Application of circular RNA circALPK2 in myocardial cell repair and regeneration
By designing the circALPK2 interference sequence and using the lentiviral system to transfect cardiomyocytes, the problem of low proliferation efficiency of central myocytes in the prior art was solved, and significant proliferation of cardiomyocytes and improvement of cardiomyocytes were achieved.
Patent Information
- Application Number
- CN202510113010.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-24
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art has problems of inefficiency, high cost and toxic side effects in promoting cardiomyocyte proliferation and heart regeneration, making it difficult to effectively repair and regenerate damaged myocardium.
By designing the circALPK2 interference sequences related to myocardial proliferation, an interference plasmid is constructed, and the lentiviral system is used to transfect the interference plasmids into cardiomyocytes to obtain interfering cells. In this way, the expression of circALPK2 in cardiomyocytes is reduced by transplanting these interfering cells, thereby promoting the proliferation of cardiomyocytes and heart regeneration.
It has achieved significant improvement in the proliferation ability of cardiomyocytes, improved cardiac function damage, and provided an effective method to promote myocardial regeneration and repair, which has potential clinical application value.
Smart Images
Figure CN119979695A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of circular RNA circALPK2 in myocardial cell repair and regeneration. Background Art
[0002] Myocardial infarction is caused by severe stenosis or blockage of the coronary arteries, which leads to local myocardial ischemia and hypoxia, and then myocardial necrosis. The myocardial cells of adult mammals lack the ability to proliferate, which makes it difficult for the heart to regenerate itself after being damaged. Once a myocardial infarction occurs, fibrosis will appear in the infarcted area, the myocardial contractility will weaken, and ventricular remodeling will occur, which may eventually lead to heart failure. In terms of clinical treatment, traditional drug therapy and mechanical treatment methods can only relieve the symptoms of myocardial infarction, but cannot fundamentally save the damaged myocardium. Although heart transplantation can solve the problem in theory, it faces many limitations due to the severe shortage of donor organs. Therefore, achieving myocardial regeneration in patients with heart damage and promoting the repair and functional reconstruction of necrotic myocardium has always been a long-standing goal in the clinical treatment of myocardial infarction.
[0003] Cardiomyocytes derived from human pluripotent stem cells have brought new hope for solving this problem. They are expected to become a long-term and feasible seed cell for regenerative medicine. At present, researchers have made significant progress in the efficient generation of cardiomyocytes from pluripotent stem cells. At present, promoting the proliferation of endogenous cardiomyocytes and the transplantation of exogenous cardiomyocytes are methods of cardiac regeneration and repair that have attracted much attention. In addition, a method for the efficient and directed differentiation of human pluripotent stem cells into cardiomyocytes has been successfully established, which can obtain a large number of seed cells for transplantation to replace damaged myocardium. However, cardiomyocytes derived from pluripotent stem cells have the problems of weak proliferation ability and low survival rate after transplantation, which greatly limits their ability to repair damaged myocardium. The prior art attempts to reverse the loss of cardiac function after myocardial infarction in rats by intraperitoneal delivery of a mixture of five small molecules, induce cardiomyocyte proliferation, and reduce myocardial fibrosis (Du, Jianyong et al. Cell Stem Cell, Volume 29, Issue 4, 545-558.); or by intramyocardial injection of a single molecule to promote endogenous cardiomyocyte proliferation in adult mice, thereby improving cardiac damage repair after myocardial infarction (Feng et al. Circulation 2024 Mar 26; 149 (13): 1004-1015.). Although these methods have improved the level of myocardial proliferation to a certain extent, they are still not enough to compensate for the large number of cardiomyocytes that have died due to damage. In addition, these molecules also have problems such as high price, poor stability, or obvious toxic side effects on individuals, which seriously restricts their clinical transformation and market development.
