CN115770296A - Application of circEYA3 in preparation of products for diagnosing/treating congenital heart disease - Google Patents
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Abstract
The invention discloses an application of circEYA3 in preparation of a product for diagnosing/treating congenital heart disease, wherein the circEYA3 regulates and controls the expression of NLRP3, GSDMD, ASC, clear-caspase-1 and clear-IL-1 beta by directly combining with downstream protein Smad5 so as to regulate myocardial cell apoptosis; in addition, the differences of the expression of the circEYA3 in congenital heart disease patients and healthy samples are obvious, and the auxiliary diagnosis of the congenital heart diseases can be accurately, quickly and simply realized by detecting the expression quantity of the circEYA 3.
Description
Technical Field
The invention relates to the technical field of biology, in particular to application of circEYA3 in preparation of a product for diagnosing/treating congenital heart disease.
Background
Congenital Heart Disease (CHD) is a cardiac abnormality caused by abnormal development of the heart and blood vessels during the development of a human fetus. CHD is the leading cause of birth defects. CHD is a serious health hazard in children and is one of the important factors leading to neonatal death and adult disability. Therefore, early diagnosis and treatment of CHD are very important. Most researchers now believe that the pathogenesis of CHD is primarily a result of genetic and environmental interactions.
As environmental pollution caused by human activities increases, the influence of Formaldehyde (FA) on the development of embryo systems is attracting attention. In addition to being present in the air, formaldehyde is also used in manufacturing industries such as construction, furniture, cigarettes, and textiles. Formaldehyde can be ingested through the gastrointestinal tract of the mother, absorbed by the skin, or inhaled into the respiratory tract, and then transmitted to the fetus through the placental circulation. It has been found that the sensitivity of the embryo to formaldehyde and its removal of formaldehyde is slower than that of the mother, and therefore formaldehyde is continuously accumulated in fetal organs. Notably, fetal congenital abnormalities in the general population may be associated with non-professional exposure of pregnant women to paint and smoking during the first trimester of pregnancy. Furthermore, exposure of pregnant women to organic dyes, glues, paints or other indoor environmental contaminants increases the risk of coronary heart disease, such as a conical septal defect. Residential environment formaldehyde exposure (>2.42microg/m 3 ) Increases the risk of congenital heart malformations by 24% (OR =1.24;95% CI 0.81-2.07). Therefore, besides the imaging early diagnosis of CHD, understanding the molecular genetic mechanism of CHD, and clarifying the toxicity mechanism of formaldehyde to embryonic cardiac development and the regulation of signal pathways thereof have important significance for the early diagnosis of CHD.
Apoptosis (apoptosis) is a form of programmed death that has been newly discovered and validated in recent years. NLRP3 (nucleotide binding oligo-like receptor family 3) is a key factor for apoptosis and interacts with ASC (apoptosis-related speckled protein containing caspase recruiting segments) to recruit caspase-1 to form inflammasome. The inflammatory corpuscle can activate Caspase-1, and the activated Caspase-1 not only cleaves pro-IL-1 beta and pro-IL-18 to form mature IL-1 beta and IL-18, but also cleaves GSDMD to enable the N end of the GSDMD to be positioned and aggregated on a cell membrane to form a pore, and intracellular contents are secreted out of the membrane through the pore. Cell apoptosis can disrupt plasma membrane integrity and the normal osmotic barrier leading to rapid cell lysis and release of inflammatory substances. Researches show that the cell scorching is widely existed in the occurrence and development processes of cardiovascular diseases, endocrine diseases and other diseases. Therefore, the research on the apoptosis of the cells also becomes a new breakthrough point for explaining the pathogenesis of various diseases.
Recently, circular RNA (circular RNA) is found abundantly in mammals. CircRNA is a novel non-coding RNA (ncRNA) that is highly enriched in cardiac tissue. The circRNAs play an important role in the development process of cardiovascular diseases and other diseases. The CircRNA is a novel endogenous ncRNA (non-coding RNA), is highly conserved, has a covalent closed-loop structure and does not have a 5 'end cap and a 3' end polyA tail, and therefore is more stable. Compared with other ncrnas, the functional mechanisms of circrnas include isolation of microRNAs (miRNAs), direct interaction with proteins, transcriptional regulation, and translation. With the gradual understanding of the structure and function of circRNA, the role of circRNA in the development process is also gradually emphasized. However, the mechanism of action of circRNA during cardiac development under formaldehyde exposure has not been reported.
