lncPostn as a biomarker for predicting cardiac fibrosis and its application

Abstract

The present invention belongs to the field of medicine, and specifically relates to a lncPostn as a biomarker for predicting cardiac fibrosis and its application. The nucleotide sequence of the lncPostn gene is shown in SEQ ID NO: 1. The present invention first discovered the lncPostn gene, found that it is homologous to humans and mice, and is expected to become a new target for the treatment of cardiac fibrosis after myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and specifically relates to lncPostn as a biomarker for predicting cardiac fibrosis and application thereof. Background Art

[0002] Heart failure is a leading cause of death, with a mortality rate of over 50% within five years of diagnosis, mainly caused by adverse ventricular remodeling, cardiac fibrosis, and reduced ventricular compliance. Cardiac fibrosis is a wound healing response caused by myocardial injury, characterized by the accumulation of extracellular matrix proteins in the myocardial interstitium. Fibrotic remodeling may disrupt the electromechanical coordination between cardiomyocytes and impair the electrical conduction system, leading to systolic and diastolic dysfunction and sudden cardiac death. The severity of cardiac fibrosis is closely related to the long-term mortality of patients with heart failure. Clinically, although the new quadruple therapy can significantly reduce the all-cause mortality of patients with heart failure, there is still a lack of effective treatments to reduce or even reverse myocardial fibrosis due to the variability of cardiac fibrosis types and their complex pathological mechanisms. Therefore, there is an urgent need to develop effective therapies that directly target cardiac fibroblast sparks and fibrotic remodeling.

[0003] Long non-coding RNAs (lncRNAs) are a heterogeneous class of long transcripts (more than 200 nucleotides). Studies have shown that lncRNAs play a key role in regulating atherosclerosis, plaque inflammation, cell hypertrophy and proliferation. Some lncRNAs have been identified as key regulators of cardiac fibroblast proliferation and regulation of fibrotic gene expression. Super-enhancer-associated lncRNA-Wisper controls the expression of fibrotic Loxl2, which contributes to collagen cross-linking and cell matrix stabilization. lncRNA-Meg3, which is enriched in cardiac fibroblasts, promotes myocardial fibrosis by regulating MMP-2. SAFB interacts with lncRNA-SAIL and regulates SAFB-mediated transcription of fibrosis-related genes. However, the current research on the molecular mechanisms underlying cardiac fibrosis and the role of lncRNAs in myocardial fibrosis is still very limited. Summary of the invention

[0004] In the present invention, the inventors screened the myocardial tissue of the proximal area after myocardial infarction in mice and the control using non-coding RNA chips, and found that lncRNA-NONMMUT043086 was upregulated from 1 day after myocardial infarction in mice, and the increase was most obvious 21 days after infarction. Compared with the proximal and distal areas, the increase in the myocardial infarction area was significant. At the same time, the inventors verified in the TGF-β-induced cell fibrosis model that lncRNA-NONMMUT043086 was also significantly increased. Compared with myocardial cells, lncRNA specifically acts on cardiac fibroblasts (Figure 1 ). Through UCSC sequence alignment, the inventors found that lncRNA-NONMMUT043086 was located in exon 17, adjacent to the coding gene Periostin (Postn), so the inventors named it lncPostn and further explored the role of lncPostn in cardiac fibrosis.

[0005] Specifically, the technical solution of the present invention is as follows:

[0006] The first aspect of the present invention discloses a lncPostn gene, whose nucleotide sequence is shown in SEQ ID NO: 1.

[0007] The second aspect of the present invention discloses a biomarker for predicting cardiac fibrosis, wherein the biomarker comprises the above-mentioned lncPostn gene.

[0008] Preferably, the biomarker is a biomarker of ventricular remodeling and cardiac fibrosis in patients with acute myocardial infarction.

[0009] The fourth aspect of the present invention discloses the use of the above-mentioned lncPostn gene as a new target for clinical improvement of cardiac fiber.

[0010] The fifth aspect of the present invention discloses a method for constructing an in vitro model for non-diagnostic and therapeutic purposes for verifying the application of the lncPostn gene as a new target for clinical improvement of cardiac fiber, comprising:

[0011] S1: A vector knocking down the lncPostn gene was constructed to detect the function of lncPostn in a TGF-β-induced mouse cardiac fibrosis model;

[0012] S2: A vector overexpressing the lncPostn gene was constructed to detect the function of lncPostn in the TGF-β-induced mouse cardiac fibrosis model.