[0004] Circular RNA (circRNA) is defined as a type of RNA transcription product that does not encode proteins and forms a complete closed loop structure with covalent bonds without a 5'-cap end and a 3'-polyA tail. It is stable and conservative. Recent studies have shown that circRNA is widely present in cardiac tissues. It mainly acts as a sponge to adsorb microRNA (miRNA), regulate protein expression, protein translation and gene transcription. It plays an important role in the pathological and physiological processes of apoptosis, autophagy, proliferation, angiogenesis, fibrosis and so on after myocardial infarction. It is expected to become a specific biomarker and therapeutic target for myocardial infarction. Summary of the invention
[0005] To solve the above technical problems, the present invention provides a new method for inducing cardiac regeneration and repair. The method first provides a circALPK2 interference sequence related to myocardial proliferation, then constructs an interference plasmid, and uses the lentiviral resuspension packaged with the interference plasmid to co-culture with pluripotent stem cell-derived cardiomyocytes to obtain interference cells, thereby improving the proliferation capacity of cardiomyocytes, and using the interference cell transplantation to achieve regeneration and repair of damaged myocardium.
[0006] The first object of the present invention is to provide the use of circular RNA circALPK2 in the preparation of cardiomyocyte repair and / or regeneration products, wherein the nucleotide sequence of the circALPK2 is shown in SEQ ID NO.1.
[0007] Human circALPK2 originates from the reverse splicing of exon 4 of its host gene ALPK2. It has been found to be expressed on the fourth day of directed differentiation of human pluripotent stem cells into cardiomyocytes and maintain a high level in cardiomyocytes. It is of great significance to develop and prepare cardiac regeneration and repair promoters targeting circALPK2 that is specifically and highly expressed in the myocardium.
[0008] The second object of the present invention is to provide a preparation for reducing the expression of circular RNA circALPK2 for use in preparing myocardial cell repair and / or regeneration products, wherein the nucleotide sequence of the circALPK2 is shown in SEQ ID NO.1.
[0009] The third object of the present invention is to provide a preparation for promoting cardiomyocyte repair and / or regeneration, wherein the preparation reduces the expression of circular RNA circALPK2 in cardiomyocytes.
[0010] Furthermore, the preparation comprises short hairpin RNA, the sense strand of the short hairpin RNA is shown as SEQ ID NO.2, and the antisense strand of the short hairpin RNA is shown as SEQ ID NO.3.
[0011] The short hairpin RNA provided by the present invention can be cleaved in cells to form siRNA, and the siRNA binds to the RNA-induced silencing complex, thereby inhibiting the expression of circALPK2.
[0012] Furthermore, the positive strand of the short hairpin RNA is 5'-GATCCGTGATCCTCCTCA GCTGTACTTCCTGTCAGATACAGCTGAGGAGGATCACTTTTTG-3'.
[0013] Furthermore, the antisense strand of the short hairpin RNA is 5'-AATTCAAAAAGTGATCC TCCTCAGCTGTATCTGACAGGAAGTACAGCTGAGGAGGATCACG-3'.
[0014] Furthermore, the preparation contains small interfering RNA, the sense strand of the small interfering RNA is shown as SEQ ID NO.4, and the antisense strand of the small interfering RNA is shown as SEQ ID NO.5.
[0015] Furthermore, the positive strand of the small interfering RNA is 5'-GTGATCCTCCTCAGCTGT A-3'.
[0016] Furthermore, the antisense strand of the small interfering RNA is 5'-TACAGCTGAGGAGGATC AC-3'.
[0017] Furthermore, the preparation also includes one or more of a plasmid, a lentivirus and an adeno-associated virus containing the short hairpin RNA.
[0018] In one embodiment of the present invention, an interfering plasmid is constructed by connecting the above-mentioned short hairpin RNA to a vector, and the interfering plasmid and the auxiliary plasmid are co-transfected into a lentivirus using a three-plasmid lentiviral system to obtain a lentivirus suspension, which is co-cultured with cardiomyocytes to obtain interfering cells, and the proliferation ability of the interfering cells is significantly improved.
[0019] The fourth object of the present invention is to provide a preparation for reducing the expression of circular RNA circALPK2 for use in the preparation of a drug for treating myocardial infarction.
