Application of targeted intervention strategy based on TRIM25-SNRK regulatory axis in treatment of non-small cell lung cancer

Abstract

The invention relates to application of a targeted intervention strategy based on a TRIM25-SNRK regulatory axis in treatment of non-small cell lung cancer. The nucleotide sequences of the mRNA of the TRIM25 gene and the mRNA of the SNRK gene are respectively as shown in SEQ ID NO.1 to SEQ ID NO.2. According to the invention, it is found for the first time that the combined targeting TRIM25-SNRK regulatory axis has a more significant cancer inhibition effect in the cell proliferation and migration process of the non-small cell lung cancer compared with single interference of TRIM25 expression level or transfection of exogenous SNRK mRNA expression plasmids. The siTRIM25 and SNRK-OE combination strategy designed by the invention can specifically knock down the level of TRIM25 mRNA and up-regulate the level of SNRK mRNA at the same time, and compared with the condition of singly transfecting siTRIM25 or SNRK-OE, a cell function experiment shows that the combination strategy has a more effective anti-tumor effect. Therefore, the TRIM25-SNRK regulation axis can be used as an intervention target to be applied to research, development and preparation of non-small cell lung cancer antitumor drugs.

Description

Technical Field

[0001] The present invention relates to application of a targeted intervention strategy based on the TRIM25-SNRK regulatory axis in the treatment of non-small cell lung cancer, and belongs to the field of biomedical technology. Background Art

[0002] Lung cancer is a highly prevalent malignancy worldwide. Its highly invasive nature and significant tumor heterogeneity pose a serious threat to human health. Pathologically, lung cancer is primarily divided into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC accounts for approximately 85% of clinically diagnosed cases and can be further subdivided into three subtypes: squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Current clinical treatment strategies for NSCLC primarily include surgical resection, adjuvant chemoradiotherapy, targeted therapy, and immunotherapy. Notably, targeted drugs targeting specific gene mutations, such as EGFR, ALK, KRAS, and BRAF, have demonstrated remarkable efficacy in personalized NSCLC treatment, effectively prolonging patient survival. However, these targeted drugs often induce drug resistance in tumor cells during treatment, which is a major factor in accelerating the progression of patients to terminal stages. Therefore, identifying novel driver genes or signaling pathways and developing novel interventional drugs based on these pathways is crucial for improving the prognosis of NSCLC patients.

[0003] Small interfering RNA (siRNA) was originally discovered by David Baulcombe's team in plant research. It is a double-stranded RNA molecule composed of 19-25 nucleotide pairs. In 2001, Thomas Tuschl's team first demonstrated the siRNA-mediated mRNA interference effect in mammalian cells. This breakthrough discovery promoted the widespread application of siRNA technology in biomedical research and drug development. In the RNA interference technology system, ensuring the specificity and effectiveness of target gene mRNA interference is both a core technical point and a core indicator for evaluating the quality of siRNA design. Therefore, designing siRNA molecules with efficient knockdown ability and high specificity for specific target genes will provide key technical support for the functional research of new driver genes in NSCLC and the development of targeted treatment strategies.

[0004] Gene expression plasmids are synthetic circular DNA molecules that, when transfected into host cells, can autonomously replicate and drive the transcription and translation of specific genes, thereby achieving efficient expression of exogenous target genes within the host cells. Plasmid vectors typically include a replication origin, multiple cloning sites, promoters, terminators, antibiotic resistance genes, and reporter genes. By inserting the coding sequence of a target gene into a plasmid vector and applying transfection techniques, target gene expression can be upregulated in target cells. This is of great significance for studying the biological functions of target genes in cancer cells and the underlying regulatory mechanisms. Summary of the Invention

[0005] In response to the deficiencies of the existing technology, the present invention provides an application of a targeted intervention strategy based on the TRIM25-SNRK regulatory axis in the treatment of non-small cell lung cancer.

[0006] The technical solutions of the present invention are as follows:

[0007] The application of TRIM25-SNRK regulatory axis as drug target in the preparation of anti-non-small cell lung cancer drugs.