Disclosure of Invention
The invention aims to provide application of circEYA3 in preparation of a product for diagnosing/treating congenital heart diseases, wherein the expression difference of the circEYA3 in congenital heart disease patients and healthy samples is obvious, and the auxiliary diagnosis of the congenital heart diseases can be accurately, quickly and simply realized by detecting the expression quantity of the circEYA 3.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention provides application of circEYA3 in preparation of a product for regulating myocardial cell scorch.
Preferably, the circEYA3 is applied to preparation of a product for regulating myocardial cell apoptosis by targeting Smad 5.
Preferably, the inhibitor of circEYA3 is applied to preparation of products for inhibiting myocardial cell apoptosis, and the nucleotide sequence of the inhibitor is shown in SEQ ID NO. 2 and SEQ ID NO. 3.
In a second aspect, the invention provides a product for inhibiting myocardial cell apoptosis, which comprises an inhibitor of circEYA3, wherein the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
The third aspect of the invention provides an application of circEYA3 in preparing a product for diagnosing/treating congenital heart diseases.
Preferably, the inhibitor of the circEYA3 is applied to preparing the treatment product of congenital heart disease, and the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
In a fourth aspect, the present invention provides a product for diagnosing congenital heart disease, comprising a marker identifying circEYA3 as described above.
Preferably, the label is a binding primer for the cDNA complementary to the circEYA3 or a biological macromolecule that binds to the circEYA 3;
the biomacromolecule includes antibodies, antibody functional fragments, RAN binding proteins and RAN binding protein functional fragments.
Preferably, the nucleotide sequence of the binding primer of the cDNA complementary to circEYA3 is shown as SEQ ID NO. 4 and SEQ ID NO. 5.
In a fifth aspect, the invention provides a medicament for treating congenital heart disease, which comprises a therapeutically effective amount of an inhibitor of circEYA3, the nucleotide sequence of which is shown as SEQ ID No. 2 and SEQ ID No. 3.
Compared with the prior art, the invention has the beneficial effects that at least:
the expression difference of the circEYA3 in congenital heart disease patients and healthy samples is obvious, and the auxiliary diagnosis of the congenital heart disease can be accurately, quickly and simply realized by detecting the expression quantity of the circEYA 3.
According to the invention, the circEYA3 directly combines with downstream protein Smad5 to regulate the expression of NLRP3, GSDMD, ASC, cleared-caspase-1 and cleared-IL-1 beta, so as to regulate myocardial cell apoptosis. In addition, myocardial cell apoptosis can be inhibited by knocking down circEYA3, the occurrence of formaldehyde-induced cardiac dysplasia is improved, and a potential therapeutic target is provided for diagnosis and treatment of CHD.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings used in the detailed description or the prior art description will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
FIG. 1 is a graph showing the effect of formaldehyde on circEYA3 in animal models, cardiomyocytes, and plasma in CHD patients in example 1 of the present invention.
FIG. 2 shows the result of the identification of the cyclic structure of circEYA3 in example 2 of the present invention.
FIG. 3 shows the results of the study of the occurrence of apoptosis of cells in heart of fetal rat induced by formaldehyde in example 3 of the present invention.
FIG. 4 shows the results of the study of the occurrence of formaldehyde-induced cardiomyocyte apoptosis in example 4 of the present invention.
FIG. 5 shows the results of the study of the inhibition of myocardial cell apoptosis by circEYA3 knock-down in example 5 of the present invention;
FIG. 6 shows the results of the study of the overexpression of circEYA3 to promote the occurrence of myocardial apoptosis in example 6 of the present invention.
FIG. 7 is a result of verifying direct binding of circEYA3 and Smad5 in example 7 of the present invention;
FIG. 8 is a result of investigation of the promotion of cardiomyocyte apoptosis by circEYA3 through the inhibition of Smad5 in example 8 of the present invention.
