Method for screening and verifying target molecules for inhibiting apoptosis of hypoxic myocardial cells

Abstract

The invention provides a target molecule screening and verification method for inhibiting apoptosis of hypoxic myocardial cells, and relates to the technical field of biology. The method comprises the following steps of: firstly, utilizing CUTamp; a potential target molecule of the NRF-1 for regulating and controlling apoptosis of hypoxic myocardial cells is analyzed by combining a Tag technology with transcriptome sequencing, whether the NRF-1 can be combined with and regulate and control a promoter region of the potential target molecule or not is verified by utilizing a ChIP-qPCR technology and a dual-luciferase reporter gene experiment, a potential target molecule Cflash is screened out, and then the potential target molecule Cflash is screened out through CUTamp; the direct regulation and control relationship between the NRF-1 and the Cflash gene is determined through Tag sequencing and a chromatin co-immunoprecipitation technology, and then the transcriptional regulation and space expression relationship between the NRF-1 and the Cflash is verified through a dual-luciferase reporter gene experiment and a cellular immunofluorescence technology. An H9C2 myocardial cell model regulated and controlled by NRF-1 and Cflar expression is constructed, the cell apoptosis rate, Caspase activity and apoptosis-related protein expression are detected under the anoxic condition, it is revealed that NRF-1 inhibits hypoxia-induced myocardial cell apoptosis by up-regulating Cflar expression, and the regulation and control relation between NRF-1 and Cflar is further verified.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for screening and verifying a target molecule for inhibiting apoptosis of hypoxic myocardial cells. Background Art

[0002] Heart failure, the terminal stage of various heart diseases, is characterized by an irreversible course, poor quality of life, poor long-term treatment outcomes, and low survival rates. With the accelerating aging of the global population, the prevalence of heart failure is increasing annually, necessitating the urgent need for effective treatments. However, even the most advanced treatments can only slow the progression of heart failure and prolong survival, but cannot achieve improvement or a cure. Numerous studies have demonstrated that cardiomyocyte apoptosis, triggered by ischemia and hypoxia, is a key factor in the massive loss of cardiomyocytes and ultimately heart failure. Related studies have shown that nuclear respiratory factor 1 (NRF-1) can enhance cell proliferation and viability, inhibit reactive oxygen species production, and alter mitochondrial membrane potential, thereby alleviating hypoxia-induced apoptosis in H9C2 cardiomyocytes. However, the specific mechanism by which NRF-1 inhibits cardiomyocyte apoptosis remains unclear. To further explore the mechanism of action of NRF-1, it is necessary to identify NRF-1 as a molecular target for the treatment of heart failure. Addressing this question will not only help to uncover the molecular mechanisms of myocardial ischemia but also provide a critical theoretical foundation for the development of new cardioprotective strategies. Summary of the Invention

[0003] The present invention provides a method for screening and verifying target molecules for inhibiting hypoxic myocardial cell apoptosis, which mainly includes:

[0004] CUT&Tag technology combined with transcriptome sequencing was used to analyze the potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis;

[0005] ChIP-qPCR technology and dual-luciferase reporter gene experiments were used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar;

[0006] In H9C2 cells overexpressing NRF-1, siRNA was used to interfere with Cflar to study its regulatory effect on cell apoptosis and downstream molecular proteins of apoptosis. Cflar was also overexpressed in H9C2 cells with sh-RNA interference of NRF-1 to study its regulatory effect on cell apoptosis and downstream molecular proteins of apoptosis for verification.

[0007] Furthermore, the use of CUT&Tag technology combined with transcriptome sequencing to analyze potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis includes:

[0008] Using lentiviral transfection and shRNA interference technology, rat H9C2 cardiomyocytes were constructed into the following groups: NRF-1 overexpression group, empty vector overexpression group, NRF-1 interference group, empty vector interference group, and normal control group. After 12 hours of hypoxia and normal culture, cells were collected and transcriptome sequencing was performed. Differential genes and pathways associated with hypoxia-induced cardiomyocyte apoptosis were analyzed, as well as differential genes and pathways induced by NRF-1 interference.

[0009] Given the transcription factor properties of NRF-1, H9C2 cardiomyocytes and NRF-1 antibodies were subjected to CUT&Tag sequencing to analyze DNA sequences that bind to the NRF-1 protein. Combined with the above transcriptome sequencing results, the mechanism by which NRF-1 regulates apoptosis of hypoxic cardiomyocytes was further analyzed and its potential target molecules were predicted.

[0010] Furthermore, the ChIP-qPCR technology and dual luciferase reporter gene experiments were used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar, including:

[0011] Based on the CUT&Tag sequencing results, the binding regions of NRF-1 and target molecules were analyzed and predicted. Chromatin immunoprecipitation was used to verify the binding of NRF-1 to the promoter region of the target molecule. Dual-luciferase reporter gene experiments were used to verify the regulatory effect of NRF-1 on the promoter activity of potential target molecule genes.

[0012] Lentiviral transfection and siRNA interference technology were used to construct the following groups of rat cardiomyocytes: NRF-1 overexpression group, target molecule interference in NRF-1 overexpression group, NRF-1 interference group, target molecule overexpression in NRF-1 interference group, and normal control group. The cells were cultured under hypoxia and normal conditions, respectively. BioTek was used to monitor cell proliferation and mortality in real time. TUNEL staining was used to detect cell apoptosis rate after 12 hours of hypoxia. Caspase-3 / -8 / -9 activity levels were detected by spectrophotometry. Death receptor pathway and mitochondrial apoptosis pathway-related molecules were detected by Western Blot. Potential target molecules were further screened. It was speculated that Cflar may be a potential target molecule of NRF-1 in regulating the apoptosis pathway of H9C2 cells.

[0013] Furthermore, the siRNA interference of Cflar in H9C2 cells overexpressing NRF-1 to study its regulatory effect on cell apoptosis and apoptosis downstream molecular proteins, and the overexpression of Cflar in H9C2 cells interfering with NRF-1 by sh-RNA to study its regulatory effect on cell apoptosis and apoptosis downstream molecular proteins are verified, including:

[0014] ChIP and dual-luciferase reporter gene experiments verified that NRF-1 can bind to and regulate the activity of the Cflar gene promoter. NRF-1 and Cflar proteins are co-expressed in the nucleus of H9C2 cells, proving that NRF-1 can target and regulate Cflar.

[0015] Interference with Cflar in H9C2 cells overexpressing NRF-1 reduced the total number of H9C2 cells and increased their mortality during hypoxia. Cell apoptosis rate (P<0.01), Caspase-8 activity (P<0.05), and protein expression of cleaved-Caspase-8 and tBid were significantly increased 12 hours after hypoxia (P<0.05). During this process, there were no statistically significant differences in the activity of Caspase-9 and Caspase-3, or the protein expression of Bcl-2 and Bax. This suggests that Cflar participates in the regulation of apoptosis in H9C2 cells overexpressing NRF-1 in response to hypoxia by affecting the death receptor pathway.

