Application of circFam120asiRNA in the preparation of products for inhibiting cardiomyocyte apoptosis and autophagy and congenital heart disease

Abstract

The present invention discloses the use of circFam120a siRNA in the preparation of a product for inhibiting myocardial cell apoptosis and autophagy and congenital heart disease. The circFam120a siRNA of the present invention can inhibit myocardial cell apoptosis and autophagy, thereby preventing and treating congenital heart disease. In addition, circFam120a is significantly differentially expressed in formaldehyde-induced animal tissues, myocardial cells, and CHD patient plasma. Therefore, by detecting the expression level of circFam120a, auxiliary diagnosis of congenital heart disease can be achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of circFam120a siRNA in preparing a product for inhibiting myocardial cell apoptosis and autophagy and congenital heart disease. Background Art

[0002] Congenital heart disease (CHD) is a common and serious birth defect and the most common cause of neonatal mortality. It occurs in approximately 1% of live births and affects millions of people worldwide. CHD is caused by structural abnormalities of the heart before birth, which occur during fetal development in the uterus. Although advances in cardiovascular medicine and cardiac surgery have enabled most patients with CHD to receive timely treatment, allowing them to reach adulthood and live longer, they face a higher risk of late complications such as arrhythmias, endocarditis, pulmonary hypertension, and heart failure as they age. Therefore, CHD is one of the most important causes of neonatal mortality and late adult complications, placing a heavy burden on families and significantly reducing quality of life. Therefore, early prevention, diagnosis, and treatment of CHD are crucial. The development of CHD is driven by both environmental and genetic factors. Existing research has demonstrated the influence of environmental factors on genetic cascades and genomic mechanisms involved in gene regulation, including post-transcriptional regulation.

[0003] Formaldehyde (FA) is a common pollutant found in the environment but is also produced endogenously through a variety of essential biological processes, including mitochondrial one-carbon metabolism, metabolite oxidation, and nuclear epigenetic modification. Disturbances in endogenous formaldehyde metabolism and high exposure to exogenous formaldehyde can lead to multisystem diseases, including cardiovascular, neurological, respiratory, immune, hematologic, and reproductive disorders. The embryotoxic and teratogenic effects of formaldehyde are of increasing concern. Exposure to formaldehyde during organogenesis can cause toxic changes in placental structure, disrupting placental function and resulting in reduced fetal weight. Neonates with congenital heart disease are at increased risk of low birth weight and a two- to three-fold increased risk of preterm birth. Previous studies have shown that chronic low-level exposure to formaldehyde during pregnancy increases the risk of miscarriage and fetal cardiac malformations. Exposure to paints, dyes, glues, and other indoor environmental pollutants during pregnancy may be a risk factor for congenital heart disease in offspring. Other studies have found that women who experience miscarriage in early pregnancy have significantly higher plasma formaldehyde levels than those who deliver at term. Therefore, elevated formaldehyde levels in the human body may be an independent risk factor for miscarriage, with higher formaldehyde levels being associated with a higher risk of miscarriage. An epidemiological case-control study demonstrated that residential formaldehyde exposure is associated with a 24% increased risk of congenital heart malformations. Another study demonstrated that subacute and subchronic formaldehyde inhalation can stimulate oxidative stress, leading to secondary toxic effects on cardiac cells and tissues. High doses of formaldehyde can also induce oxidative stress and apoptosis in cardiomyocytes of pregnant mice and their offspring. The first eight weeks of pregnancy are a critical period for embryonic heart development. Currently, fetal congenital heart disease is primarily diagnosed through imaging techniques during the second trimester, when the embryonic heart is already fully developed. Therefore, understanding the molecular genetic regulatory mechanisms underlying the development and progression of CHD, and elucidating the potential mechanisms and signaling pathways of formaldehyde toxicity during embryonic heart development, will facilitate early diagnosis and treatment of CHD.

[0004] In view of this, the present invention is proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a use of circFam120a siRNA in the preparation of a product for inhibiting cardiomyocyte apoptosis and autophagy and congenital heart disease. In the present invention, circFam120a can directly target EIF4A2 to promote formaldehyde-induced cardiomyocyte apoptosis and autophagy, thereby leading to the occurrence of congenital heart disease. Therefore, the circFam120siRNA of the present invention can inhibit cardiomyocyte apoptosis and autophagy, thereby achieving the prevention and treatment of congenital heart disease; in addition, circFam120a has significant differential expression in formaldehyde-induced animal tissues, cardiomyocytes and CHD patient plasma. Therefore, by detecting the expression level of circFam120a, auxiliary diagnosis of congenital heart disease can be achieved.

[0006] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are adopted:

[0007] A first aspect of the present invention provides a use of circFam120a siRNA in preparing a product for inhibiting cardiomyocyte apoptosis and autophagy.

[0008] Preferably, the circFam120a siRNA is used to prepare a product for inhibiting cardiomyocyte apoptosis and autophagy by targeting EIF4A2.

[0009] Preferably, the nucleotide sequence of the circFam120a siRNA is shown in SEQ ID NO: 1.

[0010] Preferably, the products include medicines and foods.

[0011] The second aspect of the present invention provides a product for inhibiting cardiomyocyte apoptosis and autophagy, comprising the circFam120a siRNA and excipients.

[0012] Preferably, the excipients include pharmaceutically acceptable excipients or food acceptable excipients.

[0013] A third aspect of the present invention provides a use of circFam120a siRNA in the preparation of a product for treating and / or preventing congenital heart disease.

[0014] Preferably, the circFam120a siRNA is used in the preparation of a product for treating and / or preventing congenital heart disease by targeting EIF4A2.

[0015] Preferably, the nucleotide sequence of the circFam120a siRNA is shown in SEQ ID NO: 1.

[0016] Preferably, the products include medicines and foods.

[0017] A fourth aspect of the present invention provides a product for treating and / or preventing congenital heart disease, characterized in that it comprises the circFam120a siRNA and excipients.

[0018] Preferably, the excipients include pharmaceutically acceptable excipients or food acceptable excipients.

[0019] A fifth aspect of the present invention provides use of a reagent for detecting circFam120a in the preparation of a product for diagnosing congenital heart disease.

