Application of eIF4A2 gene as a potential prognostic marker for colorectal cancer

Abstract

The application discloses application of an eIF4A2 gene as a potential prognostic marker of colorectal cancer, and at least has the following beneficial effects: researches prove that the eIF4A2 gene plays a role in promoting proliferation and invasion in colorectal cancer; the eIF4A2 gene is significantly up-regulated in colorectal cancer tissues, and high expression of the eIF4A2 gene is closely related to low differentiation and poor prognosis of patients; cell WB experiments show that the eIF4A2 gene is significantly higher in expression in various colorectal cancer cell lines than in normal colon epithelial cells; function experiments show that knocking down the eIF4A2 gene can significantly inhibit the proliferation and invasion ability of a colorectal cancer cell line HCT116. Therefore, the eIF4A2 gene can be used as a potential prognostic marker, is a key promoting factor in the progression of colorectal cancer, is used for evaluating the condition and prognosis of patients, provides experimental basis for becoming a potential treatment target, and provides a reference for individualized treatment.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical biotechnology, and in particular to the application of the eIF4A2 gene as a potential prognostic biomarker for colorectal cancer. Background Technology

[0002] According to 2022 data from the International Agency for Research on Cancer (IARC), colorectal cancer (CRC) accounts for 9.6% (third) of global cancer incidence and 9.3% (second) of cancer mortality. According to the latest statistics from the IARC (2024), colorectal cancer accounts for 10.8% of new cases and 9.4% of all malignant tumors in my country. RNA-binding proteins (RBPs) are key factors in post-transcriptional gene regulation, regulating cellular function through interactions with RNA. Dysregulation of RBP gene expression is widely involved in the abnormal activation of cancer driver genes and oncogenic pathways. The eukaryotic translation initiation factor 4F (eIF4F) complex is an RNA-binding protein that regulates translation initiation, composed of eIF4A, eIF4E, and eIF4G. Eukaryotic translation initiation factor 4A (eIF4A), acting as a dead-box RNA helicase, influences the translation initiation rate by regulating mRNA secondary structure. Members of this family, eIF4A1, eIF4A2, and eIF4A3, are aberrantly expressed in various cancers and correlate with clinicopathological features, demonstrating their potential as cancer biomarkers. eIF4A2 is highly homologous to eIF4A1 but functionally different, and its expression is regulated by differences in tissue and growth conditions. Studies have shown that eIF4A2 is associated with poor prognosis in colorectal cancer and non-small cell lung cancer (NSCLC), and its high expression is also associated with tumor metastasis and chemotherapy resistance (such as to oxaliplatin and paclitaxel). In summary, based on previous studies, it is hypothesized that eIF4A2 plays a promoting role in the development and progression of colorectal cancer. It could serve as a potential prognostic biomarker, used to assess patient condition and prognosis, and provide a reference for personalized treatment. However, the exact role of eIF4A2 in the development and progression of colorectal cancer and its mechanism as a potential prognostic biomarker still require further elucidation. Summary of the Invention

[0003] The purpose of this application is to provide an application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer, in order to study the expression of the eIF4A2 gene in colorectal cancer, its impact on the malignant biological behavior of tumor cells and its clinical prognostic value, and to provide a theoretical basis for the precision treatment of colorectal cancer.

[0004] To address the aforementioned technical issues, this application provides an application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer, wherein the eIF4A2 gene can promote the proliferation and invasion of colorectal cancer cells.

[0005] As a preferred embodiment, the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer is described. The eIF4A2 gene is highly expressed in colorectal cancer, and its high expression level is associated with poor differentiation and poor prognosis in patients.

[0006] As a preferred embodiment, the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer is described, in which the expression of the eIF4A2 gene is significantly higher than that of normal colonic epithelial cells in various colorectal cancer cell lines.

[0007] As a preferred embodiment, the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer shows that the positive expression rate of the eIF4A2 gene in colorectal cancer tissue is significantly higher than that in adjacent normal tissue.

[0008] As a preferred embodiment, the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer is described, whereby knocking down the eIF4A2 gene can significantly inhibit the proliferation and invasion ability of the colon cancer cell line HCT116.

[0009] The protocol further explains the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer, noting that downregulation of eIF4A2 gene expression can effectively inhibit the proliferation of HCT116 cells.

[0010] The protocol further explains the application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer, noting that downregulation of eIF4A2 gene expression can effectively inhibit the invasive ability of HCT116 cells.

