positive strand of the control siRNA was 5'-GUAUAAGUCAACUGUUGAC-3'. The siRNA was synthesized and transfected into HT29 and HCT116 cells using Lipofectamine 3000 according to the manufacturer's instructions. Samples were taken for analysis 72 hours after transfection. RT-qPCR and Western blotting were performed according to the procedures in Example 3.

[0080] Figure 8 The siRNA knockdown of CCDC78 expression was demonstrated. The efficiency of CCDC78 knockdown was detected by (AB) immunoblotting and (CD) RT-qPCR. Data are presented as mean ± standard deviation; ***p<0.001, **p<0.01. The results show that CCDC78 expression was significantly downregulated in cells transfected with siCCDC78-1 and siCCDC78-2, indicating that the designed siRNA can specifically knock down CCDC78 gene expression.

[0081] Example 9

[0082] Using the method described in Example 8, siRNA was transfected into HT29 and HCT116 cells, and the following experiments were then performed:

[0083] 1. CCK-8 cell proliferation experiment

[0084] Cell viability was assessed using a CCK-8 assay kit. HT-29 and HCT116 cells were seeded at a density of 2000 cells per well in 96-well plates and cultured until the corresponding detection time. After adding CCK-8 reagent, the cells were incubated at 37°C for 1 hour. Absorbance was measured at 450 nm using a microplate reader to evaluate cell proliferation activity.

[0085] 2. Cell invasion assay

[0086] 3×10 4 100 μL of cells were seeded into the upper chamber of a Transwell chamber and added to serum-free McCoy's 5A medium. 600 μL of McCoy's 5A complete medium containing 20% ​​fetal bovine serum (FBS) was added to the lower chamber. The Transwell chambers were incubated at 37°C in a 5% CO2 incubator for 48 h. After incubation, unmigrated cells in the upper chamber were gently wiped with a cotton swab and washed three times with PBS. Cells were then fixed with 4% paraformaldehyde for 30 min, washed, and stained with 0.1% crystal violet for 20 min. Images were acquired under a microscope, and the number of invasive cells was analyzed using image analysis software.

[0087] 3. Cell scratch assay

[0088] Inoculate 1×10⁶ cells per well in a 24-well plate. 5 Cells were collected and, after adhesion, were scratched perpendicularly to the well plate surface using a 200 μL pipette tip. The cells were washed three times with PBS to remove detached cells. The scratched area was observed at 0 h and 72 h post-scratching. Images were acquired using a microscope, and the scratch width was analyzed using image analysis software. Cell migration rate was calculated using the formula: Migration rate = (0 h scratch width - 72 h scratch width) / 0 h scratch width × 100%

[0089] 4. Colony Formation Experiment

[0090] 1500 cells were seeded per well in McCoy's 5A complete medium containing 10% FBS and cultured at 37°C in a 5% CO2 incubator for 12 days. After culture, cells were fixed with 4% paraformaldehyde for 30 min, washed with PBS, and stained with 0.1% crystal violet for 20 min. Images were acquired under a microscope, and the number of cell colonies with a diameter ≥50 μm was counted using image analysis software.

[0091] Figure 9 This study demonstrates the effect of CCDC78 knockdown expression on the proliferation, migration, and invasion of colorectal cancer cells using in vitro cell experiments. (AB) Changes in colony formation rate after CCDC78 knockdown expression. (CD) Proliferation capacity of HT29 / HCT116 cells after CCDC78 knockdown expression. (EF) Transwell migration assay to assess migration ability. (GH) Scratch assay to assess invasion ability (scale bar: 200 μm). Data are expressed as mean ± standard deviation; ***p<0.001, **p<0.01, *p<0.05.

[0092] In the CCK8 experiment, knockdown of CCDC78 significantly reduced the proliferation rates of both HT29 and HCT116 cell lines. Figure 9 AB). Clonogenesis assays showed that CCDC78 knockdown inhibited cell proliferation ( ). Figure 9 CD. Transwell migration and wound healing assays showed that knockdown significantly impaired cell migration ability. Figure 9 (EH). In summary, in vitro cell experiments confirmed that knocking down CCDC78 expression can slow down the inhibition of colon cancer cell proliferation, migration, and invasion. This indicates that CCDC78 plays a pro-oncogene role in COAD and is a potential new target for COAD treatment.

[0093] Example 10

[0094] Using the method described in Example 8, siRNA was transfected into HT29 and HCT116 cells. 72 hours post-transfection, the relative expression levels of cell cycle-related genes were detected by RT-qPCR. The results were analyzed using 2... -ΔΔCT The method involved processing. The genes detected included CDK4, CDKN1A, and E2F1. The functions of the proteins they encode are as follows:

[0095] CDK4 is a cyclin-dependent kinase 4 (CDK4) belonging to the CDK family, and is involved in the transition from G1 to S phase. Its binding to Cyclin D releases E2F via Rb phosphorylation, propelling the cell from G1 to S phase and initiating DNA replication. CDK4 can also integrate growth factor signals (such as the MAPK pathway) and nutrient status signals to determine whether the cell enters the cell cycle. It has been reported to amplify or overexpress in various cancers (such as breast cancer and melanoma), leading to cell cycle dysregulation.