[0020] Preferably, the reagent for detecting circFam120a includes a marker that recognizes circFam120a.

[0021] Preferably, the marker is a binding primer for the cDNA complementary to the circFam120a or a biomacromolecule that binds to the circFam120a;

[0022] The biomacromolecules include antibodies, antibody functional fragments, RAN binding proteins and RAN binding protein functional fragments.

[0023] The congenital heart disease in the present invention is congenital heart disease.

[0024] Compared with the prior art, the beneficial effects of the present invention include at least:

[0025] The circFam120a siRNA of the present invention can inhibit cardiomyocyte apoptosis and autophagy, thereby achieving the prevention and treatment of congenital heart disease; in addition, circFam120a has significant differential expression in formaldehyde-induced animal tissues, cardiomyocytes and CHD patient plasma. Therefore, by detecting the expression level of circFam120a, auxiliary diagnosis of congenital heart disease can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly describes the drawings required for the specific embodiments or the description of the prior art. Similar elements or parts are generally identified by similar reference numerals throughout the drawings. Elements or parts in the drawings are not necessarily drawn to scale.

[0027] Figure 1 The results of the study on the differential expression of CircFam120a in formaldehyde-induced animal tissues, cardiomyocytes and plasma of CHD patients;

[0028] Figure 2 The results of the study show that formaldehyde induces autophagy in cardiomyocytes;

[0029] Figure 3 The results show that knocking down circFam120a inhibits cardiomyocyte apoptosis and autophagy.

[0030] Figure 4 The results show that overexpression of circFam120a promotes cardiomyocyte apoptosis and autophagy;

[0031] Figure 5 To verify the direct binding experimental results of circFam120a and EIF4A2 protein;

[0032] Figure 6 This is the research result of the targeted regulation of EIF4A2 by CircFam120a;

[0033] Figure 7 The results of the study show that CircFam120a regulates formaldehyde-induced cardiomyocyte apoptosis and autophagy by targeting EIF4A2;

[0034] Figure 8 This is the research result of the animal model in the examples of the present invention, which confirmed that knocking down circFam120a in vivo alleviated formaldehyde-induced apoptosis and autophagy. DETAILED DESCRIPTION

[0035] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention and are therefore only used as examples and cannot be used to limit the scope of protection of the present invention.

[0036] It should be noted that, unless otherwise specified, the technical or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0037] The embodiments of the present invention provide a use of circFam120a siRNA in preparing a product for inhibiting cardiomyocyte apoptosis and autophagy.

[0038] Furthermore, the circFam120a siRNA is used to prepare a product for inhibiting cardiomyocyte apoptosis and autophagy by targeting EIF4A2.

[0039] In the present invention, circFam120a can directly target EIF4A2 to promote formaldehyde-induced cardiomyocyte apoptosis and autophagy, thereby leading to the occurrence of congenital heart disease. Therefore, the circFam120asiRNA of the present invention can inhibit cardiomyocyte apoptosis and autophagy, thereby achieving the prevention and treatment of congenital heart disease.

[0040] In one embodiment, the nucleotide sequence of the circFam120a siRNA is as shown in SEQ ID NO: 1, specifically CUCACUGAAGGUUGCACAG. In addition, a TT overhang can be added to the end of the above sequence to facilitate the unwinding of the double-stranded small interfering RNA after entering the cell and increase its stability.

[0041] The present invention does not limit the type of product that inhibits myocardial cell apoptosis and autophagy. For example, it can be a medicine or a food.

[0042] The embodiment of the present invention further provides a product for inhibiting cardiomyocyte apoptosis and autophagy, comprising the circFam120a siRNA and excipients.

[0043] Furthermore, the excipients include pharmaceutically acceptable excipients or food acceptable excipients.

[0044] Another embodiment of the present invention provides a use of circFam120a siRNA in preparing a product for treating and / or preventing congenital heart disease.

[0045] Furthermore, the circFam120a siRNA is used in the preparation of a product for treating and / or preventing congenital heart disease by targeting EIF4A2.

[0046] In one embodiment, the nucleotide sequence of the circFam120a siRNA is shown in SEQ ID NO: 1.

[0047] The present invention does not limit the type of product for treating and / or preventing congenital heart disease. For example, it can be a medicine or a food.

[0048] Another embodiment of the present invention provides a product for treating and / or preventing congenital heart disease, comprising the circFam120a siRNA and excipients.

[0049] Furthermore, the excipients include pharmaceutically acceptable excipients or food acceptable excipients.

[0050] In the present invention, CircFam120a is significantly differentially expressed in formaldehyde-induced animal tissues, cardiomyocytes, and plasma of CHD patients. Therefore, auxiliary diagnosis of congenital heart disease can be achieved by detecting the expression level of circFam120a.

[0051] Therefore, another embodiment of the present invention provides a use of a reagent for detecting circFam120a in the preparation of a product for diagnosing congenital heart disease.

[0052] In one embodiment, the reagent for detecting circFam120a includes a marker that recognizes circFam120a.

[0053] In one embodiment, the marker is a primer that binds to a cDNA complementary to circFam120a or a biomacromolecule that binds to circFam120a;

[0054] The biomacromolecules include antibodies, antibody functional fragments, RAN binding proteins and RAN binding protein functional fragments.

[0055] The technical solution of the present invention is further described in detail below through specific embodiments.

[0056] Example

[0057] This example is a study on the regulation of formaldehyde-induced cardiomyocyte apoptosis and autophagy by CircFam120a by targeting EIF4A2.

[0058] 1. Experimental methods

[0059] 1.1 Obtaining blood samples from CHD patients and healthy subjects

[0060] From September 2020 to September 2022, blood samples from 15 children diagnosed with CHD who visited the cardiac ultrasound department of our hospital were collected, and blood samples from 15 healthy children were used as controls. Inclusion criteria: ① diagnosed with CHD and without any treatment; ② no pulmonary function disorder; ③ no coagulation disorder; ④ no liver and kidney function abnormalities; ⑤ no other underlying diseases. Exclusion criteria: ① combined with pulmonary function disorder; ② combined with congenital immunodeficiency; ③ combined with coagulation abnormalities; ④ combined with liver and kidney function abnormalities; ⑤ combined with known hereditary diseases. Fresh blood samples were centrifuged to remove red blood cells and properly stored in liquid nitrogen for subsequent use. All blood samples were obtained with the informed consent of the patients or their legal guardians, and the informed consent form was voluntarily signed. Approved by the Ethics Committee of the Affiliated Hospital of Qingdao University.