[0011] The application of the eIF4A2 gene as a potential prognostic biomarker for colorectal cancer provided by this invention includes at least the following beneficial effects: Studies have confirmed that the eIF4A2 gene plays a role in promoting proliferation and invasion in colorectal cancer; the expression of the eIF4A2 gene is significantly upregulated in colorectal cancer tissues, and its high expression is closely related to poor differentiation and poor prognosis in patients; Western blot (WB) experiments show that the expression of eIF4A2 in various colorectal cancer cell lines is significantly higher than that in normal colonic epithelial cells; functional experiments show that knocking down eIF4A2 can significantly inhibit the proliferation and invasion ability of the HCT116 colon cancer cell line. Therefore, the eIF4A2 gene, as a key promoting factor in the progression of colorectal cancer, can serve as a potential prognostic biomarker for assessing patient condition and prognosis, providing experimental evidence for its potential therapeutic target and a reference for personalized treatment. Attached Figure Description

[0012] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0013] Figure 1 This is a schematic diagram of eIF4A2 expression and prognostic survival curves provided in an embodiment of this application;

[0014] Figure 2 This is a box plot of eIF4A2 expression levels in colorectal cancer tissues and normal tissues from the TCGA database, provided in an embodiment of this application.

[0015] Figure 3 This is a schematic diagram illustrating the expression of eIF4A2 protein in colorectal cancer tissue and adjacent normal tissue provided in the embodiments of this application;

[0016] Figure 4 This is a survival curve of eIF4A2 expression level provided in an embodiment of this application;

[0017] Figure 5 A schematic diagram illustrating the wb detection of eIF4A2 expression levels in colon cancer tissue and adjacent normal tissue provided in an embodiment of this application;

[0018] Figure 6 This is a schematic diagram illustrating the wb detection of eIF4A2 expression levels in colon cancer cells and normal cells provided in an embodiment of this application.

[0019] Figure 7 This is a representative image of HCT116 cells before and after siRNA transfection, provided in an embodiment of this application.

[0020] Figure 8This is a schematic diagram of HCT116 transfection efficiency detection provided in an embodiment of this application;

[0021] Figure 9 This is a schematic diagram of the proliferation curves of the knockdown of eIF4A2 and the control group provided in the embodiments of this application;

[0022] Figure 10 A schematic diagram showing the invasion results of HCT116 cells after knocking down eIF4A2 and the control group, as provided in an embodiment of this application.

[0023] Figure 11 The diagram shows a flow cytometry assay for apoptosis detection (Figure A) and a diagram shows a quantitative analysis of Figure A (Figure B) provided in the embodiments of this application. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0025] The core of this application is to provide an application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer, in order to study the expression of the eIF4A2 gene in colorectal cancer, its impact on the malignant biological behavior of tumor cells and its clinical prognostic value, and to provide a theoretical basis for the precision treatment of colorectal cancer.

[0026] Figure 1 This is a schematic diagram of eIF4A2 expression and prognostic survival curves provided in an embodiment of this application; Figure 2 This is a box plot of eIF4A2 expression levels in colorectal cancer tissues and normal tissues from the TCGA database, provided in an embodiment of this application. Figure 3 This is a schematic diagram illustrating the expression of eIF4A2 protein in colorectal cancer tissue and adjacent normal tissue provided in the embodiments of this application; Figure 4 This is a survival curve of eIF4A2 expression level provided in an embodiment of this application; Figure 5 A schematic diagram illustrating the wb detection of eIF4A2 expression levels in colon cancer tissue and adjacent normal tissue provided in an embodiment of this application; Figure 6 This is a schematic diagram illustrating the wb detection of eIF4A2 expression levels in colon cancer cells and normal cells provided in an embodiment of this application. Figure 7 This is a representative image of HCT116 cells before and after siRNA transfection, provided in an embodiment of this application. Figure 8 This is a schematic diagram of HCT116 transfection efficiency detection provided in an embodiment of this application; Figure 9 This is a schematic diagram of the proliferation curves of the knockdown of eIF4A2 and the control group provided in the embodiments of this application; Figure 10A schematic diagram showing the invasion results of HCT116 cells after knocking down eIF4A2 and the control group, as provided in an embodiment of this application. Figure 11 See Figure A for a schematic diagram of flow cytometry detection of apoptosis provided in the embodiments of this application, and Figure B for a schematic diagram of quantitative analysis of Figure A. Figures 1 to 11 As shown.

[0027] This application combines TCGA database analysis with 60 clinical tissue samples (detected by immunohistochemistry, IHC) to evaluate the correlation between eIF4A2 gene expression and clinicopathological features and prognosis of CRC. Western blot (WB) analysis was used to compare eIF4A2 protein levels in normal and CRC cell lines. After siRNA knockdown of the eIF4A2 gene, its effects on cell proliferation, invasion, and apoptosis were detected using CCK-8 assays, Transwell assays, and flow cytometry.