[0020] The fifth object of the present invention is to provide a drug for treating myocardial infarction, wherein the therapeutic drug targets circular RNA circALPK2 and reduces the expression of circular RNA circALPK2 in cardiomyocytes.
[0021] Furthermore, the preparation comprises short hairpin RNA and / or small interfering RNA, wherein the nucleotide sequence of the short hairpin RNA is shown in SEQ ID NO.2-3, and the nucleotide sequence of the small interfering RNA is shown in SEQ ID NO.4-5.
[0022] Furthermore, the administration method of the drug includes intramyocardial injection or intravenous injection.
[0023] In one embodiment of the present invention, the drug is administered by intramyocardial injection.
[0024] In one embodiment of the present invention, the interfering plasmid is constructed by connecting the above-mentioned short hairpin RNA to a vector, and the interfering plasmid and the auxiliary plasmid are co-transfected into the lentivirus using a three-plasmid lentiviral system to obtain a lentiviral suspension, which is co-cultured with cardiomyocytes to obtain interfering cells. The interfering cells are transplanted into mice with myocardial infarction by intramyocardial injection, effectively promoting the recovery of myocardial function in mice.
[0025] Beneficial effects of the present invention:
[0026] (1) ShRNA and siRNA designed for the circular RNA interface are used to transfect engineered cardiomyocytes with low circALPK2 expression mediated by lentivirus. Cardiomyocytes with significantly reduced circALPK2 expression can be efficiently obtained by simply infecting cardiomyocytes derived from normally cultured human pluripotent stem cells in vitro. This preparation method has a simple operation process and has the potential for large-scale promotion and application, which provides convenience for related research and clinical practice;
[0027] (2) The sustained and stable low expression of circALPK2 in cardiomyocytes was achieved, which effectively avoided the problem of transient interference expression failure that may occur after human pluripotent stem cell-derived cardiomyocytes are transplanted into the body due to the long survival time required, thus ensuring the durability and stability of the interference effect;
[0028] (3) The engineered cardiomyocytes with low expression of circALPK2 can significantly promote the proliferation of cardiomyocytes and significantly reduce the damage to cardiac function. The experimental results show that this method is effective in improving the condition of myocardial infarction. The left ventricular ejection fraction and shortening fraction used to reflect the cardiac function of mice are significantly increased compared with the control group, providing a promising new approach for the treatment of myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS
[0029] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein
[0030] Figure 1It is a schematic diagram of the principle of designing shRNA for reducing the expression of circular RNA circALPK2 in the present invention;
[0031] Figure 2 This is a result diagram of detecting the relative expression of genes in interference cells in Example 1 of the present invention, NC is a cardiomyocyte transfected with an empty vector, sh-circALPK2 is a cardiomyocyte transfected with an interference vector, wherein A is the relative expression of ALPK2 and circular RNAcircALPK2, and B is the relative expression of proliferation-related genes CCNA2 and CDK1;
[0032] Figure 3 Example 2 of the present invention uses immunofluorescence to detect the proliferation of cardiomyocytes after circALPK2-shRNA interference, NC is cardiomyocytes transfected with an empty vector, sh-circALPK2 is cardiomyocytes transfected with an interference vector, wherein A is the fluorescence microscopy result, and B is the percentage of EdU-positive cells;
[0033] Figure 4 This is the cardiac function detection after interfering cell transplantation in damaged mouse heart in Example 3 of the present invention, Sham represents the sham operation group, MI represents the myocardial infarction operation group, MI+NC represents the control cell treatment group transplanted after myocardial infarction operation, MI+sh represents the circALPK2 interfering cell treatment group transplanted after myocardial infarction operation, wherein A is the electrocardiogram, B is the change in left ventricular ejection fraction, and C is the change in shortening fraction. DETAILED DESCRIPTION
[0034] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0035] Example 1: circALPK2 interference cell construction
[0036] 1. Construction of circALPK2-shRNA vector
[0037] According to the sequence information of circALPK2, the corresponding interference sequence was designed and synthesized at Anshengda. CircRNA is circularized from an exon of the linear gene ALPK2, so the interference sequence can only be designed at the interface position of the ring, effectively interfering with the expression of circRNA without affecting the expression of the gene ALPK2. The specific process is as follows Figure 1 The sense strand of the designed circALPK2-shRNA is shown in SEQ ID NO.2, and the antisense strand is shown in SEQ ID NO.3. The sense strand of the circALPK2-siRNA obtained by cleavage is shown in SEQ ID NO.4, and the antisense strand is shown in SEQ ID NO.5.
