Application of KCNJ15 as a target in auxiliary diagnosis, prognosis and treatment drug screening of colorectal cancer

Abstract

This invention belongs to the field of biomedical technology and provides the application of the gene KCNJ15 as a target in the auxiliary diagnosis, prognosis assessment, and therapeutic drug screening of colorectal cancer. The invention also includes the application of reagents for detecting the expression level of the target gene KCNJ15 in the preparation of diagnostic reagents for colorectal cancer, wherein KCNJ15 is expressed at low levels in colorectal cancer cells and tissues. Low expression of KCNJ15 in colorectal cancer tissues is associated with poor prognosis. Furthermore, overexpression of KCNJ15 inhibits the proliferation, migration, invasion, and EMT process of cancer cell lines. Notably, KCNJ15 overexpression inhibits the EMT process through the PI3K / AKT / mTOR signaling pathway. KCNJ15's inhibition of colorectal cancer migration, invasion, and EMT through the PI3K / AKT / mTOR signaling pathway suggests its significant potential as a novel therapeutic target for colorectal cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically involving the application of gene KCNJ15 as a target in the auxiliary diagnosis, prognosis assessment and screening of therapeutic drugs for colorectal cancer. Background Technology

[0002] Colorectal cancer (CRC) is the third most common malignant tumor worldwide, and its cancer-related mortality rate is second only to lung cancer. Current treatment strategies for CRC include chemotherapy based on cytotoxic drugs, targeted therapy against vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR), and immunotherapy based on the tumor immune microenvironment. Early diagnosis and optimized treatment strategies have improved the prognosis of CRC, but targeted therapy still faces many challenges, such as high drug resistance after long-term use, insufficient drug specificity, and a limited variety of available drugs. Therefore, a deeper understanding of the molecular mechanisms of CRC occurrence and progression is of great significance for promoting innovation and progress in treatment strategies.

[0003] The KCNJ15 gene is located on human chromosome 21q22.13, and it encodes the Kir4.2 channel, which is widely distributed in human brain tissue, kidneys, and pancreas. The Kir4.2 inward rectifier potassium channel encoded by the KCNJ15 gene is a potassium-dependent activation channel composed of four subunits. Its structural features include two transmembrane domains (M1 and M2) and a pore (P) region composed of a highly conserved H5 segment in each subunit. This channel functions as a potassium sensor in the kidney, maintaining physiological potassium levels. + Balance. Studies have shown that aberrant expression of KCNJ15 is closely related to the development and progression of various tumors. In esophageal squamous cell carcinoma (ESCC), high expression of KCNJ15 is associated with poor prognosis and may promote tumor invasion through the PI3K / Akt signaling pathway, participating in the epithelial-mesenchymal transition (EMT) process. In renal cell carcinoma, KCNJ15 expression is absent, inhibiting tumor metastasis by suppressing EMT and MMP-7 expression. Notably, KCNJ15 promotes chemotherapy resistance in breast cancer by affecting lysosomal function. KCNJ15 plays an important role in maintaining cellular homeostasis and participating in tumorigenesis. Currently, the expression and distribution changes of KCNJ15 in CRC have not been explored, and its mechanism of action in CRC progression remains unclear.

[0004] EMT refers to the biological process by which epithelial cells transform into mesenchymal cells through a specific procedure, playing a crucial role in physiological and pathological processes such as embryonic development, tissue repair, chronic inflammation, and cancer metastasis. In tumorigenesis, EMT not only endows tumor cells with the ability to migrate and invade but also interacts with immune regulation within the tumor microenvironment. The PI3K / AKT / mTOR signaling pathway, as an important regulatory pathway in cancer development and progression, functions by promoting cell proliferation, angiogenesis, and mediating chemotherapy resistance. Recent studies have shown that activation of the PI3K / AKT / mTOR pathway can promote EMT. Summary of the Invention

[0005] This invention provides the application of gene KCNJ15 as a target in the auxiliary diagnosis, prognosis assessment and screening of therapeutic drugs for colorectal cancer.

[0006] The present invention is achieved by the following technical solution: the application of a reagent for detecting the expression level of the target gene KCNJ15 in the preparation of a reagent for diagnosing colorectal cancer, wherein KCNJ15 is expressed at low levels in colorectal cancer cells and tissues.

