Application of MIDN in diagnosis and treatment of glioma

Abstract

The invention relates to the technical field of biomedicine, and particularly discloses application of MIDN protein in diagnosis and treatment of glioma. Glioblastoma (GBM) is the most common malignant glioma in the central nervous system and accounts for more than 50% of all glioma, and the existing treatment means face challenges of high drug resistance, poor prognosis and the like. Based on the association between protein degradation imbalance and tumor occurrence and development, the invention finds that MIDN can capture substrate protein through a Catch structural domain thereof and degrade carcinogenic substrate molecules (such as PPDPF) through a non-ubiquitin proteasome pathway, thereby inhibiting the malignant progression of glioma.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to the application of MIDN in the diagnosis and treatment of glioma. Background Technology

[0002] Glioblastoma (GBM) is a malignant tumor located in the central nervous system. 95% of central nervous system malignancies occur in the brain, with 75% originating from glial cells. GBM accounts for more than 50% of all gliomas. Dysregulation of protein degradation is closely related to tumor development and progression. Recent studies have found that MIDN (myeloblastoma mitochondrial granuloma) undergoes ubiquitination-independent degradation via the proteasome pathway, potentially participating in tumor regulation.

[0003] In existing technologies, treatment options for gliomas are limited, and drug resistance is a significant problem. The role of MIDN in different tumors remains unclear, especially its functional mechanism in gliomas, which requires further investigation. Against this backdrop, this invention proposes a novel application of MIDN in the diagnosis and treatment of gliomas. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of MIDN in the diagnosis and treatment of gliomas.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: An application of MIDN in the diagnosis and treatment of glioma, specifically glioblastoma GBM, characterized by: assessing the presence or malignancy of glioma by detecting the expression level of MIDN in biological samples.

[0006] An application of MIDN protein in the treatment of glioma, specifically glioblastoma GBM, characterized by inhibiting glioma cell proliferation by regulating the expression or activity of MIDN.

[0007] Preferably, the method of regulating MIDN expression includes overexpressing MIDN or using a MIDN agonist.

[0008] An application of the interaction between MIDN and PPDPF in the diagnosis and treatment of glioma: glioma can be diagnosed or treatment efficacy can be evaluated by detecting the interaction level between MIDN and PPDPF.

[0009] A method for constructing MIDN knockdown or overexpression glioma cell lines, using siRNA or vector transfection technology to regulate the expression of MIDN in different types of glioma cells U251, U87 or Hs683.

[0010] A glioma treatment drug based on MIDN degradation of PPDPF, comprising MIDN protein or an active fragment thereof, for promoting PPDPF degradation.

[0011] A method for screening compounds targeting the MIDN-PPDPF pathway, and evaluating the effects of compounds on the interaction or degradation of MIDN and PPDPF using in vitro or in vivo models.

[0012] An application of MIDN as a biomarker in the prognostic assessment of glioma, combining MIDN and PPDPF expression levels to predict patient survival.

[0013] A therapeutic composition containing the MIDN gene for gene therapy of glioma, wherein the MIDN gene is delivered via a viral vector.

[0014] The beneficial effects of this invention are as follows: MIDN, as a novel tumor suppressor factor, can inhibit the malignant progression of gliomas by targeting and degrading molecules such as PPDPF, providing a new strategy for clinical diagnosis and treatment.

[0015] (It could be added that: PPDPF has been found to have a pro-cancer effect in glioma cells, and that PPDPF is a substrate protein of MIDN.) Attached Figure Description