[0061] 1.2 Construction of animal model

[0062] Sprague-Dawley (SD) rats, weighing 250–300 g, were used. Twenty females (aged: 9 weeks) and ten males (aged: 9 weeks) were housed at the SPF Animal Center of Qingdao University and maintained under a standard diet with a 12-h light and 12-h dark cycle. Female rats were randomly divided into four groups: a control group (normal saline), a formaldehyde-induced group (2.0 mg / kg), an AAV9-shNC + formaldehyde-induced group, and an AAV9-shcircFam120a + formaldehyde-induced group. Formaldehyde induction was performed starting on day 2 of housing with intraperitoneal injection of 2.0 mg / kg formaldehyde solution or normal saline once daily. On day 7 of housing, SD rats were co-housed overnight in a 2:1 ratio of male to female. The following morning, vaginal plugs were examined; the presence of a vaginal plug was counted as the first day of pregnancy. One week after co-housing, the male and female rats were separated. Pregnant rats were induced with formaldehyde until parturition. After parturition, the rats were observed for the number of offspring and the presence of stillbirths. Fetal mice underwent echocardiography, after which fetal heart tissue was collected and stored in tissue fixative or liquid nitrogen. Subsequently, relevant parameters were assessed using qRT-PCR, Western blotting, intracellular chromatin (IHC), immunofluorescence (IF), and hematoxylin and eosin staining. All animal experiments were approved by the Animal Ethics Committee of the Affiliated Hospital of Qingdao University.

[0063] 1.3 Cell culture and drug treatment

[0064] H9C2 rat cardiomyocytes were purchased from Shanghai Saibaikang Biotechnology Co., Ltd. and cultured in a standard incubator at 37°C with 5% CO2 using DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS). Cells can be passaged or processed after reaching a cell density of 95%. The formaldehyde stock solution was diluted in DMEM medium containing 10% FBS to prepare formaldehyde solutions of different concentrations: 0, 50, 100, 150, 200 μM / L, and incubated with H9C2 cells for approximately 24 hours. A formaldehyde solution with a concentration of 150 uM / L was used, and a time gradient was set: 0, 12, 24, 36, 48 hours, and incubated with H9C2 cells.

[0065] 1.4 Cell transfection

[0066] Cells were seeded into well plates and placed in an incubator until they reached 70%-90% confluency for transfection. Lipofectamine 3000 reagent was diluted with serum-free DMEM medium, mixed thoroughly, and allowed to stand for 5 minutes. A DNA premix was prepared by diluting the circFam120a DNA overexpression plasmid with serum-free DMEM medium, and then P3000 reagent was added and mixed thoroughly. The diluted DNA was added to each tube of diluted reagent at a 1:1 ratio, mixed thoroughly, and allowed to stand for 15 minutes. The DNA-liposome complex was added to the cells in the corresponding wells. The cells were incubated in a 37°C incubator for 2-4 days before subsequent analysis.

[0067] Table 1: Artificially synthesized RNA sequences for transfection are shown in the table below.

[0068]

[0069] 1.5 RNA reverse transcription and real-time fluorescence quantitative PCR

[0070] RNA from cells or tissues was extracted using the Trizol reagent method, and reverse transcription was performed using the HiScript IIIRT SuperMix for qPCR (+gDNA wiper) reagent from Novizan. qRT-PCR was then performed using the qPCR SYBR Green Master Mix reagent from Yisheng. A premix was prepared according to the manufacturer's instructions based on the number of samples. 18 μl of premix and 2 μl of cDNA were added to each well of the eight-well strip. The samples were then amplified in a qRT-PCR instrument (95°C / 30 s, annealing for 30 s, extension at 72°C / 40 s, for 40 cycles). After the reaction, the amplification and melting curves were confirmed, and the Ct values were recorded. The relative gene expression levels were calculated using the threshold cycle (Ct) method using the formula 2-Ct. PCR primers are listed in Table 2.

[0071] Table 2 PCR primer sequences

[0072]

[0073] 1.6 Western Blot (WB)

[0074] Gel Preparation: Use the YAZYME PAGE Rapid Gel Kit, select the appropriate concentration, and prepare according to the manufacturer's instructions. Electrophoresis: Prepare 1L of 1× electrophoresis buffer and add it to the electrophoresis tank. Add 30µg of protein sample to the loading well, add 5µl of protein marker to the left, and fill the remaining wells with 1× loading buffer. Perform electrophoresis at a constant voltage of 80V for 30 minutes, and then at 120V for 60 minutes. Transfer: Cut the PVDF membrane into 8×5cm sections and activate it with methanol for 30 seconds. Prepare 1L of 1× transfer buffer (containing 200ml of methanol) and pre-cool it. Place the transfer clamps in the following order from top to bottom: white plate (positive electrode) - sponge - filter paper - PVDF membrane - gel - sponge - filter paper - black plate (negative electrode) and clamp them securely. Transfer the membrane to the electrophoresis tank, add pre-cooled transfer buffer, and cool it in an ice box. Place the instrument in an ice bath, connect the electrodes, set the transfer conditions, and transfer the membrane at a constant current of 0.29A for 90 minutes. Blocking: Block the membrane in 5% skim milk (prepared in TBST) on a shaker (15 rpm) for 1 hour at room temperature, then recover the milk. Primary Antibody Incubation: Wash the membrane three times with 1× TBST buffer on a shaker (35 rpm) for 10 minutes each time, then add the primary antibody (prepared in 5% BSA) and incubate at room temperature on a shaker (15 rpm) for 2 hours or overnight at 4°C, then recover the primary antibody. Secondary Antibody Incubation: Wash the membrane three times with 1× TBST buffer on a shaker (35 rpm) for 10 minutes each time, then add the secondary antibody (prepared in 5% BSA) and incubate at room temperature on a shaker (15 rpm) for 1 hour, then recover the secondary antibody and wash the membrane three times with 1× TBST. Development: Incubate the membrane in ECL chemiluminescent solution (solution A + solution B) at room temperature in the dark for 1 minute, then place in a developer, expose using a chemiluminescent imaging system, and save the image. Image processing: ImageJ software was used for grayscale analysis, with β-Actin as the internal reference, and the ratio of the test protein to the internal reference was used to represent the relative expression level of the test protein.