[0008] Preferably according to the present invention, the TRIM25-SNRK regulatory axis includes the TRIM25 gene and the SNRK gene.

[0009] Preferably, according to the present invention, the nucleotide sequence of the mRNA of the TRIM25 gene is shown as SEQ ID NO.1, and the nucleotide sequence of the mRNA of the SNRK gene is shown as SEQ ID NO.2.

[0010] Preferably, according to the present invention, the anti-non-small cell lung cancer drug uses the TRIM25 gene and the SNRK gene as intervention targets, introduces the exogenous SNRK gene while interfering with the expression of the TRIM25 gene, and can efficiently and specifically downregulate the expression level of the TRIM25 gene while upregulating the expression level of the SNRK gene.

[0011] Preferably, according to the present invention, the anti-non-small cell lung cancer drug is a composition of an inhibitor that specifically interferes with TRIM25 gene expression and an exogenous expression plasmid that specifically upregulates SNRK gene expression.

[0012] The application of siRNA that specifically interferes with TRIM25 gene expression and exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression in the preparation of anti-non-small cell lung cancer drugs.

[0013] Preferably, according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with TRIM25 gene expression is shown as SEQ ID NO.3; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO.4.

[0014] Further preferably, the volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, unit: μL / μg.

[0015] An anti-non-small cell lung cancer drug contains siRNA that specifically interferes with TRIM25 gene expression and an exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression.

[0016] Preferably, according to the present invention, the targeting nucleotide sequence of the siRNA that specifically interferes with TRIM25 gene expression is shown as SEQ ID NO.3; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO.4.

[0017] Further preferably, the volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, unit: μL / μg.

[0018] Beneficial effects:

[0019] The present invention discovered for the first time that the combined targeting TRIM25-SNRK regulatory axis has a more significant tumor suppressor effect in the cell proliferation and migration process of non-small cell lung cancer than that of interfering with TRIM25 expression levels alone or transfecting exogenous SNRK mRNA expression plasmids. The siTRIM25 and SNRK-OE combination strategy designed by the present invention can simultaneously and specifically knock down TRIM25 mRNA levels and upregulate SNRK mRNA levels. Compared with the case of transfection of siTRIM25 or SNRK-OE alone, cell function experiments showed that the combination strategy has a more effective anti-tumor effect. Therefore, the TRIM25-SNRK regulatory axis can be used as an intervention target and applied to the research and development and preparation of anti-tumor drugs for non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A549 cells were selected and transfected with siNC+Vector or siTRIM25+SNRK-OE. The cells were collected and RNA was extracted. Reverse transcription-real-time fluorescence quantitative PCR was used to detect TRIM25 and SNRK mRNA levels. The Student's t test was used to determine the statistical significance of the differences between the groups.

[0021] Figure 2 A549 cells were transfected with siNC+Vector, siTRIM25, SNRK-OE or siTRIM25+SNRK-OE, respectively. The proliferation ability of A549 cells was detected by CCK-8 assay, and the statistical significance of the differences between the groups was verified by two-way ANOVA test.

[0022] Figure 3 A549 cells were selected and transfected with siNC+Vector, siTRIM25, SNRK-OE or siTRIM25+SNRK-OE, respectively. The migration ability of A549 cells was evaluated by Transwell assay, and the statistical significance of the differences between the groups was determined by one-way ANOVA test. DETAILED DESCRIPTION

[0023] 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.

[0024] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0025] siTRIM25 targeting sequence: 5′-ggtggagcagctacaacaa-3′;

[0026] Example 1

[0027] Previous research by the inventors has revealed that abnormal activity of the TRIM25-SNRK regulatory axis in non-small cell lung cancer tissue is closely associated with the malignant progression of the disease. Consequently, the inventors discovered that combined intervention of TRIM25 and SNRK expression levels can produce a synergistic anti-tumor effect. This combined intervention strategy provides innovative insights into the development of novel anti-tumor drugs for non-small cell lung cancer and is expected to play a significant role in clinical treatment.