[0016] Figure 1 A schematic diagram of glioma epidemiological data, showing the proportion of GBM in gliomas; Figure 2 : MIDN knockdown efficiency verification graph, showing the MIDN knockdown effect in U251 and U87 cells using Western blot; Figure 3 The decrease in MIDN expression promoted glioma cell proliferation, as verified by CCK8 cell proliferation and cell cycle assays. Figure 4 : MIDN overexpression efficiency verification graph, showing the MIDN overexpression effect in U87 and Hs683 cells by Western blot; Figure 5 The effects of upregulated MIDN expression on glioma cell proliferation were verified using CCK8 cell proliferation assays, cell cycle assays, and colony formation assays. Figure 6 Knocking down PPDPF protein expression in U87 cells inhibited glioma cell proliferation. Figure 7 Overexpression of PPDPF protein in U87 cells promotes glioma cell proliferation; Figure 8 : Validate the interaction between MIDN and its substrate protein PPDPF and the proteasome core regulatory subunit PSMD2 in the U87 cell line; Figure 9MIDN can promote the degradation of PPDPF protein; Figure 10 MIDN can inhibit the proliferative effect of PPDPF on glioma cells. Detailed Implementation

[0017] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0018] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0019] Example 1: Construction of MIDN overexpression plasmid and verification of its overexpression efficiency in glioma U87 cells. To evaluate the role of MIDN in glioma, this application constructed an overexpression plasmid of MIDN and constructed a MIDN-overexpressing U87 cell line using plasmid transfection technology. Subsequently, Western blot was used to detect the expression level of MIDN in different glioma cell lines (such as U251, U87, and Hs683), laying the foundation for subsequent functional studies.

[0020] The steps for constructing the MIDN overexpression plasmid are as follows: 1.1 PCR amplification of MIDN ORF 1. Preparation of PCR reaction system (50 μL system, high-fidelity amplification, avoid base mismatch): Add 5 μL of 10× high-fidelity PCR buffer, 4 μL of dNTP mixture (2.5 mmol / L each), 2 μL of upstream primer (10 μmol / L), 2 μL of downstream primer (10 μmol / L), 1 μL of template DNA (cDNA containing the MIDN gene), 0.5 μL of high-fidelity DNA polymerase (such as PrimeSTAR), and 35.5 μL of sterile deionized water to a sterile centrifuge tube. Mix gently, centrifuge briefly (1000 rpm, 10 s), and collect the liquid to the bottom of the tube to avoid air bubbles affecting amplification.

[0021] 1.2 PCR reaction program settings (adapted to high-fidelity enzyme to reduce mismatch): Pre-denaturation 95℃ 5 min; denaturation 95℃ 30 s, annealing (adjust according to primer annealing temperature, gradient annealing can be set for optimization) 30 s, extension 72℃ (extension time is calculated according to the target fragment length, 1 kb / min, such as MIDN ORF is 1.2 kb, then extension is 72 s), for a total of 30 cycles (high-fidelity enzyme amplification efficiency is slightly lower, avoid excessive cycles leading to non-specific bands); final extension 72℃ 10 min (ensure complete extension of the target fragment); incubate at 4℃ for later use to avoid DNA degradation.

[0022] 1.3 Gel recovery and purification of the target fragment 1. Add the PCR product to 1× loading buffer (volume ratio 5:1), mix gently, and load onto a 1% agarose gel (containing the non-toxic staining agent GoldView, replacing EB to reduce safety risks). At the same time, load a 100 bp~10 kb DNA marker and electrophoresis at 120 V for 30~40 min (adjust according to the size of the target fragment to ensure effective separation of the target band from impurities).

[0023] 2. After electrophoresis, observe the gel under a gel imaging system to find the specific band that matches the theoretical size of MIDN ORF (avoid interference from extraneous bands). Use a sterile blade to precisely cut the gel block containing the target band, remove as much excess agarose as possible (to reduce impurities), and place it in a sterile centrifuge tube.

[0024] 3. Follow the instructions of the DNA gel extraction kit to purify and recover the target fragment: Add an appropriate amount of sol solution to the gel block, heat in a 56°C water bath for 10-15 min, repeatedly inverting the centrifuge tube during this period to ensure complete gel dissolution; transfer the dissolved solution to the adsorption column of the kit, centrifuge at 12000 rpm for 30 s, and discard the waste liquid; add washing buffer (pre-added with anhydrous ethanol), centrifuge at 12000 rpm for 30 s, discard the waste liquid, and repeat the washing once; centrifuge the empty column at 12000 rpm for 2 min to completely remove residual washing buffer (to avoid affecting subsequent enzyme digestion); add 30-50 μL of sterile deionized water (preheated to 65°C to improve elution efficiency) to the center of the adsorption column, let stand for 2 min, centrifuge at 12000 rpm for 2 min, collect the eluent (containing the purified MIDN ORF fragment), and store at -20°C for later use.