[0007] Furthermore, the KCNJ15 is expressed at low levels in colorectal cancer cells that promote proliferation, migration, or invasion, as well as the EMT process.

[0008] This invention also provides the application of gene KCNJ15 as a target in the preparation of colorectal cancer cell metastasis detection reagents and drug screening.

[0009] The gene KCNJ15 is used as a target in screening drugs to inhibit or slow down the proliferation, migration, and invasion of colorectal cancer cells.

[0010] The overexpression of KCNJ15 inhibits or slows down the proliferation, migration, and invasion of colorectal cancer cells, and suppresses the EMT process.

[0011] Overexpression of KCNJ15 upregulated the expression of the epithelial marker E-cadherin, while downregulating the expression of the mesenchymal marker N-cadherin and EMT-related transcription factors Snail and ZEB1.

[0012] Overexpression of KCNJ15 significantly downregulated the phosphorylation levels of PI3K, AKT, and mTOR, and significantly reduced the activation level of the PI3K / AKT / mTOR signaling pathway.

[0013] The drugs that inhibit or slow down the proliferation, migration, and invasion of colorectal cancer cells are drugs that significantly upregulate or promote the overexpression of KCNJ15, or drugs that significantly downregulate the phosphorylation levels of PI3K, AKT, and mTOR, or drugs that significantly reduce the activation level of the PI3K / AKT / mTOR signaling pathway.

[0014] The drug is a specific PI3K inhibitor.

[0015] This invention reports for the first time that KCNJ15 expression in colorectal cancer is lower than in normal intestinal epithelial tissue, and that its expression level is closely related to histological grade, lymphovascular invasion, and prognosis. Cellular function experiments confirmed that KCNJ15 overexpression inhibits the proliferation, migration, and invasion of colorectal cancer cell lines. These results indicate that KCNJ15 affects the progression of colorectal cancer by regulating the biological behavior of tumor cells.

[0016] This invention confirms that KCNJ15 inhibits EMT in colorectal cancer by regulating the PI3K / AKT / mTOR pathway, suggesting its function as a tumor suppressor gene. KCNJ15 inhibits CRC migration, invasion, and EMT by regulating the PI3K / AKT / mTOR signaling pathway. This discovery not only deepens our understanding of the function of KCNJ15 in cancer but also provides a new potential target for targeted therapy of CRC. In the future, the development of small molecule agonists targeting KCNJ15 is expected to become a new direction for precision treatment of CRC.

[0017] KCNJ15 is expressed at low levels in colorectal cancer tissues and is associated with poor prognosis. Furthermore, KCNJ15 overexpression inhibits the proliferation, migration, invasion, and EMT process of cancer cell lines. Notably, KCNJ15 overexpression inhibits EMT through the PI3K / AKT / mTOR signaling pathway. KCNJ15's inhibition of colorectal cancer migration, invasion, and EMT via the PI3K / AKT / mTOR signaling pathway suggests its significant potential as a novel therapeutic target for colorectal cancer. Attached Figure Description

