GTF2IRD1 transcription factor, proliferation promoting agent and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a GTF2IRD1 transcription factor, a proliferation promoting agent and application of the proliferation promoting agent. The GTF2IRD1 gene is located in a chromosome 7, codes a transcription factor capable of being specifically combined with DNA and regulating and controlling the transcription activity of downstream genes, and plays a key role in establishment of craniofacial forms, cognitive functions and behavior patterns in the normal development process of an organism. The invention further discloses that the GTF2IRD1 has the functions of promoting myocardial cell proliferation and relieving heart tissue damage, provides a new intervention strategy and a potential drug target for clinical treatment of myocardial infarction, and has an important clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a GTF2IRD1 transcription factor, a proliferator, and its applications. Background Technology

[0002] Myocardial infarction is one of the leading causes of heart failure and death worldwide. Its pathogenesis involves coronary artery obstruction leading to ischemia and hypoxia of myocardial cells, resulting in massive myocardial cell death. Because adult myocardial cells lack significant regenerative capacity, they are often filled with fibrous scar tissue after injury. This pathological remodeling directly leads to a progressive decline in ventricular function and the development of heart failure. Therefore, effectively promoting the proliferation of endogenous myocardial cells to repair damage and rebuild cardiac function has become a major challenge in the field of cardiovascular regenerative medicine.

[0003] The GTF2IRD1 gene is located on human chromosome 7 and, as an important transcription factor, regulates the transcription of downstream genes by specifically binding to DNA. Existing research has established the association between GTF2IRD1 and tumors and neurological diseases, and its abnormal expression is associated with genetic diseases such as Williams syndrome. Summary of the Invention

[0004] This invention reveals and verifies for the first time that overexpression of GTF2IRD1 possesses a novel biological function in driving cell proliferation. In in vitro cell experiments, GTF2IRD1 overexpression mediated by an adenovirus vector significantly increased the percentage of cells positive for multiple proliferation markers (including PH3, Ki67, and AuroraB), clearly demonstrating its proliferative effect at the cellular level. In in vivo animal experiments, intravenous injection of a GTF2IRD1-overexpressing adenovirus into a C57 mouse model further revealed a significant increase in the number of Ki67-positive cells in myocardial tissue, accompanied by an objective improvement in cardiac systolic and diastolic function. The above in vitro and in vivo experimental results consistently demonstrate that proliferators centered on GTF2IRD1 can effectively activate the proliferation process of cardiomyocytes and improve cardiac function.

[0005] The primary objective of this invention is to provide a GTF2IRD1 transcription factor, the amino acid sequence of which is shown in SEO ID NO.1.

[0006] Furthermore, this invention provides the use of the GTF2IRD1 transcription factor in the preparation of medicaments for the prevention and treatment of myocardial infarction.

[0007] Furthermore, this invention provides the application of the GTF2IRD1 transcription factor in the preparation of drugs that promote cardiomyocyte proliferation.

[0008] Furthermore, overexpression of the GTF2IRD1 transcription factor can promote cardiomyocyte proliferation and repair cardiac damage.

[0009] Furthermore, the GTF2IRD1 transcription factor overexpression is an adenovirus vector-mediated GTF2IRD1 transcription factor overexpression.

[0010] A second objective of this invention is to provide a GTF2IRD1 proliferator, the nucleic acid sequence of which is shown in SEO ID NO.2.

[0011] Furthermore, the present invention provides the use of GTF2IRD1 proliferator in the preparation of medicaments for the prevention and treatment of myocardial infarction.

[0012] Furthermore, this invention provides the application of GTF2IRD1 proliferator in the preparation of drugs that promote cardiomyocyte proliferation.

[0013] Compared with existing technologies, this invention has the following beneficial effects: It reveals and verifies for the first time that the transcription factor GTF2IRD1 has a novel function of promoting cardiomyocyte proliferation and repairing cardiac damage. Unlike traditional therapies that focus on thrombolysis, intervention, or slowing down myocardial remodeling, this invention focuses on actively promoting myocardial regeneration, potentially leading to fundamental repair of cardiac function. This invention overcomes the limitation that this gene is only known in fields such as neurodevelopment. Its related diagnostic kits can also be used for the auxiliary diagnosis of the degree of myocardial injury, efficacy assessment, or prognosis, possessing significant translational medical value. Attached Figure Description

[0014] Figure 1 The following are Western Bolt images showing the expression of GTF2IRD1 in 30-day-old and newborn mice in this invention. Figure 2 This is statistical data on the proliferation index PH3 during GTF2IRD1 overexpression in this invention; Figure 3 This is the statistical data of the proliferation index ki67 during GTF2IRD1 overexpression in this invention; Figure 4 This is the statistical data of the proliferation index AuroraB when GTF2IRD1 is overexpressed in this invention; Figure 5 This is the statistical data of the proliferation index ki67 when GTF2IRD1 is overexpressed in the 2-week myocardial infarction mouse model constructed in this invention; Figure 6 This is the cardiac ejection fraction data when GTF2IRD1 is overexpressed in the 2-week myocardial infarction mouse model constructed in this invention; Figure 7 This is the fractional data of cardiac shortening when GTF2IRD1 is overexpressed in the 2-week myocardial infarction mouse model constructed in this invention. Detailed Implementation