[0025] 4. Purity and concentration detection of the target fragment: Take 2 μL of eluent and detect the OD260 / OD280 ratio using Nanodrop. A ratio between 1.8 and 2.0 is considered to be of acceptable purity. At the same time, verify the integrity of the target fragment by 1% agarose gel electrophoresis to ensure that there is no degradation or impurities.

[0026] 4.2 Double digestion of pcDNA plasmid and target fragment 3.4 4.2.1 Preparation of enzyme digestion system (digest plasmid and target fragment separately, 50 μL system) 1. pcDNA3.4 plasmid digestion system: Add 10 μL of pcDNA3.4 plasmid (concentration ≥100ng / μL), 5 μL of 10× digestion buffer (adapted to the two selected restriction enzymes, such as EcoRI / XhoRI universal buffer), 1 μL of the first restriction enzyme (such as EcoRI), 1 μL of the second restriction enzyme (such as XhoRI), and 33 μL of sterile deionized water to a sterile centrifuge tube. Mix gently, centrifuge briefly, and collect the liquid to the bottom of the tube.

[0027] 2. MIDN ORF fragment digestion system: Add 20 μL of purified target fragment, 5 μL of 10× digestion buffer, 1 μL of the first restriction endonuclease, 1 μL of the second restriction endonuclease, and 23 μL of sterile deionized water to a sterile centrifuge tube in sequence, mix well, and centrifuge briefly.

[0028] 4.2.2 Enzymatic digestion reaction and product purification 1. Place both enzyme digestion systems in a 37℃ constant temperature water bath and digest for 2-3 hours (ensure complete digestion; incomplete digestion of pcDNA3.4 plasmid will lead to an increase in false positive clones). After digestion, incubate at 65℃ for 10 minutes to inactivate restriction endonucleases and avoid interference with subsequent ligation reactions.

[0029] 2. Enzyme digestion product purification: Using a DNA gel extraction kit, following step 4.1.2, the digested pcDNA3.4 plasmid and MIDN ORF fragment were purified separately to remove impurities such as digestion buffer and incompletely digested plasmid / fragment. After purification, the concentrations of the plasmid and the target fragment were detected by Nanodrop to ensure that the concentrations of the two were appropriate (for a more reasonable ligation ratio).

[0030] 4.3 Ligation of pcDNA plasmid with MIDN ORF fragment after enzyme digestion 3.4 1. Preparation of ligation system (20 μL system, T4 DNA ligase, adapted for sticky end ligation): Add 2 μL of enzyme-digested and purified pcDNA3.4 plasmid, 8 μL of enzyme-digested and purified MIDN ORF fragment (the molar ratio of target fragment to plasmid should be controlled at 3:1~5:1 to improve ligation efficiency), 2 μL of 10×T4 ligation buffer, 1 μL of T4 DNA ligase, and 7 μL of sterile deionized water to a sterile centrifuge tube in sequence. Mix gently and centrifuge briefly (avoid air bubbles).

[0031] 2. Ligation reaction: Place the ligation system in a 16℃ water bath and ligate overnight (12~16 h; sufficient time is needed for ligation of pcDNA3.4 plasmid and the target fragment to improve the recovery rate of recombinant plasmid); after ligation, store at 4℃ for later use, or use directly for transformation.

[0032] 4.4 Transformation of recombinant plasmid (pcDNA3.4-MIDN) 4.4.1 Resuscitation of competent cells 1. Take out DH5α competent cells (100 μL / tube) stored at -80℃ and quickly place them on ice for slow recovery (10~15 min). Avoid repeated freeze-thaw cycles during this period to prevent a decrease in competent cell efficiency. After recovery, the competent cells will be transparent. Gently invert the centrifuge tube to mix them, avoiding violent shaking (which may damage the cells).

