Application of RBM22 in the preparation of drugs promoting myocardial regeneration

Abstract

The present invention belongs to the field of biomedical technology, and specifically relates to an application of RBM22 in the preparation of a drug for promoting myocardial regeneration. Since promoting the expression of RBM22 can promote the proliferation and regeneration of myocardial cells, it can improve myocardial ischemic damage. Based on this discovery, the present invention provides an application of RBM22 in the preparation of a drug for promoting myocardial regeneration. By promoting the expression of RBM22 by a drug for promoting myocardial regeneration, the proliferation and regeneration of myocardial cells can be promoted, thereby achieving the regeneration and repair of damaged myocardium after ischemic heart disease, inhibiting myocardial fibrosis reaction, and achieving the effect of protecting cardiac function and reducing the occurrence of heart failure after ischemic heart disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to an application of RBM22 in the preparation of a drug for promoting myocardial regeneration. Background Art

[0002] Heart failure, a terminal condition of cardiovascular disease, carries a high mortality rate, with a five-year survival rate of less than 50%. Ischemic myocardial infarction is one of the main causes of heart failure. An acute myocardial infarction (MI) can cause ischemic necrosis of cardiomyocytes in a short period of time, leading to decreased cardiac function and the development of heart failure. In the early stages of ischemia, cardiomyocytes are deprived of oxygen and nutrients, resulting in massive loss of cardiomyocytes. Multiple processes, including the release of DAMPs following cardiomyocyte injury, synergistically initiate an acute inflammatory response and activate cardiac fibroblasts to produce collagen and other extracellular matrix components to maintain the heart's basic structure. In the late stages of ischemia, a large number of myofibroblasts accumulate, reducing myocardial contractility and impairing the heart's pumping function. Due to the limited proliferation capacity of adult cardiomyocytes, they are unable to repair damaged areas, resulting in cardiac pathological remodeling and fibrosis, ultimately leading to irreversible, advanced heart failure.

[0003] Aside from heart transplantation, clinical treatments for ischemic heart disease, such as ventricular assist, currently fail to replace the large number of lost cardiomyocytes. Therefore, enhancing cardiomyocyte proliferation or stem cell transplantation to replenish lost cardiomyocytes has become a research hotspot for the treatment of heart failure after myocardial infarction.

[0004] Previous studies have reported that RBM22 belongs to the SLT11 family and contains a unique RNA zinc-finger binding domain, C-x8-C-x5-C-x3-H. It is a component of the spliceosome catalytic core and plays a crucial role in alternative splicing of pre-mRNA. Furthermore, RBM22 is involved in gene regulation, capable of binding DNA and acting as a bona fide transcription factor on numerous target genes. Studies have found that RBM22 expression is increased in glioblastoma, while decreased RBM22 expression affects cellular function: tumor cell migration is reduced, vascular endothelial growth factor (VEGF) secretion is inhibited, the number of stem / progenitor cells in tumor spheres is reduced, and apoptosis is promoted. However, whether RBM22 is involved in regeneration and repair after cardiac stress injury has not been reported. Summary of the Invention

[0005] The inventors of the present invention have discovered that promoting the expression of RBM22 can promote the proliferation and regeneration of cardiomyocytes, thereby improving myocardial ischemic damage. Based on this discovery, the present invention provides a use of RBM22 in the preparation of a drug that promotes myocardial regeneration.

[0006] In the application of RBM22 provided by the present invention in the preparation of drugs promoting myocardial regeneration, the drugs promoting myocardial regeneration may also have the following features: wherein, the drugs promoting myocardial regeneration have the following functions: promoting the proliferation and regeneration of myocardial cells, promoting the regeneration and repair of damaged myocardium after ischemic heart disease, inhibiting myocardial fibrosis reaction, protecting cardiac function, and reducing the occurrence of heart failure after ischemic heart disease.

[0007] The use of RBM22 provided by the present invention in the preparation of a drug for promoting myocardial regeneration may also have the following characteristics: wherein the drug for promoting myocardial regeneration is used to promote the expression of RBM22, thereby promoting the proliferation and regeneration of myocardial cells.