[0015] Example 1 GTF2IRD1 expression detection in 30-day-old and newborn mice This invention uses Western blotting to detect the expression of GTF2IRD1 in 30-day-old and newborn mice to verify the expression level of GTF2IRD1 in mice of different ages. The specific experimental steps are as follows: 1. Grind and lyse the treated heart tissue using RIPA lysis buffer containing the protease inhibitor PMSF for 30 minutes; after centrifugation and collection of supernatant, add SDS-PAGE protein loading buffer and boil in a 95°C metal bath to fully denature the protein. Store the resulting protein sample at -80°C for use.

[0016] 2. Perform SDS-PAGE gel electrophoresis: Load the protein sample into the gel sample wells, electrophore at 80 V for 30 minutes. After the sample is concentrated into a single line, adjust the voltage to 120 V and continue electrophoresis until the target protein is fully separated.

[0017] 3. Electrotransfer: Under a constant current of 220 mA, the protein on the gel was transferred to a PVDF membrane by wet transfer method, and the transfer time was 90 minutes.

[0018] 4. Blocking: After the transfer is complete, the PVDF membrane is blocked with 5% skim milk at room temperature for 2 hours to block non-specific binding sites.

[0019] 5. Primary antibody incubation: Dilute GTF2IRD1 antibody with 3% BSA solution at a ratio of 1:500, place the membrane in the diluted primary antibody solution, and incubate overnight at 4°C or at room temperature for 2 hours. After incubation, wash the membrane 3 times with TBST buffer for 10 minutes each time.

[0020] 6. Secondary antibody incubation: The membrane was then incubated with the corresponding species-derived HRP-labeled secondary antibody at room temperature for 1 hour. The membrane was washed three times with TBST for 10 minutes each time. Finally, an enhanced chemiluminescence detection reagent was added and spread onto the PVDF membrane for development. The expression level of GTF2IRD1 protein in the sample was analyzed based on the development results.

[0021] Using the Western blotting procedure described above, the expression level of GTF2IRD1 in the heart tissue of newborn mice was found to be significantly higher than that in 30-day-old mice. The experimental results are as follows: Figure 1 As shown, high expression of GTF2IRD1 is positively correlated with cardiomyocyte proliferation capacity, thus providing key evidence for the application of GTF2IRD1 in promoting cardiomyocyte proliferation as concluded in this invention.

[0022] Example 2 Isolation and culture of cardiomyocytes First, all surgical instruments (including forceps, scissors, Erlenmeyer flasks, and cell filters) were autoclaved, and digestion solution, 1×PBS buffer, fetal bovine serum (FBS), and DMEM / F12 culture medium were prepared in advance. One-day-old suckling mice were harvested, and after surface disinfection with alcohol, the heart was quickly removed under aseptic conditions via thoracotomy and placed in pre-chilled PBS. The heart tissue was then rinsed three times with PBS to remove residual blood, minced into approximately 1 mm³ pieces, and transferred to a digestion solution containing 0.2 mg / mL trypsin (Sigma, P-3292) and 1.0 mg / mL type II collagenase (Worthington, LS004176). Digestion was performed in fractions at 37°C, with the supernatant collected every 7 minutes and transferred to a centrifuge tube containing 5 mL FBS to terminate the reaction. This process was repeated until the tissue was completely digested. All digestion solutions were combined and centrifuged. The cell pellet was collected, filtered through a cell strainer to remove undigested tissue, and then the cell suspension was seeded in DMEM / F12 medium containing 5% FBS. The cells were cultured using the differential adhesion method for 1.5 hours to remove non-cardiomyocytes such as fibroblasts. Finally, the non-adherent cardiomyocytes were collected, resuspended in fresh complete medium, and incubated at 37°C for 24 hours for subsequent immunofluorescence experiments.

[0023] Example 3 Immunofluorescence assay First, cells or tissues were fixed with paraformaldehyde for 10 minutes and washed three times with PBS buffer (5 minutes each time). Then, 0.1% Triton X-100 was added for permeabilization at room temperature for 15 minutes to enhance membrane permeability, followed by three more washes with PBS (5 minutes each time). Next, cells were blocked with 3% BSA solution for 20 minutes to block non-specific binding sites. After blocking, the blocking solution was discarded, and an appropriate amount of diluted primary antibody solution was added. The cells were incubated overnight at 4°C or for 2 hours at room temperature. The next day, cells were thoroughly washed with PBS, and then TRITC-labeled anti-rabbit IgG (Jackson, 111-025-003, 1:200) and FITC-labeled anti-mouse IgG (Jackson, 115-095-003, 1:200) were added, respectively. The cells were incubated with a 1:200 dilution of DAPI as a secondary antibody at room temperature in the dark for 1 hour; washed three times with PBS (5 minutes each time); and finally, the cell nuclei were counterstained with DAPI solution at a 1:500 dilution for 5 minutes. After washing with PBS, anti-fluorescence quenching mounting medium was added, and the cells were observed and images were acquired under a confocal microscope and a fluorescence microscope. The results showed that Ki67 cells exhibited high positive expression of proliferation markers, confirming that cardiomyocytes overexpressing GTF2IRD1 had significant proliferative activity.