[0016] Overexpression of Cflar in H9C2 cells antagonizing NRF-1 during hypoxia increased the total number of H9C2 cells, decreased their mortality, and increased the apoptosis rate (P<0.05) and Caspase-8 activity (P<0.05) after 12 hours of hypoxia, while significantly decreasing the protein expression of cleaved-Caspase-8 and tBid (P<0.05). During this process, there were no statistically significant differences in the activities of Caspase-9 and Caspase-3, or the protein expression of Bcl-2 and Bax, demonstrating that Cflar interferes with NRF-1's regulation of apoptosis in hypoxic H9C2 cells by affecting the death receptor pathway.

[0017] The technical solution provided by the embodiment of the present invention may have the following beneficial effects:

[0018] The present invention discloses a method for screening and verifying target molecules that inhibit hypoxic cardiomyocyte apoptosis. The method first uses CUT&Tag technology combined with transcriptome sequencing to analyze potential target molecules of NRF-1 that regulate hypoxic cardiomyocyte apoptosis. ChIP-qPCR technology and dual-luciferase reporter gene experiments are used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar. Then, CUT&Tag sequencing and chromatin immunoprecipitation technology are used to determine the direct regulatory relationship between NRF-1 and the Cflar gene. Subsequently, dual-luciferase reporter gene experiments and cell immunofluorescence technology are used to verify the transcriptional regulation and spatial expression relationship of NRF-1 on Cflar. By constructing an H9C2 cardiomyocyte model regulated by NRF-1 and Cflar expression, the cell apoptosis rate, Caspase activity, and apoptosis-related protein expression are detected under hypoxic conditions. It is revealed that NRF-1 inhibits hypoxia-induced cardiomyocyte apoptosis by upregulating Cflar expression, further confirming the regulatory relationship between NRF-1 and Cflar. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a target molecule screening and verification method for inhibiting hypoxic myocardial cell apoptosis of the present invention.

[0020] Figure 2 This is a map of the NRF-1 overexpression vector and interference plasmid vector of the present invention.

[0021] Figure 3 This is a map of the Cflar overexpression vector of the present invention.

[0022] Figure 4 This is a partial result diagram of the CUT&Tag sequencing of the present invention.

[0023] Figure 5 For the present invention Figure 4 Pathway network diagram in.

[0024] Figure 6 It is a differential molecule of the present invention that overexpresses or interferes with NRF-1 to affect the cell apoptosis pathway.

[0025] Figure 7 The CUT&Tag of the present invention is combined with transcriptome sequencing to analyze the target molecules of the NRF-1-regulated cell apoptosis pathway.

[0026] Figure 8 This is a diagram of the upstream and downstream relationship between Cflar and Birc3 in the Apoptosis pathway of the present invention.

[0027] Figure 9 It is the binding site between the NRF-1 protein of the present invention and the Cflar gene promoter.

[0028] Figure 10 For the present invention Figure 9 Figure A in .

[0029] Figure 11 For the present invention Figure 9 Figure B in .

[0030] Figure 12 For the present invention Figure 9 Figure C in .

[0031] Figure 13 This figure shows the effect of interfering with Cflar on cell growth and apoptosis under hypoxic conditions in H9C2 cells overexpressing NRF-1 according to the present invention.

[0032] Figure 14 Graph showing the effect of interfering with Cflar on apoptosis of H9C2 cells subjected to 12 h of hypoxia in H9C2 cells overexpressing NRF-1 according to the present invention (n=3).

[0033] Figure 15 This is a red fluorescence image expressed during the virus packaging process of 293T cells of the present invention.

[0034] Figure 16 This is the effect of overexpressing Cflar in H9C2 cells that interfere with NRF-1 of the present invention on cell apoptosis under hypoxic conditions (n=3). DETAILED DESCRIPTION

[0035] To help those skilled in the art better understand the technical solutions in this specification, the following will provide a clear and complete description of the technical solutions in the embodiments of this specification, in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of this specification, not all of them. All other embodiments derived by those skilled in the art based on the embodiments in this specification without creative effort shall fall within the scope of protection of this specification.

[0036] NRF-1, a transcription factor originally discovered during research on mitochondrial cytochrome C transcription, plays a crucial regulatory role in cellular energy metabolism, mitochondrial biogenesis, oxidative stress, and cell growth. Previous studies by the inventors of this application have shown that NRF-1 can alleviate H9C2 cardiomyocyte apoptosis caused by CoCl2-induced chemical hypoxia and 1% O2-induced physical hypoxia by improving cellular energy metabolism, reducing mitochondrial membrane potential changes, and inhibiting ROS production [1,2,5-8]. Preliminary studies have also shown that NRF-1 can inhibit H9C2 cell apoptosis through both death receptor and mitochondrial pathways. However, the specific molecular mechanism by which NRF-1 improves cardiomyocyte apoptosis remains unclear and requires further investigation.

[0037] Because NRF-1 is a nuclear transcription factor, genome-wide chromatin immunoprecipitation studies have shown that NRF-1 binds to the promoter regions of numerous genes, revealing its broad regulatory role. Therefore, our previous studies in the in vitro hypoxic H9C2 myocardial model revealed that NRF-1 treatment resulted in changes in both the mRNA and protein levels of CASP8 and FADD-like apoptosis regulator (Cflar). Combined with CUT&Tag sequencing, we hypothesized that Cflar may be a potential target of NRF-1 in regulating apoptosis. Cflar, also known as cellular FLICE inhibitory protein (c-FLIP), is a key regulator of caspase-8 activity. By preventing the cascade of caspase-8 on the death receptor complex (DISC), it inhibits caspase-8 activation, thereby regulating death receptor-mediated apoptosis. On the other hand, activated caspase-8 can induce the cleavage of the pro-apoptotic molecule Bid in the cytoplasm to tBid. The -COOH end of tBid then translocates to the outer mitochondrial membrane, promoting the dimerization of Bax and Bad there, initiating the mitochondrial apoptosis pathway. However, can NRF-1 directly regulate the transcription of Cflar? What role does Cflar play in NRF-1-mediated apoptosis of hypoxic cardiomyocytes? In addition to regulating the death receptor pathway, does it also regulate the mitochondrial apoptosis pathway? Currently, no reports have been published. Therefore, confirming NRF-1's transcriptional regulation of Cflar and elucidating its mechanism of action in ischemic-hypoxia-induced cardiomyocyte apoptosis will lay the foundation for further exploration of NRF-1's functions and provide a theoretical basis for its role in alleviating cardiomyocyte apoptosis and treating heart failure.

[0038] like Figure 1 This embodiment provides a method for screening and verifying target molecules that inhibit hypoxic cardiomyocyte apoptosis, which may specifically include:

[0039] S101. Use CUT&Tag technology combined with transcriptome sequencing to analyze the potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis.

[0040] Here, using lentiviral transfection and shRNA interference technology, rat H9C2 cardiomyocytes were constructed into the following groups: NRF-1 overexpression group, empty vector overexpression group, NRF-1 interference group, empty vector interference group, and normal control group. After 12 hours of hypoxia and normal culture, cells were harvested and subjected to transcriptome sequencing. Differential genes and pathways associated with hypoxia-induced cardiomyocyte apoptosis were analyzed, as well as differential genes and pathways induced by NRF-1 interference.