[0008] Functions and effects of the invention

[0009] According to the present invention, an application of RBM22 in the preparation of a drug for promoting myocardial regeneration, since the expression of RBM22 can regulate the proliferation and regeneration level of myocardial cells, by promoting the expression of RBM22 through a drug that promotes myocardial regeneration, the proliferation and regeneration of myocardial cells can be promoted, thereby achieving the regeneration and repair of damaged myocardium after ischemic heart disease, inhibiting myocardial fibrosis reaction, and achieving the effect of protecting cardiac function and reducing the occurrence of heart failure after ischemic heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 3 is a graph comparing the expression of RBM22 in the sham-operated group and the apical resection model group of wild-type newborn mice in the embodiments of the present invention;

[0011] Figure 2 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in the AAV9 control group and the RBM22 overexpression group when EDU is used as a cell proliferation marker in an embodiment of the present invention;

[0012] Figure 3 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in a blank control group and an RBM22 expression interference group when EDU is used as a cell proliferation marker in an embodiment of the present invention;

[0013] Figure 4 This is a graph showing the percentage of primary cardiomyocyte proliferation in wild-type neonatal rats in a blank control group and an RBM22 expression interference group when EDU is used as a cell proliferation marker in an embodiment of the present invention;

[0014] Figure 5 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in the AAV9 control group and the RBM22 overexpression group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention;

[0015] Figure 6Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in a blank control group and an RBM22 expression interference group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention;

[0016] Figure 7 This is a graph showing the percentage of primary cardiomyocyte proliferation of wild-type neonatal rats in the blank control group and the RBM22 expression interference group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention. DETAILED DESCRIPTION

[0017] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the present invention is described in detail in the following embodiments with reference to the accompanying drawings.

[0018] <Example>

[0019] In this example, experiments were conducted on mice to verify the relationship between RBM22 and myocardial regeneration after myocardial injury, as follows:

[0020] In this example, an apical resection model was established in 3-day-old C57 mice, and a sham operation group and an apical resection model group were set up. Western blotting was performed on apical tissue samples 7 days later to detect changes in RBM22 expression. The experimental process is as follows:

[0021] Total tissue protein was extracted using TPER, Halt Protease Inhibitor Cocktail, and PMSF (Thermo Fisher Scientific). Western blotting was performed using transfer buffer, electrophoresis buffer, and TBST (Shanghai Yazyme Biopharmaceutical Technology Co., Ltd.) using an electrophoresis tank and transfer apparatus (Bio-Rad). The expression of RBM22 and β-actin in myocardial tissue of each group was analyzed using the Tianneng automated chemiluminescence fluorescence analysis system and ultrasensitive chemiluminescence developer (Thermo Fisher Scientific).

[0022] Figure 1 3 is a graph comparing the expression levels of RBM22 in the sham operation group and the apical resection model group of wild-type newborn mice in the examples of the present invention.

[0023] like Figure 1 As shown, the sham operation group (P3-Sham) did not undergo apical resection, and RBM22 was expressed normally. After apical resection of the apical resection model group (P3-AR), the expression level of RBM22 in the myocardial tissue of neonatal mice was significantly upregulated after myocardial injury.

[0024] In this example, the relationship between RBM22 and cardiomyocyte proliferation and regeneration was further verified by interfering with RBM22 expression and promoting RBM22 expression, and comparing them with the control group, as follows:

[0025] siRNA specifically interfering with RBM22 expression (siRNA-RBM22, synthesized by Dharmacon, siRNAD-055902-01: GAUGCAGGAUUAUCAUUUA; siRNAD-055902-02: GGAGAGAUCCGCACCAUCA; siRNAD-055902-03: GGAGGCGUCUGCCAACUAC; siRNAD-055902-04: UAUCAAAGACCGGUACUAU) and adeno-associated virus overexpressing RBM22 (AAV9-RBM22, GV571 vector, NheI / NheI enzyme digestion, purchased from Shanghai GeneChem Technology Co., Ltd.) were constructed. Primary cardiomyocytes from 1-3 day old mice (C57 strain, purchased from Shanghai Xipul-Bikai Laboratory Animal Co., Ltd.) were isolated and divided into a control group (NC), an RBM22 expression interference group (siRNA-RBM22), a control group (AAV9-Ctrl), and an RBM22 overexpression group (AAV9-RBM22). After 48 hours of treatment, the cardiomyocyte marker α-actinin was co-stained with various cardiomyocyte proliferation indicators such as Ki67 and EDU to detect differences in their proliferation levels in the four groups.

[0026] C57 neonatal rat cardiomyocytes were cultured in vitro and transfected with siRNA-RBM22 and AAV9-RBM22, followed by immunofluorescence staining: fixation with 4% paraformaldehyde (Boster Biotech) at room temperature for 15 minutes, followed by blocking with 1% BSA (Biofroxx) diluted in PBST prepared with PBS and Triton (Amresco) at room temperature for 1 hour. Antibodies against cardiomyocyte marker α-actinin (Abcam) and cell proliferation marker Ki67 (Abcam) were used in conjunction with an EdU kit (Biyuntian). After incubation at 4°C overnight, the cells were rinsed three times with 1X PBS (Gene Ray), incubated with Alexa 488, 594 secondary antibodies (Invitrogen) at room temperature for one hour, and rinsed again with 1X PBS three times. The cells were mounted with DAPI-containing mounting medium (Southern Biotech) and imaged using a Leica fluorescence microscope. The proportion of double-positive cells was counted to reflect cardiomyocyte proliferation.

