Method for screening interferon pathway activated by intervention of glioblastoma

Abstract

The invention discloses a method for screening interferon pathways activated by intervention of glioblastoma, and relates to the technical field of biological medicines. The invention provides a new application of Atopaxar in treatment of glioma, and the clinical application range of the medicine is widened; meanwhile, clear molecular mechanism evidence is provided, and it is proved that the cGAS-STING-1 type interferon pathway is activated to achieve the anti-tumor effect; the invention proposes that Atopaxar has good blood brain barrier penetrating power, solves the bottleneck problem of a traditional immune agonist in central nervous system tumor treatment, and provides a new target spot and strategy for precise immunotherapy of glioblastoma.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for screening interferon pathway activation in glioblastoma. Background Technology

[0002] Glioblastoma (GBM) is one of the most challenging brain tumors to treat clinically. Current clinical treatment methods mainly include: (1) Surgical resection: The tumor tissue can be safely removed to the maximum extent, but due to the high invasiveness of GBM, it is difficult to completely remove the tumor cells.

[0003] (2) Radiotherapy: Postoperative adjuvant radiotherapy, using conformal intensity-modulated radiotherapy to target the residual tumor area.

[0004] (3) Chemotherapy: Temozolomide (TMZ) is a standard chemotherapy drug that causes tumor cell death by methylating DNA.

[0005] (4) Targeted therapy: Drugs targeting specific molecular targets, such as anti-angiogenic drugs like bevacizumab.

[0006] In immunotherapy, the cGAS-STING signaling pathway, as an important component of innate immunity, has had its mechanism of action preliminarily elucidated: after cGAS recognizes cytoplasmic dsDNA, it catalyzes the synthesis of the second messenger cGAMP, which activates the STING protein and triggers downstream signaling cascades, ultimately inducing the production of type 1 interferon.

[0007] Problems and shortcomings of existing technologies: (1) Traditional treatments have limited effectiveness While current standard treatments can temporarily control tumor growth, the overall survival of GBM patients remains extremely poor. The main reasons are: The blood-brain barrier restricts drug penetration; Tumors exhibit strong heterogeneity and are prone to developing treatment resistance; The tumor microenvironment has strong immunosuppressive properties; (2) Difficulty in clinical translation of cGAS-STING targeted therapy Although preclinical studies have shown that several cGAS-STING agonists (such as flavonoid 8-acetic acid) have anti-tumor potential, none have achieved the expected results in clinical trials. The main reasons for this include: The drug has poor stability and a short half-life in vivo. Insufficient targeting can easily trigger a systemic immune response; The blood-brain barrier has limited penetration ability; The complexity of the tumor immune microenvironment affects drug efficacy; (3) The mechanism of action is unclear. The specific mechanisms of action of existing cGAS-STING agonists in gliomas have not been fully elucidated, particularly: The specific regulatory mechanisms of type 1 interferon pathway activation; Direct effects on tumor cell proliferation and apoptosis; Synergistic mechanism with other treatment methods; The existence of these problems severely restricts the clinical application of glioma treatment strategies based on the cGAS-STING pathway, thus requiring a new solution to these issues. Summary of the Invention

[0008] The purpose of this invention is to provide a method for screening interferon pathway activation in glioblastoma, so as to solve the technical problems mentioned in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a method for screening interferon pathway activation in glioblastoma, comprising at least the following steps: S1: Prepare experimental materials, human glioma cell line U251 and the test drug Atopaxar; S2: Cell treatment; S3: RNA extraction, cell collection, and extraction of total RNA using RNAiso plus reagent; S4: RNA reverse transcription, using a reverse transcription kit to reverse transcribe RNA into cDNA; S5: qPCR detection, using the qPCR method to detect the expression level of type 1 interferon-related genes; S6: Data analysis, comparing the expression differences of type 1 interferon-related genes in the Atopaxar treatment group and the control group, and evaluating the activation effect of Atopaxar on the interferon pathway.

[0010] Furthermore, S2 includes at least the following steps: U251 cells were seeded into 6-well plates at a density of 500,000 cells per well, with 3 replicates per cell type. Add 2 ml of culture medium to each well and add Atopaxar at a working concentration of 5 μM. Add an equal volume of DMSO to the control group. Incubate at a constant temperature.