[0033] 4.4.2 Conversion Operation 1. Slowly add the ligation product (20 μL) to the revived DH5α competent cells, mix gently, and incubate on ice for 30 min (to ensure that the recombinant plasmid is fully bound to the competent cells); do not shake or centrifuge during this period to avoid affecting the transformation efficiency.

[0034] 2. Heat shock treatment: After the centrifuge tubes were placed in an ice bath, they were placed in a 42°C constant temperature water bath for 90 seconds (the time and temperature should be strictly controlled; overheating will cause cell death, while too short a time will result in incomplete transformation). After the heat shock, the tubes were quickly transferred to ice and kept in an ice bath for 2 minutes to terminate the heat shock reaction.

[0035] 3. Resuscitation culture: Add 800 μL of sterile SOC medium (antibiotic-free) to the centrifuge tube and place it in a shaker at 37°C and 200 rpm for 1 h (to revive competent cells and express the ampicillin resistance gene for subsequent screening).

[0036] 4.5 Screening and Culture of Positive Clones 1. Centrifugation concentration of bacterial culture: Transfer the revived bacterial culture to a sterile centrifuge tube, centrifuge at 5000 rpm for 5 min, discard the supernatant (retain about 100 μL of supernatant), gently pipette to mix, and resuspend the bacterial pellet (concentrate the bacterial culture to increase the colony density on the plate).

[0037] 2. Plate screening: Spread the resuspended bacterial solution evenly onto LB solid medium plates containing 100 μg / mL ampicillin (pcDNA3.4 plasmid contains the Ampᵣ resistance gene, and only positive bacteria containing the recombinant plasmid can survive). After spreading, place the plate in a clean bench and let it air dry for 10-15 min (avoid bacterial flow, which may lead to colony fusion).

[0038] 3. Incubation: Invert the dried LB solid plates (to prevent condensation from dripping and contaminating the colonies), place them in a 37°C constant temperature incubator, and incubate for 12-16 hours until clear single colonies appear on the plates (avoid incubation time that may lead to oversized colonies and contamination by other microorganisms).

[0039] 4. Single colony picking and expansion culture: In a clean bench, use a sterile inoculation loop to pick single colonies with regular shape and uniform size from the plate (each colony is picked separately to avoid cross-contamination), and inoculate them into LB liquid medium containing 100 μg / mL ampicillin (5 mL / tube). Place the tubes in a shaker at 37℃ and 200 rpm for 12-16 h to obtain the bacterial culture.

[0040] 4.6 Extraction and Identification of Recombinant Plasmid (pcDNA3.4-MIDN) 4.6.1 Mini-preparation of recombinant plasmids 1. Take 5 mL of the cultured bacterial solution after shaking and add it to a sterile centrifuge tube. Centrifuge at 8000 rpm for 5 min and discard the supernatant (try to remove as much as possible to avoid residual culture medium affecting plasmid extraction).

[0041] 2. Follow the instructions of the plasmid miniprep kit to extract the recombinant plasmid: Add lysis buffer to the bacterial pellet and shake vigorously until the pellet is completely suspended; add neutralization buffer and gently invert to mix until a white flocculent precipitate appears; centrifuge at 12,000 rpm for 10 min, and transfer the supernatant to the adsorption column of the kit; subsequently, wash with washing buffer and elute with sterile deionized water according to the kit steps, collect the eluent (containing the extracted recombinant plasmid), and store at -20℃ for later use.