[0038] The circALPK2-shRNA was connected to the pLVTHM vector by T4 ligation to obtain the interference plasmid. Subsequently, the constructed interference plasmid was transformed into competent cells DH5a, and the bacterial solution was plated and cultured for 16 hours before selecting single clones for colony verification. The positive clones were sequenced and verified before plasmid extraction. Among them, circALPK2-shRNA is a hairpin structure, which can be cut into siRNA in cells. siRNA binds to RNA-induced silencing complex (RISC), thereby inhibiting the expression of circALPK2.
[0039] 2. Lentiviral packaging
[0040] The three-plasmid lentiviral system was used to co-transfect the auxiliary plasmids (psPAX2 and pMD2G) and the constructed circALPK2-shRNA plasmid into 293T cells. The virus supernatant was collected at 48h and 72h after transfection (new culture medium was added after taking the supernatant 48h after transfection), filtered through a 0.45μm filter and centrifuged for 20min (4℃, 4000g). The virus precipitate was resuspended with 500μL basal culture medium to obtain a resuspension containing lentivirus (lentivirus resuspension), which was aliquoted and stored at -80℃.
[0041] 3. Obtaining circALPK2-shRNA interference cells
[0042] After commercial human pluripotent stem cells were induced to differentiate and purified cardiomyocytes were obtained, a culture medium containing a lentiviral resuspension was added (the cardiomyocyte culture medium was RPMI1640 basal culture medium containing 500 μg / mL BSA (Sigma-Aldrich), 213 μg / mL L-ascorbic acid 2-phosphate (Sigma-Aldrich), and 5 μg / mL of polybrene was added) for culture. 48 hours after transfection, the culture medium containing the lentiviral resuspension was discarded and replaced with fresh culture medium. The green fluorescence was observed under a fluorescence microscope to determine the transfection efficiency, and puromycin was added at a final concentration of 5 μg / mL for resistance screening to obtain circALPK2-shRNA interference cells. The total RNA of the constructed interference cells was extracted using the Trizol reagent method. Primers were designed, and the internal reference gene 18S was selected. Real-time fluorescence quantitative PCR was used to detect the expression of circALPK2 and proliferation-related genes CCNA2 and CDK1 in the interference cells. By Figure 2It can be seen that the expression of ALPK2 gene in the interference cells was not affected, while the expression of circular RNA circALPK2 decreased significantly. At the same time, the proliferation-related genes CCNA2 and CDK1 in the interference cells were increased to varying degrees, indicating that inhibiting the expression of circALPK2 helps to improve the proliferation ability of cardiomyocytes.
[0043] Example 2: Effects of circALPK2 interference on cardiomyocyte proliferation
[0044] EdU (deoxyuracil) method is a method commonly used to detect DNA synthesis in cells. Its detection under a fluorescence microscope can reflect the proliferation of cells. The proliferation ability of cardiomyocytes was detected by EdU cell proliferation detection kit (Biyuntian Biotechnology Co., Ltd.). The results are as follows Figure 3 As shown in the figure, compared with the NC group, the proliferation ability of cardiomyocytes was significantly enhanced after circALPK2 interference, indicating that circALPK2 inhibits the proliferation ability of cardiomyocytes, and its inhibitor circALPK2-shRNA can effectively improve the proliferation ability of cardiomyocytes.