Detailed Description
The following describes embodiments of the present invention in detail with reference to the following embodiments. The following examples are only for illustrating the technical solutions of the present invention more clearly, and therefore are only used as examples, and the protection scope of the present invention is not limited thereby.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. The meaning and scope of a term should be clear, however, in the event of any potential ambiguity, the definition provided herein takes precedence over any dictionary or extrinsic definition. In this application, unless otherwise indicated, the use of the term "including" and other forms is not limiting.
Generally, the nomenclature used, and the techniques thereof, in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. Enzymatic reactions and purification techniques are performed according to the manufacturer's instructions, as commonly practiced in the art, or as described herein. The nomenclature used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, and the laboratory procedures and techniques thereof, are those well known and commonly employed in the art.
The embodiment of the invention provides an application of circEYA3 in preparation of a product for regulating myocardial cell apoptosis, wherein the circEYA3 is the renaming of rno _ circ _009926, and the specific nucleotide sequence is shown as SEQ ID NO:1, and specifically comprises the following steps:
ACAGUUUGUGAAGACCGCAGAGACUUGCAGUCCAGUCACAUAAGUGUGUGUAAAGAGACAGCGGUCCUCAUGGAAGAAGAGCAAGACCUACCAGAGCGACCGUUUCCAUAGCACUGUGUAUGCAAAGCCUACAUGUUAGCGAGGGGUGAAAAAGGCCAAGAUGCAGGAACCAGGAGAACAAACUUUAAGUCAAGUAAACAACCCAGACACCAGUGAUCAGAAGCCUGAGACCGCCAGCCUUGCGUCAAACCUUAGCAUGUCAGAGGAAAUUAUGACAUGCACCGAUUACAUCCCUCGCUCAUCCAAUGAUUAUACCUCACAAAUGUAUUCUGCAAAACCUUAUGCACACAUCCUCUCGGUUCCUGUUUCGGAAACCACUUAUCCUGGGCAGACUCAGUACCAGACACUGCAGCAGUCUCAACCCUACGCUGUCUACCCUCAGGCAACCCAAACUUAUGGACUACCUCCUUUCG。
research shows that the circEYA3 directly combines with downstream protein Smad5 to regulate the expression of NLRP3, GSDMD, ASC, clear-caspase-1 and clear-IL-1 beta, and further regulate myocardial cell apoptosis. Therefore, the circEYA3 can be applied to the preparation of products for regulating myocardial cell apoptosis by targeting Smad 5.
In one embodiment, the inhibitor of circEYA3 is applied to the preparation of products for inhibiting myocardial cell apoptosis, the nucleotide sequence of the inhibitor is shown in SEQ ID NO. 2 and SEQ ID NO. 3, and the nucleotide sequence shown in SEQ ID NO. 2 is CCUGUUCGGAAACCACUUT: the nucleotide sequence shown in SEQ ID NO. 3 is AAGUGGUUCCGAAACAGGTT.
The embodiment of the invention also provides a product for inhibiting myocardial cell scorching, which comprises the inhibitor. The inhibitor can knock down circEYA3, thereby inhibiting the focal death of myocardial cells.
The embodiment of the invention also provides application of circEYA3 in preparation of a product for diagnosing/treating congenital heart diseases.
The expression difference of the circEYA3 in congenital heart disease patients and healthy samples is obvious, and the auxiliary diagnosis of the congenital heart disease can be accurately, quickly and simply realized by detecting the expression quantity of the circEYA 3.
Further, the application of the inhibitor of circEYA3 in preparing the treatment product of congenital heart disease is that the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 2 and SEQ ID NO. 3. .
Yet another embodiment of the present invention provides a product for diagnosing congenital heart disease, which comprises a marker identifying circEYA3 as described above.
Further, the marker is a binding primer of a cDNA complementary to circEYA3 or a biological macromolecule binding to said circEYA 3;
biological macromolecules include antibodies, antibody functional fragments, RAN binding proteins, and RAN binding protein functional fragments.
In the present invention, the antibody or antibody functional fragment may be a fluorescently labeled antibody or antibody functional fragment; the RNA binding protein or functional fragment of RNA binding protein may be a fluorescently labeled RNA binding protein or functional fragment of RNA binding protein.
Using the above-mentioned markers, the expression level of circEYA3 can be quantitatively detected.
