Use of a lncrna in the manufacture of a medicament for treating cardiac injury

Abstract

The application provides a long non-coding RNA (lncRNA) lncRNAPlekhart sequence related to heart injury and application thereof in promoting recovery of heart injury and preparation of a drug for treating heart injury.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and more specifically, to the application of a lncRNA in the preparation of a drug for treating heart injury. Background Technology

[0002] Myocardial infarction (MI) is one of the leading causes of morbidity and mortality in cardiovascular disease patients worldwide. Due to the massive loss of cardiomyocytes, the infarcted heart undergoes infarct remodeling and eventually develops into heart failure. A major strategy in the treatment of myocardial infarction is to treat it by reducing the loss of cardiomyocytes through exogenous or endogenous stimulation of cardiomyocyte proliferation.

[0003] Recent studies have shown that neonatal rat cardiomyocytes possess regenerative capacity within 7 days, and that the hearts of rats that underwent apical resection on day 1 could fully regenerate within 21 days. This significantly enhances the possibility of repairing infarcted hearts through endogenous enhancement of cardiac regeneration. Significant efforts have been made to identify novel molecules and molecular mechanisms with therapeutic potential for addressing myocardial infarction-related cell loss. Transcription factors such as Meis1, Pitx2, Tbx20, and Gata4 play crucial roles in regulating the proliferative capacity of mammalian cardiomyocytes. The Hippo / YAP1 signaling pathway is known to regulate cardiomyocyte division and the regeneration of damaged cardiomyocytes through multiple transcriptional mechanisms.

[0004] Non-coding RNAs (ncRNAs) are RNAs that do not encode proteins. They regulate gene expression through various mechanisms and perform a wide range of biological functions, such as apoptosis, proliferation, and differentiation. New research indicates that small ncRNAs (miRNAs) and long ncRNAs (lncRNAs) play important roles in tissue development, cancer, neurodegenerative diseases, and heart disease. LncRNAs have recently been identified as key regulators of cardiac development, physiological function, and pathological processes. However, current knowledge about the regulation of heart disease by lncRNAs remains quite limited, especially regarding their potential role in regulating cardiomyocyte proliferation and cardiac repair. Summary of the Invention

[0005] To fill a gap in existing technology, the inventors unexpectedly discovered the function of lncRNA Plekhart in reactivating cardiomyocyte proliferation and promoting cardiac regeneration after myocardial injury during their research. Therefore, this invention provides the following technical solution:

[0006] A first aspect of the present invention provides the use of a lncRNA Plekhart inhibitor in the preparation of a medicament for treating cardiac injury.

[0007] A second aspect of the present invention provides the use of a lncRNA Plekhart inhibitor in the preparation of a medicament for treating cardiac fibrosis.

[0008] A third aspect of the invention provides the use of a lncRNA Plekhart inhibitor in the preparation of a medicament for treating myocardial infarction.

[0009] A fourth aspect of the present invention provides the use of a lncRNA Plekhart inhibitor in the preparation of a medicament for promoting cardiomyocyte regeneration.

[0010] In one embodiment, the lncRNAPlekhart inhibitor is an interfering sequence of lncRNAPlekhart.

[0011] In a preferred embodiment, the interfering sequence is selected from shRNA or siRNA.

[0012] A fourth aspect of the present invention provides the use of lncRNAPlekhart in the preparation of products for diagnosing cardiac injury.

[0013] In one embodiment, the product is a reagent kit or a gene chip.

[0014] Myocardial infarction and subsequent heart failure remain leading causes of death worldwide. Early reperfusion and drug therapy can reduce morbidity and mortality, but the progression of cardiac remodeling remains irreversible, primarily caused by the loss of cardiomyocytes. The adult mammalian heart possesses a limited potential for self-renewal. The neonatal mammalian heart retains a brief regenerative capacity after birth, which gradually disappears after birth. We identified a lncRNA, Plekhart, that shows progressively increasing expression in cardiomyocytes during mouse development. Attached Figure Description

[0015] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0016] Figure 1 To verify the differentially expressed lncRNAs in the myocardial tissues of 1-day-old suckling mice and 28-day-old adult mice screened by high-throughput sequencing using real-time quantitative PCR in Example 1;

[0017] Figure 2 The results of real-time quantitative PCR detection of lncRNA Plekhart expression in Example 2;

[0018] Figure 3 This is a fluorescence imaging result image from Example 3;

[0019] Figure 4 The echocardiogram in Example 4;

[0020] Figure 5 The results of Masson staining in Example 4;

[0021] Figure 6 The results of determining the infarct area using Image Pro Plus 6.0 in Example 4;

[0022] Figure 7 The results of co-focused laser scanning are shown in Example 4. Detailed Implementation

[0023] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0024] Example 1: Expression of lncRNA Plekhart during mouse heart development

[0025] Sample collection: Mice (C57BL / 6J mice, purchased from Guangzhou Yancheng Biotechnology Co., Ltd.) were anesthetized with isoflurane, and heart tissues were harvested from 1-day-old mice (n=3) and 28-day-old mice (n=3) after thoracotomy.