[0013] Preferably, detecting the function of lncPostn includes immunofluorescence or flow cytometry detection indicators, and the indicator is the ratio of α-SMA / EdU double-positive cells.

[0014] Preferably, the function of detecting lncPostn includes fluorescent quantitative PCR indicators, and the indicators include the expression levels of α-SMA, Col1a1, Col3a1, and CTGF.

[0015] The sixth aspect of the present invention discloses a method for constructing an in vivo model for non-diagnostic and therapeutic purposes for verifying the application of the lncPostn gene as a new target for clinical improvement of cardiac fiber, comprising:

[0016] The mice underwent left anterior descending coronary artery ligation surgery to construct a myocardial infarction model, and the vector for knocking down the lncPostn gene was injected into the tail vein of the mice.

[0017] The seventh aspect of the present invention discloses the application of the above-mentioned lncPostn gene, the above-mentioned biomarker, the above-mentioned application or the above-mentioned method in the medical field.

[0018] Preferably, the application is in the field of cardiac fibrosis.

[0019] Based on the common sense in the art, the above-mentioned preferred conditions can be combined arbitrarily without exceeding the concept and protection scope of the present invention.

[0020] Compared with the prior art, the present invention has the following significant advantages and effects:

[0021] The present invention discovered the lncPostn gene for the first time and found that it is homologous to humans and mice, and is expected to become a new target for the treatment of cardiac fibrosis after myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of screening differentially expressed lncRNAs in cardiac fibrosis models. AC: Chip screening of lncRNAs that changed in the post-MI remodeling model (n=5). D: lncRNAs with a change of >5 times were selected for fluorescence quantitative PCR verification (n=5) (each group in the bar graph in the figure is Sham group and MI group from left to right). E: Fluorescence quantitative PCR was performed to verify the changed lncRNAs in the cell model (n=5) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right). F: Fluorescence quantitative PCR detected that lnc043086 (lncPostn) was more highly expressed in fibroblasts (n=5) (each group in the bar graph in the figure is NMCF group and NMCM group from left to right). G: Compared with the control group, the expression of lncPostn began to increase 1 day after myocardial infarction, and the increase was most obvious on day 21 (n=4) (each group in the bar graph in the figure is Sham group and MI group from left to right).

[0023] Figure 2Schematic diagram of knocking down lncPostn to improve TGF-b-induced cell fibrosis. A: Fluorescence quantitative PCR verified the efficiency of lncPostn small interfering RNA (n=5). B: Fluorescence quantitative PCR detected that knocking down lncPostn inhibited the transformation of fibroblasts (n=5) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right). CE: Immunofluorescence detection of knocking down lncPostn inhibited the proliferation of fibroblasts (n=4) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right). FH: Flow cytometry detection of knocking down lncPostn inhibited the proliferation of fibroblasts (n=4) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right).

[0024] Figure 3 Schematic diagram of overexpression of lncPostn to promote cell fibrosis. A: Fluorescence quantitative PCR verified the efficiency of lncPostn overexpression adenovirus (n=5). B: Fluorescence quantitative PCR detected that overexpression of lncPostn promoted the transformation of fibroblasts (n=5) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right). CE: Immunofluorescence detection of overexpression of lncPostn to promote the proliferation of fibroblasts (n=4) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right). FH: Flow cytometry detection of overexpression of lncPostn to promote the proliferation of fibroblasts (n=4) (each group in the bar graph in the figure is CTL group and TGF-β1 group from left to right).

[0025] Figure 4 Schematic diagram of the fact that knocking down lncPostn under physiological conditions has no statistical effect on the cardiac function of mice. A: Fluorescence quantitative PCR to detect the efficiency of lncPostn knockdown adenovirus injected into the tail vein in vivo (n=3). BD: Echocardiography to detect the cardiac function of mice knocked down lncPostn and its control group. E: Fluorescence quantitative PCR to detect the expression of fibrosis-related indicators in myocardial tissue after knocking down lncPostn in vivo (n=3) ( Figure 4 Each group in the middle bar graph is the sh-nc group and the sh-lncPostn group from left to right).