[0042] 4.6.2 Identification of recombinant plasmids (double identification to ensure correct insertion of the target gene) 1. Enzyme digestion identification: Prepare a 20 μL enzyme digestion identification system by adding 5 μL of recombinant plasmid, 2 μL of 10× enzyme digestion buffer, 0.5 μL each of the two selected restriction endonucleases (such as EcoRI and XhoRI), and 12 μL of sterile deionized water. Digest at 37℃ for 1 h. After enzyme digestion, perform 1% agarose gel electrophoresis, simultaneously loading DNA markers, undigested recombinant plasmid, and digested pcDNA3.4 plasmid as controls. After electrophoresis, observe under a gel imaging system. If two clear bands appear (one for the pcDNA3.4 plasmid vector and one for the MIDN ORF target fragment), and the band size is consistent with the theoretical value, the enzyme digestion identification is qualified.

[0043] 2. Sequencing Identification (Gold Standard): The recombinant plasmid samples that have passed the enzyme digestion identification are sent to a gene sequencing company for sequencing using universal sequencing primers for pcDNA3.4 plasmid (such as CMV promoter downstream primers and polyA upstream primers). The sequencing results are compared with the MIDN ORF standard sequence. If the sequences are completely identical (no base mismatches, deletions, or insertions), it indicates that the recombinant plasmid (pcDNA3.4-MIDN) has been successfully constructed.

[0044] Example 2: Construction of MIDN knockdown U87 cells U251 and U87 cell lines with MIDN knockdown were constructed using siRNA transfection technology, and the knockdown efficiency was verified using Western blot.

[0045] The specific steps are as follows: 1. Cell resuscitation and passage: U251 and U87 cells were removed from liquid nitrogen and quickly placed in a 37°C water bath for resuscitation. After the cells were completely thawed, they were centrifuged at 800 rpm for 5 min and the supernatant was discarded. 2 mL of complete DMEM medium was added, and the cells were gently mixed by pipetting and transferred to cell culture flasks and cultured in a 37°C, 5% CO2 cell culture incubator. Cells were passaged every 2-3 days. During passage, cells were digested with trypsin to adjust the cell density and ensure good cell growth (regular morphology, uniform adhesion, and no bacterial contamination).

[0046] 2. Preliminary transfection conditions: Well-grown U251 and U87 cells were seeded in 6-well plates. Different final siRNA concentrations (50, 100, 150 nmol / L) and liposome-to-siRNA volume ratios (1:1, 1:2, 1:3) were set, and fluorescently labeled siRNA was used for transfection. After 48 h of transfection, the fluorescence intensity was observed under an inverted fluorescence microscope, the transfection efficiency was calculated, and the optimal transfection conditions for the two cell types were determined (recommended: final siRNA concentration 100 nmol / L, liposome-to-siRNA volume ratio 1:2).

[0047] 3. Reagent preparation: 1 h before transfection, equilibrate the liposome transfection reagent at room temperature for 30 min; preheat Opti-MEM medium and complete DMEM medium to room temperature; after the siRNA stock solution has thawed at room temperature, mix it gently, and collect the liquid to the bottom of the tube by short centrifugation.

[0048] 2.2 Transient transfection and screening of effective siRNAs 1. Cell seeding: One day before transfection, U251 and U87 cells were digested with trypsin, resuspended in complete DMEM medium, and the cell density was adjusted to 2 × 10⁶ cells / year. 5Cells were seeded at a density of 1 / mL into 6-well plates, with 2 mL of cell suspension added to each well. The plates were then incubated in a cell culture incubator for 24 h. Transfection was performed when the cell confluence reached 70%–80% (at this point, cell proliferation was active, cell membrane permeability was suitable, and transfection efficiency was the highest).

[0049] 2. Preparation of transfection complex: Each well was set up as follows: blank control group (no siRNA, no liposomes), NC-siRNA group, MIDN-siRNA-1 group, MIDN-siRNA-2 group, and MIDN-siRNA-3 group; each group was set up with 3 replicates to ensure experimental reproducibility.

[0050] 3. Complex preparation procedure: Take 125 μL of Opti-MEM medium in each well, add the corresponding siRNA (calculate the amount based on a final concentration of 100 nmol / L), gently invert to mix, and let stand at room temperature for 5 min; take another 125 μL of Opti-MEM medium, add 2 μL of liposome transfection reagent, mix well, and let stand at room temperature for 5 min; slowly mix the two solutions, gently invert to mix, and let stand at room temperature for 20 min to form a stable siRNA-liposome complex (avoid vigorous shaking to prevent complex rupture).