[0026] 1. Clean the tissue surface with pre-cooled PBS to remove residual blood, aliquot on ice, and quickly freeze in liquid nitrogen for 30 min. Store in an ultra-low temperature freezer at -80°C.

[0027] 2. Tissue RNA Preparation and Quantitative Analysis: Tissue was minced using enzyme-free ophthalmic scissors, and 1000 μl of Trizol reagent was added. The tissue was lysed using a tissue homogenizer and incubated at room temperature for 10 minutes. 200 μl of chloroform was added, the mixture was vortexed for 10 seconds, incubated at room temperature for 3 minutes, and centrifuged at 12000g for 15 minutes at 4°C. 400 μl of the supernatant was collected and 400 μl of pre-chilled isopropanol was added. The mixture was incubated at room temperature for 15 minutes and centrifuged at 2000g for 10 minutes at 4°C. The precipitate was washed with 1 ml of anhydrous ethanol and centrifuged at 7500g for 10 minutes at 4°C. The precipitate was air-dried and dissolved in 10 μl of DEPC water. PrimeScript was used for RNA analysis. TM The RT kit reversed cellular RNA into cDNA. Using the cDNA as a template, qRT-PCR was performed on the CFX96 Real-Time PCR detection system according to the TBGreen™ Premix ExTaq™ kit instructions.

[0028] 3. The primer sequences used to detect lncRNA Plekhart are as follows:

[0029] F:5-GCCAGACAGCAGCAAAGGAAGG-3

[0030] R:5-GACGGAGTGGTGATGGATTGATGG-3

[0031] The primers used for detecting GAPDH (internal reference) are as follows:

[0032] F:5'-ACCCACTCCTCCACCTTTGAC-3'

[0033] R:5'-TGTTGCTGTAGCCAAATTCGTT-3'

[0034] 4. Results:

[0035] like Figure 1 As shown, the expression level of lncRNA Plekhart in the heart tissue of 28-day-old adult mice was higher than that in the heart tissue of 1-day-old mice, and the difference was statistically significant (P<0.05).

[0036] Example 2: Effect of cardiac injury on lncRNA Plekhart expression

[0037] Newborn mice (n=5) were subjected to apical resection of the heart at 1 day old. The mice were anesthetized on ice for 4 minutes. All mice underwent thoracotomy and were divided into two groups: the surgical group (n=5) underwent approximately 10% ventricular resection to expose the left ventricle, while the control group (“sham surgery” group, n=5) underwent only thoracotomy. One day later, hearts were harvested from all mice in both groups, and tissue RNA was extracted. The cellular RNA was reverse-engineered into cDNA using a 5-PrimeScript™ RT kit. qRT-PCR was performed using a CFX96 Real-Time PCR system with the cDNA as a template, following the instructions of the TBGreen™ Premix ExTaq™ kit. The primer sequences for detecting lncRNA Plekhart and for detecting GAPDH (internal control) were the same as those described in Example 1.

[0038] The results are as follows Figure 2 As shown, the expression level of lncRNA Plekhart in the surgical animal group (which underwent apical resection) was significantly lower than that in the control group, and the difference was statistically significant (P<0.05).

[0039] Example 3: AAV9 adenovirus interference sequence can effectively reduce the expression of mouse cardiac lncRNA Plekhart.