[0041] Given the transcription factor properties of NRF-1, H9C2 cardiomyocytes and NRF-1 antibodies were subjected to CUT&Tag sequencing to analyze DNA sequences that bind to the NRF-1 protein. Combined with the above transcriptome sequencing results, the mechanism by which NRF-1 regulates apoptosis of hypoxic cardiomyocytes was further analyzed and its potential target molecules were predicted.

[0042] Specifically, using CUT&Tag technology combined with transcriptome sequencing to analyze potential target molecules for NRF-1 regulation of hypoxic cardiomyocyte apoptosis, the CUT&Tag technique first enriched NRF-1 binding sites across the genome. CUT&Tag is a highly efficient epigenetic technique that precisely captures the interaction sites between transcription factors and DNA by labeling DNA fragments bound by target proteins in living cells. In the experiment, an NRF-1-specific antibody bound to the NRF-1 protein in the nucleus of H9C2 cells was then used to cleave and label the DNA fragments using Tn5 transposase. Finally, high-throughput sequencing was used to identify NRF-1 binding sites. Combined with transcriptome sequencing data, genes with significant expression changes under hypoxia were screened and compared with the CUT&Tag sequencing results to identify potential target molecules that may be regulated by NRF-1. Ultimately, CUT&Tag sequencing identified 18 potential target molecules in the NRF-1-regulated apoptosis pathway.

[0043] S102. Use CUT&Tag technology combined with transcriptome sequencing to analyze the potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis; use ChIP-qPCR technology and dual luciferase reporter gene experiments to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar.

[0044] Based on the CUT&Tag sequencing results, the binding regions of NRF-1 and target molecules were analyzed and predicted. Chromatin immunoprecipitation was used to verify the binding of NRF-1 to the promoter region of the target molecule. Dual-luciferase reporter gene experiments were used to verify the regulatory effect of NRF-1 on the promoter activity of potential target molecule genes.

[0045] Lentiviral transfection and siRNA interference technology were used to construct the following groups of rat cardiomyocytes: NRF-1 overexpression group, target molecule interference in NRF-1 overexpression group, NRF-1 interference group, target molecule overexpression in NRF-1 interference group, and normal control group. The cells were cultured under hypoxia and normal conditions, respectively. BioTek was used to monitor cell proliferation and mortality in real time. TUNEL staining was used to detect cell apoptosis rate after 12 hours of hypoxia. Caspase-3 / -8 / -9 activity levels were detected by spectrophotometry. Death receptor pathway and mitochondrial apoptosis pathway-related molecules were detected by Western Blot. Potential target molecules were further screened. It was speculated that Cflar may be a potential target molecule of NRF-1 in regulating the apoptosis pathway of H9C2 cells.

[0046] For example, analysis revealed that the Cflar gene's expression is significantly upregulated under hypoxic conditions, and that its promoter region contains an NRF-1 binding site, suggesting it as a potential target of NRF-1. This analytical approach, combining transcriptome data with CUT&Tag results, can efficiently identify key genes associated with NRF-1 function, providing candidate targets for subsequent validation. ChIP-qPCR was used to validate NRF-1 binding to the Cflar promoter region. The NRF-1 protein-DNA complex in H9C2 cells was first fixed by formaldehyde cross-linking, followed by sonication to release the DNA fragments. NRF-1-bound DNA fragments were immunoprecipitated using an NRF-1-specific antibody, and qPCR was used to quantify the enrichment of the Cflar promoter region. Specific primers were designed to amplify the NRF-1 binding site in the Cflar promoter region. Results showed that this region was significantly more enriched in the NRF-1 immunoprecipitation group than in the control group, indicating that NRF-1 directly binds to the Cflar promoter region.

[0047] For example, experimental data showed that the enrichment of the Cflar promoter region was more than 5 times, which was statistically significant. ChIP-qPCR technology can directly verify the binding of transcription factors to DNA, providing an experimental basis for NRF-1 to regulate Cflar. When using a dual-luciferase reporter gene experiment to verify NRF-1's regulation of Cflar promoter activity, a luciferase reporter gene vector containing the Cflar promoter region was first constructed and transfected into H9C2 cells. At the same time, an NRF-1 overexpression vector and an empty vector were transfected as controls. The effect of NRF-1 on the transcriptional activity of the Cflar promoter region was evaluated by detecting luciferase activity. The experimental results showed that the luciferase activity in the NRF-1 overexpression group was significantly higher than that in the control group, indicating that NRF-1 can enhance the transcriptional activity of the Cflar promoter.

[0048] For example, the luciferase activity in the NRF-1 overexpression group increased by more than three times compared to the control group, and the difference was statistically significant. The dual-luciferase reporter gene experiment can intuitively reflect the regulatory effect of transcription factors on promoter activity, further verifying the transcriptional regulatory function of NRF-1 on Cflar. Through the above experiments, Cflar was screened and verified as a potential target molecule for NRF-1 to regulate hypoxic cardiomyocyte apoptosis. CUT&Tag technology combined with transcriptome sequencing provides an efficient method for target molecule screening. ChIP-qPCR technology directly verifies the binding of NRF-1 to the Cflar promoter region. The dual-luciferase reporter gene experiment further confirms NRF-1's regulation of Cflar transcriptional activity. The combination of these experimental methods can not only systematically reveal the regulatory mechanism of NRF-1, but also provide reliable technical support for in-depth research on the role of NRF-1 in cardiomyocyte apoptosis.

[0049] S103. siRNA interference of Cflar in H9C2 cells overexpressing NRF-1 was used to study its regulatory effects on apoptosis and downstream apoptosis proteins. Cflar was also overexpressed in H9C2 cells with sh-RNA interference of NRF-1 to study its regulatory effects on apoptosis and downstream apoptosis proteins. This includes:

[0050] ChIP and dual luciferase reporter gene experiments verified that NRF-1 can bind to and regulate the activity of the Cflar gene promoter, and NRF-1 and Cflar proteins are co-expressed in the nucleus of H9C2 cells, proving that NRF-1 can target and regulate Cflar.

[0051] ChIP technology was used to extract DNA bound by NRF-1 protein in H9C2 cells. Primers were designed based on the predicted TFBS in the Cflar promoter region for RT-PCR to verify the binding relationship between NRF-1 and the Cflar promoter region.

[0052] GIV software was used to analyze the CUT&Tag sequencing results and identify peak sequences where NRF-1 protein binds to the Cflar gene. HOMER software and the JASPAR database were used to predict the transcription factor binding site (TFBS) of NRF-1 on the Cflar gene.

[0053] Chromatin immunoprecipitation assay (ChIP) verified the binding of NRF-1 to the Cflar promoter region.

[0054] ChIP technology was used to extract DNA bound by NRF-1 protein in H9C2 cells. Primers were designed based on the predicted TFBS in the Cflar promoter region for RT-PCR to verify the binding relationship between NRF-1 and the Cflar promoter region.

[0055] Based on the predicted TFBS of NRF-1, wild-type (WT) and mutant (MUT) dual-luciferase reporter gene vectors were constructed and inserted into the Cflar promoter. The NRF-1 expression vector and the dual-luciferase reporter gene vector were co-transfected into 293T cells, and the regulatory effect of NRF-1 on Cflar promoter activity was analyzed using a dual-luciferase reporter gene assay.