In one embodiment, the nucleotide sequence of the binding primer of the cDNAs complementary to circEYA3 is shown in SEQ ID NO. 4 and SEQ ID NO. 5, specifically, the nucleotide sequence shown in SEQ ID NO. 4 is: GGACTACCTTTTTCGACAGTT; the nucleotide sequence shown in SEQ ID NO. 5 is GTCGCTCTCTGGTAGGTCTTGC.
Another embodiment of the present invention provides a medicament for treating congenital heart disease, which comprises a therapeutically effective amount of the above-mentioned inhibitor.
The technical solution of the present invention is further described in detail by the following specific examples, however, it should be understood that these examples are for the purpose of more detailed description only and are not to be construed as limiting the present invention in any manner.
Example 1
This example is the effect of formaldehyde on circEYA3 and apoptosis in animal models and in plasma from cardiomyocytes and CHD patients:
the expression of circEYA3 was detected in rat Cardiomyocytes (CM) and Cardiac Fibroblasts (CF) using qRT-PCR method, and it was found that the expression of circEYA3 in CM was significantly increased (P < 0.05) compared to CF (fig. 1, a);
collecting organs of adult rat and fetal rat, detecting the expression of circEYA3 by qRT-PCR, and finding that the expression of circEYA3 is highest in fetal rat heart (P < 0.05) compared with other organs (B in figure 1);
constructing a formaldehyde-exposed fetal rat heart model, harvesting hearts of newborn rats after an intraperitoneal injection control group (normal saline) and a formaldehyde-exposed group (2.0 mg/kg), detecting the expression of circEYA3 by adopting qRT-PCR (quantitative reverse transcription-polymerase chain reaction), and finding that the expression of the circEYA3 in the hearts is obviously increased (P < 0.05) after formaldehyde induction (C in figure 1);
plasma of CHD patients and normal persons confirmed clinically by B-ultrasonography was collected, and the expression of circEYA3 was detected by qRT-PCR, showing that the expression of circEYA3 was significantly higher than that of normal persons (P < 0.05) (D in fig. 1).
In fig. 1, a: detecting the circEYA3 expression level in rat cardiac muscle cells and fibroblasts by using a qRT-PCR experiment;
b: the qRT-PCR experiment detects the circEYA3 expression level in heart, kidney, muscle, colon, liver, skin of adult rats and neonatal mice.
C: the qRT-PCR experiment detected circEYA3 expression levels in the control group (n = 8) and the formaldehyde treated group (n = 8).
D: the qRT-PCR experiment detected circEYA3 expression levels in the plasma of normal humans and CHD patients.
ns indicates no significant difference; * P <0.01; * P <0.001; # P <0.0001.
In summary, as shown in fig. 1, circEYA3 can preliminarily show that circEYA3 may have pathogenic effect on CHD. It can be used as a diagnostic marker for CHD.
Example 2
This example is to identify the cyclic structure of circEYA3
By Sanger sequencing, circEYA3 was found to have a cyclic structure (A, B in FIG. 2). When H9C2 cells were treated with actinomycin D, a significant reduction in EYA3 expression was found without significant change in circEYA3, demonstrating the stability of the cyclic structure of circEYA3 (C in fig. 2). In addition, it was also found by FISH experiment that circEYA3 is not only present in the nucleus but also expressed in the cytoplasm (D in FIG. 2). In FIG. 2, A-B: circEYA3 head-to-tail connected ring structure. C: expression levels of circEYA3 and EYA3 mrnas after treatment with actinomycin D. D: expression of circEYA3 in H9C2 nuclei, cytoplasm. Scale bar: 50 μm; * P <0.05.
Example 3
This example is a study of the occurrence of formaldehyde-induced apoptosis in fetal rat hearts:
in the formaldehyde-induced fetal mouse heart model, pathological changes such as structural disorder, VSD, myocardial thinning, left ventricular hypertrophy and the like were observed in the fetal mouse heart after intraperitoneal injection of formaldehyde by HE staining (a in fig. 3). In addition, using immunohistochemical examination of formaldehyde-induced fetal rat hearts, it was found that the expression of the pyro-death related factors NLRP3, GSDMD, ASC, clear-caspase-1 and clear-IL-1 β was significantly increased (P < 0.05) compared to the control group (B in fig. 3); in fig. 3, a: fetal mouse heart HE sections in control and formaldehyde-induced groups; the arrow positions indicate chamber, right Ventricular (RV), left Ventricular (LV), ventricular septum (IVS); scale bar: 1000 μm; b: expression of the tar death-related factor in the heart of fetal rats in the control group and the formaldehyde-induced group; scale bar: 200 mu m; # P <0.0001.