[0018] Figure 1 The expression levels and survival curves of KCNJ15 in colorectal cancer tissues and cancer cell lines are shown. (A, B) Representative images of KCNJ15 immunohistochemical analysis in clinical samples, A indicates high expression, B indicates low expression; magnification: 40×, 200×; (C, D) Expression levels of KCNJ15 in colorectal cancer tissues and normal epithelial tissues; (E, F) Expression levels of KCNJ15 in colorectal cancer cell lines and normal epithelial cells analyzed by Western blotting and qRT-PCR; (G, H) Survival curves of KCNJ15 in colorectal cancer. P <0.05、 P <0.01、 P <0.001 and P <0.0001; Figure 2 KCNJ15 was used to inhibit the proliferation, migration, invasion, and EMT of colorectal cancer cells. (A) CCK8 assay to verify the effect of KCNJ15 on cell proliferation; (B) Clonogenic assay to verify the effect of KCNJ15 on cell proliferation; (C) Wound healing assay to verify the effect of KCNJ15 on cell migration; (D) Transwell assay to verify the effect of KCNJ15 on cell migration and invasion; (E) Western blot and qRT-PCR were used to detect the levels of E-cadherin, N-cadherin, Snail, and ZEB1 to verify the effect of KCNJ15 on the EMT process. P <0.05、 P <0.01、 P <0.001 and P <0.0001; Figure 3 To investigate the effect of KCNJ15 on EMT in colorectal cancer via the PI3K / AKT / mTOR pathway. (A) The expression levels of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR were detected by Western blot to verify the effect of KCNJ15 on the PI3K / AKT / mTOR pathway; (B) The levels of PI3K / AKT / mTOR and EMT markers in KCNJ15 overexpressing cell lines after 740-YP treatment were detected by Western blot; (C) The levels of PI3K / AKT / mTOR and EMT markers in KCNJ15 low-expressing cell lines after LY294002 treatment were detected by Western blot. P <0.05、 P <0.01、 P <0.001 and P <0.0001; Figure 4KCNJ15 inhibits the proliferation, migration, and invasion of colorectal cancer by suppressing the PI3K / AKT / mTOR-mediated EMT process. (A) CCK8 assay to detect changes in cell proliferation after exposure to 740-YP and LY294002; (B) Clonogenic assay to detect changes in cell proliferation after exposure to 740-YP and LY294002; (C) Wound healing assay to detect changes in cell migration after exposure to 740-YP and LY294002; (D) Transwell assay to detect changes in cell migration and invasion after exposure to 740-YP and LY294002. P <0.05、 P <0.01、 P <0.001 and P <0.0001; Figure 5 To investigate the effect of KCNJ15 on tumor cell proliferation in vivo. (A) After subcutaneous inoculation of nude mice with SW620-OE-KCNJ15 cells and SW620-NC cells, the effect of KCNJ15 on tumor growth in vivo was observed; (B) Tumor volume was measured in the OE-KCNJ15 group and the NC group; (C) Tumor weight was measured in the OE-KCNJ15 group and the NC group. P <0.05、 P <0.01、 P <0.001 and P <0.0001; Figure 6 Western blot (WB) and quantitative real-time PCR (qRT-PCR) were used to detect cell transfection efficiency; in the figure: (a) transfection efficiency was detected by WB; (b) transfection efficiency was detected by qRT-PCR. P<0.05, P<0.01, P<0.001, P<0.0001; Figure 7 For KCNJ15 gene functional enrichment analysis; Figure: (a) KCNJ15 gene interaction network constructed using GeneMania database; (b) KCNJ15 protein interaction network constructed using STRING database; (c) Bubble chart showing GO and KEGG enrichment analysis results of 27 KCNJ15 associated genes. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0021] Equivalent technologies of the specific embodiments described herein that are apparent to those skilled in the art through routine experimentation are included in this application.

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0023] I. Materials and Methods 1. Patient tissue samples: This invention included 116 patients diagnosed with CRC at Shanxi Cancer Hospital between January 2017 and December 2018. The inclusion criteria were as follows: (1) pathologically confirmed diagnosis of CRC; (2) complete clinicopathological data; (3) radical surgical treatment. The exclusion criteria were: (1) preoperative antitumor treatment; (2) other tumors; (3) incomplete clinical information and follow-up data. Paraffin-embedded tissues of cancerous and normal tissues were collected from 116 patients. This invention follows the Declaration of Helsinki. Informed consent was obtained for all tissue samples and data before inclusion in the study, and the data was approved by the Ethics Committee of Shanxi Cancer Hospital (No.: KY2023005).

[0024] 2. Immunohistochemical staining (IHC): Paraffin-embedded tissue was cut into 3 μm thick sections and incubated at 60°C for 10-15 min, followed by cooling at 4°C. Incubation with KCNJ15 (1:500; 15988-1-AP; Wuhan Sanying Biotechnology Co., Ltd.) antibody was performed overnight at 4°C. Incubation with horseradish peroxidase-labeled goat anti-mouse secondary antibody (Shanghai Roche Pharmaceuticals Co., Ltd.) was carried out at 37°C for 30 min, followed by staining with 3,3′-diaminobenzidine (DAB, Shanghai Roche Pharmaceuticals Co., Ltd.) at room temperature for 2 min. Staining was performed using an automated immunohistochemistry bench (Shanghai Roche Pharmaceuticals Co., Ltd.).