[0011] Furthermore, the isothermal culture is performed by culturing the cells in a 37°C cell incubator for 48 hours.

[0012] Furthermore, the type 1 interferon-related genes include at least IFNA1, IFNA2, and IFNB1.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention proposes a new use for Atopaxar in the treatment of glioma, broadening the clinical application scope of this drug; it also provides clear molecular mechanism evidence, confirming that it exerts its anti-tumor effect by activating the cGAS-STING-1 interferon pathway; and it proposes that Atopaxar has good blood-brain barrier penetration ability, solving the bottleneck problem of traditional immune agonists in the treatment of central nervous system tumors, providing a new target and strategy for the precision immunotherapy of glioblastoma. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 A statistical graph of RNA expression of type I interferon-related genes; Figure 2 This is a statistical graph showing the change in the proliferation capacity of U251 cells over time after drug treatment. Detailed Implementation

[0016] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0017] A screening method for interferon pathway activation in glioblastoma includes at least the following steps: S1: Prepare experimental materials, human glioma cell line U251 and the test drug Atopaxar; S2: Cell treatment; S3: RNA extraction, cell collection, and extraction of total RNA using RNAiso plus reagent; S4: RNA reverse transcription, using a reverse transcription kit to reverse transcribe RNA into cDNA; S5: qPCR detection, using the qPCR method to detect the expression level of type 1 interferon-related genes; S6: Data analysis, comparing the expression differences of type 1 interferon-related genes in the Atopaxar treatment group and the control group, and evaluating the activation effect of Atopaxar on the interferon pathway.

[0018] S2 includes at least the following steps: U251 cells were seeded into 6-well plates at a density of 500,000 cells per well, with 3 replicates per cell type. Add 2 ml of culture medium to each well and add Atopaxar at a working concentration of 5 μM. Add an equal volume of DMSO to the control group. Incubate at a constant temperature.

[0019] The isothermal culture was performed in a 37°C cell incubator for 48 hours.

[0020] The type 1 interferon-related genes include at least IFNA1, IFNA2, and IFNB1.

[0021] Based on the above implementation, the following specific operations are proposed: Cellular experiments: Preparation before the experiment: ① Human glioma cell line U251 was cultured in DMEM medium with 10% serum. ② Dissolve 1 mg of Atopaxar in 0.2084 mL of DMSO to prepare a 10 mM solution and store it in a -80°C refrigerator.

[0022] U251 cells were seeded into 6-well plates at a density of 500,000 cells per well, with each cell type seeded in 3 replicates. After adding 2 ml of culture medium to each well, Atopaxar was added at a working concentration of 5 μM. An equal volume of DMSO was added to the control group.

[0023] The cells were placed in a 37°C incubator and cultured for 48 hours. Their morphology and proliferation were observed under a microscope.

[0024] The experiment was repeated three times.

[0025] RNA extraction experiment Sample preparation: Discard the supernatant culture medium from adherent cells, wash twice with 1 mL PBS, add 500 μl of 0.25% trypsin and incubate at 37°C for 1 min, then stop digestion with 1 mL of LDM medium. After rinsing the dish walls with pipette tips, collect the cells in 1.5 mL EP tubes and centrifuge at 4000 rpm at 4°C for 5 min. Discard the supernatant, add 1 mL of RNAiso plus, and incubate for 5 min.

[0026] Phase separation: Add 200 μl of chloroform to each 1 mL of RNAiso plus, shake vigorously for 20 seconds, let stand at room temperature for 5 min, centrifuge at 12000 rpm at 4℃ for 5 min, and separate into three layers (the upper layer is a colorless solid phase containing RNA, the middle layer is a protein, and the lower layer is a pink organic phase).

[0027] RNA precipitation: Carefully transfer the upper aqueous phase to a new centrifuge tube, add an equal volume of isopropanol, gently invert to mix, let stand at room temperature for 10 min, centrifuge at 12000 rpm for 10 min at 4℃, and a white RNA precipitate will appear at the bottom of the tube.

[0028] RNA washing: After discarding the supernatant, add 1 mL of 75% ethanol (prepared with DEPC water), vortex to suspend the precipitate, and incubate at 7500 rpm for 10 min at 4℃. Discard the supernatant and dry at room temperature for 5-10 dishes (avoid over-drying).