[0051] 4. Transfection procedure: Carefully aspirate the complete DMEM medium from the 6-well plate, gently wash the cells twice with PBS buffer to remove residual medium; add 250 μL of the prepared siRNA-liposome complex to each well, gently shake the culture plate to ensure the complex evenly covers the cell surface; incubate at 37℃, 5% CO2 for 4-6 h, then aspirate the complex, add 2 mL of fresh complete DMEM medium, and continue culturing for 48-72 h.

[0052] 5. Screening of effective siRNAs: 72 h after transfection, cells from each group were collected, and total RNA and total protein were extracted. The mRNA expression level of the MIDN gene was detected by qRT-PCR, with β-actin as an internal reference, and the relative expression level of MIDN mRNA in each group was calculated. The expression level of MIDN protein was detected by Western blot, and the gray value of the protein band was analyzed. Using the NC-siRNA group as a control, the pair of MIDN-siRNAs with the highest knockdown efficiency (knockdown efficiency ≥70% was considered effective) was screened for subsequent stable cell line construction.

[0053] 2.3 Screening and culture of stable knockdown cell lines 1. Stable transfection: According to the optimal transfection conditions determined in 4.2, the selected effective MIDN-siRNA and NC-siRNA were transfected into U251 and U87 cells, respectively. After 48 h of transfection, the cells were digested with trypsin, resuspended in complete DMEM medium, passaged into new 6-well plates at a ratio of 1:10, and cultured for another 24 h to allow the cells to adhere and grow.

[0054] 2. Puromycin screening: After 24 h of subculturing, discard the complete DMEM medium and add puromycin-containing medium for selection. Example 3: Effect of MIDN on glioma cell proliferation CCK8 cell proliferation and cell cycle assays revealed that knocking down MIDN expression promoted the proliferation of U251 and U87 cells, while overexpression of MIDN inhibited cell proliferation. This indicates that MIDN has the function of inhibiting the malignant progression of glioma.

[0055] The specific steps of the CCK8 cell proliferation experiment are as follows: 1. Experimental preparation: The four groups of cells that passed the verification (MIDN knockdown group, MIDN overexpression group, negative control group, and blank control group) were revived separately; the MIDN knockdown group (MIDN-siRNA) and the negative control group (NC-siRNA) were cultured in complete DMEM medium containing puromycin, and the MIDN overexpression group (pcDNA3.4-MIDN transfected stable cell lines) and the blank control group (untransfected U251 and U87 cells) were cultured in complete DMEM medium without puromycin. All cells were cultured to the logarithmic growth phase. The CCK8 reagent was equilibrated at room temperature for 30 min in advance to avoid repeated freeze-thaw cycles. The 96-well plates were sterilized by UV irradiation for 30 min in advance for later use.

[0056] 2. Cell seeding: Digest the four groups of cells with trypsin, resuspend them in the corresponding complete DMEM medium, and after thorough mixing, count the cells using a cell counting chamber to adjust the cell density to 5 × 10³ cells / mL. Add 100 μL of cell suspension to each well of a 96-well plate, with three replicates per group. Also, set up blank control wells (containing only 100 μL of complete DMEM medium, cell-free, used to remove background values). After seeding, gently shake the 96-well plate to distribute the cells evenly and avoid cell aggregation.

[0057] 3. Cell culture: Place the 96-well plate in a 37℃, 5% CO2 cell culture incubator and culture for 0 h, 24 h, 48 h, 72 h, and 96 h respectively (adjust the culture time points according to the cell growth rate to cover the logarithmic growth phase of the cells); avoid moving the culture plate during culture to prevent cell detachment and affect the experimental results.