[0025] 3. Cell Culture: NCM460, HCT-116, and HT-29 cell lines were purchased from Wuhan Sanying Biotechnology Co., Ltd.; T84, RKO, and SW620 cell lines were purchased from Jiangsu Kaiji Biotechnology Co., Ltd. The culture conditions for SW620 were L-15 + 10% FBS; for HT-29 and HCT-116, McCoy's 5A + 10% FBS; for T84, DMEM / F12 + 10% FBS; for RKO, MEM + 10% FBS + 1% NEAA; and for NCM460, RMPI-1640 + 10% FBS. Cells were cultured in a humidified incubator at 37℃ and 5% CO2.

[0026] 4. Western Blot (WB): Proteins were extracted from CRC cells at 4°C using RIPA buffer (KGI Biotech Co., Ltd., Jiangsu, China) containing protease and phosphatase inhibitors. After sonication, the samples were incubated on ice for 30 min, followed by centrifugation to collect the supernatant. Protein concentration was determined using a BCA protein assay kit (KGI Biotech Co., Ltd., Jiangsu, China). The total protein fraction was separated by SDS-PAGE (10%), transferred to a PVDF membrane, blocked with 5% skim milk powder, and incubated overnight at 4°C with primary antibody. Finally, the membrane, washed with TBST, was incubated with secondary antibody, and the proteins were visualized using an ECL kit (KGI Biotech Co., Ltd., Jiangsu, China). The grayscale values ​​of the images were analyzed using ImageJ software. Detailed antibody information is shown in Table 1.

[0027] Table 1: Antibody Information List 5. Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR): Total RNA was extracted using the TRIzol kit (KGI Biotechnology Co., Ltd., Jiangsu, China). cDNA synthesis and real-time quantitative PCR were performed using the cDNA reverse transcription kit (TaKaRa, Japan) and TB Green® Premix Ex Taq™ II (Tli RNaseH Plus, TaKaRa, Japan). Primer sequences for the target gene and reference gene are shown in Table 2.

[0028] Table 2: Primer sequences for real-time quantitative PCR Lentiviral transfection: KCNJ15 gene overexpression and knockdown were achieved using KCNJ15 overexpression lentivirus and KCNJ15 short hairpin RNAs (sh-KCNJ15-1, sh-KCNJ15-2, and sh-KCNJ15-3) provided by Beijing Qingke Biotechnology Co., Ltd. Overexpression cells and the control group were designated OE-KCNJ15 and NC, while KCNJ15 knockdown cells and the control group were designated KD-KCNJ15 and NC. After culturing cells in 6-well plates for 12 h, 20 μL of viral working solution (1×10⁻⁶) was added to each well. 8 Transfection was performed using TU / mL and Polybrene (5 μg / mL). After 24 hours, the medium was replaced with fresh medium and cultured for another 72 hours. Transfection efficiency was detected by qRT-PCR and Western blot. The specific KCNJ15 short hairpin RNA (shRNA) sequence is shown in Table 3.

[0029] Table 3: KCNJ15-shRNA-1, 2, and 3 sequences 6. Rescue experiment: SW620 negative control cell line (SW620-NC group) and SW620 KCNJ15 overexpressing cell line (SW620-OE-KCNJ15 group) were treated with 740-YP; HT-29 negative control cell line (HT-29-NC group) and HT-29 KCNJ15 knockdown cell line (HT-29-KD-KCNJ15 group) were treated with LY294002. The specific treatment methods are shown in Table 4.

[0030] Table 4: Drug administration methods 7. Cell Counting Kit-8 (CCK-8) assay: CRC cells were counted at 4 × 10⁶ cells per well. 3 Cells were seeded into 96-well plates and cultured for 24 hours. Then, 10 μL of CCK-8 reagent (KGI Biotechnology Co., Ltd., Jiangsu, China) was added to each well. After culturing for another 2 hours, the absorbance (OD) value at 450 nm was measured using a microplate reader (TECAN, Switzerland).

[0031] 8. Colony formation assay: Seed cells in 6-well plates and incubate at 37°C for 10 days, then terminate the culture. Wash cells twice with phosphate-buffered saline (PBS), fix with 5 mL of anhydrous ethanol for 15 min, and stain with Giemsa stain for 10-30 min. After photographing, count the number of colonies; each cluster of more than 50 cells is considered one colony.