[0026] Figure 5 Schematic diagram of in vivo knockdown of lncPostn to improve the cardiac function of mice one day after myocardial infarction. AB: Changes in electrocardiogram and TTC staining (infarct size) one day before and after myocardial infarction, indicating that the model was successfully established (n=3). CF: Echocardiography to detect the cardiac function of mice with in vivo knockdown of lncPostn and their control group one day before and after myocardial infarction (n=10). G: TTC staining to detect the infarct size of mice with in vivo knockdown of lncPostn and their control group one day before and after myocardial infarction (n=3) ( Figure 5 Each group in the middle bar graph from left to right is sham+sh-nc group, MI+sh-nc group and MI+sh-lncPostn group).

[0027] Figure 6 Schematic diagram of in vivo knockdown of lncPostn to reduce cardiac fibrosis and improve cardiac function after myocardial infarction. AE: Echocardiography detected cardiac function and heart weight / body weight at different time points after myocardial infarction, including 1, 7, 14, and 21 days (n=9) (the curves in Figure CE are from top to bottom for the sham+sh-nc group, MI+sh-nc group, and MI+sh-lncPostn group). FG: Masson staining was used to detect the degree of cardiac fibrosis at different time points after myocardial infarction (n=3) (the curves in Figure G are from top to bottom for the MI+sh-nc group, MI+sh-lncPostn group, and sham+sh-nc group). H: Collagen volume was used to detect the collagen activity of the three groups 21 days after myocardial infarction (n=3). I: Immunoblotting detected the changes in fibrosis-related indicators after in vivo knockdown of lncPostn (n=3) ( Figure 6 The bars in H are sham+sh-nc group, MI+sh-nc group and MI+sh-lncPostn group from left to right).

[0028] Figure 7 Schematic diagram of knocking down lncPostn in vivo to reduce the proliferation of fibroblasts. AB: Immunofluorescence staining of PDGFRa / Ki67 / DAPI to detect the effect of knocking down lncPostn on the proliferation of fibroblasts (n=3). CD: Immunofluorescence staining of PDGFRa / EGFP / EdU / DAPI to detect the effect of knocking down lncPostn on the proliferation of fibroblasts (n=3) (the curves in the figure are MI+sh-nc group, MI+sh-lncPostn group and sham+sh-nc group from top to bottom).

[0029] Figure 8 Schematic diagram of in vivo knockdown of lncPostn to reduce fibroblast activation. AD: Fluorescence quantitative PCR detection of the effect of knockdown of lncPostn on fibroblast activation-related indicators (n=4). EF: Masson staining detection of the effect of knockdown of lncPostn on cardiac fibrosis at 7 days and 14 days (n=3).

[0030] Fig. 9Schematic diagram of increased serum lncPostn levels in patients with acute myocardial infarction. AC. Serum lncPostn (A), ST-2 (B), and TIMP-1 (C) levels in patients with acute myocardial infarction (n=42) and healthy subjects (n=41) after logarithmic transformation. DE. Correlation analysis between serum lncPostn levels (Log transformation) and ST-2 (D) and TIMP-1 (E) levels (Log transformation) in patients with acute myocardial infarction and healthy subjects (n=83). DETAILED DESCRIPTION

[0031] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but the present invention is not limited to the scope of the embodiments.

[0032] The experimental methods in the following examples without specifying specific conditions are selected according to conventional methods and conditions, or according to the product specifications. The reagents and raw materials used in the present invention are all commercially available.

[0033] Example 1 Function of lncPostn in cardiac fibrosis

[0034] 1. Knockdown of lncPostn reduces the proliferation and activation of fibroblasts

[0035] In order to clarify the function of lncPostn in the in vitro fibrosis model, the inventors constructed knockdown minimal interfering RNA (si-lncPostn) and its control (si-nc). The inventors isolated 1-3 day old newborn mouse fibroblasts, transfected si-lncPostn and its control, and verified its knockdown efficiency by fluorescence quantitative PCR. It was found that the knockdown efficiency of si-lncPostn was about 50% ( Figure 2 A). Next, the inventors tested the function of lncPostn in a TGF-β-induced mouse cardiac fibroblast fibrosis model. The inventors found that knocking down lncPostn reduced fibroblast proliferation (immunofluorescence and flow cytometry showed a decrease in the proportion of α-SMA / EdU double-positive cells) and activation (fluorescence quantitative PCR detected fibrosis-related indicators, including decreased expression of α-SMA, Col1a1, Col3a1, and CTGF) under both physiological conditions (CTL group) and TGF-β-stimulated fibrosis conditions. Figure 2 BH).

