Use of E3 ubiquitin ligase TRIM40 in preparation of a drug for treating myocardial hypertrophy

Abstract

The application belongs to the technical field of biological medicine, and provides an application of E3 ubiquitin ligase TRIM40 in preparation of a drug for treating myocardial hypertrophy, wherein the application first discloses that E3 ubiquitin ligase TRIM40 is highly expressed in pathological myocardial hypertrophy and promotes myocardial cell hypertrophy, thereby providing a theoretical basis for the E3 ubiquitin ligase TRIM40 as a therapeutic target. In addition, the application finds that the E3 ubiquitin ligase TRIM40 regulates the function of STAT3 by directly combining and ubiquitinating STAT3, thereby expanding the upstream regulation mechanism of STAT3 in myocardial hypertrophy, and providing a potential target and a theoretical basis for developing a novel anti-myocardial hypertrophy drug targeting the TRIM40-STAT3 pathway.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of E3 ubiquitin ligase TRIM40 in preparation of a drug for treating myocardial hypertrophy. BACKGROUND

[0002] Myocardial hypertrophy is a compensatory response of myocardial tissue to cope with the increase of heart load, and is involved in the common pathological process of various cardiovascular diseases, such as heart valve disease, cardiomyopathy, hypertensive heart disease, etc., and is one of the independent risk factors of cardiovascular disease, and seriously leads to the occurrence of cardiovascular disease. Myocardial hypertrophy can be divided into physiological hypertrophy and pathological hypertrophy. Short-term physiological myocardial hypertrophy refers to myocardial hypertrophy caused by pregnancy, physical exercise, etc. Long-term pathological myocardial hypertrophy is a decompensated change of the heart when resisting a series of pathological stimuli (such as angiotensin II, Ang II). Pathological myocardial hypertrophy can cause cardiac systolic and diastolic dysfunction, and eventually lead to risks such as heart failure, arrhythmia, myocardial ischemia and even sudden death, and is specifically manifested in the enlargement of myocardial cell volume, the over-activation of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), beta-myosin heavy chain (beta-MyHC) and other fetal series genes, accompanied by myocardial cell apoptosis and necrosis, etc.

[0003] At present, the drugs for treating myocardial hypertrophy in clinical practice mainly include angiotensin-converting enzyme inhibitors, angiotensin II receptor 1 blockers and beta receptor inhibitors, etc. These drug treatment measures can temporarily alleviate the symptoms of patients, and some can slow down the development process of myocardial hypertrophy to heart failure, but these drugs are accompanied by different degrees of toxic side effects. This shows that the pathological mechanism of myocardial hypertrophy is still not completely clear, and higher requirements are put forward for further revealing the pathological process of myocardial hypertrophy, finding new targets and new drugs. Therefore, starting from the key proteins in the regulation of myocardial cell pathophysiological function in cells, it has important theoretical and transformation significance to explore new mechanisms and new targets for treating myocardial hypertrophy.

[0004] TRIM40 is one of the members of the TRIM family in the E3 ubiquitin ligase RING domain family. E3 ubiquitin ligase TRIM40 is involved in the progression of various tumor diseases, such as targeted TRIM40 signal transduction can reduce the development of esophageal cancer, TRIM40 inhibits the activity of NF-κB through NF-κB kinase subunit gamma inhibitor, and TRIM40 can prevent gastrointestinal inflammation-related carcinogenesis. However, the role of TRIM40 in myocardial hypertrophy induced by hypertension is still unclear. SUMMARY

[0005] The purpose of the present application is to provide the application of E3 ubiquitin ligase TRIM40 in the preparation of a drug for treating myocardial hypertrophy, and to solve the problems raised in the background art.

[0006] In order to solve the above problems, the application is achieved by providing an application of a substance for inhibiting expression of E3 ubiquitin ligase TRIM40 in the preparation of a drug for treating myocardial hypertrophy, wherein the E3 ubiquitin ligase TRIM40 is used as a therapeutic target.

[0007] Preferably, the myocardial hypertrophy is pathological myocardial hypertrophy.

[0008] Preferably, the myocardial hypertrophy is pathological myocardial hypertrophy induced by hypertension.

[0009] Preferably, the substance for inhibiting expression of E3 ubiquitin ligase TRIM40 comprises siRNA; the sequence of the sense strand and the antisense strand of the siRNA is shown in SEQ ID NO. 1-2.

[0010] Another object of the application is to provide a drug for treating myocardial hypertrophy, which comprises a pharmaceutically acceptable carrier and further comprises a substance for inhibiting expression of E3 ubiquitin ligase TRIM40.