[0028] The application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer was confirmed through analysis of the TCGA database and 60 clinical tissue samples. eIF4A2 promoted the proliferation and invasion of colorectal cancer cells. The expression of the eIF4A2 gene was significantly upregulated in colorectal cancer tissues, and its high expression was closely associated with poor differentiation and poor prognosis (P<0.05). Multivariate Cox regression analysis indicated that the eIF4A2 gene was a prognostic factor (P<0.05) but not an independent risk factor (P>0.05). Western blot analysis showed that the expression of the eIF4A2 gene in various colorectal cancer cell lines was significantly higher than that in normal colonic epithelial cells (P<0.001). Functional experiments showed that knocking down the eIF4A2 gene significantly inhibited the proliferation and invasion of the HCT116 colon cancer cell line (P<0.05).

[0029] 1. Materials and Methods

[0030] 1.1 Materials

[0031] Paraffin-embedded tissue specimens of cancerous and adjacent tissues from 60 patients with pathologically confirmed colorectal cancer (CRC) between June 2019 and January 2021 were collected. Inclusion criteria: 1) Pathologically confirmed CRC, all patients scheduled for CRC surgery; 2) Complete clinical and pathological data; 3) No preoperative radiotherapy or chemotherapy intervention; 4) Postoperative gene testing and IHC; 5) ≥12 lymph nodes detected postoperatively. Exclusion criteria: 1) Carcinoid tumors, appendiceal adenocarcinoma, or familial adenomatous polyposis; 2) Other malignant tumors besides primary CRC; 3) Perioperative mortality. Based on the inclusion and exclusion criteria, the 60 CRC patients included had the following clinical characteristics: 43 patients were <65 years old, and 17 patients were ≥65 years old; 40 were male and 20 were female; tumors were located in the rectum (34 cases), right colon (15 cases), and left colon (11 cases); tumor diameter was <5 cm in 34 cases and ≥5 cm in 26 cases; histological differentiation was low, intermediate, and high in 15, 38, and 7 cases, respectively; T stage was T1–T2 in 14 cases and T3–T4 in 46 cases; lymph node metastasis was positive in 24 cases and negative in 36 cases; distant metastasis was positive in 6 cases and negative in 54 cases; according to the AJCC 8th edition TNM staging: stage I in 7 cases, stage II in 26 cases, stage III in 22 cases, and stage IV in 5 cases. These characteristics are consistent with the general epidemiological distribution of colorectal cancer.

[0032] 1.2 Main Reagents and Instruments

[0033] Key antibodies and reagents included: anti-human eIF4A2 antibody (Boster Biological Technology), immunohistochemistry PV-8000 kit (Zhongshan Jinqiao), Western blotting-related antibodies (Proteintech), CCK-8 kit (MCE), apoptosis detection kit (Yisheng), Lipofectamine™ RNAiMAX transfection reagent (Invitrogen), Matrigel (BD), and various cell culture media (Gibco, Procell). Major instruments included: paraffin microtome (Shanghai Zhixin), microscope (Olympus), gel imaging system (Bio-rad), quantitative PCR instrument (ABI), microplate reader (Thermo), flow cytometer (Beckman CytoFlex S), and a clean bench (Sujing Antai). All cell lines (NCM460, HCT116, SW480, SW620) were purchased from ATCC and routinely cultured at 37℃ and 5% CO2. All siRNA duplexes were purchased from Gemma (Suzhou, China). Among them, Zhongshan Jinqiao refers to Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Yisheng refers to Yisheng Biotechnology (Shanghai) Co., Ltd., Shanghai Zhixin refers to Shanghai Zhixin Instrument Co., Ltd., Sujing Antai refers to Suzhou Sujing Antai Air Technology Co., Ltd., and ATCC refers to the American Center for Type Culture Collection.

[0034] 1.3 Methods

[0035] 1.3.1 Bioinformatics Analysis

[0036] This application grouped 270 colorectal cancer cases from the TCGA database based on the 50th percentile (P50) gene sequencing results, resulting in two subgroups: high eIF4A2 expression and low eIF4A2 expression. Kaplan-Meier survival analysis was used to plot survival curves for each group.

[0037] 1.3.2 Immunohistochemical methods

[0038] 1.3.2.1 Immunohistochemical staining procedure

[0039] After three dewaxing steps with xylene, gradient ethanol hydration, and washing with phosphate-buffered saline (PBS), tissue paraffin sections underwent high-pressure antigen retrieval (citrate buffer, pH 6.0). Endogenous peroxidase activity was blocked with 3% H₂O₂, followed by blocking with 5% goat serum at room temperature for 10 minutes. Diluted eIF4A2 primary antibody (1:1000, Boster Biological Technology) was added and incubated overnight at 4°C. The next day, the sections were washed three times with PBS, and HRP-labeled secondary antibody (PV-8000 kit) was added and incubated at room temperature for 30 minutes. DAB staining, hematoxylin counterstaining, and routine gradient ethanol dehydration and clearing were performed, followed by mounting with neutral resin. Images were then acquired for subsequent analysis.

