CN116590400A - Application of lncRNA-GRCm38 in preparation of drugs for detecting, preventing and/or treating heart failure - Google Patents
Application of lncRNA-GRCm38 in preparation of drugs for detecting, preventing and/or treating heart failure Download PDFInfo
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Abstract
The application discloses an application of lncRNA-GRCm38 in preparing a medicine for detecting, preventing and/or treating heart failure, and belongs to the technical fields of molecular biology and medicine. The lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, and experimental research shows that the improvement of the expression of the lncRNA-GRCm38 can improve heart failure by inhibiting myocardial fibrosis of mice, the lncRNA-GRCm38 provides potential targets for diagnosis, prevention and/or treatment of heart failure, and the lncRNA-GRCm38 can be used as a new molecular target of heart failure to guide screening and research of related drugs.
Description
Technical Field
The application belongs to the technical field of molecular biology and medicine, and particularly relates to application of lncRNA-GRCm38 in preparation of a medicine for detecting, preventing and/or treating heart failure.
Background
With lifestyle improvement and social aging, the incidence of cardiovascular disease is continuously rising. Reporting by the world health organization: the number of people who die from cardiovascular disease worldwide accounts for 1/3 of the various causes of death, more than half of which die from myocardial infarction. Although the application of interventional procedures and bypass surgery improves myocardial blood flow supply, it is not possible to rescue already dead cardiomyocytes, reverse ventricular remodeling and subsequent heart failure. The incidence rate of heart failure after myocardial infarction reaches 31.9% in 5 years, and the survival rate of heart failure after myocardial infarction is similar to that of malignant tumors, wherein the 1-year mortality rate of patients with New York cardiac function grade IV is as high as 50%. Therefore, promoting repair and functional reconstruction of myocardial injury after heart failure, inhibiting myocardial fibrosis and improving myocardial infarction curative effect have become key scientific problems in the field of cardiovascular research.
Myocardial fibrosis refers to excessive proliferation of fibroblasts or excessive deposition of extracellular matrix in myocardial tissue, and the concentration and volume of collagen are significantly increased, and the proportion and arrangement of various types of collagen are disordered. The pathological changes exist in various cardiovascular diseases such as hypertension, myocardial infarction, heart failure and the like, are important risk factors of sudden cardiac death, seriously threaten the health level of the whole population and reduce the quality of life, and also become a heavy economic burden of families and society. However, due to the complex mechanism of occurrence and development of myocardial fibrosis, no completely targeted therapeutic targets are found at present.
Long non-coding RNA (longnon-codingRNA, lncRNA) refers to a class of non-coding RNA that is greater than 200 nucleotides in length and that does not or weakly encodes a protein. They can be classified according to their location: (1) sense lncRNA, (2) antisense lncRNA, (3) bidirectional lncRNA, (4) intergenic lncRNA and (5) intronic lncRNA. More and more studies have shown that non-coding genes (including rRNA, tRNA, tiRNA, lncRNA, circRNA and RNAi) that occupy more than 97% of the human genome play important biological functions. Although research into non-coding RNAs (such as micrornas and pirnas) of less than 50 bases in length has made breakthrough progress, the manner in which lncrnas, which are more numerous and can fold into a specific spatial structure, interact with other biomolecules is different from micrornas and of greater significance. lncRNA is involved in gene regulation by the following pathways: a) Temporally and spatially regulating the expression of genes; b) Preventing binding of transcription factors or other proteins to chromatin; c) Detaching microRNAs from the target; d) Positioning a chromatin modifying enzyme to a cis or trans position of a target gene; e) Helper proteins assemble into ribosomal protein complexes. In recent years, the lncRNA is found to be related to growth, metastasis and invasion of tumor cells, transcription disorder of neurodegenerative diseases and cardiovascular diseases. The total expression amount of lncRNA in human heart tissues reaches 18480, but the functional characteristics of most lncRNAs are not analyzed yet. A new lncRNA was found by research: chaer, which is largely enriched in cardiac muscle, is an epigenetic regulatory molecule for myocardial hypertrophy. In the field of myocardial fibrosis research, researchers use gene chips and bioinformatics methods to generalize a few lncRNAs and mRNAs which are likely to regulate myocardial fibrosis, but lack identification and subsequent molecular biology and functional studies. Therefore, the lncRNA related to myocardial fibrosis is found and identified, the action target of the lncRNA in the heart failure process is studied in depth, the molecular mechanism is discussed, the novel drug target related to heart failure is favorably searched and identified, and the method is particularly important for early attack of cardiovascular diseases.