[0045] Example 3: Effects of circALPK2 interference on impaired cardiac function
[0046] 1. Establishment of Mouse Myocardial Infarction Model
[0047] Eight-week-old female SCID mice were anesthetized by intraperitoneal injection of 4% chloral hydrate (200 μL / mouse). After depilatory cream was used to remove hair from the mouse chest, the mouse was fixed on the operating table, intubated and connected to a ventilator, and the 3rd and 4th intercostal spaces on the left side of the mouse sternum were found under a stereoscope. A parallel incision was made between the ribs with a scalpel, the chest cavity was opened, the pericardium was cut open to expose the heart, and the upper 2 / 5 of the left coronary artery was ligated with a 6-0 suture. At the moment of ligation, the anterior wall of the left ventricle of the mouse was observed to be white, and the myocardial activity was weakened, and the myocardial infarction model was completed.
[0048] 2. circALPK2-shRNA interference cell transplantation
[0049] The mice were divided into four groups: sham group, myocardial infarction (MI) group, control cardiomyocytes transplanted after myocardial infarction (MI+CM-NC) treatment group, and interference cells transplanted after myocardial infarction (MI+CM-sh) treatment group. After the myocardial infarction model was successfully established, the MI+CM-NC group and MI+CM-sh group were injected with cells at three points in the myocardium. The myocardial muscle turned white, indicating that the myocardial cell transplantation was successful. After the injection, the chest cavity was closed layer by layer using 3-0 sutures, the ventilator was disconnected, and the mice were placed on a warm blanket for observation and awakening.
[0050] 3. Heart function test
[0051] Thoracic echocardiography was performed 28 days after surgery, and visualsonic analysis software was used to calculate left ventricular ejection fraction (EF) and fractional shortening (FS) to reflect cardiac function. Figure 4 As shown in the figure, compared with the sham group, the cardiac function of the MI group was significantly decreased, and the cardiac function of the cardiomyocyte transplantation treatment group was improved. Compared with the MI+NC group, the treatment effect of the MI+sh group was the most significant, indicating that circALPK2 interference cells can significantly promote the functional repair of damaged myocardium.
[0052] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.
Claims
1. Use of circular RNA circALPK2 in the preparation of a cardiomyocyte repair and / or regeneration product, characterized in that: The nucleotide sequence of circ-ALPK2 is shown in SEQ ID NO.
1.
2. Use of a preparation for reducing the expression of circular RNA circALPK2 in the preparation of a cardiomyocyte repair and / or regeneration product, characterized in that: The nucleotide sequence of circ-ALPK2 is shown in SEQ ID NO.
1.
3. A preparation for promoting myocardial cell repair and / or regeneration, characterized in that: The preparation reduces the expression of circular RNA circALPK2 in cardiomyocytes.
4. The preparation according to claim 3, characterized in that: The preparation comprises short hairpin RNA, the sense strand of the short hairpin RNA is shown as SEQ ID NO.2, and the antisense strand of the short hairpin RNA is shown as SEQ ID NO.
3.
5. The preparation according to claim 3 or 4, characterized in that: The preparation contains small interfering RNA, the sense strand of the small interfering RNA is shown as SEQ ID NO.4, and the antisense strand of the small interfering RNA is shown as SEQ ID NO.
5.
6. The preparation according to claim 4, characterized in that: The preparation also includes one or more of a plasmid, a lentivirus and an adeno-associated virus containing the short hairpin RNA.
7. Application of preparations that reduce the expression of circular RNA circALPK2 in the preparation of drugs for the treatment of myocardial infarction.
8. A drug for treating myocardial infarction, characterized in that: The therapeutic drug targets circular RNA circALPK2 and reduces the expression of circular RNA circALPK2 in cardiomyocytes.
9. The drug according to claim 8, characterized in that: The preparation comprises short hairpin RNA and / or small interfering RNA, the nucleotide sequence of the short hairpin RNA is shown as SEQ ID NO.2-3, and the nucleotide sequence of the small interfering RNA is shown as SEQ ID NO.4-5.
10. The drug according to claim 8, characterized in that: The administration method of the drug includes intramyocardial injection or intravenous injection.