[0040] 1.3.2.2 Interpretation of Immunohistochemical Results

[0041] Staining results showed that the cell nuclei appeared blue, the cytoplasm of positive expression cells was brownish-yellow, while the cytoplasm of the negative control group showed no staining. Two pathologists independently evaluated the cells under double-blind conditions, randomly selecting five high-power fields (×400). The cytoplasmic staining intensity was scored as follows: 0 points for almost no staining, 1 point for light yellow, 2 points for yellow, and 3 points for brownish-yellow; and the percentage of positive cells was scored as follows: <5% for 0 points, 1 point for 5%-25%, 2 points for 26%-50%, 3 points for 51%-75%, and 4 points for 76%-100%. A semi-quantitative score was calculated by multiplying the two scores. A comprehensive score of ≥8 points was considered eIF4A2 positive expression, and <8 points was considered negative expression.

[0042] 1.3.3 Western blot detection of eIF4A2 gene protein expression

[0043] 1.3.3.1 Cell Culture and Treatment

[0044] Normal colonic epithelial cells NCM460, colorectal orthotopic carcinoma lines HCT116 and SW480, and colonic metastatic carcinoma line SW620 (all purchased from ATCC) were used in the experiments. Cells were routinely cultured in medium containing 40% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (NCM460 in McCoy's 5A medium, HCT116 in DMEM medium, and SW480 and SW620 in 1640 medium) at 37°C and 5% CO2. After cell resuscitation, when cell confluence reached 80%-90%, cells were digested with 0.25% trypsin (TrypLE digestion solution) and passaged. All experiments were performed during the logarithmic growth phase. Cells in the logarithmic growth phase were collected by digestion and centrifugation. Resuspend cells in cell cryopreservation solution (50% complete culture medium, 40% FBS, 10% dimethyl sulfoxide DMSO) and adjust the density to 5 × 10⁻⁶ cells / mL. 6 / mL to 1×10 7 / mL. Aliquot into cryovials, freeze using a programmed cooling method, and finally transfer to liquid nitrogen for long-term storage.

[0045] 1.3.3.2 Western Blot detection of eIF4A2 protein expression in colon cancer cell lines and tissues

[0046] First, cellular protein samples were prepared [separating gel (concentration determined according to protein molecular weight) and stacking gel (5%)]. Separation was performed by SDS-PAGE (80V / 120V constant voltage), followed by wet transfer to a PVDF membrane (200-250 mA constant current, 60-90 min). After blocking with 5% skim milk powder for 2 hours, the samples were sequentially incubated with primary antibody (anti-eIF4A2, 1:1000, 4°C overnight) and HRP-labeled secondary antibody (1:5000, room temperature for 1 hour), and thoroughly washed with TBST. Finally, ECL chemiluminescence was used for development, and protein expression levels were analyzed using β-actin or GAPDH as internal controls. All experiments were independently repeated three times.

[0047] Fresh surgical specimens were washed with phosphate-buffered saline (PBS) within 30 minutes, mechanically minced, and homogenized with lysis buffer. The supernatant was collected by centrifugation to obtain the total protein extract. After quantification, the protein sample was mixed with loading buffer (1:2), denatured at 100°C for 3-5 minutes, centrifuged again for 10 minutes, and finally transferred to a new test tube for later use. The reagent preparation and operation procedure for Western blotting experiments are the same as described above.

[0048] 1.3.4 Constructing an eIF4A2 knockdown HCT116 cell model

[0049] 1.3.4.1 Cell Culture and Plating

[0050] HCT116 cells were routinely cultured in McCoy's 5A medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO2. Cells in the logarithmic growth phase were harvested, trypsinized, and counted, at a density of 8 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 24-well plates and cultured overnight until cell confluence reached 60 to 70%.

[0051] 1.3.4.2 siRNA transfection

[0052] siRNA sequences: The non-target control siRNA (siNegative) sequence is: 5'-UUCUCCGAACGUGUCACGUTT-3' (positive strand). The EIF4A2-targeting siRNA (siEIF4A2) sequence is: 5'-GAGCUAUUAUUCCCUGUAUTT-3' (positive strand).

[0053] Transfection procedure: Dilute 20 pmol siRNA and 1.5 μL Lipofectamine™ RNAiMAX separately in 50 μL Opti-MEM, let stand for 5 minutes, mix, and incubate at room temperature for 20 minutes to form a complex. Add the complex to the cells, incubate at 37°C for 6 hours, then replace with complete culture medium and continue culturing for 48 hours for subsequent detection.

[0054] a) After overnight plating, cells were transfected using siRNA. The specific procedure is as follows: b) Mix the calculated volume of siRNA with an appropriate amount of serum-free culture medium and let stand for 5 minutes; c) Simultaneously, mix an equal volume of Lipofectamine™ RNAiMAX transfection reagent with the same volume of serum-free culture medium and let stand for 5 minutes; then add the solution obtained in step a to the mixture in step b and continue incubation for 20 minutes; d) Add the prepared transfection complex evenly to the cell culture system and incubate at 37°C with 5% CO2 for 6 hours. Then replace with fresh complete culture medium and continue culturing under the same conditions for 48 hours. Finally, collect cell samples for subsequent experimental analysis.