The application discovers a new lncRNA: GRCm38, to identify and characterize it, and to verify its role in myocardial fibrosis, to provide new research targets and ideas for screening new drugs associated with heart failure.
Disclosure of Invention
In view of the above, the application aims to provide an application of lncRNA-GRCm38 in preparing medicines for detecting, preventing and/or treating heart failure.
In order to achieve the purpose, the application adopts the following technical scheme:
the application provides an application of lncRNA-GRCm38 in screening or preparing a drug target for detecting, preventing and/or treating heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
Further, the drug is a drug for improving heart failure cardiac function.
Further, the drug is a drug for inhibiting myocardial fibrosis.
Further, the treatment of heart failure is achieved by increasing expression of lncRNA-GRCm 38.
The application provides an application of lncRNA-GRCm38 in preparing medicaments for detecting, preventing and/or treating heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
Further, the drug is a drug for improving heart failure cardiac function.
Further, the drug is a drug for inhibiting myocardial fibrosis.
Further, the treatment of heart failure is achieved by increasing expression of lncRNA-GRCm 38.
The third aspect of the application provides application of lncRNA-GRCm38 in preparing a kit, a reagent and/or a chip for detecting heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
Compared with the prior art, the application has the following advantages: by providing a new heart failure treatment target, the difficult problem of heart failure treatment is solved, and a reference is provided for clinical heart failure treatment; using aortic arch constriction (TAC) to cause a mouse heart failure model, and using RNA-seq to analyze a sham operation group and a heart failure group to find that lncRNA-GRCm38 is low-expressed in heart failure; in vitro experiments show that the overexpression of the lncRNA-GRCm38 can inhibit the TGF-beta induced myocardial fibroblast transdifferentiation, and in vivo experiments also prove that the overexpression of the lncRNA-GRCm38 can significantly improve the heart function of a mouse with a TAC model and inhibit myocardial fibrosis, thus indicating that the lncRNA-GRCm38 is one of therapeutic targets of heart failure.
Drawings
FIG. 1 is a diagram showing the detection of heart function and hemodynamics of a mouse model with heart failure due to aortic arch stenosis (TAC);
FIG. 2 is a diagram showing myocardial tissue pathology staining of a TAC model mouse according to the present application;
FIG. 3 is a diagram showing RNA sequencing and lncRNA-GRCm38 expression level verification of a mouse myocardial tissue of a TAC model according to the present application;
FIG. 4 is a representation and identification of lncRNA-GRCm38 of the application;
FIG. 5 is a graph showing inhibition of TGF- β induced cardiomyocyte transdifferentiation by lncRNA-GRCm38 of the application;
FIG. 6 is a graph of lncRNA-GRCm38 of the application for improving heart function and inhibiting myocardial fibrosis in mice with TAC model.
Detailed Description
In order to more clearly illustrate the technical scheme of the embodiment of the present application, the application of lncRNA-GRCm38 of the present application in preparing a medicament for detecting, preventing and/or treating heart failure will be described in detail with reference to the accompanying drawings and detailed embodiments
The lncRNA-GRCm38 related to the application is long non-coding RNA (longnon-codingRNA, lncRNA), named GRCm38 and positioned at 6E3;652.73cM, no host genes.