[0036] 2. Overexpression of lncPostn promotes proliferation and activation of fibroblasts

[0037] In order to clarify the function of lncPostn in the in vitro fibrosis model, the inventors also constructed adenovirus overexpressing lncPostn (lncPostn) and its control (nc). The inventors isolated 1-3 day old newborn mouse fibroblasts, transfected lncPostn and its control, and verified its overexpression efficiency by fluorescence quantitative PCR. It was found that the expression of lncPostn overexpressing adenovirus increased by about 15 times ( Figure 3 A). Next, the inventors tested the function of lncPostn in a TGF-β-induced mouse cardiac fibroblast fibrosis model and found that overexpression of lncPostn promoted fibroblast proliferation (immunofluorescence and flow cytometry assays indicated an increase in the proportion of α-SMA / EdU double-positive cells) and activation (fluorescence quantitative PCR assays detected fibrosis-related indicators, including increased expression of α-SMA, Col1a1, Col3a1, and CTGF) under physiological conditions. Figure 3 BH), and compared with TGF-β-stimulated fibroblasts, overexpression of lncPostn did not further promote the proliferation and activation of fibroblasts.

[0038] 3. In vivo knockdown of lncPostn reduces post-MI fibrosis in mice and improves post-MI remodeling and cardiac function

[0039] 1. Knockdown of lncPostn in vivo under physiological conditions does not affect cardiac function

[0040] In order to verify the role of lncPostn in the cardiac fibrosis model in animals, the inventors constructed lncPostn short hairpin knockdown adeno-associated virus 9 (sh-lncPostn) and its control, and injected them into mice through the tail vein. After 7 days, cardiac function was detected by echocardiography, and myocardial tissue RNA was extracted. Fibrosis-related indicators were detected by fluorescent quantitative PCR. The results showed that the knockdown efficiency of sh-lncPostn-AAV9 was about 50% ( Figure 4 A). Compared with the control group (sh-nc), the fibrosis-related indicators of mice in the sh-lncPostn group decreased (fluorescence quantitative PCR detected fibrosis-related indicators, including decreased expression of α-SMA, Col1a1, Col3a1, and CTGF, Figure 4 E), but echocardiography showed no significant statistical difference in cardiac function (EF%, FS%, Figure 4 BD).

[0041] 2. In vivo knockdown of lncPostn does not improve infarct size 1 day after myocardial infarction

[0042] To further verify the function of lncPostn in the pathological cardiac fibrosis model, 7 days after the tail vein injection of (sh-lncPostn) and its control adenovirus virus, the inventors performed left anterior descending coronary artery ligation (MI) on the mice to construct a myocardial infarction model. The successful construction of the model was verified by electrocardiogram and TTC staining ( Figure 5 AB). Secondly, the inventors performed echocardiography, cardiac pathological Masson staining, immunofluorescence detection of fibroblast proliferation, and fluorescence quantitative PCR or immunoblotting on mice at different time points (including 1 day, 7 days, 14 days and 21 days after MI) to detect fibrosis-related indicators. The results showed that compared with the control group, the heart function of the mice in the MI+sh-lncPostn group improved 1 day after MI ( Figure 5 CF), further improved cardiac function and cardiac remodeling 7, 14, and 21 days after myocardial infarction ( Figure 6 AE). However, TTC staining showed that there was no statistical difference in infarction area between the MI+sh-lncPostn group and the control group ( Figure 5 G).

[0043] 3. Knockdown of lncPostn in vivo reduces the proliferation of fibroblasts after myocardial infarction

[0044] In animal experiments, the inventors have preliminarily proved that in vivo knockdown of lncPostn improves cardiac function and ventricular remodeling starting from one day after myocardial infarction and lasts until 21 days after surgery, but the regulation of lncPostn on fibrosis during the remodeling process is still unclear. To further verify the effect of lncPostn on fibroblasts at different time points in the in vivo fibrosis model, the inventors injected EdU reagent into mice intraperitoneally in advance and detected the effect of lncPostn on fibroblast proliferation by immunofluorescence (Ki67 / PDGFRα / DAPI, EdU / PDGFRα / DAPI / EGFP). The results showed that compared with the MI+sh-nc group, the proliferation of fibroblasts stained in frozen sections of the hearts of MI+sh-lncPostn mice decreased from one day after surgery and lasted until 21 days (Ki67 + PDGFRα + Cell ratio and EdU + PDGFRα + The proportion decreases, Figure 7 AD).

