Combined up-regulation of SNRK gene and GSK3B gene expression level in preparation of anti-non-small cell lung cancer drugs

Abstract

The application relates to application of combined up-regulation of SNRK gene and GSK3B gene expression levels in preparation of anti-non-small cell lung cancer drugs. The nucleotide sequences of the mRNA of the SNRK gene and the mRNA of the GSK3B gene are shown in SEQ ID NO. 1-2. The application first discloses the key role of combined up-regulation of SNRK and GSK3B mRNA expression levels in non-small cell lung cancer proliferation and migration. Experimental results show that, compared with single transfection of an exogenous SNRK mRNA expression plasmid or an exogenous GSK3B mRNA expression plasmid, the strategy of combined transfection of the SNRK-OE and GSK3B-OE exogenous expression plasmids designed by the application can more significantly inhibit the proliferation and migration ability of tumor cells. The synergistic effect indicates that SNRK and GSK3B mRNA can be used as a new anti-tumor target, the mode of combined up-regulation of SNRK and GSK3B mRNA expression levels can be used as a new non-small cell lung cancer treatment method, and a theoretical basis is provided for drug development of non-small cell lung cancer.

Description

TECHNICAL FIELD

[0001] The application relates to application of combined up-regulation of SNRK gene and GSK3B gene expression levels in preparation of anti-non-small cell lung cancer drugs and belongs to the biomedical technology field. BACKGROUND

[0002] Globally, lung cancer is the highest incidence of malignant tumor and the primary cause of cancer-related death. Epidemiological data in China show that lung cancer ranks first in male cancer incidence and second in female population, and is the primary cause of cancer-related death. In terms of pathological types, about 85% of lung cancer belongs to non-small cell lung cancer (NSCLC), which specifically includes lung adenocarcinoma, lung squamous cell carcinoma and lung large cell carcinoma. The currently clinically applied targeted drugs mainly target EGFR sensitive mutations, however, nearly half of NSCLC patients do not carry the mutation, and drug resistance problems commonly exist in the treatment process, which greatly restricts the clinical application effect and treatment benefit of the targeted drugs. Therefore, finding new treatment targets and establishing the corresponding intervention strategies have become key scientific problems to be broken through in the field of NSCLC targeted therapy.

[0003] Gene expression plasmid is a circular DNA molecule artificially designed, which can self-replicate after being transfected into a host cell and accurately regulate the transcription and translation process of an exogenous gene, so as to realize efficient expression of the target gene. Its function realization depends on the following key design elements: replication origin, multiple cloning site, promoter, terminator, antibiotic resistance gene and reporter gene, etc. The gene expression plasmid has become an important tool for studying gene function and treating genetic diseases and malignant tumors, and its high efficiency, flexibility and low immunogenicity make it have wide application prospects in the fields of basic research and clinical transformation. SUMMARY

[0004] In view of the deficiencies of the prior art, the application provides application of combined up-regulation of SNRK gene and GSK3B gene expression levels in preparation of anti-non-small cell lung cancer drugs.

[0005] The technical scheme of the application is as follows:

[0006] Application of SNRK gene and GSK3B gene as drug targets in preparation of anti-non-small cell lung cancer drugs.

[0007] According to the application, 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 GSK3B gene is shown in SEQ ID NO. 2.

[0008] According to the application, the anti-non-small cell lung cancer drug takes SNRK gene and GSK3B gene as therapeutic targets, and can efficiently and specifically up-regulate the expression levels of SNRK gene and GSK3B gene by introducing exogenous SNRK gene and GSK3B gene.

[0009] The exogenous SNRK mRNA expression plasmid for specifically up-regulating SNRK mRNA expression and the exogenous GSK3B mRNA expression plasmid for specifically up-regulating GSK3B mRNA expression are used for preparing the anti-non-small cell lung cancer drug.