Experiments prove that the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2. The specific information is as follows:
5’RACE：
GGAGGCTGCCGGGGCCGCCTAAAGAAGAGGCTGTGCTTTGGGGCTCCGGCTCCTCAGAGAGCCTCGGCTAGGTAGGGGATCGGGACTCTGGCGGGAGGGCGGCTTGGTGCGTTTGCGGGGATGGGCGGCCGCGGCAGGCCCTCCGAGCGTGGTGGAGCCGTTCTGTGAGACAGCCGGATCATTCCTTGAGGACAGGACAGTGCTTGTTTAAGGCTATATTTCTGCTGTCTGAGCAGCAACAGGTCTTCGAGATCAACATGATGTTCATAATCCCAAGATGTTGCCATTTATGTTCTCAGAAGCAAGCAGAGGCATGATGGTCAGTGACAGTAATGTCACTGTGTTAAATGTTGCTATGCAGTTTGGATTTTTCTAATGTAGTGTAGGTAGAACATATGTGTTCTGTATGAATTAAACTCTTAAGTTACACCTTGTATAATCCATGCAATGTGTTATGCAATTACCATTTTAAGTATTGTAGCTTTCTTTGTATGTGAGGATAAAGGTGTTTGTCATAAAATGTTTTGAACATTTCCCCAA
3’RACE：
GACATGTAGAAGTGTTTGTCCAGAACATTTCTTAAATGTATACTGTCTTTAGAGAGTTTAATATAGCATGTCTTTTGCAACATACTAACTTTTGTGTTGGTGCGAGCAATATTGTGTAGTCATTTTGAAAGGAGTCATTTCAATGAGTGTCAGATTGTTTTGAATGTTATTGAACATTTTAAATGCAGACTTGTTCGTGTTTTAGAAAGCAAAACTGTCAGAAGCTTTGAACTAGAAATTAAAAAGCTGAAGTATTTCAGAAGGGAAATAAGCTACTTGCTGTATTAGTTGAAGGAAAGTGTAATAGCTTAGAAAATTTAAAACCATATAGTTGTCATTGCTGAATATCTGGCAGATGAAAAGAAATACTCAGTGGTTCTTTTGAGCAATATAACAGCTTGTTATATTAAAAATTTTCCCCACAGATATAAACTCTAATCTATAACTCATAAATGTTACAAATGGATGAAGCTTACAAATGTGGCTTGACTTGTCACTGTGCTTGTTTTAGTTATGTGAAAGTTTGGCAATAAACCTATGTCCTAAATAGTCAAAAAAAAAAAAAAAAAA
the NCBIReferenceSequences (RefSeq) database shows that there are three transcripts at GRCm38 transcript level of 1230bp, 1124bp and 613bp, respectively.
Further, 3'RACE and 5' RACE experiments prove that the length of the main sequence of the lncRNA-GRCm38 in the myocardial fibroblasts is 1230bp, and Northern-blot experiments prove that the lncRNA-GRCm38 takes 1230bp as the main form in the myocardial fibroblasts. Prediction of the UCSC database shows that the GRCm38 does not have coding capability. The GRCm38 nuclear mass distribution was detected by real-time quantitative PCR and found to be distributed throughout the nuclear mass, but was most abundant in the nucleus.
Example 1: construction of aortic arch constriction (TAC) induced heart failure mouse model
Male 9-10 week old C57 mice (18 g-22 g), SPF grade, provided by Zhejiang university animal experiment center, are fed under standard conditions (room temperature 23+ -1deg.C, humidity 50-60%,12:12h day-night alternation)), and drinking water diet is free.
TAC model mice were prepared: the C57 mice clean the surgical area, shear the skin from the 4 th and 5 th ribs of the left chest of the mice, separate the muscles, prop open the rib space, separate the thymus, and expose the aortic arch. The aortic arch and the brachiocephalic trunk branch are cushioned by a 27G needle, the aorta is narrowed by a 6-0 non-absorbable surgical suture, the cushioned is drawn out after the puncture, the thoracic cavity is closed, and the patient is fed conventionally.
Sham group (Sham) mice preparation: only open chest and do not treat.
Heart function was examined with a small animal sonicator at 4, 8, and 12 weeks after molding, respectively, and the results are shown in fig. 1A, in which the TAC model mice were significantly thinner in left ventricular wall thickness and significantly reduced in Ejection Fraction (EF) and short axis shortening (FS) compared to Sham group (#p < 0.01).
The results of the hemodynamics were examined 12 weeks after molding, as shown in FIG. 1B, and compared with the Sham group, TAC model mice were significantly decreased in Left Ventricular Systolic Pressure (LVSP), left Ventricular Diastolic Pressure (LVDP), maximum rate of increase in left ventricular pressure (+dp/dtmax) and maximum rate of decrease in left ventricular pressure (-dp/dtmax) (#p <0.05, #p < 0.01).