Example 4
This example is the occurrence of formaldehyde-induced cardiomyocyte apoptosis:
NLRP3, circEYA3 expression was highest (P < 0.05) in H9C2 cells treated with formaldehyde at concentrations of 150. Mu.M/ml (A, B in FIG. 4) stimulated by formaldehyde at different concentrations. In addition, H9C2 cells were treated with 150 μ M/ml formaldehyde at different times and both NLRP3 and circEYA3 were found to be highly expressed at 24H (P < 0.05) (C, D in fig. 4). In fig. 4, a: the expression level of NLRP3 treated by different formaldehyde gradient concentrations; b: expression levels of circEYA3 after treatment with different formaldehyde gradient concentrations; c: expression levels of NLRP3 at different time points after 150. Mu.M/ml formaldehyde treatment. D: expression levels of circEYA3 at different time points after 150 μ M/ml formaldehyde treatment; ns indicates no significant difference; * P <0.05; * P <0.01; * P <0.001.
As shown in FIG. 4, formaldehyde induced the onset of myocardial cell apoptosis.
Example 5
This example is a study of the knock-down of circEYA3 in inhibiting apoptosis of H9C2 cells:
in order to study whether knocking down circEYA3 regulates myocardial cell apoptosis, a circEYA3 inhibitor designed by gimerap corporation is used for transfecting H9C2 cells for 24H, and the expression of the circEYA3 and the EYA3 is detected by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), so that the expression of the circEYA3 is obviously reduced (P < 0.05), but the expression of the EYA3 is not obviously changed (A in figure 5);
to investigate the effect of circEYA3 inhibitors on apoptosis regulation, H9C2 cells were transfected with circEYA3 inhibitors and subsequently stimulated with 150 μ M/ml formaldehyde, WB experimental results showed a decrease in the expression of apoptosis-related factors in the circEYA3 inhibitor group (B in fig. 5). Caspase-1 activity assay showed the same results, transfection of circEYA3 inhibitor resulted in a decrease of apoptosis (P < 0.05) after formaldehyde induction (C in fig. 5).
LDH release experiments showed that transfection of the circEYA3 inhibitor reduced LDH release levels following formaldehyde stimulation and decreased the occurrence of cellular apoptosis (P < 0.05) (fig. 5D). The Hoechst/PI experiment also showed the same result, transfection of the circEYA3 inhibitor resulted in a decrease in formaldehyde-induced apoptosis (P < 0.05) (FIG. 5, E). In fig. 5, a: expression levels of circEYA3 and EYA3 after transfection of a circEYA3 inhibitor; b: transfection of circEYA3 inhibitor, expression level of apoptosis-related factor after formaldehyde treatment; c: detecting the expression level of Caspase-1 after formaldehyde treatment of a transfection circEYA3 inhibitor by a Caspase-1 activity experiment; d: transfecting a circEYA3 inhibitor, and detecting LDH activity of cell supernatant through an LDH release experiment after formaldehyde treatment; e: results of staining experiments with Hoechst 33342/PI after formaldehyde treatment (PI is red and Hoechst 33342 is blue) were tested by transfection with circEYA3 inhibitor. Scale bar: 50 μm; ns indicates no significant difference; * P <0.05; * P <0.01; * P <0.001; # P <0.0001.
As can be seen from fig. 5, knocking down circEYA3 can inhibit the production of cardiomyocyte apoptosis.
Example 6
This example is a study of the promotion of apoptosis in H9C2 cells by overexpression of circEYA 3:
to investigate whether overexpression of circEYA3 aggravated myocardial cell apoptosis, H9C2 cells were transfected with circEYA3 mimic designed by gimerap, and the expression of circEYA3 and EYA3 was detected by qRT-PCR, which showed that the expression of circEYA3 was increased (P < 0.05), but no significant effect on the expression of EYA3 was seen (a in fig. 6).