[0075] 1.7 Apoptosis Assay

[0076] (1) TUNEL method: ① Using a 96-well plate, remove the treated cells from the incubator, discard the old culture medium, and wash the cells once with PBS. ② Fix the cells with 4% paraformaldehyde for 30 minutes and wash once with PBS. ③ Add PBS containing 0.3% TritonX-100 and incubate at room temperature for 5 minutes. ④ Prepare TUNEL detection solution: Add 5μl TdT enzyme and 45μl fluorescent labeling solution to each well to make 50μl detection solution. ⑤ Wash the cells twice with PBS, add the detection solution, evenly cover the cells, and incubate at 37℃ in the dark for 60 minutes. ⑥ Discard the detection solution, wash the cells three times with PBS, add DAPI and incubate for 20 minutes, and wash twice with PBS. ⑦ Observe under a fluorescence microscope and save the image.

[0077] (2) Cell flow cytometry: ① Using a six-well plate, remove the treated cells from the incubator, discard the old culture medium, wash the cells once with pre-cooled PBS, add an appropriate amount of EDTA-free trypsin to digest the cells at room temperature for about 1 minute, and discard the trypsin. ② Add 1 ml of culture medium to gently blow down the cells, transfer them to a 1.5 ml eppendorf tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, collect the cells, wash the cells twice with PBS, resuspend and count. ③ Take 5×104-1×105 resuspended cells, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 100 μl 1×Annexin V binding buffer and gently resuspend the cells. ④ Add 5 μl Annexin V-APC and mix gently. Add 5 μl PI and mix gently. ⑤ Incubate at room temperature in the dark for 10-15 minutes. ⑥ Flow cytometric analysis: After incubation, add 400 μl of 1× Annexin V binding buffer to resuspend the cells and analyze within 1 hour. Annexin V-APC is excited at 633 nm and the fluorescence emission spectrum is detected at 660 nm. PI is excited at 488 nm and the emission spectrum is detected at approximately 617 nm.

[0078] 1.8 RNA pull-down experiment

[0079] (1) Bead preparation: ① Use streptavidin agarose beads, mix the beads, prepare two 1.5ml centrifuge tubes, mark them as NC group and target group, aspirate 40μl beads for each group, centrifuge (1000g, 4℃, 5min), and discard the supernatant. ② Add 1ml lysis buffer to each group to wash the beads, centrifuge (1000g, 4℃, 5min), and discard the supernatant. ③ Add 1ml blocking buffer to each group and rotate at 4℃ for 1-3h, centrifuge (1000g, 4℃, 5min), and discard the supernatant. Wash each group three times with lysis buffer, centrifuge (1000g, 4℃, 5min), and discard the supernatant. Add 40μl lysis buffer to each group.

[0080] (2) Bead-probe binding: NC and target biotin probe were added to corresponding centrifuge tubes respectively, and incubated with rotation at 4°C for 2-3 h to allow the beads to fully bind to the probe.

[0081] (3) Probe binding to target protein: 100 μl of the previously prepared protein sample was taken as input, and the remaining protein sample was evenly added to the corresponding centrifuge tubes of the NC group and the target group, and incubated with rotation at 4°C overnight.

[0082] (4) Washing: The next day, the samples were centrifuged (1000 g, 4 ° C, 5 min), the supernatant was discarded, and the beads-probe-protein complex was washed 3 times with 1 ml high-salt buffer for each group to remove unbound proteins.

[0083] (5) Elution: After centrifugation (1000 g, 4 °C, 5 min), 36 μl of low-salt buffer was added to each group to elute the bound proteins, and the protein eluate was collected after centrifugation.

[0084] (6) Add 5× protein loading buffer to the protein eluate at a 1 / 4 volume ratio, denature in a metal bath at 95°C for 15 min, and separate by electrophoresis on a polyacrylamide gel. Perform subsequent mass spectrometry analysis or target protein detection.

[0085] 1.9 RNA-binding protein immunoprecipitation (RIP)

[0086] (1) Magnetic bead pretreatment: Prepare two 1.5 ml centrifuge tubes, labeled as IgG group and target protein group respectively. Thoroughly suspend the magnetic beads, take 30 μl of magnetic beads from each tube, place them on a magnetic rack, magnetically separate them, aspirate and discard the magnetic bead protection solution, add 400 μl binding / washing buffer, thoroughly suspend and wash the magnetic beads, place them on a magnetic rack, magnetically separate them, aspirate and discard the supernatant; repeat the above washing steps twice.

[0087] (2) Binding of antibodies to magnetic beads: ① Antibody pretreatment: Use binding / washing buffer to dilute the antibody to a final concentration of 25 μg / ml. ② Antibody-magnetic bead binding: Add 400 μl of diluted antibody to the pretreated magnetic beads, fully suspend, place in a flip mixer for incubation (4°C, 2h), magnetically separate, collect the magnetic beads, and collect the supernatant in a new EP tube for subsequent use. ③ Washing: Add 500 μl of binding / washing buffer, fully suspend the magnetic beads, magnetically separate, and discard the supernatant; repeat washing 4 times.

[0088] (3) Antigen-antibody-magnetic bead complex binding: ① Antigen-antibody-magnetic bead complex binding: Add 500 μl of the previously prepared protein sample, thoroughly suspend, incubate on a rotary mixer (4°C, 8 h), magnetically separate, and discard the supernatant. ② Washing: Use 500 μl of binding / wash buffer to thoroughly resuspend the magnetic beads, magnetically separate, and discard the supernatant; repeat washing 4 times.

[0089] (4) Extraction of protein-bound RNA: ① Resuspend the magnetic beads with 100 μl of binding / washing buffer, add 30 μg of proteinase K, mix thoroughly, and place in a 55°C metal bath for 30 min (tap the centrifuge tube with your fingertips during this period) to separate the protein from the magnetic beads, perform magnetic separation, and collect the supernatant. ② Take 100 μl of the previously prepared protein sample as the input group, and use the trizol method to extract RNA from the input, IgG, and target groups, respectively. The method is the same as that for cell RNA extraction. In the isopropanol step, 5 μl of glycogen should be added and incubated at -20°C overnight to help RNA precipitation.