[0056] The expression and co-localization of NRF-1 and Cflar proteins in normally cultured H9C2 cells were observed under a laser confocal microscope using cell immunofluorescence.

[0057] Interference with Cflar in H9C2 cells overexpressing NRF-1 during hypoxia reduced the total number of H9C2 cells and increased their mortality rate. Cell apoptosis rate (P < 0.01), Caspase-8 activity (P < 0.05), and protein expression of cleaved-Caspase-8 and tBid significantly increased 12 hours after hypoxia (P < 0.05). There were no statistically significant differences in Caspase-9 and Caspase-3 activity, or in Bcl-2 and Bax protein expression. This suggests that Cflar regulates apoptosis in H9C2 cells overexpressing NRF-1 by affecting the death receptor pathway.

[0058] Experimental groups: NRF-1 overexpression group (pCDH-NRF-1), NRF-1 overexpression group with interference of Cflar (pCDH-NRF-1+siRNA-Cflar) and normal group (H9C2).

[0059] Lentiviral transfection and flow cytometry were used to construct an NRF-1-overexpressing H9C2 cell line. SiRNA was then used to interfere with Cflar to obtain the cell models described above. Cells were cultured in an oxygen-rich incubator with 1% O₂, 5% CO₂, and 94% N₂. Cell proliferation and cell death were monitored using a BioTek real-time imaging system. TUNEL staining was used to determine apoptosis rates. Caspase-3, 8, and 9 activities were measured spectrophotometrically. Proteins involved in the death receptor and mitochondrial pathways were analyzed by Western blot.

[0060] Overexpression of Cflar in H9C2 cells antagonizing NRF-1 during hypoxia increased the total number of H9C2 cells, decreased their mortality, and increased the apoptosis rate (P<0.05) and Caspase-8 activity (P<0.05) after 12 hours of hypoxia, while significantly decreasing the protein expression of cleaved-Caspase-8 and tBid (P<0.05). During this process, there were no statistically significant differences in the activities of Caspase-9 and Caspase-3, or the protein expression of Bcl-2 and Bax, demonstrating that Cflar interferes with NRF-1's regulation of apoptosis in hypoxic H9C2 cells by affecting the death receptor pathway.

[0061] Experimental groups: NRF-1 interference group (sh-NRF-1), NRF-1 interference group with overexpression of Cflar (sh-NRF-1+pLVX-Cflar) and normal group (H9C2).

[0062] Lentiviral transfection and flow cytometry were used to construct H9C2 cell lines expressing shRNA targeting NRF-1. Lentiviral transfection was then used to overexpress Cflar to generate the cell models described above. Cells were cultured in an oxygen-depleted incubator with a three-gas atmosphere of 1% O₂, 5% CO₂, and 94% N₂ for 12 hours. Cell apoptosis was assessed by TUNEL staining, the activities of caspase-3, 8, and 9 were measured spectrophotometrically, and proteins involved in the death receptor and mitochondrial pathways were analyzed by Western blot.

[0063] The specific experimental operations and experimental results are introduced below:

[0064] 1. Experimental Operation

[0065] Find NRF-1 and Cflar gene binding sequences and predict TFBS

[0066] GIV software was used to analyze the CUT&Tag sequencing results and identify the peak sequence of the Cflar gene. DNA sequences were extracted by widening the peak region by 200 bp in both the upstream and downstream directions. Motifs were predicted from these sequences using the findMotifs Genome.pl tool in HOMER software. The predicted motifs were matched with existing motif data in the JASPAR database to identify TFBSs of NRF-1 on the Cflar gene.

[0067] ChIP experiments

[0068] ① Crosslink and lyse cells: Prepare 37% formaldehyde solution in advance (prepare immediately before use). Add 550 μL to 20 mL of cell culture medium and mix thoroughly. Crosslink at room temperature for 10 minutes. Add 2 mL of 10× Glycine to the culture dish, mix thoroughly, and incubate at room temperature for 5 minutes to terminate the reaction. Add 2 mL of pre-chilled Protease Inhibitor Cocktail II dilution to the dish, scrape the cells, and collect them in a 15 mL centrifuge tube. Centrifuge (4°C, 700 g, 5 minutes) to pellet the cells. Aspirate the supernatant and lyse the cells with 1 mL of SDSLysis Buffer (containing 5 μL of Protease Inhibitor Cocktail II).

[0069] ② Sonicate DNA fragmentation: Sonicate on ice (sonication conditions: 20-second pulses, 50-second rests, 10 cycles) to fragment chromatin to fragments of 200-1000 bp in length. Centrifuge (4°C, 15,000 × g, 10 min) to remove any undissolved precipitate. Aliquot 100 μL of the supernatant into fresh centrifuge tubes.

[0070] ③ Immunoprecipitation of cross-linked protein / DNA complexes: Add 900 μL of dilution buffer (containing 4.5 μL of Protease Inhibitor Cocktail II) and 60 μL of Protein A agarose beads to 100 μL of chromatin sample. Add 1 μg of NRF-1 antibody (using the corresponding IgG as a negative control) and incubate with rotation (4°C overnight). Add 60 μL of Protein G agarose beads to each IP reaction and incubate with rotation (4°C, 1 hour). Centrifuge (5000 × g, 1 minute) to pellet the Protein G agarose beads. Elute the Protein A bead-antibody / protein / chromatin complex with 1 mL of pre-chilled buffer.

[0071] ④ Elute the protein / DNA complex: Add 100 μL of elution buffer to the collection tube and incubate at room temperature for 15 minutes. Centrifuge (5000 × g, 1 minute) to collect the agarose and transfer the supernatant to a new collection tube. Repeat steps 4-6 to collect the eluate (approximately 200 μL).

[0072] ⑤ Decrosslinking of protein / DNA complexes: Add 8 μL of 5 M NaCl to all collection tubes (pre-add 200 μL of elution buffer to the input tubes) and incubate in a water bath (65°C, overnight). Decrosslink the DNA-protein complexes. Add 1 μL of RNase to all collection tubes and incubate at 37°C for 30 minutes. Add 4 μL of 0.5 M EDTA, 8 μL of 1 M Tris-HCl, and 1 μL of Proteinase K, mix well, and incubate in a water bath (42°C, 2 hours).

[0073] ⑥ Purify DNA: Elute and purify DNA according to the steps, collect the purified DNA by centrifugation (15000×g, 30s), and store at -20℃.

[0074] ⑦RT-qPCR: Design ChIP primers based on the predicted NRF-1 and Cflar gene binding sites, and perform subsequent RT-PCR experiments.

[0075] Dual-luciferase reporter gene assay

[0076] Based on the predicted NRF-1 motif site, a dual-luciferase reporter gene vector was constructed. Sequences 1000bp upstream and 400bp downstream of the Cflar gene transcription start site (TSS) were inserted to replace the Renilla luciferase promoter region in the dual-luciferase vector, thereby constructing a wild-type vector (WT). On this basis, the predicted NRF-1 motif site was mutated to construct a mutant vector (MUT).