[0058] 4. CCK8 reagent incubation: Before each time point, add 10 μL of CCK8 reagent to each well (avoid contact with the well wall to prevent contamination), gently shake the 96-well plate to mix the reagent with the culture medium thoroughly; place it in a 37℃, 5% CO2 cell culture incubator and incubate in the dark for 2 h (the incubation time can be adjusted according to cell viability to ensure that the amount of formazan produced is linearly related to the number of cells).

[0059] 5. Absorbance detection: After incubation, remove the 96-well plate and place it at room temperature for 10 min to equilibrate and eliminate the influence of temperature difference on absorbance; use a microplate reader to detect the OD value of each well at a wavelength of 450 nm and record the experimental data; avoid the formation of air bubbles in the wells during detection. If air bubbles are present, gently puncture them with a sterile pipette tip.

[0060] 6. Data Processing: Calculate the average OD value of the three replicates in each group, subtract the OD value of the blank control well, and obtain the actual OD value of the cells in each group; plot the cell proliferation curve with culture time as the x-axis and OD value as the y-axis; clarify the effect of MIDN knockdown and overexpression on the proliferation of U251 and U87 cells by comparing the differences in OD values ​​between the MIDN knockdown group, overexpression group, negative control group, and blank control group (the higher the OD value, the more cells there are and the stronger the proliferation ability).

[0061] 7. Experimental Conclusion: After the test is completed, properly dispose of the cells and culture medium in the 96-well plate, clean and sterilize the experimental equipment and store it; organize the experimental data, perform statistical analysis, and ensure that the experimental results are statistically significant (P<0.05 is considered significant).

[0062] The specific steps of the cell cycle experiment are as follows: 1. Experimental preparation: Four groups of cells consistent with the CCK8 experiment were selected (MIDN knockdown group, MIDN overexpression group, negative control group, and blank control group). After thawing, the cells were cultured to the logarithmic growth phase under the corresponding culture conditions (knockdown group and negative control group contain puromycin, overexpression group and blank control group do not contain puromycin); the PI staining kit (containing PI staining solution, RNase A, and fixative) was thawed at 4°C in advance to avoid repeated freeze-thaw cycles; sterile PBS buffer, centrifuge tubes, pipettes, etc. were sterilized in advance for use; the flow cytometer was preheated and adjusted to normal working condition in advance.

[0063] 2. Cell synchronization treatment (optional, to optimize experimental reproducibility): To reduce the randomness of cell cycle distribution, four groups of cells were seeded into 6-well plates and cultured until the cell confluence reached 50%~60%. Then, the medium was replaced with serum-free DMEM and starved for 24 h to synchronize the cells to arrest in the G0 / G1 phase. After starvation, the medium was replaced with the corresponding complete DMEM and cultured for another 24 h. Cells were then collected for subsequent operations (if synchronization is not performed, cells in the logarithmic growth phase can be collected directly).

[0064] 3. Cell collection: Digest each group of cells with trypsin (avoid over-digestion and cell damage), add the corresponding complete DMEM medium to stop digestion, gently pipette to mix, and transfer to sterile centrifuge tubes; centrifuge at 800 rpm and 4℃ for 5 min, discard the supernatant; gently resuspend the cell pellet with pre-cooled sterile PBS buffer, centrifuge at 800 rpm and 4℃ for 5 min, wash twice to thoroughly remove residual culture medium and trypsin to avoid affecting the staining effect.

[0065] 4. Cell fixation: Slowly add pre-cooled 70% ethanol fixative (prepared with enzyme-free deionized water) to the washed cell pellet, gently pipette to mix, and disperse the cells evenly (avoid cell aggregation); fix at 4°C in the dark for 12-24 h (fixation time should not be too long to prevent cell rupture; insufficient fixation time will lead to uneven staining); after fixation, centrifuge at 800 rpm and 4°C for 5 min, discard the fixative, and wash once with pre-cooled PBS to remove residual ethanol.