[0032] 9. Wound Healing Assay: Seed cells in the logarithmic growth phase into 6-well plates. When the cells reach 80%-85% confluence, draw a straight line using a 200μL pipette tip. Take images (100× magnification) at 0h and 24h respectively, and measure the width of the cell scratch.

[0033] 10. Migration and invasion assay: 100µL cell suspension (1×10⁻⁶ cells / mL) 5 Cells / mL were added to serum-free medium and placed in the upper chamber of a Transwell (Corning Incorporated, USA). 500 µL of FBS-containing medium was added to the lower chamber. After 24 h of culture, the matrix gel and cells in the upper chamber were wiped away with cotton swabs. Cells that had crossed the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were taken under a microscope (200× magnification), and the number of migrating or invading cells was counted.

[0034] 11. In vivo tumor growth assay: BALB / c nude mice (5 weeks old, female) were provided by Suzhou Spefol Biotechnology Co., Ltd., Animal Production License: SCXK (Su) 2022-0006. 5 × 10⁶ 6 Five SW620-OE-KCNJ15 cells and five SW620-NC cells were subcutaneously injected into the right axilla of mice. Tumor volume (0.5 × length × width) was measured every 5 days after injection. 2 The mice were recorded and weighed until 25 days after injection. After the recording was completed, all mice were deeply anesthetized with isoflurane and then euthanized by cervical dislocation. All procedures involving animals and their care in this invention were performed in accordance with the National Institutes of Health's Guidelines for the Care and Use of Laboratory Animals, Animal Ethics Number NAALH-W-2505008.

[0035] 12. Statistical Analysis: In vitro experiments were repeated three times, and in vivo experiments used five mice per group. All data were analyzed using SPSS 22.0 and GraphPad Prism 9. Wilcoxon test or t-test was used to assess differences between two groups, and analysis of variance (ANOVA) was used for multiple group comparisons. Statistical significance was set to [value missing]. P <0.05, respectively using P <0.05、 P <0.01、 P <0.001 and P <0.0001 indicates.

[0036] II. Experimental Results: 1. KCNJ15 is expressed at low levels in colorectal cancer tissues and cell lines, and is associated with poor prognosis: Immunohistochemical staining of cancerous and normal tissues from 116 colorectal cancer patients with KCNJ15 was performed. Figure 1 A, B). At the same magnification, three images were randomly captured from each slice. The immunohistochemical staining intensity of KCNJ15 was analyzed using Image Pro Plus, and the average staining intensity of each pathological slice was calculated. Based on the average value, 116 cancer tissues were divided into a high-expression group (n=40) and a low-expression group (n=76). The results showed that KCNJ15 was localized in the cell membrane and cytoplasm of cancer cells, and its expression level in cancer tissues was lower than that in normal epithelial tissues (n=76). Figure 1 C, D). Further analysis of colorectal cancer cell lines and normal epithelial cell lines revealed that KCNJ15 was expressed at low levels in cancer cell lines, consistent with the expression trend in cancer tissues. Figure 1 E, F).

[0037] Subsequently, we explored the relationship between KCNJ15 and clinicopathological features and prognosis of colorectal cancer. As shown in Table 5, KCNJ15 expression levels were significantly correlated with histological grade (P=0.010), lymphovascular invasion (P=0.038), and prognosis (P=0.017), but not with gender, age, BMI, tumor size, T stage, N stage, M stage, peripheral nerve invasion, MSI, and P53 status. KCNJ15 survival curves were plotted. Figure 1 G), low KCNJ15 expression was associated with decreased overall survival (OS) (P=0.028). Finally, the Kaplan-Meier database was used to further analyze the impact of KCNJ15 expression on the prognosis of colorectal cancer. Figure 1 H). The results showed that low expression of KCNJ15 was significantly associated with progression-free survival (PFS) and post-progression survival (PPS) in colorectal cancer (P < 0.05).