[0055] 1.3.4.3 Real-time quantitative PCR (qPCR) verification of knockdown efficiency

[0056] Total RNA was extracted from cells using the Trizol method, and its concentration was determined by Nanodrop and its integrity was verified by 1% agarose gel electrophoresis. 1 µg of RNA was reverse transcribed into cDNA using the PrimeScript™ RT kit. Using the cDNA as a template, qPCR amplification was performed on a QuantStudio 5 system using the SYBR Green method. The reaction volume was 20 µL: 10 µL SYBR Premix Ex Taq™, 0.8 µL each of forward and reverse primers, and 2 µL cDNA template. The reaction program was: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. EIF4A2 primer sequences are detailed in Table 1; GAPDH primer sequences are also detailed in Table 1. The relative expression level of EIF4A2 mRNA was calculated using the 2−ΔΔCt method, with the transfected negative control siRNA (siNC) group serving as a calibration control. Each experiment was independently repeated three times.

[0057] 1.3.4.3.1 Primer Information

[0058] Table 1 Primer Information

[0059] 1.3.4.3.2 Preparation of siRNA cell lines

[0060] Table 2 siRNA cell lines

[0061]

[0062] 1.3.4.3.3 RT Experiment Information

[0063] Table 3 RT Experiment Information

[0064]

[0065] 1.3.4.4 Western blot verification of protein expression

[0066] Protein samples were mixed with SDS loading buffer and denatured at 99°C for 5 minutes. An equal volume of the denatured protein sample was then separated by electrophoresis using a 10% SDS-PAGE gel (stacking gel 60-80V, separating gel 80-120V). The proteins were then transferred to a PVDF membrane via wet transfer (200 mA constant current, 90 minutes). The transferred PVDF membrane was blocked with TBST containing 5% skim milk at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4°C. Primary antibodies included anti-eIF4A2 (1:1000) and internal control anti-β-actin (1:5000), both diluted with TBST containing 5% skim milk. After washing with TBST, the membrane was reacted with HRP-labeled secondary antibody (1:5000, incubated at room temperature for 45 minutes) and washed again. Development was performed using ECL chemiluminescence substrate, and signals were acquired on a chemiluminescence imaging system. All experiments were independently repeated three times.

[0067] 1.3.5 Transwell cell invasion assay

[0068] Cell invasion ability was assessed using Transwell chambers (8 μm pores) coated with matrix gel. Diluted matrix gel was spread onto the upper chamber of each chamber and polymerized at 37°C for 2 hours, after which excess liquid was removed. Cells in the logarithmic growth phase were harvested, digested with trypsin, washed with phosphate-buffered saline (PBS), resuspended in serum-free medium, and the cell density was adjusted to 6.5 × 10⁻⁶ cells / mL. 4 Add 200 μL of cell suspension (containing 1.3 × 10⁻⁶ cells / mL) to the upper chamber. 4 Cells were cultured at 37°C and 5% CO2 for 48 hours. The cells were then removed from the chamber, washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.1% crystal violet for 10 minutes. Uninvaded cells in the upper chamber were gently wiped away with a cotton swab. Three fields of view were randomly selected under an inverted microscope for photographing and counting of invading cells. Each group was tested in triplicate, and the experiment was repeated three times independently. 600 μL of complete culture medium containing 10% FBS was added to the lower chamber as a chemokine. After culturing the cells at 37°C and 5% CO2 for 48 hours, the chamber was removed, washed with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.1% crystal violet for 10 minutes.

[0069] 1.3.6 CCK-8 assay for cell proliferation

[0070] Cells were cultured at 37°C and 5% CO2 for 24, 48, and 72 hours, respectively. Then, 10 µL of CCK-8 reagent was added to each well, and incubation continued for 3 hours. The absorbance (OD450) at 450 nm was measured using a microplate reader. Cell growth curves were plotted with culture time on the x-axis and the mean OD450 value on the y-axis. The experiment was independently repeated three times.

[0071] 1.3.7 Flow cytometry detection of apoptosis

[0072] Cells from each group were collected, washed with PBS, and resuspended in 100 μL of 1×Binding Buffer. 5 μL of Annexin V-Alexa Fluor 647 and 10 μL of propidium iodide (PI) were added sequentially, and the cells were incubated at room temperature in the dark for 15 minutes. After the reaction was complete, 150 μL of 1×Binding Buffer was added, and the cells were analyzed by flow cytometry within 1 hour. Annexin V⁺ PI⁻ cells were identified as early apoptotic cells, and Annexin V⁺ PI⁺ cells were identified as late apoptotic / necrotic cells. The proportion of apoptotic cells was analyzed using FlowJo software. Each group was divided into three replicates, and the experiment was independently repeated three times.

