Application of targeted TRIM25-CTNNB1 signal channel in preparation of medicine for treating non-small cell lung cancer

Abstract

The invention relates to an application of a targeted TRIM25-CTNNB1 signal channel in preparation of a medicine for treating non-small cell lung cancer. The TRIM25-CTNNB1 signal channel comprises a TRIM25 gene and a CTNNB1 gene, and the nucleotide sequences of the mRNA (messenger ribonucleic acid) of the TRIM25 gene and the mRNA of the CTNNB1 gene are respectively as shown in SEQ ID NO.1-2. The regulatory relation between the TRIM25 and the CTNNB1 and the core function of the TRIM25 and the CTNNB1 in malignant progression of the lung cancer are identified in a breakthrough mode. Compared with traditional single-target intervention, the combined strategy that siTRIM25 and siCTNNB1 simultaneously inhibit a TRIM25-CTNNB1 signal channel in a targeted mode is applied, and more excellent anti-tumor activity is shown in in-vivo and in-vitro experiments. The discovery not only clarifies a new mechanism of occurrence and development of the lung cancer, but also provides a new thought for clinical transformation: the TRIM25-CTNNB1 axis can be used as a drug target, a novel combined treatment strategy is developed, and a more effective treatment choice is provided for non-small cell lung cancer patients.

Description

Technical Field

[0001] The present invention relates to application of targeting the TRIM25-CTNNB1 signaling pathway in preparing a drug for treating non-small cell lung cancer, and belongs to the technical field of biomedicine. Background Art

[0002] Lung cancer is a malignant tumor with high morbidity and mortality worldwide. Its highly invasive and heterogeneous characteristics pose a serious threat to human health. Pathologically, lung cancer is primarily divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for over 85% of lung cancer cases and can be further subdivided into three major subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Current clinical treatment strategies for NSCLC include surgical resection, adjuvant chemoradiotherapy, targeted therapy, and immunotherapy. In recent years, targeted drugs targeting specific gene mutations, such as EGFR, KRAS, and ALK, have demonstrated clear survival benefits in personalized NSCLC treatment. However, the widespread development of tumor cell resistance during targeted therapy often leads to recurrence or metastasis after treatment, significantly shortening patient survival. Therefore, further elucidating new driver genes or key signaling pathways and developing novel interventional drugs based on these pathways are of great clinical significance for improving the prognosis of NSCLC patients and will also have significant socioeconomic benefits.

[0003] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of 19-25 nucleotide pairs and is the core effector molecule of RNA interference technology. In the cell, after siRNA enters the target cell through transfection, it binds to the RNA-induced silencing complex (RISC). Its positive strand is then degraded, and the remaining antisense strand guides RISC to specifically recognize and bind to the target gene mRNA through base complementary pairing, ultimately inducing mRNA degradation and achieving precise inhibition of target gene expression. Therefore, the development and design of siRNA molecules with both high specificity and high interference efficiency will provide new technical support and treatment strategies for targeted treatment of NSCLC. Summary of the Invention

[0004] In view of the deficiencies of the existing technology, the present invention provides an application of targeting the TRIM25-CTNNB1 signaling pathway in the preparation of a drug for treating non-small cell lung cancer.

[0005] The technical solutions of the present invention are as follows:

[0006] Application of the TRIM25-CTNNB1 signaling pathway as a drug target in the preparation of drugs for the treatment of non-small cell lung cancer.

[0007] Preferably according to the present invention, the TRIM25-CTNNB1 signaling pathway includes the TRIM25 gene and the CTNNB1 gene.

[0008] Preferably, according to the present invention, the mRNA nucleotide sequence of the TRIM25 gene is shown as SEQ ID NO.1, and the mRNA nucleotide sequence of the CTNNB1 gene is shown as SEQ ID NO.2.