[0038] Table 5: Clinical and Pathological Data 2. KCNJ15 inhibits the proliferation, migration, invasion, and EMT of colorectal cancer cells in vitro: Based on the expression of KCNJ15 in colorectal cancer cells, two cell groups with low expression levels (SW620 and RKO) were selected for stable overexpression treatment, while two cell groups with high expression levels (HT-29 and T84) were selected for stable knockdown treatment. Transfection efficiency was detected by Western blot and qRT-PCR. The results are as follows: Figure 6As shown in the figure. HT-29-KD-KCNJ15-3 and T84-KD-KCNJ15-1, which have higher transfection efficiency, were selected for subsequent experiments. Based on the research results on KCNJ15 and the clinicopathological characteristics of colorectal cancer, the effects of KCNJ15 on the biological function of colorectal cancer cells were further explored.

[0039] In SW620 and RKO cell lines overexpressing KCNJ15, CCK-8 assays and colony formation assays showed that KCNJ15 overexpression significantly reduced cell viability and proliferation. Figure 2 A, B). Wound healing and Transwell assays showed that KCNJ15 overexpression inhibited cell migration and invasion. Figure 2 C, D). This was subsequently validated in HT-29 and T84 cell lines with low KCNJ15 expression. Conversely, KCNJ15 knockdown enhanced cell proliferation, unlike overexpression experiments. Figure 2 A, B), migration and invasion capabilities were also significantly enhanced ( Figure 2 C, D).

[0040] EMT is one of the key drivers of colorectal cancer progression. To investigate the regulatory role of KCNJ15 in EMT, SW620 cell lines overexpressing KCNJ15 and HT-29 cell lines with KCNJ15 knockdown were selected. The expression levels of EMT-related markers and transcription factors were detected by Western blot and qRT-PCR. Figure 2 As shown in Figure EF, KCNJ15 overexpression significantly upregulated the expression of the epithelial marker E-cadherin, while downregulating the expression of the mesenchymal marker N-cadherin and EMT-related transcription factors Snail and ZEB1. Conversely, KCNJ15 knockdown decreased E-cadherin expression, while significantly increasing the expression of N-cadherin, Snail, and ZEB1. These results are consistent with the aforementioned finding that KCNJ15 inhibits cell proliferation, migration, and invasion, collectively indicating that KCNJ15 exerts a tumor-suppressive function through negative regulation of the EMT process.

[0041] 3. KCNJ15 Inhibits EMT in Colorectal Cancer via the PI3K / AKT / mTOR Signaling Pathway: To further investigate the regulatory mechanism of KCNJ15 in colorectal cancer, a gene-gene interaction network of KCNJ15 and its related genes was constructed based on the GeneMania platform. The results are as follows: Figure 7As shown in (a), the results revealed that the 20 genes most frequently associated with KCNJ15 included KCNJ16, PATJ, GNGT2, GNG8, GNG7, GNG4, GNG3, GNG10, GNG12, GNG5, KCNJ5, CACNA1B, CACNB3, CACNB4, GNB3, GNB2, CLCNKB, CACNA1E, KCNJ1, and CACNA1A. A protein-protein interaction (PPI) network for KCNJ15 was further generated using the STRING database. The results are as follows... Figure 7 (b) shows that the network contains 55 interacting edges and 11 nodes, specifically including KCNJ10, KCNJ16, GNB3, GNG11, GNGT1, GNG13, GNB4, KCNJ5, KCNJ2 and GABBR2.

[0042] Subsequently, functional enrichment analysis was performed on the 27 genes involved in the two databases mentioned above using the DAVID website, and the results were visualized on the ImageGP website. The enrichment results are shown below. Figure 7 As shown in (c), the results indicate that the biological processes (BP) involved by this group of genes mainly include the G-protein-coupled receptor signaling pathway, regulation of ion transmembrane transport, signal transduction, chemical synaptic transmission, and potassium ion transmembrane transport. KEGG enrichment results show that this group of genes is involved not only in signal transduction-related signaling pathways but also in cancer-related signaling pathways. Among them, the PI3K / AKT signaling pathway attracted our attention. To verify the effect of KCNJ15 on this pathway, the expression levels of p-PI3K, PI3K, p-AKT, AKT, p-mTOR, and mTOR were examined. The results showed that KCNJ15 overexpression significantly downregulated the phosphorylation levels of PI3K, AKT, and mTOR; while KCNJ15 knockdown led to a significant upregulation of the expression levels of p-PI3K, p-AKT, and p-mTOR.

