Application of intervention SNRK-MTA1 signal channel axis in preparation of non-small cell lung cancer targeted therapy drug

Abstract

The invention relates to an application of an intervention SNRK-MTA1 signal channel axis in preparation of a non-small cell lung cancer targeted therapy drug. The nucleotide sequences of the mRNA of the SNRK gene and the mRNA of the MTA1 gene are respectively as shown in SEQ ID NO.1-2. The invention innovatively provides a strategy for treating the non-small cell lung cancer through double-target combined intervention. According to the strategy, SNRK gene expression is improved through exogenous gene overexpression plasmids, and meanwhile MTA1 gene expression is silenced through the siRNA technology. In a non-small cell lung cancer model, the strategy of combined application of the SNRK-OE plasmid and siMTA1 can specifically up-regulate the SNRK mRNA level and knock down the MTA1 mRNA level at the same time, and the combined strategy shows a better anti-tumor effect than single intervention, and can more effectively inhibit the growth and migration of tumor cells. Based on the discovery, the SNRK-MTA1 signal pathway axis can be developed into a novel therapeutic target for non-small cell lung cancer, and is used for designing a drug combination scheme or a composite targeted drug.

Description

Technical Field

[0001] The present invention relates to application of intervening in the SNRK-MTA1 signaling pathway axis in preparing a targeted therapeutic drug for non-small cell lung cancer, and belongs to the technical field of biomedicine. Background Art

[0002] Lung cancer is one of the most common malignancies worldwide and the leading cause of cancer-related death. Among the various pathological types of lung cancer, non-small cell lung cancer (NSCLC) has the highest prevalence, accounting for approximately 85% of all confirmed cases. Traditional treatment options primarily include surgical resection, radiotherapy, and chemotherapy. In recent years, with the rapid development of high-throughput sequencing technologies and the discovery of oncogenic driver genes, targeted drugs targeting specific gene mutations such as EGFR, KRAS, and ALK have been gradually introduced into clinical practice, bringing significant survival benefits to NSCLC patients. However, due to the high tumor heterogeneity of NSCLC, acquired drug resistance often occurs during targeted therapy, ultimately leading to tumor recurrence and distant metastasis. Therefore, the continued discovery of new NSCLC driver genes and in-depth analysis of their oncogenic molecular mechanisms are of great significance for the development of a new generation of targeted therapeutics.

[0003] A gene expression plasmid is an artificially constructed circular DNA vector that can enter host cells through transfection and achieve autonomous replication, while precisely controlling the transcription and translation of exogenous genes, thereby efficiently expressing the target gene. This vector system achieves its function of regulating gene expression by integrating core functional elements such as replication origins, multiple cloning sites, promoters, terminators, antibiotic selection markers, and reporter genes. With its characteristics of efficient expression, flexible design, and good biocompatibility, gene expression plasmids have become key experimental tools for exploring gene function, the mechanisms of genetic diseases, and treatment strategies for malignant tumors. They are widely used in basic scientific research and clinical application transformation.

[0004] Small interfering RNA (siRNA), also known as silencing RNA or short interfering RNA, has numerous applications in biological research, the most extensively studied and widely used of which is its ability to regulate gene expression. siRNA is a double-stranded RNA molecule consisting of 19-25 nucleotide pairs. After entering target cells through transfection reagents, it assembles with the RNA-induced silencing complex (RISC) to form a complex. It specifically recognizes the target gene mRNA through complementary base pairing, thereby inducing mRNA degradation or inhibiting its translation, ultimately reducing the expression level of the target protein.

[0005] In NSCLC-related basic research and potential targeted therapy applications, how to comprehensively utilize gene expression plasmids and siRNA to achieve the effect of targeted therapy for NSCLC has always been a key issue that needs to be addressed. Summary of the Invention

[0006] In view of the deficiencies of the existing technology, the present invention provides an application of intervening in the SNRK-MTA1 signaling pathway axis in the preparation of targeted therapeutic drugs for non-small cell lung cancer.

[0007] The technical solutions of the present invention are as follows:

[0008] Application of the SNRK-MTA1 signaling pathway axis as a drug target in the preparation of targeted therapeutic drugs for non-small cell lung cancer.

[0009] Preferably according to the present invention, the SNRK-MTA1 signaling pathway axis includes the SNRK gene and the MTA1 gene.

[0010] Preferably, according to the present invention, the nucleotide sequence of the mRNA of the SNRK gene is shown as SEQ ID NO.1, and the nucleotide sequence of the mRNA of the MTA1 gene is shown as SEQ ID NO.2.

[0011] Preferably, according to the present invention, the non-small cell lung cancer targeted therapeutic drug uses the SNRK gene and the MTA1 gene as intervention targets, introduces the exogenous SNRK gene and interferes with the expression of the MTA1 gene, and can efficiently and specifically upregulate the expression level of the SNRK gene while downregulating the expression level of the MTA1 gene.