[0077] The cells were divided into 4 groups: NRF-1 overexpression vector co-transfected with Cflar gene promoter wild-type luciferase vector (pCDH-NRF-1+Cflar-WT-psiCheck2); NRF-1 overexpression vector co-transfected with Cflar gene promoter mutant luciferase vector (pCDH-NRF-1+Cflar-MUT-psiCheck2); empty overexpression vector co-transfected with Cflar gene promoter wild-type luciferase vector (pCDH-vector+Cflar-WT-psiCheck2); empty overexpression vector co-transfected with Cflar gene promoter mutant luciferase vector (pCDH-vector+Cflar-MUT-psiCheck2).

[0078] 293T cells were seeded in 24-well plates (5×10 4 / well), and co-transfected with vectors using Lip3000. The specific operation method is the same as the first part. The medium was changed 24 hours after transfection, and the luciferase assay was performed 48 hours later. The specific operation method is as follows: Thaw the reagent to room temperature in advance, prepare Dual-Glo luciferase detection reagent, and mix it with cell culture medium at a volume ratio of 1:1 to prepare the detection working solution. Aspirate the cell culture medium in the 24-well plate, add 500μL of the detection working solution in step ① to each well, and fully lyse the cells for 15 minutes. Transfer 150μL / well of the lysate in step ② to an opaque 96-well all-white plate (be careful to avoid bubbles that affect the detection), and detect firefly fluorescence on the machine. Add 75μL of stop reaction solution (Dual-GloStop&Glo detection reagent and buffer diluted at a volume ratio of 1:100) to each well, mix gently, and protect from light for 10 minutes (room temperature) to stop firefly fluorescence. Detect sea cucumber fluorescence on the machine. The calculation results were expressed as the ratio of Renilla fluorescence to Firefly fluorescence in each well. The values ​​of each experimental group were normalized and then analyzed.

[0079] Cell immunofluorescence

[0080] H9C2 cells were seeded in 6-well cell culture plates (1×10 5 / well). Stain after the cells have fully attached and expanded. Aspirate the culture medium and wash three times with PBS. Add 1 mL of 4% paraformaldehyde and fix for 30 minutes. Aspirate the paraformaldehyde and wash three times with PBS. Add 1 mL of 0.5% Triton X-100 (prepared with PBS) and permeabilize for 20 minutes. Aspirate the permeabilization solution and wash three times with PBS. Add 2 mL of 3% H2O2 (prepared with PBS) and let it stand at room temperature for 15 minutes to inactivate endogenous peroxidase. Wash three times with PBS, add 10% BSA and block for 30 minutes. Aspirate the blocking solution and add a mixture of NRF-1 and Cflar's primary antibodies to cover the cell surface and incubate at 4°C overnight to prevent the liquid from evaporating. (Note: The primary antibody for NRF-1 is rabbit-derived and diluted 1:500, and the primary antibody for Cflar is mouse-derived and diluted 1:300). Aspirate the primary antibody and wash five times with PBS. Add the corresponding secondary antibody mixture (NRF-1 secondary antibody is goat anti-rabbit green fluorescent labeling, Cflar secondary antibody is donkey anti-mouse red fluorescent labeling) and incubate at room temperature in the dark for 1 hour. Aspirate the secondary antibody and wash the cells five times with PBS. Add a fluorescent antifade reagent dropwise and cover the cell surface with a coverslip. Observe the expression of NRF-1 and Cflar proteins in H9C2 cells under a fluorescence microscope as green and red fluorescence, respectively.

[0081] Construction of stably transfected H9C2 cell lines overexpressing and interfering with NRF-1

[0082] ① Vector construction:

[0083] The CDS region sequence of rat NRF-1 gene (NM_001100708.1) (full length 1605 bp) was inserted into the overexpression vector pCDH-CMV-MCS-EF1-GFP-puro ( Figure 2 Left), the corresponding empty vector is used as a control. The restriction sites are EcoRI and BamHI respectively.

[0084] Based on the NRF-1 interference target sequence verified in the laboratory in the early stage: 5'-GCAAGTTCAGCA GGTCCATGT-3', the sequence of the shRNA oligonucleotide is as follows:

[0085]

[0086] Insert into pGreenPuro shRNA vector ( Figure 2 Right) Construction of NRF-1 interference vector, and the corresponding empty vector as a control.

[0087] ② Virus packaging: Seed 293T cells in a 6-well plate one day in advance and allow the cells to adhere to a confluency of 80%-90%. Virus packaging is most effective if performed within 24 hours of passage. Use Lipfectamine 3000 as the transfection reagent.

[0088] ③ Cell infection: H9C2 cells were seeded in a 6-well plate in advance and infected when the cells reached 70%-80% confluence. The packaged virus stock solution was added to the culture dish. After 24 hours, the medium was changed. Successfully infected cells were observed to express green fluorescence 48-72 hours after infection. Positive cells were selected for 7 consecutive days using culture medium containing 2μg / μl puromycin.

[0089] ④ Flow cytometric screening of stably transfected cells: Using a flow cytometer, H9C2 cells not transfected with any vector served as a negative control. Stably transfected cell lines exhibiting green fluorescence were screened at 488 nm excitation wavelength for expansion and subsequent experiments. Before each experiment, flow cytometric analysis was performed to ensure that the percentage of positive cells exceeded 99%.

[0090] Construction of siRNA interference Cflar cell model

[0091] Shanghai Jima Gene Biotechnology Co., Ltd. was commissioned to design and synthesize siRNA interference sequences for the Cflar gene. Subsequent experiments identified the two most effective siRNA oligos. These oligos were transfected into NRF-1-overexpressing H9C2 cells and into an empty vector-overexpressing control group. The specific procedures were as follows: Each cell group was plated in a 6-well plate. Transfection was performed when the cell density reached 60%, and the medium was replaced before transfection. In a 1.5 mL centrifuge tube, 125 μL of serum-free medium and 5 μg of siRNA oligo were added. In another 1.5 mL centrifuge tube, 125 μL of serum-free medium and 8 μL of GP-transfect-Mate transfection reagent were added and allowed to stand (room temperature, 5 minutes). The mixture was added dropwise to the centrifuge tube in step 2 and allowed to stand (room temperature, 15 minutes). The mixture was added dropwise to the cell culture medium. After 6 hours, the medium was replaced. mRNA and protein expression levels were measured 48 hours after transfection, or subsequent hypoxia experiments were performed. The siRNA sequences for CFLAR are shown in Table 1.

[0092] Table 1 CFLAR siRNA sequences

[0093] siRNA Sequence (5'-3') siRNA-CFLAR-1F CCUCCUGGAUUGUUUAAGUTT siRNA-CFLAR-1R ACUUAAACAAUCCAGGAGGTT siRNA-CFLAR-2F GAGCCAGUGUGUGGAAUAUTT siRNA-CFLAR-2R AUAUUCCACACACUGGCUCTT siRNA-NCF UUCUUCGAACGUGUCACGUTT siRNA-NCR ACGUGACACGUUCGGAGAATT

[0094] Construction of Cflar overexpressing cell model

[0095] The CDS region sequence of the rat Cflar gene (NM_001033864.4) was searched on the NCBI website. The full length is 1442 bp and inserted into the lentiviral overexpression vector pLVX-IRES-mCherry. The vector map is as follows Figure 3 , the restriction enzyme cutting sites were EcoRⅠ and XhoⅠ, and the corresponding empty vector was used as control.