To further investigate whether circEYA3 modulates apoptosis, H9C2 cells were transfected with circEYA3 mimetics and subsequently stimulated with 150 μ M/ml formaldehyde, and changes in expression of apoptosis-related factors were detected using the WB assay, which showed a significant increase in expression of apoptosis-related factors in the group of transfected circEYA3 mimetics (B in fig. 6). Caspase-1 activity kit experiments showed the same results, with the group of transfected circEYA3 mimetics showing a significant increase in cell apoptosis (P < 0.05) upon stimulation with formaldehyde (C in figure 6).
LDH release experiments showed that transfection of circEYA3 inhibitor could increase LDH release levels after formaldehyde stimulation and promote the occurrence of cellular apoptosis (P < 0.05) (fig. 6D). The Hoechst/PI experiment showed the same result, with the circEYA3 mock group transfected with a significant increase in cell apoptosis (P < 0.05) upon stimulation with formaldehyde (E in FIG. 6). In fig. 6, a: expression levels of circEYA3 and EYA3 after transfection of a circEYA3 mimic; b: transfecting circEYA3 simulants, and expressing the expression level of the apoptosis-related factor after formaldehyde treatment; c: detecting the expression level of Caspase-1 after formaldehyde treatment of a transfection circEYA3 simulant by a Caspase-1 activity experiment; d: transfecting a circEYA3 simulant, and detecting LDH activity of cell supernatant through an LDH release experiment after formaldehyde treatment; e: the circEYA3 mimic was transfected and tested in the Hoechst 33342/PI staining assay after formaldehyde treatment (PI is red and Hoechst 33342 is blue). Scale bar: 50 μm; ns indicates no significant difference; * P <0.01; * P <0.001; # P <0.0001
As can be seen in fig. 6, overexpression of circEYA3 promoted the production of cardiomyocyte apoptosis.
Example 7
This example is a study of the binding of circEYA3 to Smad 5:
to determine the downstream mRNA of circEYA3, an RNA pull-down experiment of circEYA3 was performed using the biotin probe of circEYA3 designed by gimara corporation (a in fig. 7). In addition, proteins that bind to circEYA3 were identified by mass spectrometry, the most abundant of which was Smad5 (B in fig. 7). Meanwhile, it was found that circEYA3 binds to Smad5 by RNA pull-down experiment of circEYA3 (C in fig. 7). Then, by adopting RIP experiments, the combination of Smad5 and circEYA3 is reversely verified (D in FIG. 7), which shows that the circEYA3 can directly target the Smad5, and the expression of both circEYA3 and Smad5 in nucleus and cytoplasm is found by FISH experiments (E in FIG. 7); in fig. 7, a: performing RNAsull-down experiment on circEYA3 and control protein by using a biotin probe; b: analyzing the result of RNA pull-down experiment mass spectrum; c: an RNA pull-down experiment verifies that circEYA3 is combined with Smad5; d: RIP experiments were performed with Smad5 or IgG antibodies; carrying out qRT-PCR experimental detection on the purified RNA; e: co-localization of circEYA3 and Smad5 in H9C2 cells (circEYA 3 is red, smad5 is green, DAPI is blue). Scale bar: 50 μm.
As can be seen in fig. 7, circEYA3 directly targets Smad5; by FISH experiments, circEYA3 and Smad5 were found to be expressed in both the nucleus and cytoplasm.