[0040] Adult mice (n=5) were intubated and anesthetized with 2% isoflurane. A myocardial infarction model was then induced by ligating the left anterior descending coronary artery with 7-Oprolene sutures. After myocardial infarction, the mice were divided into two groups. The first group (n=5) received an intramyocardial injection of 1×10⁻⁶ oz. 11 Adenovirus Plekhart shRNA (synthesized and produced by Paizhen Biotechnology Co., Ltd.) was designated as the interference group, and another group (n=5) of mice were injected with empty viral vector, designated as the shNC group. The sequence of the adenovirus Plekhart shRNA is as follows:

[0041] shRNA positive strand: 5-GCAAGGAGTTGGAAGTTTAAT-3,

[0042] shRNA antisense strand: 5-UAAACUUCCAACUCCUUGCTT-3;

[0043] (AAV9 Adenovirus Pagin Biotech)

[0044] After surgery, the thoracic cavity was closed. The mice were then warmed on a heat-generating blanket for several minutes until their body temperature returned to normal. Twenty-eight days post-surgery, the mice were placed in an imaging darkroom platform (AniView100 multi-modal animal in vivo imaging system) after routine anesthesia (air or needle anesthesia). The software controlled the platform's elevation to a suitable field of view, automatically turning on the illumination (bright field) to capture the first background image. Subsequently, the illumination was automatically turned off, and background images of specific photons emitted from within the mouse's body were captured under conditions without external light (dark field). The superimposed images visually show the location and intensity of specific photons within the animal, completing the imaging operation. It is worth noting that appropriate excitation and emission filters should be selected for fluorescence imaging, while bioluminescence requires pre-implantation of a substrate for excitation. Fluorescence imaging conditions were: two groups of mice, three mice per group; excitation wavelength 625 nm; scattered wavelength 680 nm; exposure time 200 ms. Results are as follows. Figure 3 As shown in the left figure, the virus targets the mouse heart. Mouse hearts were collected for further qRT-PCR analysis, using the same method as in Example 2.

[0045] The results are as follows Figure 3 As shown in the right figure, the expression level of AAV9 adenovirus Plekhart shRNA in mice was significantly lower than that in mice in the empty vector group, and the differences were statistically significant (P<0.05).

[0046] Example 4: AAV9 adenovirus Plekhart shRNA promotes cardiac function recovery in mice after myocardial infarction.

[0047] Two groups of mice (interference group and control group) from Example 3 were evaluated for cardiac function using two-dimensional echocardiography 1 day before surgery and 1, 14, and 28 days after surgery. Echocardiography was performed using a Vevo 1100 VisualSonics device (VisualSoNIC, Toronto, Canada). Short-axis shortening (FS) and ejection fraction (EF) were measured using M-mode images in a parasternal short-axis view.

[0048] The heart was harvested 28 days after the surgery for the following experiments:

[0049] (1) Fix with 4% paraformaldehyde, embed in paraffin, section every 6 μm, and then perform Masson staining according to standard procedure;

[0050] (2) Cut it into 3 mm thick slices using a metal slicer. Incubate the slices in 1% TTC dissolved in PBS at room temperature for 15 minutes. Wash with PBS to stop the staining process, take pictures, and then use Image Pro Plus 6.0 to determine the infarct area.

[0051] (3) Hearts were rapidly frozen in liquid nitrogen with embedding medium and cut into 6 μm sections. These sections were fixed in paraformaldehyde, washed with PBS, blocked with goat serum, and incubated overnight with the primary antibody against myocardial proliferation markers PH3 and Ki67 (1:200) (abcam). The incubation mixture was then incubated with secondary antibodies, goat anti-mouse IgG and goat anti-rabbit IgG. DAPI staining was used to visualize cell nuclei. Images were acquired using a confocal laser scanning microscope (BX53; Olympus, Japan).

[0052] The results are as follows Figure 4-7 As shown, injection of AAV9 adenovirus Plekhart shRNA significantly improved both ejection fraction and shortening fraction in mice after myocardial infarction compared to the NC group. Figure 4 Furthermore, the area of ​​cardiac fibrosis and infarct size in mice were significantly reduced. Figure 5-6 Furthermore, an increase in the markers of myocardial proliferation, PH3 and Ki67, was detected, indicating a significant increase in myocardial proliferation. Figure 7 ).

[0053] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of a lncRNA Plekhart inhibitor in the preparation of a drug for treating cardiac fibrosis, characterized in that: The lncRNA Plekhart inhibitor is an interfering sequence of lncRNA Plekhart, and the interfering sequence is shRNA. shRNA positive strand: 5-GCAAGGAGTTGGAAGTTTAAT-3, shRNA antisense strand: 5-UAAACUUCCAACUCCUUGCTT-3.

2. The use of a lncRNA Plekhart inhibitor in the preparation of a drug for treating myocardial infarction, characterized in that: The lncRNA Plekhart inhibitor is an interfering sequence of lncRNA Plekhart, and the interfering sequence is shRNA. shRNA positive strand: 5-GCAAGGAGTTGGAAGTTTAAT-3, shRNA antisense strand: 5-UAAACUUCCAACUCCUUGCTT-3.

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