[0096] The virus packaging method was the same as described above, and the NRF-1 interfered H9C2 cell group and the corresponding empty vector group were infected for subsequent experiments.

[0097] Bio-Tek monitors cell growth and death in real time

[0098] Cells from each group were plated in a 12-well plate until the confluence reached approximately 50% after attachment. Following the instructions for the Hochest33342 / PI double staining reagent, 3 μL of Hochest33342 and 1 μL of PI detection reagent were added to each milliliter of culture medium and mixed thoroughly. The mixture was then added to the assay wells using a replacement flow cytometer. Cells were then placed in a Bio-Tek Cell Imaging Multi-Mode Detection System and assayed under conditions of 1% O₂, 5% CO₂, 94% N₂, and 37°C. Blue and red fluorescence were measured every 30 minutes for 24 hours, and the results were analyzed using Gen 5 software.

[0099] TUNEL assay for cell apoptosis

[0100] The cells (1×10 5 Cells (cells per well) were plated in 12-well plates and stained using the Biyuntian TUNEL Cell Apoptosis Detection Kit (chromogenic method). The following procedures were performed according to the experimental instructions. Cells were washed once with PBS and fixed with 4% paraformaldehyde (room temperature, 30 minutes). Cells were washed three times with PBS and fixed with 0.3% Triton X-100 solution (incubated at room temperature for 5 minutes). Cells were washed once with PBS and incubated with 0.3% H2O2 solution (incubated at room temperature for 20 minutes). Cells were washed three times with PBS and added with 50 μl of pre-prepared biotin standard solution (prepared immediately, TdT: Bio-dUTP ratio 1:9 by volume). Cells were incubated in a 37°C incubator in the dark for 1 hour to prevent evaporation. Cells were washed once with PBS and 0.3 mL of labeling reaction stop solution was added dropwise and incubated for 10 minutes. Cells were washed three times with PBS and excess liquid was aspirated. Cells were then visualized under a microscope (prepared immediately, mixing equal volumes of Solution A and Solution B). The cells were washed three times with PBS and counterstained with hematoxylin. The cells were washed three times with PBS and photographed under an Olympus CKX41 inverted microscope. The percentage of TUNEL-positive cells in each group, representing the cell apoptosis rate, was calculated.

[0101] Detection of intracellular Caspase-3 / -8 / -9 activity

[0102] Caspase-3 / -8 / -9 activity was measured spectrophotometrically. Cells were trypsinized at 0, 3, 6, 12, and 24 hours of hypoxia and collected by centrifugation (300 × g, 5 min). 200 μl of lysis buffer (containing 1% DTT) was added and incubated on ice for 30 minutes (vortexed 4-5 times for 20 seconds each). The supernatant protein solution was collected by centrifugation (13,000 × g, 10 min). 50 μl of protein solution (100-200 μg) was added to a 96-well microplate. PBS was used as a blank control. 50 μl of reaction substrate diluent was added and incubated for 1 hour (37°C, protected from light). Absorbance at 405 nm was measured using a SkanIt@Software microplate reader, and caspase activity was calculated. Results are expressed as the ratio of each hypoxic treatment group to the control group (hypoxia at 0 hour).

[0103] Western blot

[0104] Cellular protein was extracted using a Keygi total protein extraction kit. A standard curve was generated using a BCA protein concentration assay kit to calculate protein concentration. A 12% SDS-PAGE gel with a thickness of 1.5 mm was prepared. The protein concentration was adjusted by mixing the sample with the loading buffer in a 1:5 volume ratio. The gel was then placed in a 100°C metal bath for 10 min to denature the protein. Equal amounts of denatured protein (30 μg) were added to the wells of a 12% SDS-PAGE. 3 μl of a protein marker (molecular weight 10 kDa-180 kDa) was added to each of the extra wells at either end. The protein was passed through the stacking gel at 120 V, then switched to 180 V to fully resolve the protein bands. A 0.45 μm PVDF membrane was pre-activated in methanol for 10 min. The membrane was transferred using an SD semi-dry electrotransfer device at a constant voltage of 15 V for 70 min. The PVDF membrane was washed with PBST and blocked with 5% bovine serum albumin in PBST for 2 h. Crop the membrane according to the band size of the target protein. Wash three times with PBST, place the membrane in diluted primary antibody, and incubate overnight at 4°C on a shaker. Wash three times with PBST, place the membrane in diluted secondary antibody (HRP-conjugated), and incubate for 2 hours at room temperature. Wash three times with PBST, and place the membrane on the ChemiDoc MP System. Add ECL working solution to cover the membrane surface. Use the automatic exposure program for image acquisition and data analysis.

[0105] 2. Experimental Results

[0106] Partial results of CUT&Tag sequencing

[0107] See Figure 4 In the figure, A. Peaks neighboring gene GO enrichment BP analysis diagram; B. KEGG enrichment pathway analysis diagram; C. Pathway network diagram; D. Venn diagram of genes that bind to NRF-1 in the cell apoptosis pathway. BP (Biological Process) analysis results show that genes bound by NRF-1 protein are mainly involved in various biological processes such as cell metabolism, protein modification, cell localization, and synthesis of intracellular components ( Figure 4 A). KEGG pathway analysis results showed that the genes bound by NRF-1 protein were mainly involved in pathways such as RNA transport, cell cycle, endoplasmic reticulum protein processing, cell metabolism, apoptosis and autophagy ( Figure 4 B). The results of the top 50 enriched pathway network analysis showed that the apoptosis pathway and the cell cycle pathway were key pathway entries ( Figure 4 C). There are 56 genes in the Apoptosis pathway (Pathway ID: 04210) that are associated with NRF-1 protein. Figure 4 D).

[0108] Some transcriptome sequencing results

[0109] The differentially expressed genes in each group of samples were analyzed based on the FPKM values ​​(Table 2). In this experiment, we focused on the regulatory effect of NRF-1 on apoptosis-related pathways under hypoxic conditions. KEGG pathway analysis was used to screen for differentially expressed molecules among 96 molecules related to the Apoptosis pathway (Pathway ID: 04210). Figure 6 In the figure: A. Venn diagram of the difference in molecules in the apoptosis pathway caused by overexpression of NRF-1 between normoxia and hypoxia for 12 hours; B. Venn diagram of the difference in molecules in the apoptosis pathway caused by interference with NRF-1 between normoxia and hypoxia for 12 hours; C. Heat map of the difference in molecules in the apoptosis pathway caused by overexpression of NRF-1; D. Heat map of the difference in molecules in the apoptosis pathway caused by interference with NRF-1. Through Venn analysis under control and hypoxic conditions, compared with the H9C2 group and the empty vector overexpression group, there are 4 different molecules in the apoptosis pathway caused by overexpression of NRF-1 (see Figure 6 A). Under control and hypoxic conditions, compared with the H9C2 group and the interference empty vector group, the interference of NRF-1 caused 11 differentially expressed molecules (see Figure 6 B). Figure 6 C and 6D are heat maps obtained by Log10 transformation of the FPKM values ​​of the above differentially expressed genes.