[0009] Preferably, according to the present invention, the drug for treating non-small cell lung cancer uses TRIM25 and CTNNB1 genes as intervention targets, based on interfering with TRIM25 mRNA and CTNNB1 mRNA, and can efficiently and specifically inhibit the expression levels of TRIM25 and CTNNB1 genes.

[0010] Preferably, according to the present invention, the drug for treating non-small cell lung cancer is siRNA, shRNA, microRNA or chemical inhibitors of TRIM25 and CTNNB1 mRNA.

[0011] The invention relates to the use of siRNA that specifically interferes with TRIM25 gene expression and siRNA that specifically interferes with CTNNB1 gene expression in the preparation of drugs for treating non-small cell lung cancer.

[0012] Preferably, according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown as SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the CTNNB1 gene is shown as SEQ ID NO.4.

[0013] Preferably, according to the present invention, the molar ratio of the siRNA that specifically interferes with the expression of the TRIM25 gene to the siRNA that specifically interferes with the expression of the CTNNB1 gene is 1:1.

[0014] A drug for treating non-small cell lung cancer contains siRNA that specifically interferes with TRIM25 gene expression and siRNA that specifically interferes with CTNNB1 gene expression.

[0015] Preferably, according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown as SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the CTNNB1 gene is shown as SEQ ID NO.4.

[0016] Further preferably, the molar ratio of the siRNA that specifically interferes with the expression of the TRIM25 gene to the siRNA that specifically interferes with the expression of the CTNNB1 gene is 1:1.

[0017] Beneficial effects:

[0018] This invention has groundbreakingly identified the regulatory relationship between TRIM25 and CTNNB1 and their core role in the malignant progression of lung cancer. Compared with traditional single-target interventions, the combined strategy of simultaneously targeting and inhibiting the TRIM25-CTNNB1 signaling pathway using siTRIM25 and siCTNNB1 designed in this invention has demonstrated superior anti-tumor activity both in vitro and in vivo. This discovery not only clarifies a new mechanism for the development and progression of lung cancer, but also provides new ideas for clinical translation: the TRIM25-CTNNB1 axis can be used as a drug target to develop new combination therapy strategies, providing more effective treatment options for patients with non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 A549 cells were selected and transfected with the same dose of siNC or siTC. The levels of TRIM25 and CTNNB1 mRNA were detected by reverse transcription-real-time fluorescence quantitative PCR. The statistical significance of the differences between the groups was tested using the Student's t test.

[0020] Figure 2 The same dose of siNC, siTRIM25, siCTNNB1 or siTC was transfected into A549 cells, and the proliferation ability of A549 cells was detected by CCK-8 assay. The statistical significance of the differences among multiple groups was verified by two-way ANOVA test.

[0021] Figure 3 A549 cells were selected and transfected with siNC, siTRIM25, siCTNNB1 or siTC, respectively. The transfection dose of each siRNA was the same. The migration ability of A549 cells was detected by Transwell assay, and the statistical significance of the differences between the groups was determined by one-way ANOVA. DETAILED DESCRIPTION

[0022] The technical solution of the present invention will be further described below in conjunction with specific experimental examples, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the examples are all common commercial products unless otherwise specified.

[0023] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0024] siTRIM25 targeting sequence: 5′-gctctgtcagagtgctata-3′;

[0025] siCTNNB1 targeting sequence: 5′-aagctttagtaaatataatgagg-3′.

[0026] SiTRIM25 and siCTNNB1 were synthesized by Qingke Biotechnology according to the sequence information.

[0027] Example 1

[0028] In their previous research on non-small cell lung cancer, the inventors discovered abnormal activation of the TRIM25-CTNNB1 signaling pathway. Further mechanistic investigation revealed that this pathway plays a key role in cancer cell proliferation and metastasis. Based on this, the inventors propose that inhibiting the TRIM25-CTNNB1 signaling pathway could provide a new direction for targeted treatment strategies for non-small cell lung cancer.