[0012] Preferably, according to the present invention, the non-small cell lung cancer targeted therapeutic drug is a combination of an inhibitor that specifically interferes with MTA1 gene expression and an exogenous expression plasmid that specifically upregulates SNRK gene expression.

[0013] The application of exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression and siRNA that specifically interferes with MTA1 gene expression in the preparation of targeted therapeutic drugs for non-small cell lung cancer.

[0014] Preferably, according to the present invention, the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3; the targeting nucleotide sequence of the siRNA that specifically interferes with MTA1 gene expression is shown in SEQ ID NO.4.

[0015] Further preferably, the mass-to-volume ratio of the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression to the siRNA that specifically interferes with MTA1 gene expression is 2:3, unit: μg / μL.

[0016] A targeted therapeutic drug for non-small cell lung cancer, comprising an exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression and an siRNA that specifically interferes with MTA1 gene expression.

[0017] Preferably, according to the present invention, the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3; the targeting nucleotide sequence of the siRNA that specifically interferes with MTA1 gene expression is shown in SEQ ID NO.4.

[0018] Further preferably, the mass-to-volume ratio of the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression to the siRNA that specifically interferes with MTA1 gene expression is 2:3, unit: μg / μL.

[0019] Beneficial effects:

[0020] This invention innovatively proposes a dual-target combined intervention strategy for the treatment of non-small cell lung cancer: using an exogenous gene overexpression plasmid to elevate SNRK gene expression while simultaneously silencing MTA1 gene expression through siRNA technology. In a non-small cell lung cancer model, the combined application of the SNRK-OE plasmid designed in this invention and siMTA1 was able to simultaneously and specifically upregulate SNRK mRNA levels and knock down MTA1 mRNA levels. This combined strategy demonstrated a stronger anti-tumor effect than either single intervention, more effectively inhibiting tumor cell growth and migration. Based on this discovery, the SNRK-MTA1 signaling pathway axis could be developed as a novel therapeutic target for non-small cell lung cancer, useful in designing combination drug regimens or complex targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A549 cells were selected and transfected with Vector+siNC or SNRK-OE+siMTA1. The cells were collected and RNA was extracted. Reverse transcription-real-time fluorescence quantitative PCR was used to detect SNRK and MTA1 mRNA levels. The Student's t test was used to determine the statistical significance of the differences between the groups.

[0022] Figure 2 Vector+siNC, SNRK-OE, siMTA1 or SNRK-OE+siMTA1 were transfected into A549 cells respectively, and the proliferation ability of A549 cells was detected by CCK-8 assay. The statistical significance of the differences between the groups was verified by two-way ANOVA test.

[0023] Figure 3A549 cells were selected and transfected with Vector+siNC, SNRK-OE, siMTA1 or SNRK-OE+siMTA1, 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

[0024] 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.

[0025] Human non-small cell lung cancer cell line A549 cells are available from Shanghai Fuheng Biotechnology Co., Ltd.

[0026] siMTA1 targeting sequence: 5′-gcatcattgagtactacta-3′.

[0027] Example 1

[0028] The inventors' previous research found that SNRK expression levels were significantly reduced in non-small cell lung cancer tissues, while MTA1 expression levels were abnormally elevated. In-depth mechanistic studies have shown that there is a negative regulatory relationship between SNRK and MTA1, and that abnormal expression of both is closely associated with the malignant progression of non-small cell lung cancer. Based on this finding, the inventors proposed a strategy of simultaneously enhancing SNRK expression and inhibiting MTA1 expression, that is, jointly intervening in the SNRK-MTA1 signaling pathway axis, to produce a more significant anti-tumor effect. This synergistic regulatory scheme is expected to provide important guidance for the development and clinical application of anti-tumor drugs for non-small cell lung cancer.

[0029] Example 2

[0030] The nucleotide sequences of SNRK mRNA and MTA1 mRNA were obtained from the NCBI database, as shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. Based on this, an exogenous SNRK mRNA expression plasmid (SNRK-OE) that can specifically upregulate the expression of SNRK mRNA was designed and synthesized. The SNRK-OE plasmid contains the nucleotide sequence shown in SEQ ID NO.3, and its control plasmid is an empty vector plasmid (Vector); siRNAs (siMTA1) that can specifically interfere with the expression of MTA1 mRNA were designed and synthesized, and its targeting sequence is shown in SEQ ID NO.4. The control siRNA is siNC. Among them, 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 siMTA1 was synthesized by Qingke Bio according to the sequence information.

[0031] Vector+siNC or SNRK-OE+siMTA1 were transfected into A549 cells with a growth density of 60%. 48 hours after transfection, the cells were collected and total RNA was extracted. The expression levels of SNRK mRNA and MTA1 mRNA were detected by reverse transcription-real-time fluorescence quantitative PCR. The specific results are shown in the figure. Figure 1 shown.

[0032] Depend on Figure 1 It can be seen that compared with transfection with Vector+siNC, transfection with SNRK-OE+siMTA1 into A549 cells can significantly upregulate the expression level of SNRK mRNA and significantly downregulate the expression level of MTA1 mRNA.