[0011] The application discloses, for the first time, that E3 ubiquitin ligase TRIM40 is highly expressed in pathological myocardial hypertrophy and promotes hypertrophy of myocardial cells, thereby providing a theoretical basis for the E3 ubiquitin ligase TRIM40 as a therapeutic target. In addition, the application discloses that E3 ubiquitin ligase TRIM40 regulates the function of STAT3 by directly binding to and ubiquitinating STAT3, thereby expanding the upstream regulation mechanism of STAT3 in myocardial hypertrophy and providing a potential target and a theoretical basis for developing a new type of anti-myocardial hypertrophy drug targeting the TRIM40-STAT3 pathway. BRIEF DESCRIPTION OF DRAWINGS

[0012] Figure 1 Figure 1 shows experimental results that E3 ubiquitin ligase TRIM40 may be involved in the occurrence and development of myocardial hypertrophy. In the figure, A shows the results of transcriptome sequencing, which reveals the expression profile of E3 ubiquitin ligase in the heart tissue of Ang II-induced mice, wherein the difference is statistically significant when the difference ratio is greater than 2 and p is less than 0.05; B shows a representative immunoblotting image of TRIM40 in the heart tissue of Ang II-induced mice, with GAPDH as a control; and C shows representative immunofluorescence staining pictures of TRIM40 (red), alpha-actinin (green) or vimentin (green) in the heart section of Ang II-induced mice.

[0013] Figure 2Figure 1 is a graph of experimental results for silencing TRIM40 significantly alleviates Ang II-induced cardiomyocyte hypertrophy in H9c2 cells. In the figure, A is a graph of rhodamine-phalloidin staining to assess cardiomyocyte size after Ang II induction (scale bar = 50 pm); B is a graph of quantification of cell size changes in H9c2 cells in response to Ang II, at least 100 cells were measured in different fields of 3 samples per group; C is a representative immunoblot image of b-MyHC in H9c2 cells, with GAPDH as a control; D is a density quantification of the immunoblot; E is a graph of mRNA levels of Myh7 gene detected in H9c2 cells, with Actb as a control.

[0014] Figure 3 Figure 2 is a graph of experimental results for overexpression of TRIM40 significantly aggravates Ang II-induced cardiomyocyte hypertrophy in H9c2 cells. In the figure, A is a graph of rhodamine-phalloidin staining to assess cardiomyocyte size after Ang II induction (scale bar = 50 pm); B is a graph of quantification of cell size changes in H9c2 cells in response to Ang II, at least 100 cells were measured in different fields of 3 samples per group; C is a representative immunoblot image of b-MyHC in H9c2 cells, with GAPDH as a control; D is a density quantification of the immunoblot; E is a graph of mRNA levels of Myh7 gene detected in H9c2 cells, with Actb as a control.

[0015] Figure 4 Figure 3 is a graph of experimental results for TRIM40 binds STAT3. In the figure, A is a schematic diagram of quantitative proteomic screening to identify proteins binding to TRIM40; B is a schematic diagram of mass spectrometry results showing the structure of STAT3 protein; C, D, and E are Co-IP results showing the interaction of TRIM40 with STAT3 in neonatal rat primary cardiomyocytes (NRVMs), HEK-293T tool cells, and mouse heart tissue, respectively.

[0016] Figure 5 Figure 4 is a graph of experimental results for TRIM40 binds and ubiquitinates STAT3. In the figure, A is a graph of Western blot analysis of STAT3 ubiquitination levels in HEK-293T cells co-transfected with HA STAT3 plasmid, Flag TRIM40, and different types of Myc-Ub plasmids, CO-IP experiments were performed 24 hours after transfection; B is a graph of Western blot analysis of STAT3 ubiquitination levels in HEK-293T cells co-transfected with HA STAT3 plasmid, Flag TRIM40, and different types of Myc-Ub plasmids (WT, K48, and K63), CO-IP experiments were performed 24 hours after transfection; Note: cells were pretreated with 10 µM of MG132 for 6 h before collection.

[0017] Figure 6 Schematic diagram of the molecular mechanism of TRIM40 regulating STAT3 ubiquitination. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with the drawings and examples. It should be understood that the specific examples described herein are only used to explain the present application and do not limit the present application.

[0019] So far, cardiovascular disease has been a growing public health problem worldwide. Although great progress has been made in the prevention and treatment of hypertension heart failure (i.e. pathological hypertrophy of myocardium induced by hypertension) nowadays, there are still some limitations due to the unclear mechanism of occurrence. Therefore, it is of great significance to clarify the mechanism of occurrence and development of hypertension heart failure and to find possible therapeutic targets for the prevention and treatment of hypertension heart failure.