[0073] 1.4. Statistical Methods

[0074] Data analysis was performed using SPSS 24 (Statistical Product and Service Solutions) software, and the data were categorized and organized according to clinical indicators such as age, gender, TNM stage, and tumor location. The correlation between eIF4A2 and clinical parameters was validated using the chi-square test or Fisher's exact test, and the survival status of colorectal cancer patients was analyzed using the Kaplan-Meier method. To explore independent factors influencing the prognosis of colorectal cancer, Cox univariate and multivariate regression models were used for screening. Intergroup differences were compared using unpaired t-tests, and a p-value less than 0.05 was considered statistically significant. Western blot image analysis was performed using ImageJ 1.54a software, and flow cytometry data processing was performed using FlowJo™ Software v10.9. All graphs were plotted using Prism 9.0 software.

[0075] 2. Based on the above experimental verification, the following conclusions can be drawn.

[0076] 2.1 eIF4A2 is highly expressed in colorectal cancer and is associated with poor patient prognosis.

[0077] 2.1.1 Survival Analysis

[0078] This application grouped 270 colorectal cancer cases from the TCGA database based on the 50th percentile (P50) gene sequencing results, resulting in two subgroups: high eIF4A2 expression and low eIF4A2 expression. Survival curves for each group were plotted using Kaplan-Meier survival analysis (e.g.,...). Figure 1The results showed that differential expression of eIF4A2 in colorectal cancer was associated with poor prognosis. The difference was statistically significant (P < 0.05).

[0079] Figure 1 In the diagram: n(high)=135 and n(low)=135 represent the sample size of the high expression group and the low expression group, respectively, with 135 samples in each group; HR(high) is the hazard ratio of the high expression group; p(HR) is the p-value of the hazard ratio, indicating that the hazard ratio is statistically significant; Logrank p is used to compare whether there is a significant difference between the survival curves of the two groups; p < 0.05 indicates that the difference is statistically significant.

[0080] 2.1.2 eIF4A2 is highly expressed in colorectal cancer

[0081] Due to database updates, this application uses expression level data from 286 colorectal cancer cases (270 samples before the update) and 41 normal cases obtained from TCGA (e.g., Figure 2 The results showed that eIF4A2 was highly expressed in colorectal cancer tissues compared with normal tissues (P<0.05).

[0082] Figure 2 In the middle: the vertical axis represents the value of expression per million units (TPM) transformed by log(TPM+1) to stabilize variance and facilitate statistical analysis. * indicates P<0.05.

[0083] 2.2 Immunohistochemical detection showed that the expression of eIF4A2 protein differed between colorectal cancer and adjacent non-cancerous tissues.

[0084] IHC analysis showed significant tissue-specific differences in eIF4A2 expression among 60 colorectal cancer specimens. Obvious brownish-yellow granules were observed in cancer tissue sections; this protein was mainly located in the cytoplasm and showed diffuse expression. In contrast, adjacent normal tissues showed pale yellow or colorless staining, and the protein was also mainly distributed in the cytoplasm (e.g., ...). Figure 3 Quantitative analysis based on established scoring criteria revealed that the positive expression rate of eIF4A2 in colorectal cancer tissues was significantly higher than that in adjacent normal tissues, specifically 55.0% (33 / 60) and 13.3% (8 / 60), respectively. This difference in expression further confirms the specific upregulation of eIF4A2 in colorectal cancer.

[0085] Table 4. Differences in eIF4A2 protein expression between cancerous and adjacent normal tissues.

[0086]

[0087] In Table 4: ***Expression P < 0.001, the difference was statistically significant; Ca group was the cancer group; Pa group was the adjacent normal group.

[0088] Figure 3 A. A1. eIF4A2 is positively expressed in cancer tissue; B. B1. eIF4A2 is negatively expressed in cancer tissue; C. C1. eIF4A2 is positively expressed in adjacent tissue; D. D1. eIF4A2 is negatively expressed in adjacent tissue; E. Scatter plot comparing the expression of eIF4A2 in cancer and adjacent tissue.

[0089] 2.3 Relationship between eIF4A2 protein tissue expression and clinicopathological features of colorectal cancer

[0090] Based on immunohistochemical detection of eIF4A2 protein expression in colon cancer samples, subjects were divided into positive and negative groups using a pre-defined scoring system. Statistical analysis revealed a significant correlation between eIF4A2 expression and patient survival and tumor differentiation (Table 5), but no statistical significance with demographic characteristics such as age and sex. Furthermore, clinicopathological parameters such as tumor invasion depth, lymph node metastasis, distant metastasis, TNM stage, lesion size, anatomical location, vascular invasion, nerve invasion, and microsatellite instability did not show a correlation with eIF4A2 expression levels.