[0110] Table 2. Differentially expressed genes obtained by comparison analysis between the two groups (FDR<0.05)

[0111]

[0112] The differentially expressed molecules in the apoptosis pathway identified by CUT&Tag sequencing and transcript sequencing were subjected to Venn analysis, and two potential target molecules for NRF-1 to regulate H9C2 cell apoptosis under hypoxia were screened, namely Cflar and Birc3 ( Figure 7 ). Search the upstream and downstream genes and regulatory pathways of the two genes on the KEGG website ( Figure 8 ) found that Cflar is an inhibitory molecule of Caspase-8 in the extrinsic death receptor pathway of cell apoptosis, inhibiting Caspase-8 activation and thus inhibiting cell apoptosis. Birc3 belongs to the IAP family and regulates cell apoptosis by inhibiting Caspase-3, Caspase-7, and Caspase-9.

[0113] Regulatory effect of NRF-1 on the Cflar gene promoter

[0114] See also Figure 9-12 , Figure 9A. IGV software analysis of peak positions near the Cflar gene; B. ChIP-qPCR verification of PCR product electrophoresis, fluorescence quantitative amplification curve, and melting curve of NRF-1 binding to the Cflar gene promoter; C. Dual luciferase assay verification of NRF-1 regulation of Cflar gene promoter activity (n=3), *** indicates P<0.001, NS indicates no statistically significant difference. In the CUT&Tag sequencing results analyzed by IGV software, there are two peak binding positions of NRF-1 protein binding to the Cflar gene ( Figure 9 A), where the peaks7594 / 7455 sequence is located in the promoter region. HOMER software was used to predict the NRF-1 transcription factor binding site in the peaks7594 / 7455 sequence. ChIP-qPCR results demonstrated that NRF-1 protein can bind to the promoter region of the Cflar gene ( Figure 9 B).

[0115] The Cflar gene promoter sequence was inserted into the dual luciferase reporter gene vector (WT-psiCheck2) and co-transfected into 293T cells with the NRF-1 overexpression vector (pCDH-NRF-1) or the empty overexpression vector (pCDH-vector). Figure 9 As shown in Figure C, the relative fluorescence ratio of the pCDH-NRF-1+WT group was significantly higher than that of the pCDH-vector+WT group (P < 0.001), further demonstrating that NRF-1 can enhance the promoter activity of the Cflar gene. Based on software predictions, NRF-1 binding sites were verified by mutating different regions on the WT-psiCheck2 vector. The results showed that the predicted sequence was not the NRF-1 binding site, but rather located in the mutated region of MUT3.

[0116] Effects of siRNA knockdown of Cflar in H9C2 cells overexpressing NRF-1 on cell proliferation and cell death under hypoxia

[0117] See also Figure 13 , Figure 13 A. Changes in total cell number; B. Changes in the number of dead cells; C. Apoptosis in each group after 12 hours of hypoxia, magnification 10×4, blue represents cell nuclei stained with Hochest33342, and red fluorescence represents dead cells stained with PI. Cflar was interfered with in H9C2 cells overexpressing NRF-1 (pCDH-NRF-1 group), and the growth and death of each group of cells under hypoxia were monitored in real time using the Bio-Tek cell imaging system ( Figure 13 The results showed that the number of H9C2 cells increased slightly in the early stage and then gradually decreased as the hypoxia time prolonged. Figure 13 A), the proportion of dead cells gradually increased ( Figure 13 B). Compared with the empty vector overexpression group (pCDH-vector group), the total cell number in the pCDH-NRF-1 group increased and the proportion of dead cells decreased. In H9C2 cells overexpressing NRF-1, Cflar was interfered with (pCDH-NRF-1+siRNA-Cflar-1 and pCDH-NRF-1+siRNA-Cflar-2). Compared with the interference negative control (pCDH-NRF-1+siRNA-NC) group and the pCDH-NRF-1 group, the total cell number decreased and the proportion of dead cells increased. This demonstrates that interfering with Cflar in H9C2 cells overexpressing NRF-1 inhibits cell proliferation and promotes cell death under hypoxic conditions.

[0118] Effects of siRNA knockdown of Cflar in H9C2 cells overexpressing NRF-1 on cell apoptosis under hypoxia

[0119] See also Figure 14 Figure 1. A. TUNEL assay for cell apoptosis, magnification 10×20; B. Changes in caspase-8, caspase-9, and caspase-3 activity levels; C. Expression of apoptosis-related proteins; * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, NS indicates no statistical significance. Cflar was knocked down in H9C2 cells overexpressing NRF-1, and the apoptosis rate was assessed by TUNEL staining after 12 hours of hypoxia. Figure 14 A results showed that the apoptosis rate of cells in the pCDH-NRF-1 group was significantly lower than that in the empty vector (pCDH-vector) overexpression group (P<0.01). Compared with the interference negative control (pCDH-NRF-1+siRNA-NC group), the apoptosis rate of cells in the pCDH-NRF-1+siRNA-Cflar-1 group (P<0.01) and the pCDH-NRF-1+siRNA-Cflar-2 group (P<0.001) was significantly increased. The activity level of Caspase-8 was significantly increased (P<0.05), while the activity level of Caspase-9 and Caspase-3 had no statistically significant difference ( Figure 14 B). Under hypoxia for 12 h, the protein expressions of CflarL and Cflars were significantly decreased, while the protein expressions of cleaved-Caspase-8 and tBid were significantly increased. There was no statistically significant difference in the protein expressions of NRF-1, Bcl-2, and Bax ( Figure 14 C) This suggests that Cflar is the target molecule of NRF-1 in regulating the death receptor pathway. Cflar overexpression lentivirus was packaged and infected into the H9C2 cell line that interferes with NRF-1.

[0120] See also Figure 15, Red fluorescence image of the virus packaging process in 293T cells (magnification 10×10) Construction of Cflar overexpression vector (pLVX-Cflar), with the corresponding empty vector (pLVX-vector) as control, using 293T cells to package lentivirus, Figure 15 The red fluorescence is shown in the process of virus packaging in 293T cells. The packaged viruses were used to infect the H9C2 cell line that successfully interfered with NRF-1 (sh-NRF-1 group) and the interference empty vector cell line (sh-vector group).

[0121] Effect of overexpression of Cflar in H9C2 cells with NRF-1 interference on cell apoptosis under hypoxia

[0122] See also Figure 16 In the figure, A. TUNEL detection of cell apoptosis rate, magnification 10×10; B. Changes in the activity levels of Caspase-8, Caspase-9 and Caspase-3; C. Expression of apoptosis-related proteins; * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.001, NS indicates no statistically significant difference. Cflar was overexpressed in the H9C2 cell line with interference of NRF-1 (sh-NRF-1 group) and the corresponding interference empty vector cell line (sh-vector group). Figure 16 As shown in A, the apoptosis rate of cells overexpressing Cflar (sh-vector+pLVX-Cflar) under hypoxia for 12 hours in the sh-vector group was not significantly different from that in the empty vector control group (sh-vector+pLVX-vector). However, the apoptosis rate of cells overexpressing Cflar (sh-NRF-1+pLVX-Cflar) under hypoxia for 12 hours in the sh-NRF-1 group was significantly lower than that in the sh-NRF-1+pLVX-vector control group (P<0.05). Figure 16 As shown in Figure B, compared with overexpression of empty vector, overexpression of Cflar in the sh-vector group can significantly reduce the activity levels of Caspase-3, Caspase-8 and Caspase-9 in H9C2 cells after 12h hypoxia (P<0.05). In H9C2 cells with NRF-1 interference, overexpression of Cflar can significantly reduce the activity level of Caspase-8 (P<0.001), but there is no statistically significant difference in the activity levels of Caspase-9 and Caspase-3. In H9C2 cells with NRF-1 interference, overexpression of Cflar can significantly reduce the activity level of Caspase-8 (P<0.001), but there is no statistically significant difference in the activity levels of Caspase-9 and Caspase-3. L and Cflar sThe protein expression of cleaved-Caspase-8 and tBid was significantly increased, while the protein expression of NRF-1, Bcl-2 and Bax was significantly decreased. Figure 16 C). This demonstrates that Cflar may be the target molecule of NRF-1 in regulating the death receptor pathway.