[0028] Example 2

[0029] The nucleotide sequences of TRIM25 mRNA and SNRK mRNA were obtained from the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Based on this, siRNAs (siTRIM25) that can specifically interfere with the expression level of TRIM25 mRNA were designed and synthesized, the targeting sequence of which is shown in SEQ ID NO.3, and the control siRNA is siNC; as well as an exogenous SNRK mRNA expression plasmid (SNRK-OE) that can specifically upregulate SNRK mRNA. The SNRK-OE plasmid contains the nucleotide sequence shown in SEQ ID NO.4, and the control plasmid is an empty vector plasmid (Vector); wherein the empty vector plasmid (Vector) is a conventional existing plasmid, and siNC is a conventional existing sequence, both of which can be obtained commercially. The SNRK-OE plasmid was constructed by the Miaoling plasmid platform according to the sequence information, and siTRIM25 was synthesized by Qingke Biotechnology according to the sequence information.

[0030] siNC+Vector or siTRIM25+SNRK-OE were transfected into A549 cells at a growth density of 60%. 48 hours after transfection, the cells were collected and total RNA was extracted. The expression levels of TRIM25 mRNA and SNRK mRNA were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific results are shown in the figure. Figure 1 shown.

[0031] Depend on Figure 1 It can be seen that compared with transfection of siNC+Vector, transfection of siTRIM25+SNRK-OE into A549 cells can significantly downregulate the expression level of TRIM25 mRNA and significantly upregulate the expression level of SNRK mRNA.

[0032] The specific implementation process is as follows:

[0033] (1) A549 cells were plated in 6-well plates. When the cell growth density reached about 60%, siNC + Vector or siTRIM25 + SNRK-OE were transfected into A549 cells using Lipofectamine RNAiMAX Reagent (Lifetechnologies, 13778-150) and Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000015) at a ratio of 3 μL siRNA (10 μM) to 2 μg plasmid.

[0034] (2) 48 hours after transfection, the cells obtained in step (1) were collected and RNA was extracted using the RNA-Quick Purification Kit (Shanghai Yishan, RN001). The extracted RNA concentration was detected using NanoDrop2000, and the integrity of the extracted RNA was evaluated by agarose gel electrophoresis.

[0035] (3) Using the RNA obtained in step (2) as a template, the reverse transcription kit LunaScriptTM RT SuperMix Kit (NEB, E3010) was used to perform the reverse transcription step to synthesize the cDNA product. Green Master Mix (Thermo Fisher Scientific, 4367659) kit and QuantStudioTM 5System (Thermo Fisher Scientific) PCR instrument were used for fluorescence quantitative PCR, and the level of ACTB mRNA was used as the internal reference. -ΔΔCT The relative expression levels of TRIM25 mRNA and SNRK mRNA were calculated using the formula.

[0036] Example 3

[0037] A549 cells were selected and transfected with the same dose of siNC+Vector, SNRK-OE, siTRIM25 or siTRIM25+SNRK-OE, and the proliferation ability of the transfected cells was evaluated by CCK-8 assay. The experimental results are shown in the figure. Figure 2 shown.

[0038] Depend on Figure 2 It can be seen that transfection with siTRIM25, SNRK-OE, or siTRIM25+SNRK-OE significantly weakened the growth of A549 cells compared to siNC+Vector, but co-transfection with siTRIM25+SNRK-OE showed a more significant proliferation inhibitory effect than siTRIM25 or SNRK-OE. This indicates that combined intervention of TRIM25 and SNRK expression levels has a better inhibitory effect on tumor cell proliferation than targeting the expression level of either gene alone.

[0039] The specific implementation process is as follows:

[0040] (1) A549 cells were transfected with siNC+Vector, SNRK-OE, siTRIM25, or siTRIM25+SNRK-OE, respectively. The doses of Vector and SNRK-OE were 2 μg / well, and the doses of siNC and siTRIM25 were 3 μL (concentration 10 μM) / well. The transfection method and reagents were the same as in Example 2. After 48 hours, the cells were collected and seeded into 96-well plates at a density of 2000 cells / well. The cells were divided into 5 groups (Day 0, 1, 2, 3, 4), with 4 transfected cells (Vector+siNC, SNRK-OE, siTRIM25, or siTRIM25+SNRK-OE) in each group, and 3 replicate wells were plated for each cell type.