[0091] Table 5. Correlation between eIF4A2 protein expression levels and clinical data

[0092]

[0093] Continued from Table 5

[0094]

[0095] Table 5 shows that, according to the American Joint Committee on Cancer (AJCC) 8th edition guidelines, tumor staging is assessed using the TNM system. At the molecular pathology level, mismatch repair function is detected using the MMR (Mismatch Repair) index, where dMMR (Mismatch Repair Deficient) indicates a lack of repair function, and pMMR (Proficient Mismatch Repair) indicates intact repair function. In the statistical analysis, * indicates a significance level (P-value less than 0.05), and ** indicates a P-value less than 0.01, both of which are statistically significant.

[0096] 3. Survival Analysis

[0097] 3.1 Using the Kaplan-Meier method to plot survival curves

[0098] Follow-up data were primarily collected via telephone, and the 3-year survival rate of the 60 patients was statistically estimated at 85%. Based on eIF4A2 protein expression levels, the study subjects were divided into two subgroups: high expression and low expression. Survival curves were constructed using the Kaplan-Meier method. Figure 4 The results showed that there was a significant difference in survival time between the positive and negative expression groups of eIF4A2, and the difference was statistically significant (P<0.05).

[0099] Figure 4 In the diagram: n(high)=33 and n(low)=27 represent the sample size of the high expression group and the low expression group, respectively. There are 33 samples in the high expression group and 27 samples in the low expression group. Logrank p is used to compare whether there is a significant difference between the survival curves of the two groups. p<0.05 indicates that the difference is statistically significant.

[0100] 3.2 Cox regression model analysis of prognostic factors

[0101] This application used a Cox proportional hazards model to explore prognostic factors in colorectal cancer patients (results are shown in Table 6). Univariate analysis showed that elevated eIF4A2 expression, differentiation grade, lymph node metastasis, distant metastasis, and TNM stage were significantly correlated with patient prognosis, while other factors did not show statistical significance. In multivariate Cox regression analysis, tumor differentiation grade and distant metastasis were statistically significant.

[0102] Table 6. Cox regression analysis of prognosis in 60 cases of colorectal cancer

[0103]

[0104] In Table 6: * indicates a significance level P-value less than 0.05, and ** indicates a P-value less than 0.01, both of which are statistically significant.

[0105] 4. Western blot analysis of eIF4A2 protein expression in colorectal cancer and adjacent normal tissues.

[0106] This application collected cancer and adjacent normal tissues from four patients and used Western blot to detect the expression of eIF4A2 in colorectal cancer histological lineages and adjacent normal tissues. Gray-scale analysis showed that in groups Y and B, Pa expression was lower than Ca expression, while in group S, Ca expression was slightly higher than Pa expression (e.g., ...). Figure 5 ).

[0107] Western blotting (WB) was used to quantitatively analyze the expression levels of eIF4A2 mRNA transcripts and corresponding proteins in the NCM460 normal colonic mucosal epithelial cell line and three colon cancer cell lines: HCT116, SW620, and RSW480. Figure 6 Experimental data showed that, compared to the NCM460 cell line, the HCT116, SW620, and SW480 cancer cell lines all exhibited significantly high expression of the eIF4A2 protein. Because HCT116 cells are easily transfected with viral vectors, and based on the above findings, this application selected the HCT116 cell line as the main research object for subsequent experiments.

[0108] Figure 5 In the diagram: Figure A shows the protein expression level; Figure B is a bar chart of protein expression grayscale values; Figure C is a statistical analysis chart of grayscale values, where Y-1, B-1, and S-1 represent adjacent normal tissue; and Y-2, B-2, and S-2 represent cancerous tissue.

[0109] Figure 6 Figure A shows the eIF4A2 protein expression levels in different cell lines; Figure B is a bar chart of statistical analysis after multiple measurements of protein expression levels; Ncm460: normal colonic epithelial cell line; HCT116: colonic in situ carcinoma cell line; SW480: rectal in situ carcinoma cell line; SW620: colonic metastatic carcinoma cell line.

[0110] 5. HCT116 Transfection Efficiency Detection

[0111] A cell model of eIF4A2 gene repression was established using small interfering RNA (siRNA) technology. After 72 hours of siRNA transfection, cell morphology was observed using an optical microscope. Compared with the control group transfected with empty vector, the cell proliferation rate of the experimental group transfected with Si eIF4A2 was significantly slowed (see...). Figure 7 This phenomenon confirms the effectiveness of siRNA transfection.

[0112] Further research results indicate that knockdown of HCT116 significantly decreased at the mRNA level and slightly decreased at the protein level (e.g., Figure 8 SI1, SI2, and SI3 are three groups of HCT116 cells that were transfected with Si eIF4A2. Figure 8 -B in SI refers to the HCT116 cell grouping after integrating the above 3 groups.