[0090] (5) Agarose gel electrophoresis detection: ① The extracted RNA was subjected to conventional reverse transcription, followed by PCR amplification. The amplified products were placed on ice for later use. The preparation system and reaction conditions are shown in Table 6. ② Agarose gel preparation: Weigh 0.9g agarose, add it to 1×TAE, mix well, and heat it in a microwave oven to 100°C. Then add 5μl nucleic acid dye to prepare 1.8% agarose gel. ③ Electrophoresis: Place the prepared gel in an electrophoresis tank containing 1×TAE, add 5μl of marker and each group of amplified products to the sample well in sequence, 120V, and electrophoresis for 30 minutes. ④ Use ultraviolet light in the developer to develop and save the image.

[0091] 1.10 Fluorescence in situ hybridization (FISH) of cells or tissues

[0092] Use 48-well plate, put in the slide, according to 1×10 4H9C2 cells were seeded into 48-well plates at a density of cells / well and cultured overnight in an incubator; the culture medium was aspirated and washed twice with PBS, each for 5 minutes; the PBS was aspirated and 100 μl of 4% paraformaldehyde was added to each well and fixed at room temperature for 15 minutes; the 4% paraformaldehyde was aspirated and 100 μl of 0.1% TritonX-100 was added to each well and treated at room temperature for 15 minutes; the 0.1% TritonX-100 was aspirated and washed twice with PBS, each for 5 minutes; the PBS was aspirated and 100 μl of 0.1% TritonX-100 was added to each well. 2×SSC, placed in a 37°C incubator for 30 minutes; hybridization buffer was incubated in a 73°C water bath in advance for 30 minutes until clear and translucent; probe dilution: prepared to a concentration of 100μM according to the instructions; 100μl of probe mixture was prepared with hybridization buffer, concentration of 5μM, and denatured at 73°C for 5 minutes; 2×SSC was discarded, 100μl of denatured probe mixture was added to each well, and after taking measures to protect from light, it was placed in a 37°C incubator for hybridization overnight; the next day of hybridization, the sample was removed from the 37°C incubator, the probe mixture was discarded, and 100μL of 42°C preheated 0.1% Tween 20 was added to each well for washing for 5 minutes; 0.1% Tween 20 was discarded, and 100μl of 42°C preheated 2×SSC was added to each well for washing for 5 minutes; 2×SSC was discarded, and 100μl of Wash in 42°C preheated 1× SSC for 5 min, then discard the wash solution. Remove the slide from the well plate, let it dry, and add 3 μl of mounting medium containing DAPI to the slide. Attach the slide to the mounting medium to complete the sealing process. Observe under a laser confocal microscope.

[0093] 1.11 LC3 autophagy double-labeled adenovirus detection of autophagy

[0094] H9C2 cells were seeded into confocal dishes and placed in a cell culture incubator overnight. When the cell density reached approximately 30-50%, the LC3 autophagy double-labeled adenovirus was transfected into the cells. 10 μl of diluted virus was added to each dish. After placing the dish in the incubator for 12 hours, the old virus-containing culture medium was removed for subsequent operations. After processing, the dish was observed under a laser confocal microscope.

[0095] 1.12 Reactive oxygen species (ROS) detection

[0096] ① Probe loading: Dilute DCFH-DA at a ratio of 1:1000 with serum-free culture medium to a final concentration of 10 μM / L. Using a six-well plate, remove the treated cells from the incubator, discard the old culture medium, and add 1 ml of diluted DCFH-DA. Incubate in a 37°C cell culture incubator for 30 minutes. Wash the cells three times with serum-free DMEM to fully remove any DCFH-DA that has not entered the cells. Set up a positive control well and stimulate the cells with a positive reactive oxygen species control for 30 minutes. ② Observe and record using a fluorescence microscope.

[0097] 2. Statistical methods

[0098] Data for each experiment are presented as the mean ± standard error of the mean (SEM) of three independent replicates. Student's t-test was used for comparisons between two independent groups, and one-way ANOVA was used for comparisons between multiple groups. GraphPad Prism 8.0 software was used for statistical analysis and graphing. Statistical significance was considered to be established when the P value was < 0.05.

[0099] 3. Experimental results

[0100] 3.1 Study on the differential expression of CircFam120a in formaldehyde-induced animal tissues, cardiomyocytes, and plasma of CHD patients

[0101] Through the previously constructed formaldehyde-exposed rat model, the whole transcriptome sequencing was performed with the healthy group as the control, from which several circRNAs with the most significant differential expression were screened. By constructing a formaldehyde-induced H9C2 cell model, Qrt-PCR was used to verify the expression trend of circRNA, and circFam120a with the most obvious trend was screened out. In the sequencing results, circFam120a was upregulated in the formaldehyde-exposed group. By verifying in the formaldehyde-induced cell model, it was found that circFam120a showed a gradual upregulation trend in the cell model treated with formaldehyde concentration gradient for 24 hours, and the upregulation was most significant at a concentration of 150μM (P<0.05) ( Figure 1 Subsequently, H9C2 cells were treated with 150 μM formaldehyde solution for a time gradient, and the expression level of circFam120a was verified by QRT-PCR. It was found that the expression level of circFam120a gradually increased with the extension of treatment time (P<0.05) ( Figure 1 The expression of circFam120a was also verified in animal tissues. Consistent with the sequencing results, the expression of circFam120a was increased in the formaldehyde exposure group (P<0.05) ( Figure 1 C). Blood samples from 15 patients with congenital heart disease were collected clinically, and 15 blood samples from healthy controls were used to detect the expression of circFam120a. It was found that the expression of circFam120a in the blood samples of CHD patients was significantly higher than that in normal controls (P<0.05) ( Figure 1 In addition, qRT-PCR was used to detect the expression of circFam120a in various organs and tissues of adult rats and suckling mice, and it was found that its expression level in heart tissue was the most significant (P<0.05) ( Figure 1In addition, to eliminate the interference of fibroblasts in cardiac tissue, RNA was extracted from cardiac fibroblasts (CF) and cardiomyocytes (CM), and the expression of circFam120a was detected by qRT-PCR. It was found that its expression in cardiomyocytes was significantly higher than that in fibroblasts (P<0.05) ( Figure 1 F). We conducted a conservation analysis of circFam120a in humans and rats. By sequence alignment, we found that circFam120a is conserved in humans and rats ( Figure 1 Fluorescence in situ hybridization (FISH) revealed that circFam120a was expressed in the nucleus and cytoplasm of H9C2 cells ( Figure 1 Middle H).