[0033] The specific implementation process is as follows:

[0034] (1) A549 cells were plated in 6-well plates. When the cell growth density reached about 60%, vector + siNC or SNRK-OE + siMTA1 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 2 μg of plasmid and 3 μL of siRNA (10 μM).

[0035] (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.

[0036] (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. -ΔΔCTThe relative expression levels of SNRK mRNA and MTA1 mRNA were calculated using the formula.

[0037] Example 3

[0038] A549 cells were selected and transfected with the same dose of Vector+siNC, SNRK-OE, siMTA1 or SNRK-OE+siMTA1, 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.

[0039] Depend on Figure 2 It can be seen that transfection with SNRK-OE, siMTA1, or SNRK-OE+siMTA1 can slow down the growth rate of A549 cells compared with Vector+siNC, and the cell proliferation inhibitory effect of simultaneous transfection with SNRK-OE+siMTA1 is more obvious than that of single transfection with SNRK-OE or siMTA1. This indicates that the combined intervention of SNRK and MTA1 expression levels has a more prominent inhibitory effect on tumor cell proliferation than targeting the expression level of either gene alone.

[0040] The specific implementation process is as follows:

[0041] (1) A549 cells were transfected with Vector+siNC, SNRK-OE, siMTA1, or SNRK-OE+siMTA1, respectively. The doses of Vector and SNRK-OE were 2 μg / well, and the doses of siNC and siMTA1 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 five groups (Day 0, 1, 2, 3, 4), with four transfected cells (Vector+siNC, SNRK-OE, siMTA1, or SNRK-OE+siMTA1) in each group, and three replicate wells were plated for each cell type.

[0042] (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);

[0043] (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.

[0044] Example 4

[0045] A549 cells were selected and transfected with the same dose of Vector+siNC, SNRK-OE, siMTA1 or SNRK-OE+siMTA1, and the migration ability of the transfected cells was evaluated by Transwell assay. The specific results are shown in the figure. Figure 3 shown.

[0046] Depend on Figure 3 As shown, transfection with SNRK-OE+siMTA1 significantly inhibited A549 cell migration compared to transfection with either SNRK-OE or siMTA1 alone. This result suggests that simultaneously interfering with SNRK and MTA1 gene expression levels exhibits a more effective tumor suppressor effect than targeting either gene alone.

[0047] The specific implementation process is as follows:

[0048] (1) A549 cells were selected and cultured in 6-well plates. When the cell growth density reached about 60%, they were transfected with Vector+siNC, SNRK-OE, siMTA1, or SNRK-OE+siMTA1, respectively. The specific method was as described in Example 3.

[0049] (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.

[0050] (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;

[0051] (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. The migrated cells were observed under an inverted microscope and photographed for recording, and the cells were counted and statistically analyzed.

Claims

1. Application of the SNRK-MTA1 signaling pathway axis as a drug target in the preparation of targeted therapeutic drugs for non-small cell lung cancer, characterized in that: The SNRK-MTA1 signaling pathway axis includes the SNRK gene and the MTA1 gene; the nucleotide sequence of the mRNA of the SNRK gene is shown in SEQ ID NO.1, and the nucleotide sequence of the mRNA of the MTA1 gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The non-small cell lung cancer targeted therapeutic drug uses the SNRK gene and the MTA1 gene as intervention targets, introduces the exogenous SNRK gene and interferes with the expression of the MTA1 gene, and can efficiently and specifically upregulate the expression level of the SNRK gene while downregulating the expression level of the MTA1 gene.

3. The use according to claim 1, characterized in that The non-small cell lung cancer targeted therapeutic drug is a composition of an inhibitor that specifically interferes with MTA1 gene expression and an exogenous expression plasmid that specifically upregulates SNRK gene expression.

4. Use of an exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression and an siRNA that specifically interferes with MTA1 gene expression in the preparation of a targeted therapeutic drug for non-small cell lung cancer, characterized in that: The exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3; the targeting nucleotide sequence of the siRNA that specifically interferes with the expression of the MTA1 gene is shown in SEQ ID NO.

4.

5. The use according to claim 4, characterized in that The mass-to-volume ratio of the exogenous SNRKmRNA expression plasmid that specifically upregulates SNRK gene expression to the siRNA that specifically interferes with MTA1 gene expression is 2:3, unit: μg / μL.

6. A targeted therapeutic drug for non-small cell lung cancer, characterized in that: The non-small cell lung cancer targeted therapeutic drug contains an exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression and an siRNA that specifically interferes with MTA1 gene expression; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3; the targeting nucleotide sequence of the siRNA that specifically interferes with MTA1 gene expression is shown in SEQ ID NO.

4.

7. The use according to claim 5, characterized in that In the non-small cell lung cancer targeted therapeutic drug, the mass volume ratio of the exogenous SNRK mRNA expression plasmid that specifically upregulates SNRK gene expression to the siRNA that specifically interferes with MTA1 gene expression is 2:3, unit: μg / μL.

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