[0066] 5. PI staining: Add 100 μL of RNase A solution (final concentration 100 μg / mL) to the cell pellet and incubate at 37°C for 30 min to degrade intracellular RNA (avoid RNA binding to PI, which would interfere with DNA staining results); after incubation, place the centrifuge tube on ice to cool for 5 min, add 400 μL of PI staining solution (final concentration 50 μg / mL), mix gently, and stain at 4°C in the dark for 30 min (avoid repeated shaking during staining to prevent uneven staining, and strictly avoid light to prevent PI fluorescence quenching).

[0067] 6. Flow cytometry: After staining, the cell cycle distribution of each group was detected by flow cytometry. The excitation wavelength was 488 nm and the emission wavelength was 610 nm. 1×10⁻⁶ cells were collected from each sample. 4 The proportion of cells in each phase of the cell cycle (G0 / G1, S, G2 / M) was recorded. Before the test, the cell suspension was filtered through a 300-mesh sterile filter to remove cell aggregates and impurities to avoid clogging the flow cytometer tubing.

[0068] 7. Data Processing: Flow cytometry software (such as ModFit) was used to analyze the percentage of cells in the G0 / G1, S, and G2 / M phases of each group, and the proliferation index was calculated (PI = (S phase + G2 / M phase) cell percentage / total cell percentage × 100%). A higher proliferation index indicates stronger cell proliferation activity, consistent with experimental conclusions (the proliferation index of the knockdown group should be higher than that of the control group, and the proliferation index of the overexpression group should be lower than that of the control group). Data were organized and statistically analyzed to ensure that the differences between groups were statistically significant (P < 0.05 was considered significant).

[0069] 8. Experimental Conclusion: After the detection is completed, the stained cell suspension is properly disposed of with waste liquid containing disinfectant; experimental equipment (centrifuge tubes, pipette tips, 6-well plates, etc.) are cleaned, sterilized and stored; the flow cytometer is shut down according to the operating procedures and maintained; experimental data are compiled, a cell cycle distribution bar chart is plotted, and combined with the CCK8 experimental results, the regulatory role of MIDN on the proliferation and cell cycle of U251 and U87 cells is clarified.

[0070] Example 4: Interaction and Degradation Mechanism between MIDN and PPDPF The interaction between MIDN and its substrate protein PPDPF was verified using Co-IP and Western blot techniques. Experiments showed that in U87 cells, MIDN overexpression promoted PPDPF degradation, while MIDN knockdown slowed down the degradation. Further domain analysis identified key interaction regions.

[0071] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. An application of MIDN in the diagnosis and treatment of glioma, wherein the glioma is glioblastoma GBM, characterized in that: The presence or malignancy of gliomas can be assessed by detecting the expression level of MIDN in biological samples.

2. The application of a MIDN protein in the treatment of glioma, specifically glioblastoma GBM, characterized by: Inhibit glioma cell proliferation by regulating the expression or activity of MIDN.

3. The application according to claim 2, characterized in that: The methods for regulating MIDN expression include overexpressing MIDN or using MIDN agonists.

4. An application of the interaction between MIDN and PPDPF in the diagnosis and treatment of glioma, characterized in that: The interaction level between MIDN and PPDPF can be used to diagnose gliomas or assess treatment efficacy.

5. A method for constructing MIDN knockdown or overexpression glioma cell lines, characterized in that: The expression of MIDN in different types of glioma cells, U251, U87, or Hs683, was regulated using siRNA or vector transfection technology.

6. A glioma treatment drug based on MIDN-degraded PPDPF, characterized in that: It contains MIDN protein or its active fragment, which promotes PPDPF degradation.

7. A method for screening compounds targeting the MIDN-PPDPF pathway, characterized in that: The effects of compounds on the interaction or degradation of MIDN and PPDPF were evaluated using in vitro or in vivo models.

8. An application of MIDN as a biomarker in the prognostic assessment of glioma, characterized in that: Combined expression levels of MIDN and PPDPF to predict patient survival.

9. A therapeutic composition comprising the MIDN gene, characterized in that: For gene therapy of glioma, the MIDN gene is delivered via a viral vector.

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