[0010] According to the application, the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO. 3, and the exogenous GSK3B mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO. 4. By overexpressing the two sequences shown as SEQ ID NO. 3-4, the SNRK and GSK3B mRNA expression levels can be jointly up-regulated.

[0011] Further preferably, the mass ratio of the exogenous SNRK mRNA expression plasmid to the exogenous GSK3B mRNA expression plasmid is 1:1.

[0012] An anti-non-small cell lung cancer drug contains an exogenous SNRK mRNA expression plasmid and an exogenous GSK3B mRNA expression plasmid.

[0013] Further preferably, the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO. 3, and the exogenous GSK3B mRNA expression plasmid contains the nucleotide sequence shown as SEQ ID NO. 4.

[0014] Most preferably, the mass ratio of the exogenous SNRK mRNA expression plasmid to the exogenous GSK3B mRNA expression plasmid is 1:1.

[0015] Advantages:

[0016] The application first discloses a key role of jointly up-regulating SNRK and GSK3B mRNA expression levels in non-small cell lung cancer proliferation and migration. The experimental results show that, compared with separately transfecting an exogenous SNRK mRNA expression plasmid or an exogenous GSK3B mRNA expression plasmid, a strategy of jointly transfecting the SNRK-OE and GSK3B-OE exogenous expression plasmids designed in the application can more significantly inhibit the proliferation and migration ability of tumor cells. The synergistic effect indicates that SNRK and GSK3B genes can be used as new anti-tumor targets, and the method of jointly up-regulating the SNRK and GSK3B mRNA expression levels can be used as a new non-small cell lung cancer treatment method, and provides a theoretical basis for non-small cell lung cancer drug development. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Vector plasmid or SG-OE plasmid was transfected into A549 cells, RNA was extracted, and reverse transcription-real-time fluorescent quantitative PCR was used to detect the SNRK and GSK3B mRNA levels, and Student's t test was used to determine the statistical significance of the differences between groups.

[0018] Figure 2 A549 cells were selected, and Vector, SNRK-OE, GSK3B-OE or SG-OE plasmids were transfected, respectively, CCK-8 experiment was used to evaluate the proliferation ability of A549 cells, and the statistical significance of the differences between multiple groups was verified by two-way variance test.

[0019] Figure 3 A549 cells were selected, and Vector, SNRK-OE, GSK3B-OE or SG-OE plasmids were transfected, respectively, Transwell experiment was used to detect the migration ability of A549 cells, and one-way variance test was used to determine the statistical significance of the differences between groups. DETAILED DESCRIPTION

[0020] The technical solutions of the application will be further described below in combination with specific experimental examples, but the protection scope of the application is not limited to this. The reagents and materials involved in the examples are all ordinary commercially available products if not otherwise specified.

[0021] Human non-small cell lung cancer cell line A549 cells are commercially available from Shanghai Fuheng Biological Technology Co., Ltd.

[0022] Example 1

[0023] The inventors found in previous studies that the expression levels of SNRK gene and GSK3B gene in non-small cell lung cancer are significantly down-regulated. Further comprehensive study on the mechanism of the two genes found that they are related to the malignant progression of non-small cell lung cancer. Therefore, the inventors believe that up-regulating the expression levels of SNRK gene and GSK3B gene will play a better anticancer role in non-small cell lung cancer, and the intervention strategy of jointly up-regulating the expression levels of SNRK and GSK3B mRNA will play an important role in the research and development of anti-tumor drugs for non-small cell lung cancer.

[0024] Example 2

[0025] The nucleotide sequences of SNRK mRNA and GSK3B mRNA were obtained from the NCBI database, as shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively, and based on this, the exogenous SNRK mRNA expression plasmid (SNRK-OE) and the exogenous GSK3B mRNA expression plasmid (GSK3B-OE) that can specifically up-regulate the expression of SNRK mRNA and GSK3B mRNA were designed and synthesized. The exogenous SNRK mRNA expression plasmid contains a nucleotide sequence as shown in SEQ ID NO. 3, and the exogenous GSK3B mRNA expression plasmid contains a nucleotide sequence as shown in SEQ ID NO. 4. The two exogenous expression plasmids SNRK-OE and GSK3B-OE were mixed in a mass ratio of 1:1 to prepare the joint expression plasmid SG-OE. The SNRK-OE and GSK3B-OE plasmids were both entrusted to the Moliang plasmid platform for construction according to the sequence information.