[0123] The present invention discloses a method for screening and verifying target molecules that inhibit hypoxic cardiomyocyte apoptosis. The method first uses CUT&Tag technology combined with transcriptome sequencing to analyze potential target molecules of NRF-1 that regulate hypoxic cardiomyocyte apoptosis. ChIP-qPCR technology and dual-luciferase reporter gene experiments are used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar. Then, CUT&Tag sequencing and chromatin immunoprecipitation technology are used to determine the direct regulatory relationship between NRF-1 and the Cflar gene. Subsequently, dual-luciferase reporter gene experiments and cell immunofluorescence technology are used to verify the transcriptional regulation and spatial expression relationship of NRF-1 on Cflar. By constructing an H9C2 cardiomyocyte model regulated by NRF-1 and Cflar expression, the cell apoptosis rate, Caspase activity, and apoptosis-related protein expression are detected under hypoxic conditions. It is revealed that NRF-1 inhibits hypoxia-induced cardiomyocyte apoptosis by upregulating Cflar expression, further confirming the regulatory relationship between NRF-1 and Cflar. The present invention clarifies the molecular mechanism by which NRF-1 inhibits hypoxic cardiomyocyte apoptosis through Cflar, and provides a new target and strategy for the prevention and treatment of myocardial ischemia injury.

[0124] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for screening and validating target molecules for inhibiting apoptosis of hypoxic cardiomyocytes, characterized in that: include: CUT&Tag technology combined with transcriptome sequencing was used to analyze the potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis; ChIP-qPCR technology and dual-luciferase reporter gene experiments were used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar; In H9C2 cells overexpressing NRF-1, siRNA was used to interfere with Cflar to study its regulatory effect on cell apoptosis and downstream molecular proteins of apoptosis. Cflar was also overexpressed in H9C2 cells with sh-RNA interference of NRF-1 to study its regulatory effect on cell apoptosis and downstream molecular proteins of apoptosis for verification.

2. The method according to claim 1, wherein The use of CUT&Tag technology combined with transcriptome sequencing to analyze potential target molecules of NRF-1 in regulating hypoxic cardiomyocyte apoptosis includes: Using lentiviral transfection and shRNA interference technology, rat H9C2 cardiomyocytes were constructed into the following groups: NRF-1 overexpression group, empty vector overexpression group, NRF-1 interference group, empty vector interference group, and normal control group. After 12 hours of hypoxia and normal culture, cells were collected and transcriptome sequencing was performed. Differential genes and pathways associated with hypoxia-induced cardiomyocyte apoptosis were analyzed, as well as differential genes and pathways induced by NRF-1 interference. Given the transcription factor properties of NRF-1, H9C2 cardiomyocytes and NRF-1 antibodies were subjected to CUT&Tag sequencing to analyze DNA sequences that bind to the NRF-1 protein. Combined with the above transcriptome sequencing results, the mechanism by which NRF-1 regulates apoptosis of hypoxic cardiomyocytes was further analyzed and its potential target molecules were predicted.

3. The method according to claim 2, wherein The ChIP-qPCR technology and dual luciferase reporter gene experiments were used to verify whether NRF-1 can bind to and regulate the promoter region of potential target molecules and screen out the potential target molecule Cflar, including: Based on the CUT&Tag sequencing results, the binding regions of NRF-1 and target molecules were analyzed and predicted. Chromatin immunoprecipitation was used to verify the binding of NRF-1 to the promoter region of the target molecule. Dual-luciferase reporter gene experiments were used to verify the regulatory effect of NRF-1 on the promoter activity of potential target molecule genes. Lentiviral transfection and siRNA interference technology were used to construct the following groups of rat cardiomyocytes: NRF-1 overexpression group, target molecule interference in NRF-1 overexpression group, NRF-1 interference group, target molecule overexpression in NRF-1 interference group, and normal control group. The cells were cultured under hypoxia and normal conditions, respectively. BioTek was used to monitor cell proliferation and mortality in real time. TUNEL staining was used to detect cell apoptosis rate after 12 hours of hypoxia. Caspase-3 / -8 / -9 activity levels were detected by spectrophotometry. Death receptor pathway and mitochondrial apoptosis pathway-related molecules were detected by Western Blot. Potential target molecules were further screened. It was speculated that Cflar may be a potential target molecule of NRF-1 in regulating the apoptosis pathway of H9C2 cells.

4. The method according to claim 3, wherein The method involves interfering with Cflar by siRNA in H9C2 cells overexpressing NRF-1 to study its regulatory effect on apoptosis and downstream apoptosis molecules, and overexpressing Cflar in H9C2 cells interfering with NRF-1 by sh-RNA to study its regulatory effect on apoptosis and downstream apoptosis molecules for verification, including: ChIP and dual-luciferase reporter gene experiments verified that NRF-1 can bind to and regulate the activity of the Cflar gene promoter. NRF-1 and Cflar proteins are co-expressed in the nucleus of H9C2 cells, proving that NRF-1 can target and regulate Cflar. Interference with Cflar in H9C2 cells overexpressing NRF-1 reduced the total number of H9C2 cells and increased their mortality during hypoxia. Cell apoptosis rate (P<0.01), Caspase-8 activity (P<0.05), and protein expression of cleaved-Caspase-8 and tBid were significantly increased 12 hours after hypoxia (P<0.05). During this process, there were no statistically significant differences in the activity of Caspase-9 and Caspase-3, or the protein expression of Bcl-2 and Bax. This suggests that Cflar participates in the regulation of apoptosis in H9C2 cells overexpressing NRF-1 in response to hypoxia by affecting the death receptor pathway. Overexpression of Cflar in H9C2 cells antagonizing NRF-1 during hypoxia increased the total number of H9C2 cells, decreased their mortality, and increased the apoptosis rate (P<0.05) and Caspase-8 activity (P<0.05) after 12 hours of hypoxia, while significantly decreasing the protein expression of cleaved-Caspase-8 and tBid (P<0.05). During this process, there were no statistically significant differences in the activities of Caspase-9 and Caspase-3, or the protein expression of Bcl-2 and Bax, demonstrating that Cflar interferes with NRF-1's regulation of apoptosis in hypoxic H9C2 cells by affecting the death receptor pathway.

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