[0020] The present application fully clarifies the role and specific mechanism of TRIM40 in hypertension heart failure. Based on these studies, it is speculated that the high expression of TRIM40 in myocardial tissue and the abnormal activation of STAT3 may have the potential to become a biomarker molecule for predicting the prognosis of patients with hypertension heart failure. In addition, from the perspective of TRIM40 specifically catalyzing STAT3 to mediate hypertension heart failure, some small molecule inhibitors specific to TRIM40 / STAT3 complex are designed to prevent and treat hypertension heart failure, which can further improve the quality of life and prognosis of patients with hypertension heart failure when combined with the current clinical blood pressure management drugs.

[0021] Example 1: E3 ubiquitin ligase TRIM40 may be involved in the occurrence and development of myocardial hypertrophy: In the previous work of the present application, a mouse myocardial hypertrophy model was constructed by subcutaneous micro-pump of angiotensin II (Ang II), and the transcription level of E3 ubiquitin ligase gene was analyzed in the heart tissue transcriptome sequencing data, and the results are shown in Figure 1 The results show that among the E3 ubiquitin ligase genes, one E3 ubiquitin ligase gene has no report on myocardial hypertrophy, the gene is up-regulated and the change fold is more than 2 times, i.e. TRIM40. The expression of TRIM40 in the myocardial tissue of heart failure mice was also detected by the present application, as shown in Figure 1 B, compared with healthy mice (CON group), the level of TRIM40 in the heart tissue of Ang II model mice was increased, while the level of GAPDH had no obvious change. Then, the expression and cell source of TRIM40 in the heart section were further analyzed by fluorescence double staining, and the results Figure 1TRIM40 expression was mainly increased in a-actinin positive cardiomyocytes, but relatively weak in vimentin positive fibroblasts. Therefore, it can be considered that cardiomyocyte TRIM40 can be a potential regulator of myocardial hypertrophy.

[0022] Example 2: Silencing TRIM40 in cardiomyocytes significantly alleviates Ang II-induced myocardial hypertrophy: To confirm the role of TRIM40 in cardiomyocytes in vitro, the following experiment was performed: first, siRNA sequences were screened and transfected in H9c2 cells to silence TRIM40, to find the best siRNA for silencing TRIM40; the sequences of the sense and antisense strands of the best siRNA are shown in SEQ ID NO. 1-2, specifically, the nucleotide sequence of the sense strand of the best siRNA is: GAAAGACUCAAUCGGAGAA, and the nucleotide sequence of the antisense strand is: UUCUCCGAUUGAGUCUUUC. The above-mentioned best siRNA was used for transfection to silence TRIM40, and Si TRIM40 was obtained.

[0023] Then, the cells were stimulated with Ang II for 48h, and rhodamine-phalloidin staining was performed. Phalloidin (also known as phalloidin) is a toxic cyclic heptapeptide obtained from the poisonous mushroom phalloidin, which can selectively bind to actin in animals and plants, and its effect is exactly the opposite of cytochalasin, which only binds to polymerized microfilaments, not to actin monomer molecules, making phalloidin staining a powerful tool for studying intracellular actin microfilaments, and facilitating the staining of the cytoskeleton. Under a fluorescence microscope, the fluorescence-labeled phalloidin can clearly show the morphology and distribution of intracellular microfilaments. Rhodamine-phalloidin staining showed that silencing TRIM40 can prevent Ang II-induced cell hypertrophy (as shown in Figure 2 A and B). In addition, this protective effect was seen when the hypertrophy-related factor β-MyHC was detected by Western blot (as shown in Figure 2 C and D). This protective effect was also seen when the hypertrophy-related factor Myh7 was detected by RT-PCR (as shown in Figure 2 E). The above experimental results confirmed that silencing TRIM40 can significantly inhibit Ang II-induced myocardial hypertrophy in vitro.

[0024] Example 3: TRIM40 overexpression exacerbates Ang II-induced hypertrophic response in cardiomyocytes: The results of the above example have shown that TRIM40 silencing significantly inhibited Ang II-induced hypertrophy and other damages. In this example, we investigated whether overexpression of TRIM40 in H9c2 cells has the opposite results. First, we exposed TRIM40 overexpressing (Flag-TRIM40) cells to Ang II for 48 h. Similar experiments as those described above for TRIM40 silencing were performed. Rhodamine-phalloidin staining showed that overexpression of TRIM40 exacerbated Ang II-induced hypertrophy in H9c2 cells (as shown in A and B of Figure 3 ). Similarly, Western blot and RT-PCR experiments showed that overexpression of TRIM40 exacerbated the hypertrophic response of H9c2 cells to Ang II (as shown in C, D, E of Figure 3 ). These results suggest that TRIM40 is involved in the expression of Ang II-induced hypertrophic factors in H9c2 cells.