[0102] Figure 1 These are the results of a study on the differential expression of CircFam120a in formaldehyde-induced animal tissues, cardiomyocytes, and plasma of CHD patients. Figure 1 Figure 2: A: qRT-PCR detection of circFam120a expression in H9C2 cells treated with formaldehyde concentration gradients. B: qRT-PCR detection of circFam120a expression in H9C2 cells treated with formaldehyde time gradients. C: qRT-PCR detection of circFam120a expression in rat fetal heart tissue. D: qRT-PCR detection of circFam120a expression in blood of CHD patients and healthy controls (n=15). E: qRT-PCR detection of circFam120a expression in skin, muscle, heart, brain, liver, kidney, and colon of adult and fetal rats. F: qRT-PCR detection of circFam120a expression in rat cardiac fibroblasts and cardiomyocytes. G: Conservation analysis of circFam120a in humans and rats. H: FISH detection of circFam120a localization in H9C2 cells. ns: not significantly different; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0103] Depend on Figure 1 It can be seen that circFam120a plays a certain regulatory role under formaldehyde induction.

[0104] 3.2 Study on the occurrence of formaldehyde-induced cardiomyocyte autophagy

[0105] H9C2 cells were treated with formaldehyde concentration gradient for 24 h, and it was found that the number of cells decreased significantly with the increase of formaldehyde concentration ( Figure 2Previous studies have shown that formaldehyde can induce apoptosis in H9C2 cells. There is also some crosstalk between apoptosis and autophagy, so we considered whether formaldehyde could induce autophagy in H9C2 cells. Western blot was used to detect the expression of autophagy-related proteins LC3Ⅰ / Ⅱ and p62 in formaldehyde-treated H9C2 cells. It was found that with increasing formaldehyde concentration, LC3Ⅱ expression levels increased significantly, while p62 expression levels decreased significantly, indicating the occurrence of autophagy ( Figure 2 Middle B). H9C2 cells were treated with 150 μM formaldehyde, and autophagy double-labeled adenovirus assay also showed that the autophagy level increased after formaldehyde treatment (P<0.05) ( Figure 2 Middle C).

[0106] Figure 2 These are the research results on the occurrence of formaldehyde-induced autophagy in cardiomyocytes. Figure 2 Middle, A: H9C2 cells treated with formaldehyde gradients. Scale bar: 200 μm. B: Expression of autophagy-related proteins in H9C2 cells treated with formaldehyde gradients. C: Autophagy levels in H9C2 cells detected by autophagy dual-labeling adenovirus. ****P < 0.0001.

[0107] Depend on Figure 2 It can be seen that formaldehyde can induce autophagy in H9C2 cells.

[0108] 3.3 Knockdown of circFam120a inhibits cardiomyocyte apoptosis and autophagy

[0109] To verify the function of circFam120a, its small interfering RNA (sequence shown in SEQ ID NO: 1) was designed and transfected into H9C2 cells to knock down circFam120a. The knockdown efficiency of circFam120a was verified by QRT-PCR, and its expression level was found to be significantly reduced (P < 0.05) ( Figure 3 A, left), while knockdown of circFam120a had no significant effect on the expression level of its parent gene Fam120a ( Figure 3 To observe whether formaldehyde stimulation would lead to oxidative stress in cells, cells were stimulated with 150 μM / L formaldehyde solution for 24 h, and the level of reactive oxygen species (ROS) in the cells was detected. It was found that the level of ROS in cells increased after formaldehyde stimulation, while the level of ROS in cells decreased after knockdown of circFam120a ( Figure 3Middle B). Subsequently, the expression levels of intracellular apoptosis and autophagy-related factors were detected by WB. It was found that after formaldehyde stimulation, the expression levels of cleaved caspase3 and LC3Ⅱ increased, and the expression level of p62 decreased, indicating the occurrence of apoptosis and autophagy. Knockdown of circFam120a alleviated this phenomenon, and the levels of apoptosis and autophagy decreased ( Figure 3 Tunel and flow cytometry experiments also showed the same results. The level of cell apoptosis increased after formaldehyde stimulation, and apoptosis was alleviated after knockdown of circFam120a ( Figure 3 At the same time, the autophagy level detected by autophagy double-labeled adenovirus also found that knocking down circFam120a alleviated the autophagy phenomenon induced by formaldehyde stimulation ( Figure 3 Middle F).

[0110] Figure 3 The results show that knocking down circFam120a inhibits cardiomyocyte apoptosis and autophagy. Figure 3 Figure 2: A: qRT-PCR analysis of circFam120a knockdown efficiency and the effect on Fam120a expression. B: Detection of ROS levels in cells. C: Western blot analysis of the expression levels of apoptosis- and autophagy-related proteins in cells. D: Tunel analysis of cell apoptosis (Tunel in green, DAPI in blue). E: Flow cytometry analysis of cell apoptosis. F: Autophagy double-labeled adenovirus assay to detect cell autophagy levels. ns indicates no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

[0111] Depend on Figure 3 It can be seen that knocking down circFam120a can reduce the occurrence of cell apoptosis and autophagy.