[0045] 4. Knockdown of lncPostn in vivo reduces activation of fibroblasts after myocardial infarction

[0046] Secondly, to verify the regulatory effect of lncPostn on cardiac fibroblast activation in vivo, the inventors also performed pathological Masson staining, fluorescence quantitative PCR or immunoblotting on the heart at different time points (including 1 day, 7 days, 14 days and 21 days after myocardial infarction) to detect fibrosis-related indicators. The results showed that compared with the MI+sh-nc group, pathological Masson staining of the MI+sh-lncPostn group mice showed a significant reduction in cardiac fibrosis starting from 7 days after myocardial infarction ( Figure 6 F, Figure 8 EF), which lasted until 21 days. Fluorescence quantitative PCR showed that cardiac fibrosis-related indicators (including α-SMA, Col1a1, Col3a1, and CTGF) began to increase 1 day after myocardial infarction, reached the highest peak on 14 days, and decreased on 21 days compared with 14 days. Compared with the MI+sh-nc group, the myocardial fibrosis indicators of mice in the MI+sh-lncPostn group had no statistical difference 1 day after myocardial infarction, and began to decrease from 7 days to 21 days ( Figure 8 AD). In summary, combined with the results of cardiac function, degree of cardiac fibrosis, proliferation and activation of fibroblasts in mice after MI at different time points, the inventors considered that: 1 day after MI, knocking down lncPostn in vivo reduced fibroblast proliferation, but did not affect fibroblast activation. Starting from 7 days after MI, knocking down lncPostn in vivo reduced fibroblast proliferation and activation, thereby reducing cardiac fibrosis, improving ventricular remodeling and cardiac function.

[0047] Example 2 lncPostn is expected to become a new target for clinical improvement of cardiac fibrosis

[0048] Mouse animal and cell experiments The inventors have confirmed that lncPostn begins to be upregulated 1 day after myocardial infarction, with the most significant increase at 21 days. Knockdown of lncPostn improves cardiac fibrosis and ventricular remodeling after myocardial infarction. Through bioinformatics homology analysis, the inventors found that the homology of lncPostn in mice and humans is 57%, suggesting that lncPostn can be used as a new target for improving cardiac fibrosis clinically. From January to December 2020, the inventors recruited patients and healthy controls 7 days after acute myocardial infarction in the Third Affiliated Hospital of Soochow University (Changzhou First People's Hospital) with written informed consent. Inclusion criteria were 1) 40-70 years old, 2) diagnosed with ST-segment elevation myocardial infarction, 3) complete revascularization by percutaneous coronary intervention (PCI), and 4) receiving standard medical treatment at the discretion of the attending physician. Exclusion criteria were 1) pregnancy, 2) the following comorbidities, including tumors, thyroid dysfunction, liver and kidney dysfunction, immune and rheumatic diseases, or major mental illness. The control group consisted of healthy volunteers with no history of cardiovascular disease. The clinical data of patients with acute myocardial infarction and healthy subjects were obtained from their medical records. The relative expression of lncPostn in serum was determined by qRT-PCR. Fig. 9 As shown in A: Compared with healthy people, the expression of lncPostn in the serum of patients with acute myocardial infarction is significantly increased, which is consistent with the results of mouse animal and cell experiments. Therefore, combined with the results of animal, cell and clinical sample studies, the inventors believe that lncPostn is homologous to humans and mice and is expected to become a new target for the treatment of cardiac fibrosis after myocardial infarction.

[0049] Previous studies have shown that ST-2 and TIMP-1 can be used as serological biomarkers of cardiac fibrosis. In this study, compared with healthy subjects, the expression of ST-2 and TIMP-1 in the serum of patients with acute myocardial infarction was significantly increased. Correlation analysis showed that the expression of lncPostn was positively correlated with ST-2 and TIMP-1 ( Fig. 9 BE), suggesting that serum lncPostn expression could serve as a potential biomarker for ventricular remodeling and cardiac fibrosis in patients with acute myocardial infarction.

[0050] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting lncPostn expression in the preparation of a product for diagnosing ventricular remodeling and cardiac fibrosis in patients with acute myocardial infarction, wherein the nucleotide sequence of the lncPostn gene is shown in SEQ ID NO.1.

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