[0096] CDKN1A, or p21, is a CDK inhibitor belonging to the CIP / KIP family. It binds to and inhibits the CDK4 / 6-Cyclin D complex, blocking Rb protein phosphorylation, leading to E2F inactivation and G1 phase arrest.

[0097] E2F1, a transcription factor belonging to the E2F family, is divided into activating and repressor types. As an activating member, it can target and regulate genes such as DNA synthases (e.g., DHFR), Cyclin E / A, and CDK1, promoting the transcription of genes in the S phase. It has a dual role in tumors: excessive activation leads to uncontrolled proliferation (e.g., in Rb-mutant tumors), but it can also induce apoptosis in genomically unstable cells.

[0098] The primers used are as follows: CDK4 upstream primer: 5'-ATGGCTACCTCTCGATATGAGC-3', downstream primer: 5'-CATTGGGGACTCTCACACTCT-3'; CDKN1A upstream primer: 5'-TGTCCGTCAGAACCCATGC-3', downstream primer: 5'-AAAGTCGAAGTTCCATCGCTC-3'; E2F1 upstream primer: 5'-CATCCCAGGAGGTCACTTCTG-3', downstream primer: 5'-GACAACAGCGGTTCTTGCTC-3'.

[0099] Figure 10 Preliminary exploration of the mechanism by which CCDC78 functions. (A, B) RT-qPCR analysis of cell cycle-related genes after knockdown of CCDC78 expression in HT29 and HCT116 cells. Data are presented as mean ± standard deviation; ***p<0.001, **p<0.01, *p<0.05. Results showed that knockdown of CCDC78 expression in HT29 and HCT116 cells induced significant upregulation of the key cell cycle regulators CDKN1A / p21 (4.8-fold and 2.2-fold, respectively, p<0.001). CDKN1A can block G1 / S phase transition by inhibiting the activity of the CDK4 / 6-cyclin D complex. Simultaneously, CDK4 expression was downregulated (60.6% and 57.5%, respectively). Further analysis revealed that CCDC78 may exert its function by regulating the master transcription factor E2F1, which enters the S phase. Knockdown of CCDC78 significantly inhibited E2F1 expression (inhibition rates of 82.1% and 57.8%), suggesting that it promotes the G1 / S phase transition through E2F1-mediated transcriptional activation. Considering the regulatory roles of CDK4, CDKN1A, and E2F1 in the cell cycle, this suggests that CCDC78 drives cell cycle progression by inhibiting CDKN1A expression and / or maintaining CDK4-E2F1 axis activity. Its loss of function triggers G1 / S phase checkpoint activation, indicating potential tumor suppressor capabilities.

Claims

1. Application of the CCDC78 gene as a diagnostic marker, prognostic marker, or therapeutic target for colorectal cancer.

2. The application according to claim 1, characterized in that, The prognostic assessment is an evaluation of one or more of the following: overall survival, disease-specific survival, and progression-free survival.

3. The application according to claim 1, characterized in that, The CCDC78 gene is used to prepare kits for the diagnosis and / or prognosis of colorectal cancer.

4. The application according to claim 3, characterized in that, The kit uses CCDC78 RNA for detection.

5. The application according to claim 3, characterized in that, The kit uses the CCDC78 protein as the detection target.

6. The application according to claim 1, characterized in that, The CCDC78 gene expression inhibitor is used to prepare drugs for treating colon cancer.

7. The application according to claim 6, characterized in that, The application of the CCDC78 gene expression inhibitor in the preparation of a drug that blocks the G1 / S phase transition of colon cancer by inhibiting the activity of the CDK4 / 6-cyclin D complex.

8. The application according to claim 6, characterized in that, The application of the CCDC78 gene expression inhibitor in the preparation of a drug that blocks G1 / S phase transition in colon cancer by inhibiting CDK4-E2F1.

9. The application according to any one of claims 6 to 8, characterized in that, The expression inhibitor of the CCDC78 gene is siCCDC78-1, wherein the sense strand of siCCDC78-1 is shown in SEQ ID NO.1, and the antisense strand of siCCDC78-1 is shown in SEQ ID NO.

2.

10. The application according to any one of claims 6 to 8, characterized in that, The expression inhibitor of the CCDC78 gene is siCCDC78-2, wherein the sense strand of siCCDC78-2 is shown in SEQ ID NO.3, and the antisense strand of siCCDC78-2 is shown in SEQ ID NO.4.

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