[0026] The SG-OE group and the vector control Vector group were set up, and SG-OE (2 μg) and empty vector plasmid (Vector, 2 μg) were transfected into A549 cells with a growth density of 60%. After 48 hours of transfection, the cells were collected and total RNA was extracted, and the expression levels of SNRK mRNA and GSK3B mRNA were detected by reverse transcription-real-time fluorescent quantitative PCR. The specific results are shown in Figure 1 . The empty vector plasmid (Vector) is a conventional existing plasmid and can be obtained commercially.

[0027] It can be seen from Figure 1 that transfection of the joint expression plasmid SG-OE can significantly up-regulate the expression levels of SNRK mRNA and GSK3B mRNA in A549 cells.

[0028] The specific operation process is as follows:

[0029] (1) A549 cells were seeded in 6-well plates. When the cell density reached about 60% confluence, 2 μg of SG-OE or Vector plasmid was transfected into A549 cells using Lipofectamine 3000 transfection reagent (Thermo Fisher Scientific, L3000015).

[0030] (2) Cells were collected 48 hours after transfection. 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.

[0031] (3) Using the extracted RNA as a template, LunaScript was used. TM cDNA was synthesized using the RT SuperMix Kit (NEB, E3010) and Power. Green Master Mix (Thermo Fisher Scientific, 4367659) in QuantStudio TM Real-time quantitative PCR analysis was performed on a 5-System (Thermo Fisher Scientific). ACTB was selected as an internal control gene, and the results were analyzed using 2... -ΔΔCT The relative expression levels of SNRK mRNA and GSK3B mRNA were calculated using this method.

[0032] Example 3

[0033] The exogenous expression plasmids SNRK-OE and GSK3B-OE were mixed at a 1:1 mass ratio to prepare the co-expression plasmid SG-OE. A549 cells were selected, and equal masses (2 μg) of the SNRK-OE, GSK3B-OE, and SG-OE plasmids, along with the control Vector plasmid, were transfected into A549 cells. Cell proliferation was assessed using a CCK-8 assay. Specific results are shown below. Figure 2 As shown.

[0034] Depend on Figure 2 It was found that transfection with SNRK-OE, GSK3B-OE, or SG-OE significantly inhibited A549 cell growth compared to Vector, but the weakening effect of SG-OE on A549 cell proliferation was more significant than that of SNRK-OE or GSK3B-OE. This indicates that combined upregulation of SNRK and GSK3B mRNA expression levels exhibits superior tumor cell proliferation inhibition compared to upregulating either gene expression level alone.

[0035] The specific implementation process is as follows:

[0036] (1) Equal amounts (2 μg) of SNRK-OE, GSK3B-OE, SG-OE plasmids and control Vector plasmids were respectively transfected into A549 cells, and the specific method is described in Example 2. The transfected cells were collected 48 hours after transfection, and different transfected cells were re-cultured in 96-well plates (2000 cells / well), which were divided into 5 groups (Day 0, 1, 2, 3, and 4). Each group included Vector, SNRK-OE, GSK3B-OE, and SG-OE, a total of 4 kinds of transfected cells, and each cell was plated in 3 replicate wells.

[0037] (2) After the cells obtained in step (1) adhered (about 2-4 hours), 10 μL of CCK-8 reagent (TargetMol, C0005) was added to the Day 0 group, and incubated in a 37°C incubator for 1 hour. After simple shaking, the absorbance value at 450 nm was measured in an enzyme marker. This is the initial planting cell (Day 0) absorbance value.