[0025] Example 4: TRIM40 directly interacts with STAT3: E3 ubiquitin ligases regulate biological activities by affecting the degradation or function of substrate proteins. To identify potential substrate proteins regulated by TRIM40, we used TRIM40 immunoprecipitation and mass spectrometry to find potential binding proteins of TRIM40 in neonatal rat primary cardiomyocyte NVRMs. The schematic diagram of quantitative proteomic screening is shown in A of Figure 4 . Among the many TRIM40 binding proteins, we focused on the top-ranking STAT3 protein that binds to TRIM40. And our previous studies have shown that it plays a key role in regulating Ang II-induced hypertrophic remodeling after abnormal activation. Representative mass spectrum of STAT3 is shown in B of Figure 4 . To confirm this potential interaction between TRIM40 and STAT3, we transfected Flag-labeled TRIM40 into neonatal rat primary cardiomyocyte NVRMs and HEK-293T cells, and used Co-IP to confirm the interaction between STAT3 and TRIM40 again (as shown in C and D of Figure 4 ), and the binding of TRIM40 and STAT3 in mouse heart tissue was also confirmed (as shown in E of Figure 4 ). Therefore, it can be speculated that STAT3 may mediate cardiac hypertrophy as a candidate substrate of TRIM40 in cardiomyocytes.

[0026] Example 5: TRIM40 binds and ubiquitinates STAT3: The previous research results have confirmed that TRIM40 is involved in AngII-induced cardiomyocyte hypertrophy, and the preliminary results of the co-immunoprecipitation experiment have confirmed the interaction of TRIM40 and STAT3 (as shown in Figure 4 Considering that TRIM40 is an E3 ubiquitin ligase, therefore, the embodiment of the present application verifies the type of STAT3 ubiquitinated by TRIM40 by co-transfecting HA STAT3 plasmid, Myc-Ub plasmid and Flag TRIM40 plasmid, Co-IP (IP: HA-beads), and the preliminary results show that TRIM40 mediates the polyubiquitination of STAT3 (as shown in Figure 5 As shown in FIG. 1A), and the results show that TRIM40 mediates the polyubiquitination of STAT3 K63 chain (as shown in Figure 5 FIG. 1B).

[0027] As shown in Figure 6 The embodiment of the present application discloses that the E3 ubiquitin ligase TRIM40 is a new target in myocardial hypertrophy (the previous research on the E3 ubiquitin ligase TRIM40 mainly focuses on diseases such as tumors, and has not been reported in heart diseases). The embodiment of the present application constructs a cardiomyocyte-specific TRIM40 knockout mouse by tail vein injection of AAV, and further clarifies the mechanism of cardiomyocyte TRIM40 in myocardial hypertrophy from the aspects of animals, cells and molecules, expands the new role and function of TRIM40, and provides a new biological mechanism explanation and treatment target for clarifying the occurrence and development of myocardial hypertrophy.

[0028] In the embodiment of the present application, the specific molecular mechanism of TRIM40 in regulating the substrate protein STAT3 in cardiomyocyte signal transduction will be further clarified. TRIM40 specifically catalyzes and regulates STAT3 ubiquitination, and the embodiment of the present application will reveal a new mechanism of TRIM40-STAT3-myocardial hypertrophy pathological changes, and also provides a new upstream regulator for STAT3 regulating myocardial hypertrophy.

[0029] The above-described embodiments only express several embodiments of the present application, which are described in detail and in detail, but cannot be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. The application of a substance that inhibits the expression of E3 ubiquitin ligase TRIM40 in the preparation of drugs for treating myocardial hypertrophy, characterized in that, The E3 ubiquitin ligase TRIM40 is used as a therapeutic target; the substance that inhibits the expression of E3 ubiquitin ligase TRIM40 is siRNA; the sense and antisense strand sequences of the siRNA are shown in SEQ ID NO.1-2, respectively.

2. The application according to claim 1, characterized in that, The myocardial hypertrophy mentioned is pathological myocardial hypertrophy.

3. The application according to claim 2, characterized in that, The myocardial hypertrophy mentioned refers to pathological myocardial hypertrophy induced by hypertension.

4. A drug for treating myocardial hypertrophy, comprising a pharmaceutically acceptable carrier, characterized in that, It also includes a substance that inhibits the expression of E3 ubiquitin ligase TRIM40; the substance that inhibits the expression of E3 ubiquitin ligase TRIM40 is siRNA; the sense and antisense strand sequences of the siRNA are shown in SEQ ID NO.1-2, respectively.

Images