[0043] To explore the upstream regulatory mechanism of KCNJ15 in inhibiting EMT in colorectal cancer, the KCNJ15-overexpressing colorectal cancer cell line SW620 was first treated with 740-YP (a specific PI3K activator). Western blot analysis revealed the following results: Figure 3 As shown in Figure A, in KCNJ15-overexpressing cells, the activation level of the PI3K / AKT / mTOR signaling pathway was significantly reduced, and the EMT process was also significantly inhibited. Notably, treatment with 740-YP significantly reversed the inhibition of PI3K / AKT / mTOR signaling caused by KCNJ15 overexpression, while the EMT process was also significantly activated. Figure 3 B).

[0044] To verify the reliability of the above results, colorectal cancer cell lines with low KCNJ15 expression were treated with LY294002 (a specific PI3K inhibitor). The results showed that in KCNJ15 knockdown cells, the PI3K / AKT / mTOR signaling pathway and EMT process were significantly activated, and this activation effect was effectively inhibited after LY294002 treatment. Figure 3 C). The above results strongly suggest that KCNJ15 exerts its biological role in inhibiting EMT in colorectal cancer by regulating the PI3K / AKT / mTOR signaling pathway.

[0045] 4. KCNJ15 inhibits the proliferation, migration, and invasion of colorectal cancer by suppressing the PI3K / AKT / mTOR-mediated EMT process: To further verify the above findings, a series of functional experiments were conducted. The results showed that in 740-YP-treated KCNJ15-overexpressing cell lines, the inhibitory effect of KCNJ15 overexpression on cell proliferation was significantly alleviated. Figure 4 A, B). In wound healing and Transwell assays, KCNJ15 overexpression inhibited cell migration, but this inhibitory effect was weakened after exposure to 740-YP. Figure 4 C, D). Similarly, in KCNJ15-low expression cell lines, LY294002 treatment reversed the enhanced cell proliferation induced by KCNJ15 knockdown. Figure 4 A, B), and significantly inhibited cell migration and invasion abilities ( Figure 4 (C, D). In summary, these findings indicate that KCNJ15 inhibits the malignant behavior of colorectal cancer cells by suppressing the EMT process mediated by the PI3K / AKT / mTOR signaling pathway.

[0046] 5. KCNJ15 Inhibits Tumor Cell Proliferation in Vivo: To explore the effect of KCNJ15 on the growth of colorectal cancer cells in vivo, a stably transfected KCNJ15-overexpressing cell line was constructed. Stably transfected SW620-OE-KCNJ15 cells and SW620-NC cells were injected subcutaneously into the right axilla of nude mice. The mice were observed for 25 consecutive days, and tumor volume and weight were recorded. The mouse body weight statistics are shown in Table 6. The effect of KCNJ15 on tumor growth in vivo after subcutaneous inoculation of nude mice with SW620-OE-KCNJ15 cells and SW620-NC cells is shown in Table 6. Figure 5 As shown in A, the tumor volume detection data are shown in Table 7. Figure 5 As shown in B, the tumor weight is shown in Table 8. Figure 5 As shown in Figure C. Consistent with in vitro studies, KCNJ15 overexpression resulted in a reduction in both tumor volume and weight compared to the control group. Figure 5 (B, C). In summary, these findings suggest that KCNJ15 can inhibit the tumorigenic potential of colorectal cancer.

[0047] Table 6: Mouse body weight (unit: grams) Table 7: Tumor volume detection data (unit: cubic centimeters) Note: Length (L); Width (W).

[0048] Table 8: Tumor Weight (Unit: grams) In recent years, colorectal cancer has become a major challenge in global public health, with an increasing trend towards younger age of onset. Thanks to optimized early screening programs and the application of novel treatment strategies, cancer-related mortality rates among middle-aged individuals are declining. However, the prognosis for patients with recurrent, metastatic, and advanced colorectal cancer remains a serious challenge. The inward rectifier potassium channel (Kir), encoded by the KCNJ gene, is widely distributed in cardiomyocytes, neurons, glial cells, endothelial cells, epithelial cells, blood cells, and oocytes, and is characterized by allowing K... + They are more likely to enter cells than flow out. Studies have shown that Kir channels not only participate in maintaining the resting potential of cells and regulating excitability, but also play a role in the development and progression of various cancers.