[0029] Example 2

[0030] The nucleotide sequences of TRIM25 mRNA and CTNNB1 mRNA were obtained using the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. siRNAs (siTRIM25 and siCTNNB1) that specifically interfere with the expression levels of TRIM25 mRNA or CTNNB1 mRNA were designed for the above sequences, and their targeting sequences are shown in SEQ ID NO.3 and SEQ ID NO.4. The above two siRNAs were mixed at a molar ratio of 1:1 to obtain siTC, and the conventional sequence siNC was used as a negative control. A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC (siRNAs negative control, a conventional existing sequence) and siTC, respectively. After 48 hours of transfection, the cells were collected and RNA was extracted. The expression levels of TRIM25 mRNA and CTNNB1 mRNA were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific results are shown in the figure below. Figure 1 shown.

[0031] Depend on Figure 1 It can be seen that transfection of siTC can significantly downregulate the expression levels of TRIM25 and CTNNB1 mRNA in A549 cells.

[0032] The specific implementation process is as follows:

[0033] (1) A549 cells were collected and plated in 6-well plates. When the cell growth density reached about 60%, siTC and siNC were transfected into A549 cells using the transfection reagent Lipofectamine RNAiMAX Reagent (Life technologies, 13778-150). The siRNA dose used was the same (volume 3 μL, concentration 10 μM). The cells were cultured for 48 hours.

[0034] (2) The cells obtained in step (1) were collected and total RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001). RNA integrity was assessed by 1% agarose gel electrophoresis, and RNA concentration and purity were determined using NanoDrop2000.

[0035] (3) Using the RNA obtained in step (2) as a template, reverse transcription kit LunaScript TM RT SuperMix Kit (NEB, E3010) was used for reverse transcription and synthesis of cDNA products. Green Master Mix (ThermoFisher Scientific, 4367659) kit in QuantStudio TM Fluorescence quantitative PCR was performed on a 5System (Thermo Fisher Scientific) PCR instrument, and ACTB was selected as the internal reference gene. -ΔΔCT The relative expression levels of TRIM25 and CTNNB1 mRNA were calculated by the method.

[0036] Example 3

[0037] A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siTRIM25, siCTNNB1 and siTC, respectively. The proliferation ability of the transfected cells was detected by CCK-8 assay. The specific results are shown in the figure. Figure 2 shown.

[0038] Depend on Figure 2 It can be seen that transfection with siTRIM25, siCTNNB1, or siTC significantly weakened the proliferation ability of A549 cells compared to siNC, while transfection with siTC weakened the viability of A549 cells more significantly than the same dose of siTRIM25 or siCTNNB1. These results indicate that compared to interfering with the expression levels of TRIM25 or CTNNB1 alone, combined interference with the expression of TRIM25 and CTNNB1 genes has a more effective inhibitory effect on tumor cell proliferation.

[0039] The specific implementation process is as follows:

[0040] (1) The same dose (volume 3 μL, concentration 10 μM) of siNC, siTRIM25, siCTNNB1, and siTC were transfected into A549 cells, respectively. The specific method is detailed in Example 2. 48 hours after transfection, the transfected cells were collected and re-cultured in 96-well plates (2,000 cells / well). They were divided into five groups (Day 0, 1, 2, 3, 4). Each group included four transfected cells: siNC, siTRIM25, siCTNNB1, and siTC. Three replicate wells were set for each cell type.

[0041] (2) After the cells were cultured in a cell incubator for 2-4 hours to allow them to adhere, 10 μL of CCK-8 reagent (TargetMol, C0005) was added to each well of the Day 0 group. The cells were placed in a 37°C incubator for 1 hour. After brief shaking, the absorbance (450 nm) was measured using a microplate reader. This was the absorbance value of the initial seeded cells (Day 0).

[0042] (3) After completing the initial cell absorbance measurement, measure the absorbance values ​​of Day 1, 2, 3, and 4 groups every 24 hours, and calculate the proliferation changes of cells in each group with Day 0 group as the reference.