[0027] Figure 2Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in the AAV9 control group and the RBM22 overexpression group when EDU is used as a cell proliferation marker in an embodiment of the present invention; Figure 3 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in a blank control group and an RBM22 expression interference group when EDU is used as a cell proliferation marker in an embodiment of the present invention; Figure 4 This is a graph showing the percentage of primary cardiomyocyte proliferation of wild-type neonatal rats in the blank control group and the RBM22 expression interference group when EDU is used as a cell proliferation marker in an embodiment of the present invention.

[0028] Figure 2 and Figure 3 From left to right in the middle are the staining results of α-actinin (cardiomyocytes), the staining results of EDU (proliferating cells) and the overall fluorescence staining results.

[0029] like Figure 2 As shown, the proliferating cells in the RBM22 overexpression group (AAV9-RBM22) were significantly more than those in the AAV9 control group (AAV9-Ctrl).

[0030] like Figure 3 and Figure 4 As shown, the proliferative cells in the RBM22 interference expression group (siRNA-RBM22) were significantly less than that in the blank control group (NC), and the proportion of proliferative cells in the RBM22 interference expression group was also significantly lower than that in the blank control group.

[0031] Figure 5 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in the AAV9 control group and the RBM22 overexpression group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention; Figure 6 Schematic diagram of the fluorescence staining results of primary cardiomyocytes of wild-type neonatal rats in a blank control group and an RBM22 expression interference group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention; Figure 7 This is a graph showing the percentage of primary cardiomyocyte proliferation of wild-type neonatal rats in the blank control group and the RBM22 expression interference group when Ki67 is used as a cell proliferation marker in an embodiment of the present invention.

[0032] Figure 5 and Figure 6 From left to right in the middle are the staining results of α-actinin (cardiomyocytes), the staining results of Ki67 (proliferating cells), and the overall fluorescence staining results.

[0033] like Figure 5 As shown, the proliferating cells in the RBM22 overexpression group (AAV9-RBM22) were significantly more than those in the AAV9 control group (AAV9-Ctrl).

[0034] like Figure 6 and Figure 7 As shown, the proliferative cells in the RBM22 interference expression group (siRNA-RBM22) were fewer than those in the blank control group (NC), and the proportion of proliferative cells in the RBM22 interference expression group was also significantly lower than that in the blank control group.

[0035] In summary, interfering with RBM22 expression can inhibit cardiomyocyte proliferation, while promoting RBM22 expression can promote cardiomyocyte proliferation and regeneration. Therefore, promoting RBM22 expression can promote cardiomyocyte proliferation and regeneration, thereby promoting the regeneration and repair of damaged myocardium and improving myocardial ischemic injury.

[0036] Furthermore, methods for promoting RBM22 expression include but are not limited to AAV9 and mRNA technology. Accordingly, drugs that promote RBM22 expression are not limited to gene drugs prepared based on AAV9 and / or mRNA technology; as long as they are drugs that can promote RBM22 overexpression, they can promote the proliferation and regeneration of myocardial cells, promote the regeneration and repair of damaged myocardium after ischemic heart disease, inhibit myocardial fibrosis reaction, protect cardiac function, and reduce the occurrence of heart failure after ischemic heart disease.

[0037] Functions and Effects of the Embodiments

[0038] According to the application of RBM22 in the preparation of a drug for promoting myocardial regeneration involved in this embodiment, since the expression of RBM22 can regulate the proliferation and regeneration level of myocardial cells, by promoting the expression of RBM22 through drugs that promote myocardial regeneration, the proliferation and regeneration of myocardial cells can be promoted, thereby achieving the regeneration and repair of damaged myocardium after ischemic heart disease, inhibiting myocardial fibrosis reaction, and achieving the effect of protecting cardiac function and reducing the occurrence of heart failure after ischemic heart disease.

[0039] The above embodiments are preferred examples of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. The use of RBM22 in the preparation of a drug for promoting myocardial regeneration, characterized in that: The drug for promoting myocardial regeneration is used to promote the expression of RBM22, thereby promoting the proliferation and regeneration of myocardial cells.

2. The use of RBM22 according to claim 1 in the preparation of a drug for promoting myocardial regeneration, characterized in that: in, The drug for promoting myocardial regeneration has the following functions: promoting the proliferation and regeneration of myocardial cells, promoting the regeneration and repair of damaged myocardium after ischemic heart disease, inhibiting myocardial fibrosis reaction, protecting cardiac function and reducing the occurrence of heart failure after ischemic heart disease.

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