[0112] 3.4 Overexpression of circFam120a promotes cardiomyocyte apoptosis and autophagy

[0113] Then, we constructed a circFam120a overexpression plasmid and transfected it into H9C2 cells. Qrt-PCR was used to verify the overexpression efficiency of circFam120a, and found that its expression level was significantly increased (P<0.05) ( Figure 4 A, left), while overexpression of circFam120a had no significant effect on the expression level of its parent gene Fam120a ( Figure 4 ROS detection showed that overexpression of circFam120a further increased the intracellular ROS level induced by formaldehyde stimulation, indicating that circFam120a can aggravate the intracellular oxidative stress level ( Figure 4Then, the expression levels of intracellular apoptosis and autophagy-related factors were detected by Western blotting. It was found that overexpression of circFam120a further increased the intracellular apoptosis and autophagy levels induced by formaldehyde stimulation, as shown by the further increase in the expression levels of cleaved caspase3 and LC3Ⅱ and the further decrease in the expression level of p62 ( Figure 4 Tunel and flow cytometry experiments also showed the same results. Overexpression of circFam120a further aggravated the apoptosis induced by formaldehyde stimulation ( Figure 4 At the same time, autophagy double-labeled adenovirus detection also found that overexpression of circFam120a further aggravated the level of cellular autophagy induced by formaldehyde stimulation ( Figure 4 Middle F).

[0114] Figure 4 The results show that overexpression of circFam120a promotes cardiomyocyte apoptosis and autophagy. Figure 4 A: qRT-PCR analysis of circFam120a and Fam120a expression levels after transfection with a circFam120a overexpression plasmid. B: Detection of intracellular ROS levels. C: Western blotting analysis of apoptosis- and autophagy-related protein expression levels. D: Tunel analysis of intracellular apoptosis levels (Tunel in green, DAPI in blue). E: Flow cytometry analysis of apoptosis levels. ns indicates no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0115] Depend on Figure 4 It can be seen that overexpression of circFam120a can cause cell apoptosis and autophagy.

[0116] 3.5 Verification of direct binding of circFam120a to EIF4A2 protein

[0117] To explore whether circFam120a exerts its regulatory effect by directly binding to proteins, we constructed and used a biotin probe for circFam120a to perform RNA pull-down experiments and mass spectrometry analysis ( Figure 5 The results showed that EIF4A2 was one of the most abundant proteins that bound to circFam120a. Combining bioinformatics prediction analysis with existing literature on EIF4A2, we speculated that EIF4A2 is a potential downstream target of circFam120a. CircFam120a RNA pull-down experiments further confirmed that circFam120a can bind to EIF4A2 ( Figure 5C). RIP experiments also confirmed that EIF4A2 can bind to circFam120a ( Figure 5 This indicates that circFam120a can directly target EIF4A2. FISH experiments were used to detect the co-localization of circFam120a and EIF4A2, and the results showed that circFam120a and EIF4A2 were expressed in both the nucleus and cytoplasm of H9C2 cells ( Figure 5 Middle E).

[0118] Figure 5 To verify the direct binding experimental results of circFam120a and EIF4A2 protein. Figure 5 A: RNA pull-down assay, followed by PAGE gel electrophoresis and Coomassie blue staining to determine RNA-binding proteins. B: RNA pull-down assay analysis of RNA-binding proteins. C: RNA pull-down assay in which EIF4A2 was pulled down by a biotinylated probe targeting circFam120a, and Western blotting verified their binding. D: Reverse immunoprecipitation (RIP) assay to verify the binding of circFam120a to EIF4A2. E: Colocalization of circFam120a and EIF4A2 in H9C2 cells (circFam120a in red, EIF4A2 in green, and DAPI in blue). Scale bar: 10 μm.

[0119] Depend on Figure 5 It can be seen that circFam120a can directly bind to EIF4A2 and may play a regulatory role by directly targeting EIF4A2.

[0120] 3.6 Study on the targeted regulatory effect of CircFam120a on EIF4A2

[0121] H9C2 cells were stimulated with formaldehyde concentration gradients. WB results showed that formaldehyde stimulation reduced the expression level of EIF4A2, and the expression level gradually decreased with the increase of formaldehyde concentration ( Figure 6 In addition, H9C2 cells were stimulated with formaldehyde at a concentration of 150 μM / L, and samples were collected at different time points according to the time gradient. The results of WB experiments showed that the expression level of EIF4A2 gradually decreased with the extension of formaldehyde stimulation time ( Figure 6 Middle B). Under physiological conditions, H9C2 cells were transfected with circFam120a small interfering RNA. WB results showed that the expression level of EIF4A2 was significantly increased after knockdown of circFam120a compared with the control group ( Figure 6Middle C); H9C2 cells were transfected with circFam120a overexpression plasmid, and WB results showed that the expression level of EIF4A2 after overexpression of circFam120a was significantly decreased compared with the control group ( Figure 6 Then, H9C2 cells were transfected with circFam120a small interfering RNA and overexpression plasmids, respectively, and then stimulated with formaldehyde. Under pathological conditions, WB was used to detect the expression of EIF4A2. It was also found that knocking down circFam120a could increase the expression level of EIF4A2 ( Figure 6 Middle E), overexpression of circFam120a can reduce the expression level of EIF4A2 ( Figure 6 Middle F).

[0122] Figure 6 These are the research results on the targeted regulatory effect of CircFam120a on EIF4A2. Figure 7 A: EIF4A2 expression levels in H9C2 cells after formaldehyde concentration gradient treatment. B: EIF4A2 expression levels in H9C2 cells after 150 μM / ml formaldehyde time gradient treatment. C: EIF4A2 expression levels after circFam120a knockdown under physiological conditions. D: EIF4A2 expression levels after circFam120a overexpression under physiological conditions. E: EIF4A2 expression levels after circFam120a knockdown under pathological conditions. F: EIF4A2 expression levels after circFam120a overexpression under pathological conditions. ns indicates no significant difference; *P < 0.05; ***P < 0.001.

[0123] Depend on Figure 6 It can be seen that circFam120a has a targeted regulatory effect on EIF4A2.