[0024] Figure 2This is an immunofluorescence graph showing the proliferation index PH3 when GTF2IRD1 is overexpressed in this invention. The results indicate that the number of Ki67-positive cells increases.

[0025] Figure 3 This is an immunofluorescence graph showing the proliferation index ki67 when GTF2IRD1 is overexpressed in this invention. The results indicate that the number of ki67-positive cells increases.

[0026] Figure 4 This is an immunofluorescence graph of the proliferation marker AuroraB when GTF2IRD1 is overexpressed in this invention. The results show that the number of Ki67 positive cells increases.

[0027] Figure 5 The images show immunofluorescence patterns and statistical graphs of tissue sections containing the proliferation marker ki67 during GTF2IRD1 overexpression in this invention. The results indicate an increase in ki67-positive cells.

[0028] The results showed that GTF2IRD1 overexpression could promote cardiomyocyte proliferation.

[0029] Example 4 Establish a 2-week mouse model of myocardial infarction Anesthetized mice were intubated and connected to a small animal ventilator to maintain intraoperative ventilation. The left chest wall was incised layer by layer between the 3rd and 4th ribs to open the thoracic cavity, and the heart was fully exposed using a thoracic dilator. Subsequently, the distal segment of the left anterior descending coronary artery (LAD) was ligated using 6-0 nylon sutures (Ningbo Medical Suture Needle Co., Ltd., China). Confirmation of pallor and weakened motility of the left ventricular anterior wall indicated successful myocardial infarction modeling. Finally, the chest wall incision was sutured layer by layer. Once the mouse's spontaneous breathing stabilized, it was extubated and continuously monitored until it fully recovered.

[0030] Example 5 Cardiac function index testing Two weeks after establishing a mouse model of myocardial infarction, mice were anesthetized, and ultrasound images were acquired using a Vevo 2100 imaging system (VisualSonics Inc., Canada) and a 40-MHz MS-250 high-frequency scanning probe, taking views along the long axis of the left ventricle and the short axis at the level of the papillary muscles. Results are as follows: Figure 4 , 5 As shown, compared with wild-type controls, mice treated with GTF2IRD1 proliferators showed significantly enhanced left ventricular systolic function, demonstrating that the proliferator can effectively improve cardiac pumping function after myocardial infarction.

[0031] Figure 6 In this invention, when GTF2IRD1 is overexpressed, the cardiac function index ejection fraction is significantly increased compared with the control group.

[0032] Figure 7 In this invention, when GTF2IRD1 is overexpressed, the cardiac function index shortening score is significantly increased compared with the control group.

[0033] The results showed that injection of GTF2IRD1 proliferators could significantly improve cardiac function and alleviate cardiac damage.

[0034] Example 6 Tail vein injection The viral load was diluted with physiological saline to 1×10^ 11 The PFU was used to overexpress adenovirus at a concentration of GTF2IRD1. Next, the mice were restrained and placed under a heat lamp to appropriately warm the tail vessels. After disinfecting the tail with an alcohol swab, the thicker tail vein on the back was selected, and the diluted viral suspension was slowly injected into the vein using an insulin needle. After injection, brief pressure was applied to the injection site to stop bleeding. The entire procedure was performed under sterile conditions and strictly adhered to animal ethics and biosafety protocols.

[0035] The results showed that intravenous injection of GTF2IRD1-overexpressing adenovirus promoted cardiomyocyte proliferation.

Claims

1. A GTF2IRD1 transcription factor, characterized in that: The amino acid sequence of the GTF2IRD1 transcription factor is shown in SEO ID NO.

1.

2. The use of the GTF2IRD1 transcription factor as described in claim 1 in the preparation of a medicament for the prevention and treatment of myocardial infarction.

3. The use of the GTF2IRD1 transcription factor as described in claim 1 in the preparation of drugs that promote cardiomyocyte proliferation.

4. The application as described in claim 3, characterized in that: Overexpression of the GTF2IRD1 transcription factor can promote cardiomyocyte proliferation and repair cardiac damage.

5. The application as described in claim 4, characterized in that: The GTF2IRD1 transcription factor overexpression was mediated by an adenovirus vector.

6. A GTF2IRD1 proliferator, characterized in that: The nucleic acid sequence of the GTF2IRD1 proliferator is shown in SEO ID NO.

2.

7. The use of the GTF2IRD1 proliferator as described in claim 6 in the preparation of a medicament for the prevention and treatment of myocardial infarction.

8. The use of the GTF2IRD1 proliferator as described in claim 6 in the preparation of a drug that promotes cardiomyocyte proliferation.

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