[0113] Figure 7 In the diagram: A, B, and C are HCT116 cells without siRNA; A1, B1, and C1 are cells transfected with the empty vector, serving as the transfected cell control group; A2, B2, and C2 are cells transfected with Si eIF4A2, serving as the transfected cell experimental group.

[0114] Figure 8: A and B show the grayscale analysis and WB results of HCT116 protein expression after eIF4A2 knockdown (SI in Figure B represents the HCT116 cell grouping after integrating the above 3 groups); C shows the expression level of HCT116 cell mRNA after eIF4A2 knockdown, NC represents cells transfected with empty vector; SI, SI-1, SI-2, and SI-3 represent cells transfected with Si eIF4A2.

[0115] 6. Effects of eIF4A2 knockdown on cell proliferation

[0116] This application used the CCK8 reagent kit to measure the absorbance of the experimental group and the control group at different time points, and plotted the proliferation kinetic curves accordingly (see...). Figure 9 The experimental results showed that, compared with the SI-NC group, SI-eIF4A2 did not show significant changes in proliferation 24 hours after transfection; however, when the transfection time was extended to 48 hours and 72 hours, the proliferation level of this experimental group was significantly reduced, and statistical analysis confirmed that the difference was significant (P<0.05). The above experimental data indicate that downregulation of eIF4A2 gene expression can effectively inhibit the proliferation activity of HCT116 cells.

[0117] Figure 9 In the diagram: OD value: absorbance, reflecting the number of cells; the X-axis represents the growth time of cancer cells, and the Y-axis represents the OD value.

[0118] 7. Knocking down eIF4A2 can inhibit the invasive ability of HCT116 cells.

[0119] The Transwell invasion assay was used to evaluate the regulatory effect of eIF4A2 gene knockdown on the invasive ability of HCT116 cells. Experimental data showed that, compared with the untreated group, the number of cells crossing the matrix gel in the eIF4A2 knockout group was significantly reduced (P<0.05). Figure 10 As shown in the figure. Based on the above experimental results, it can be concluded that downregulation of eIF4A2 expression can effectively inhibit the invasive ability of colon cancer cells.

[0120] Figure 10 In the diagram: A, B, and C represent three groups of samples transfected with si-NC; A1, B1, and C1 represent three groups of samples transfected with si-eIF4A2; D is a bar chart for cell invasion detection analysis.

[0121] 8. Effect of eIF4A2 knockdown on HCT116 apoptosis

[0122] In HCT116 cells subjected to flow cytometry apoptosis detection, 48 h after transfection, there was no statistically significant difference between the SI-eIF4A2 group and the SI-NC group (P > 0.05). Therefore, this application found that knockdown of eIF4A2 had no significant effect on cell apoptosis (e.g., Figure 11 ).

[0123] Figure 11 In Chinese: Annexin V: commonly used for early apoptotic cells; PI: Propidium Iodide is used to label cell nuclei.

[0124] Other embodiments of this application will readily occur to those skilled in the art upon consideration of the specification and practice of the application disclosed herein. This application is intended to cover any variations, uses, or adaptations of this application that follow the general principles of this application and incorporate common knowledge or customary techniques in the art disclosed herein. The specification and examples are to be considered exemplary only, and the true scope of this application is indicated by the claims.

[0125] It should be understood that this application is not limited to the precise structure described above and shown in the accompanying drawings, and various modifications and changes can be made without departing from its scope. The embodiments of this application described above do not constitute a limitation on the scope of protection of this application.

Claims

1. The application of the eIF4A2 gene as a potential prognostic biomarker for colorectal cancer, characterized in that, The eIF4A2 gene can promote the proliferation and invasion of colorectal cancer cells.

2. The application of the eIF4A2 gene as a potential prognostic biomarker for colorectal cancer according to claim 1, characterized in that, The eIF4A2 gene is highly expressed in colorectal cancer, and its high expression level is associated with poor differentiation and poor prognosis in patients.

3. The application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer according to claim 1, characterized in that, The eIF4A2 gene is expressed significantly higher in various colorectal cancer cell lines than in normal colonic epithelial cells.

4. The application of the eIF4A2 gene as a potential prognostic biomarker for colorectal cancer according to claim 1, characterized in that, The positive expression rate of the eIF4A2 gene in colorectal cancer tissues is significantly higher than that in adjacent normal tissues.

5. The application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer according to claim 1, characterized in that, Knocking down the eIF4A2 gene can significantly inhibit the proliferation and invasion of the colon cancer cell line HCT116.

6. The application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer according to claim 5, characterized in that, Downregulation of eIF4A2 gene expression can effectively inhibit the proliferation of HCT116 cells.

7. The application of the eIF4A2 gene as a potential prognostic marker for colorectal cancer according to claim 5, characterized in that, Downregulation of eIF4A2 gene expression can effectively inhibit the cell invasion ability of HCT116 cells.

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