Example 8
This example is a study of CircEYA3 in promoting the occurrence of cardiomyocyte apoptosis by inhibiting Smad 5:
the WB experiment results showed that the expression of Smad5 gradually decreased with increasing formaldehyde concentration by treating H9C2 cells with different concentrations of formaldehyde (a in fig. 8). Samples were collected at various time points after treatment of H9C2 cells with 150 μ M/ml formaldehyde, and WB experimental results showed that the expression level of Smad5 gradually decreased with increasing formaldehyde treatment time (B in fig. 8). H9C2 cells were transfected with circEYA3 mimic and WB experimental results showed a significant reduction in expression of Smad5 in the circEYA3 mimic transfected group compared to the control group (C in fig. 8). H9C2 cells were transfected with the circEYA3 inhibitor, and WB experimental results showed a significant increase in Smad5 expression in the circEYA3 inhibitor transfected group compared to the control group (D in fig. 8). H9C2 cells were transfected with Smad5 inhibitors and qRT-PCR experimental results showed a significant decrease in Smad5 expression (P < 0.05) (fig. 8, E). LDH release experiments showed that inhibition of Smad5 expression increased LDH release levels following formaldehyde stimulation, exacerbating cell apoptosis, while circEYA3 inhibited Smad 5-induced decrease in cell apoptosis (P < 0.05) (fig. 8F). The Hoechst/PI experiment showed the same result (P < 0.05) (G in FIG. 8). In fig. 8, a: expression levels of Smad5 after treatment with formaldehyde gradients of different concentrations; b: smad5 expression levels at various time points after 150 μ M/ml formaldehyde treatment; c: expression level of Smad5 following transfection with circEYA3 mimic; d: expression level of Smad5 following transfection with circEYA3 inhibitor; e: expression levels of Smad5 following transfection with Smad5 inhibitors; f: transfecting Smad5 inhibitor and circEYA3 inhibitor, and treating cell supernatant LDH activity after formaldehyde treatment; g: and (3) detecting results of Hoechst 33342/PI staining experiments after transfection of Smad5 inhibitors and circEYA3 inhibitors and formaldehyde treatment (PI is red, and Hoechst 33342 is blue). Scale bar: 50 μm; ns indicates no significant difference; * P <0.05; * P <0.001.
As can be seen from fig. 8, circEYA3 can regulate the occurrence of formaldehyde-induced cardiomyocyte apoptosis through Smad 5.
The experiments show that the expression difference of the circEYA3 in congenital heart disease patients and healthy samples is obvious, and the auxiliary diagnosis of the congenital heart disease can be accurately, quickly and simply realized by detecting the expression quantity of the circEYA 3.
In addition, the circEYA3 regulates and controls the expression of NLRP3, GSDMD, ASC, cleared-caspase-1 and cleared-IL-1 beta by directly combining with downstream protein Smad5, thereby regulating and controlling the myocardial cell apoptosis. In addition, myocardial cell apoptosis can be inhibited by knocking down circEYA3, the occurrence of formaldehyde-induced cardiac dysplasia is improved, and a potential therapeutic target is provided for diagnosis and treatment of CHD.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.
Claims (10)
- Application of circEYA3 in preparation of products for regulating myocardial cell scorching.
- 2. The use according to claim 1, wherein the circEYA3 is used for preparing a product for regulating myocardial cell apoptosis by targeting Smad 5.
- 3. The use of claim 1, wherein the inhibitor of circEYA3 has a nucleotide sequence shown in SEQ ID NOs 2 and 3 for preparing a product for inhibiting myocardial cell apoptosis.
- 4. A product for inhibiting myocardial cell apoptosis is characterized by comprising an inhibitor of circEYA3, wherein the nucleotide sequence of the inhibitor is shown as SEQ ID NO. 2 and SEQ ID NO. 3.
- Application of circEYA3 in preparation of products for diagnosing/treating congenital heart diseases.
- 6. The use according to claim 5, wherein the inhibitor of circEYA3 has a nucleotide sequence shown in SEQ ID NO. 2 and SEQ ID NO. 3 for the preparation of a product for treating congenital heart disease.
- 7. A product for diagnosing congenital heart disease, characterized in that it comprises a marker identifying circEYA3 according to claim 5.
- 8. The product of claim 7, wherein the label is a binding primer for the cDNAs complementary to the circEYA3 or a biomacromolecule that binds to the circEYA 3;the biomacromolecule comprises an antibody, an antibody functional fragment, a RAN binding protein and a RAN binding protein functional fragment.
- 9. The product according to claim 8, wherein the nucleotide sequence of the primer binding of the cDNAs complementary to circEYA3 is shown in SEQ ID NO. 4 and SEQ ID NO. 5.
- 10. A medicament for treating congenital heart disease, comprising a therapeutically effective amount of an inhibitor of circEYA3 according to claim 5, the nucleotide sequence of said inhibitor being shown in SEQ ID No. 2 and SEQ ID No. 3.
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