[0043] Example 4

[0044] A549 cells were selected and transfected with the same dose (volume 3 μL, concentration 10 μM) of siNC, siTRIM25, siCTNNB1 and siTC, respectively. The migration ability of the transfected cells was detected by Transwell assay. The specific results are shown in the figure. Figure 3 shown.

[0045] Depend on Figure 3 It can be seen that compared with single transfection of siTRIM25 or siCTNNB1, transfection of siTC has a more significant inhibitory effect on the migration ability of A549 cells. These results indicate that compared with single interference of TRIM25 or CTNNB1 gene expression, simultaneous knockdown of TRIM25 and CTNNB1 gene expression exhibits a more effective tumor suppressor effect.

[0046] The specific implementation process is as follows:

[0047] (1) A549 cells were selected and cultured in a 6-well plate. When the cell growth density reached about 60%, siNC, siTRIM25, siCTNNB1, and siTC were transfected respectively. The specific method was as described in Example 2.

[0048] (2) After 48 hours of transfection, the transfected cells obtained in step (1) were collected and counted, and the cell concentration was adjusted to 100,000 cells / mL; complete culture medium (600 μL / well) was added to a 24-well plate, and a Transwell chamber (Corning, 3422) was placed in the well plate;

[0049] (3) 200 μL of cell suspension was added to each Transwell chamber, and the chamber was placed in a cell culture incubator (37°C, 5% CO2) for 48 hours.

[0050] (4) The Transwell chamber was removed and washed with PBS buffer. The cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes and then stained with crystal violet (0.1%) overnight. The non-migrated cells on the surface of the chamber were removed. The migrated cells were observed under an inverted microscope and photographed for recording. The cells were counted and statistically analyzed.

Claims

1. Application of the TRIM25-CTNNB1 signaling pathway as a drug target in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The TRIM25-CTNNB1 signaling pathway includes the TRIM25 gene and the CTNNB1 gene; the mRNA nucleotide sequence of the TRIM25 gene is shown in SEQ ID NO.1, and the mRNA nucleotide sequence of the CTNNB1 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The drug for treating non-small cell lung cancer uses TRIM25 and CTNNB1 genes as intervention targets, and is based on interfering with TRIM25 mRNA and CTNNB1 mRNA, and can effectively and specifically inhibit the expression levels of TRIM25 and CTNNB1 genes.

3. The use according to claim 2, characterized in that The drug for treating non-small cell lung cancer is siRNA, shRNA, microRNA or TRIM25 and CTNNB1 mRNA chemical inhibitor.

4. Use of siRNA that specifically interferes with TRIM25 gene expression and siRNA that specifically interferes with CTNNB1 gene expression in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown in SEQ ID NO.3, and the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the CTNNB1 gene is shown in SEQ ID NO.

4.

5. The use according to claim 4, characterized in that The molar ratio of the siRNA that specifically interferes with the expression of the TRIM25 gene to the siRNA that specifically interferes with the expression of the CTNNB1 gene is 1:

1.

6. A drug for treating non-small cell lung cancer, characterized in that: The drug for treating non-small cell lung cancer contains siRNA that specifically interferes with TRIM25 gene expression and siRNA that specifically interferes with CTNNB1 gene expression; The nucleotide sequence of the siRNA that specifically interferes with the expression of the TRIM25 gene is shown in SEQ ID NO.3, and the nucleotide sequence of the siRNA that specifically interferes with the expression of the CTNNB1 gene is shown in SEQ ID NO.

4.

7. The drug for treating non-small cell lung cancer according to claim 6, wherein In the drug for treating non-small cell lung cancer, the molar ratio of the siRNA that specifically interferes with the expression of the TRIM25 gene to the siRNA that specifically interferes with the expression of the CTNNB1 gene is 1:1.

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