[0041] (2) After the cells obtained in step (1) have attached to the wall (approximately 2-4 hours), add 10 μL of CCK-8 reagent (TargetMol, C0005) to the Day 0 group, place in a 37°C incubator for 1 hour, shake briefly, and then place in a microplate reader to measure the absorbance at 450 nm. This is the absorbance value of the initial seeded cells (Day 0);

[0042] (3) After completing the initial cell absorbance measurement, the absorbance values ​​of the cells in the Day 1, 2, 3, and 4 groups were measured every 24 hours using the same method. The absorbance value of the Day 0 group was used as a reference to calculate the changes in cell viability and cell proliferation trends in each group.

[0043] Example 4

[0044] A549 cells were selected and transfected with the same dose of siNC+Vector, SNRK-OE, siTRIM25 or siTRIM25+SNRK-OE, and the migration ability of the transfected cells was evaluated by Transwell assay. The specific results are shown in the figure. Figure 3 shown.

[0045] Depend on Figure 3 It can be seen that the inhibitory effect of co-transfection of siTRIM25 + SNRK-OE on the migration ability of A549 cells is more significant than that of transfection of siTRIM25 or SNRK-OE alone. This result shows that co-intervention of TRIM25 and SNRK gene expression levels exhibits a more superior tumor suppressor effect than targeting the expression of either gene alone.

[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%, they were transfected with siNC+Vector, SNRK-OE, siTRIM25, or siTRIM25+SNRK-OE, respectively. The specific method was as described in Example 3.

[0048] (2) After 48 hours of transfection, collect and count the transfected cells obtained in step (1) and adjust the cell concentration to 100,000 cells / mL. Add complete medium (600 μL / well) to a 24-well plate and place a Transwell chamber (Corning, 3422) in the well plate.

[0049] (3) Add 200 μL of cell suspension to each Transwell chamber and continue culturing in a cell culture incubator (37°C, 5% CO2) for 48 hours;

[0050] (4) The Transwell chamber was collected, the culture medium was discarded, and the cells were washed with PBS. 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, and the migrated cells were observed under an inverted microscope and photographed and recorded. The photographs were recorded and statistically analyzed.

Claims

1. Application of the TRIM25-SNRK regulatory axis as a drug target in the preparation of anti-non-small cell lung cancer drugs, characterized in that: The TRIM25-SNRK regulatory axis includes the TRIM25 gene and the SNRK gene; the nucleotide sequence of the mRNA of the TRIM25 gene is shown in SEQ ID NO.1, and the nucleotide sequence of the mRNA of the SNRK gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The anti-non-small cell lung cancer drug uses the TRIM25 gene and the SNRK gene as intervention targets, introduces the exogenous SNRK gene while interfering with the expression of the TRIM25 gene, and can efficiently and specifically downregulate the expression level of the TRIM25 gene while upregulating the expression level of the SNRK gene.

3. The use according to claim 1, characterized in that The anti-non-small cell lung cancer drug is a composition of an inhibitor that specifically interferes with TRIM25 gene expression and an exogenous expression plasmid that specifically upregulates SNRK gene expression.

4. Use of siRNA that specifically interferes with TRIM25 gene expression and exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression in the preparation of an anti-non-small cell lung cancer drug, characterized in that: The targeting nucleotide sequence of the siRNA that specifically interferes with TRIM25 gene expression is shown as SEQ ID NO.3; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO.

4.

5. The use according to claim 4, characterized in that The volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, unit: μL / μg.

6. An anti-non-small cell lung cancer drug, characterized in that: The anti-non-small cell lung cancer drug contains siRNA that specifically interferes with TRIM25 gene expression and an exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression; the targeting nucleotide sequence of the siRNA that specifically interferes with TRIM25 gene expression is shown in SEQ ID NO.3; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.

4.

7. The use according to claim 5, characterized in that In the anti-non-small cell lung cancer drug, the volume-to-mass ratio of the siRNA that specifically interferes with TRIM25 gene expression to the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression is 3:2, unit: μL / μg.

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