[0049] EMT, or the process by which epithelial cells transform into motile mesenchymal cells, is characterized by the downregulation of E-cadherin and the upregulation of N-cadherin expression, leading to decreased cell adhesion. During this process, cytoskeleton reorganization, changes in cell shape, formation of cell processes, and degradation of the extracellular matrix all create favorable conditions for cancer cell migration and invasion. EMT, by enhancing cell migration, invasion, and anti-apoptosis capabilities, becomes a key step in the cancer process. In this invention, KCNJ15 overexpression significantly upregulated E-cadherin expression in colorectal cancer cells while downregulating the expression of N-cadherin, Snail, and ZEB1, suggesting that KCNJ15 influences colorectal cancer metastasis and invasion by inhibiting the EMT process.

[0050] In this invention, KCNJ15 overexpression reduced the phosphorylation levels of PI3K, AKT, and mTOR in colorectal cancer cells, suggesting that KCNJ15 exerts its anti-tumor effect by inhibiting the PI3K / AKT / mTOR signaling pathway. Reversal of function assays further confirmed the regulatory role of KCNJ15 in the PI3K / AKT / mTOR pathway, providing important evidence for a deeper understanding of the molecular mechanism of KCNJ15 in colorectal cancer. Exposure to 740-YP reversed the inhibition of PI3K / AKT / mTOR and EMT caused by KCNJ15 overexpression. This result indicates that KCNJ15 inhibits EMT through the PI3K / AKT / mTOR pathway.

[0051] This invention reports for the first time that KCNJ15 expression in colorectal cancer is lower than in normal intestinal epithelial tissue, and that its expression level is closely related to histological grade, lymphovascular invasion, and prognosis. Cellular function experiments confirmed that KCNJ15 overexpression inhibits the proliferation, migration, and invasion of colorectal cancer cell lines. These results indicate that KCNJ15 affects the progression of colorectal cancer by regulating the biological behavior of tumor cells.

[0052] This invention confirms that KCNJ15 inhibits EMT in colorectal cancer by regulating the PI3K / AKT / mTOR pathway, suggesting its function as a tumor suppressor gene. KCNJ15 inhibits CRC migration, invasion, and EMT by regulating the PI3K / AKT / mTOR signaling pathway. This discovery not only deepens our understanding of the function of KCNJ15 in cancer but also provides a new potential target for targeted therapy of CRC. In the future, the development of small molecule agonists targeting KCNJ15 is expected to become a new direction for precision treatment of CRC.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a reagent for detecting the expression level of the target gene KCNJ15 in the preparation of reagents for diagnosing colorectal cancer, characterized in that: The KCNJ15 is expressed at low levels in colorectal cancer cells and tissues.

2. The application of a reagent for detecting the expression level of the target gene KCNJ15 in the preparation of a prognostic assessment reagent for colorectal cancer, characterized in that: The KCNJ15 is expressed at low levels in colorectal cancer cells, which promote proliferation, migration, or invasion, as well as the EMT process.

3. Application of gene KCNJ15 as a target in the preparation of colorectal cancer cell metastasis detection reagents and drug screening.

4. The application according to claim 3, characterized in that: The gene KCNJ15 is used as a target in screening drugs to inhibit or slow down the proliferation, migration, and invasion of colorectal cancer cells.

5. The application according to claim 4, characterized in that: The overexpression of KCNJ15 inhibits or slows down the proliferation, migration, and invasion of colorectal cancer cells, and suppresses the EMT process.

6. The application according to claim 5, characterized in that: Overexpression of KCNJ15 inhibits the activation level of the PI3K / AKT / mTOR signaling pathway and suppresses the EMT process.

7. The application according to claim 4, characterized in that: Overexpression of KCNJ15 significantly downregulated the phosphorylation levels of PI3K, AKT, and mTOR, and significantly reduced the activation level of the PI3K / AKT / mTOR signaling pathway.

8. The application according to claim 7, characterized in that: The drugs that inhibit or slow down the proliferation, migration, and invasion of colorectal cancer cells are drugs that significantly upregulate or promote the overexpression of KCNJ15, or drugs that significantly downregulate the phosphorylation levels of PI3K, AKT, and mTOR, or drugs that significantly reduce the activation level of the PI3K / AKT / mTOR signaling pathway.

9. The application according to claim 8, characterized in that: The drug is a specific PI3K inhibitor.

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