[0038] (3) After the initial cell absorbance value measurement was completed, the absorbance values of the cells in the Day 1, 2, 3, and 4 groups were measured every 24 hours according to the same method. The absorbance value of the Day 0 group was used as a reference to calculate the cell viability changes of each group, and the cell proliferation trend of each group was calculated.

[0039] Example 4

[0040] SNRK-OE and GSK3B-OE two exogenous plasmids were mixed in a mass ratio of 1:1 to prepare the joint expression plasmid SG-OE. A549 cells were selected, and the same mass (2 μg) of SNRK-OE, GSK3B-OE, SG-OE plasmids and control Vector plasmids were respectively transfected into A549 cells. The Transwell experiment was used to evaluate the migration ability of the transfected cells, and the specific results are shown in Figure 3 .

[0041] As can be seen from Figure 3 , the inhibitory effect of transfection of the SG-OE plasmid on the migration ability of A549 cells is more significant than that of transfection of SNRK-OE or GSK3B-OE alone. This indicates that compared to the single up-regulation of the expression level of any one of the genes, the joint up-regulation of the expression level of SNRK and GSK3B mRNA exhibits a more optimal tumor cell migration inhibition effect. Combined with the results obtained in Example 3, it can be concluded that compared to the single targeting up-regulation of any one of the genes, the joint up-regulation of the expression level of SNRK and GSK3B mRNA exhibits a more effective anticancer effect.

[0042] The specific implementation process is as follows:

[0043] (1) Select A549 cells and culture in 6-well plates, when the cell density reaches about 60%, respectively transfect Vector, SNRK-OE, GSK3B-OE and SG-OE plasmid. The specific method is described in Example 2;

[0044] (2) Add complete culture medium (600 μL / well) in 24-well plates, and place the Transwell chamber (Corning, 3422) in the well plate;

[0045] (3) After 48 hours of transfection, collect the transfected cells obtained in step (2) and count, adjust the cell concentration to 100,000 / mL, add 200 μL of cell suspension into each Transwell chamber, and place in the cell culture box (37℃, 5% CO2) for further culture for 48 hours;

[0046] (4) Collect the Transwell chamber and discard the culture medium, wash with PBS buffer, fix the cells at room temperature using 4% paraformaldehyde for 15 minutes, then stain with crystal violet (0.1%) overnight, remove the cells that have not migrated on the inner surface of the chamber, observe the migrated cells under an inverted microscope and take pictures, count and statistically analyze.

Claims

1. The application of a co-expression plasmid co-expressing the SNRK gene and the GSK3B gene in the preparation of a drug for non-small cell lung cancer, wherein the nucleotide sequence of the SNRK gene mRNA is shown in SEQ ID NO.1, and the nucleotide sequence of the GSK3B gene mRNA is shown in SEQ ID NO.2; the co-expression plasmid is prepared by mixing the SNRK expression plasmid and the GSK3B expression plasmid at a 1:1 mass ratio.

2. The application of exogenous SNRK mRNA expression plasmids that specifically upregulate SNRK mRNA expression and exogenous GSK3B mRNA expression plasmids that specifically upregulate GSK3B mRNA expression in the preparation of drugs for treating non-small cell lung cancer, characterized in that, The exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3, and the exogenous GSK3B mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.4; the mass ratio of the exogenous SNRK mRNA expression plasmid to the exogenous GSK3B mRNA expression plasmid is 1:

1.

3. A drug for treating non-small cell lung cancer, characterized in that, The anti-non-small cell lung cancer drug contains an exogenous SNRK mRNA expression plasmid and an exogenous GSK3B mRNA expression plasmid; the exogenous SNRK mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.3, and the exogenous GSK3B mRNA expression plasmid contains the nucleotide sequence shown in SEQ ID NO.4; in the anti-non-small cell lung cancer drug, the mass ratio of the exogenous SNRK mRNA expression plasmid to the exogenous GSK3B mRNA expression plasmid is 1:1.

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