[0029] RNA dissolution: Add 25 μl of RNase-free water, incubate for 10 min to dissolve, and store at -80℃ for long-term storage.

[0030] RNA concentration and purity determination (spectrophotometry): Take 1 μl of RNA sample and use NanoDrop to determine the A260 / A280 and A260 / A230 values. Acceptable standards: A260 / A280 = 1.8-2.1 (no protein contamination); A260 / A230 ≥ 2.0 (no organic residue). The experiment was repeated three times. RNA reverse transcription Reverse transcription system: Prepare the reverse transcription system according to Table 1. Table 1 Reverse transcription program: Set up the reverse transcription program according to Table 2. Table 2 Dilute the PCR product 30 times and store the resulting cDNA at -20°C. The experiment was repeated three times. qPCR system: Prepare the qPCR system according to Table 3. Table 3 qPCR program: Set up the qPCR reaction program according to Table 4. Table 4 The experiment was repeated three times.

[0031] The experimental results showed that after the addition of 5 μM atopaxar, the type 1 interferon-related pathway was activated, and the gene expression levels of interferons IFNA1, IFNA2, and IFNB1 were significantly increased. Figure 1 ).

[0032] Furthermore, a CCK8 cell viability assay was proposed. First, count the number of cells in the prepared cell suspension using a cell counting chamber. Seed the cells into 96-well plates at a density of 4000 U251 cells per well (100 μL / well). Pre-culture the plates in an incubator for a period of time (37°C, 5% CO2).

[0033] Add 10 μL of CCK-8 solution to each well (be careful not to generate air bubbles in the wells, as they will affect the OD value reading).

[0034] Incubate the culture plate in an incubator for 1.5 hours.

[0035] The absorbance at 450 nm was measured using an ELISA reader.

[0036] Cell viability calculation: Cell viability * (%) = [A(drug-added) - A(blank)] / [A(0-drug-added) - A(blank)] × 100 A(drug-added): Absorbance of wells containing cells, CCK-8 solution, and drug solution A(blank): Absorbance of wells containing culture medium and CCK-8 solution but no cells A(0-drug-added): Absorbance of wells containing cells and CCK-8 solution but no drug solution The experiment was repeated three times.

[0037] The experimental results show that, as detected by CCK8 assay, the viability of U251 cells treated with Atopaxar decreased compared to the control group, and this phenomenon was concentration-dependent. Figure 2 ).

[0038] In summary, the present invention has the following characteristics: This provides a novel drug application strategy that can effectively activate the type 1 interferon pathway in glioma cells; To elucidate the specific mechanism of action of Atopaxar in the treatment of glioma; To develop a cGAS-STING pathway-targeted therapy strategy with good blood-brain barrier penetration capability; This provides a new approach for the precision treatment of glioblastoma based on the activation of the type 1 interferon pathway.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for screening interventions that activate the interferon pathway in glioblastoma, characterized in that: At least the following steps are included: S1: Prepare experimental materials, human glioma cell line U251 and the test drug Atopaxar; S2: Cell treatment; S3: RNA extraction, cell collection, and extraction of total RNA using RNAiso plus reagent; S4: RNA reverse transcription, using a reverse transcription kit to reverse transcribe RNA into cDNA; S5: qPCR detection, using the qPCR method to detect the expression level of type 1 interferon-related genes; S6: Data analysis, comparing the expression differences of type 1 interferon-related genes in the Atopaxar treatment group and the control group, and evaluating the activation effect of Atopaxar on the interferon pathway.

2. The method for screening interferon pathway activation in glioblastoma according to claim 1, characterized in that: S2 includes at least the following steps: U251 cells were seeded into 6-well plates at a density of 500,000 cells per well, with 3 replicates per cell type. Add 2 ml of culture medium to each well and add Atopaxar at a working concentration of 5 μM. Add an equal volume of DMSO to the control group. Incubate at a constant temperature.

3. The method for screening interferon pathway activation in glioblastoma according to claim 2, characterized in that: The isothermal culture was performed in a 37°C cell incubator for 48 hours.

4. The method for screening interferon pathway activation in glioblastoma according to claim 1, characterized in that: The type 1 interferon-related genes include at least IFNA1, IFNA2, and IFNB1.

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