[0124] 3.7 CircFam120a regulates formaldehyde-induced cardiomyocyte apoptosis and autophagy by targeting EIF4A2

[0125] To investigate the mutual regulation between circFam120a and EIF4A2, small interfering RNA of EIF4A2 was constructed and transfected into H9C2 cells to knock down EIF4A2. qRT-PCR and WB results showed that the expression level of EIF4A2 was decreased (P<0.05) ( Figure 7A and B). CircFam120a small interfering RNA or overexpression plasmid was co-transfected with EIF4A2 small interfering RNA and stimulated with formaldehyde. Western blotting was used to detect the expression levels of apoptosis- and autophagy-related proteins. The results showed that circFam120a knockdown partially reversed the apoptosis and autophagy caused by EIF4A2 knockdown, as shown by decreased expression levels of cleaved caspase3 and LC3Ⅱ and increased expression level of p62 (P<0.05) ( Figure 7 Middle C); Overexpression of CircFam120a further aggravated the apoptosis and autophagy caused by EIF4A2 knockdown, as shown by increased expression levels of cleaved caspase3 and LC3Ⅱ and decreased expression level of p62 (P<0.05) ( Figure 7 To further verify the occurrence of apoptosis, Tunel and flow cytometry were used to detect cell apoptosis under formaldehyde stimulation after co-transfection of circFam120a small interfering RNA or overexpression plasmid and EIF4A2 small interfering RNA. The results also showed that circFam120a knockdown partially reversed the apoptosis caused by EIF4A2 knockdown ( Figure 7 E and G), overexpression of circFam120a further aggravated the apoptosis induced by EIF4A2 knockdown ( Figure 7 To further verify the occurrence of autophagy, H9C2 cells were transfected with autophagy double-labeled adenovirus, and then co-transfected with circFam120a small interfering RNA or overexpression plasmid and EIF4A2 small interfering RNA. The cell autophagy was detected under formaldehyde stimulation. The results showed that circFam120a knockdown partially reversed the autophagy caused by EIF4A2 knockdown ( Figure 7 Overexpression of circFam120a further aggravated the autophagy induced by EIF4A2 knockdown ( Figure 8 (J).

[0126] Figure 7 These are the research results showing that CircFam120a regulates formaldehyde-induced cardiomyocyte apoptosis and autophagy by targeting EIF4A2. Figure 7 A: qRT-PCR detection of EIF4A2 knockdown efficiency. B: Western blotting detection of EIF4A2 knockdown efficiency. C and D: Western blotting detection of autophagy and apoptosis-related protein expression in each group. E and F: Flow cytometry detection of apoptosis in each group. G and H: Tunel detection of apoptosis in each group. I and J: Autophagy double-labeled adeno-associated virus detection of autophagy levels in each group. ns indicates no significant difference; *P < 0.05; ***P < 0.001.

[0127] Depend on Figure 7It can be seen that circFam120a can target EIF4A2 to regulate cardiomyocyte apoptosis and autophagy.

[0128] 3.8 Animal Model Confirms that circFam120a Knockdown in Vivo Alleviates Formaldehyde-Induced Apoptosis and Autophagy

[0129] Previously, we studied the regulatory effect of circFam120a on EIF4A2 in H9C2 cells under formaldehyde stimulation. In order to further confirm the above results in animals, we constructed animal models of formaldehyde-induced disease group and circFam120a knockdown treatment group. According to the results of previous studies, 0.2 mg / kg formaldehyde concentration was selected as the pathological condition to induce rats. Adeno-associated virus with circFam120a knockdown was constructed and injected into the tail vein of rats as the treatment group. Echocardiography was performed on the newborn rats in each group, and it was found that the offspring of rats stimulated by formaldehyde had abnormal cardiac function, which was manifested as lower EF% and FS% than the healthy group, and a slower heart rate. The offspring of rats treated with circFam120a knockdown had normal cardiac function and heart rate ( Figure 8 The hearts of neonatal rats in each group were collected and the expression level of circFam120a was detected by qRT-PCR. The results showed that formaldehyde stimulation increased the expression level of circFam120a, while after knocking down circFam120a, its expression level was significantly reduced ( Figure 8 Middle E). Hematoxylin staining (HE) results showed that the offspring of rats stimulated by formaldehyde had abnormal cardiac structure, manifested as loose and disordered arrangement of cardiomyocytes, and some had defects in the ventricular septum. However, the offspring of rats treated with circFam120a knockdown showed no obvious abnormalities in cardiac structure ( Figure 8 To verify the regulatory effect of circFam120a on EIF4A2, the expression levels of EIF4A2 in the hearts of neonatal rats in each group were detected by Western blotting. It was found that the expression level of EIF4A2 decreased after formaldehyde stimulation, while the expression level of EIF4A2 increased after knockdown of circFam120a ( Figure 8 In order to observe the apoptosis and autophagy levels in the hearts of neonatal rats in each group, WB was used to detect the expression levels of apoptosis- and autophagy-related proteins in the hearts of neonatal rats in each group. It was found that formaldehyde induction increased the expression of cleaved caspase3 and LC3Ⅱ and decreased the expression of p62. Knockdown of circFam120a reversed this phenomenon ( Figure 8 Middle G).

[0130] Figure 8 The results of the study confirmed that knockdown of circFam120a in vivo alleviated formaldehyde-induced apoptosis and autophagy in animal models. Figure 8Figures A-D: Representative echocardiograms of neonatal rats in each group. E: QRT-PCR analysis of circFam120a expression in each group. F: HE staining of cardiac sections from neonatal rats in each group. G: Western blot analysis of the expression levels of apoptosis- and autophagy-related proteins in the hearts of neonatal rats in each group. ns indicates no significant difference; *P < 0.05; ***P < 0.001.

[0131] These results are consistent with those from cell-based experiments, showing that certain concentrations of formaldehyde induce cardiomyocyte apoptosis and autophagy. Knockdown of CircFam120a can partially reverse this phenomenon.

[0132] The above experiments of the present invention are sufficient to prove that circFam120a siRNA can inhibit cardiomyocyte apoptosis and autophagy, thereby achieving the prevention and treatment of congenital heart disease.

[0133] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or make equivalent replacements for some or all of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. Use of circFam120a siRNA in the preparation of a medicament for treating and / or preventing congenital heart disease, wherein the nucleotide sequence of the circFam120a siRNA is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that The circFam120a siRNA treats and / or prevents congenital heart disease by inhibiting cardiomyocyte apoptosis and autophagy by targeting EIF4A2.

3. The use according to claim 1, characterized in that The circFam120a siRNA is used in the preparation of medicines for treating and / or preventing congenital heart disease by targeting EIF4A2.

4. A drug for treating and / or preventing congenital heart disease, characterized in that: Comprising the circFam120a siRNA according to claim 1 and excipients.

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