Heart was cut longitudinally 12 weeks after molding, paraffin was cut, HE stained and photographed, and the result was shown in fig. 2A, in which the TAC model mice had a larger heart overall and a thinner ventricular wall compared to Sham group.
Hearts were taken 12 weeks after molding, paraffin sections were cut and photographed after HE staining and Siriusred staining, and the results are shown in fig. 2B, in which TAC model mice had increased cardiomyocyte loss, disordered myocardial arrangement, and massive collagen fibril formation compared to Sham group.
Hearts were taken 12 weeks after molding, frozen sections were subjected to WGA staining, SMA staining, and Collagen-1 staining, and photographs were taken, as shown in FIG. 2C, and compared with Sham group, mice with TAC model had myocardial cell hypertrophy and increased Collagen production.
Example 2: RNA sequencing (RNA-seq) revealed significantly low expression of lncRNA-GRCm38 in TAC model mouse myocardial tissue
The TAC model mice and Sham mice successfully modeled in example 1 were randomly selected 8 each.
Heart tissue was taken 12 weeks after molding, detected using RNA-seq technology and analyzed for results using bioinformatics, the most distinct genes were found: lncRNA-GRCm38 (fig. 3A-B). By KEGG pathway enrichment analysis, it was found that the differentially expressed genes were mostly enriched in fibrosis-associated pathways (fig. 3C).
Sequencing analysis results are verified by using a realtimeepcr, and the results are shown in fig. 3D, and compared with Sham group, the expression level of lncRNA-GRCm38 in myocardium of TAC model mice is significantly reduced.
Example 3: identification and characterization of lncRNA-GRCm38
Nucleotide sequence information was determined by RACE experiments on lncRNA-GRCm38 detected in example 2, as shown in FIG. 4A.
The protein encoding ability of lncRNA-GRCm38 was predicted by bioinformatics, and the results are shown in fig. 4B, which show that lncRNA-GRCm38 has a weak protein encoding ability.
The size of GRCm38 was determined by Northern-blot analysis on the lncRNA-GRCm38 detected in example 2, and the results are shown in FIG. 4C.
The distribution of the lncRNA-GRCm38 nucleoplasm was found by nucleoplasm analysis experiments and immunofluorescent staining, and the nuclei were slightly more, and the results are shown in FIGS. 4D-E.
Example 4: lncRNA-GRCm38 significantly inhibited TGF- β -induced cardiomyocyte transdifferentiation by observation of TGF- β -induced cardiomyocyte transdifferentiation by phalloidin immunofluorescence staining, and the results are shown in FIG. 5, in which normal group (CON) cells are irregularly triangular or fusiform, and individual TGF- β -induced constituent fibroblasts are rounded, indicating that after TGF- β induction, cardiomyocytes transdifferentiate to myofibroblasts. Compared with the independent TGF-beta induction group, the lncRNA-GRCm38 knockout group (GRCm 38-KD) cells have larger cell volume and circular shape after being induced by TGF-beta, which indicates that myocardial fibroblasts are transdifferentiated to myofibroblasts. lncRNA-GRCm38 overexpressing cells were constructed after infection of cardiomyocyte cells by synthesis of lncRNA-GRCm38 sequences, ligation to adeno-associated virus (AAV) vectors. Compared with the independent TGF-beta induction group, after the TGF-beta induction of the lncRNA-GRCm38 over-expression group (GRCm 38-OE) cells, the cell volume is not increased, and the morphology is fusiform, so that the myocardial fibroblasts are not differentiated into myofibroblasts.
Example 5: the overexpression of lncRNA-GRCm38 obviously improves the heart function of mice with TAC models and inhibits myocardial fibrosis
The successful TAC model mice from example 1 were randomly selected and randomly split into lncRNA-GRCm38 over-expression (GRCm 38-OE) and viral empty (Vector) groups.
The heart function was examined with a small animal ultrasonic instrument 12 weeks after molding, as shown in FIG. 6A, the left ventricular wall thickness of the mice in the Vector group was significantly thinner, and the Ejection Fraction (EF) and short axis shortening (FS) were significantly reduced, compared with those in the GRCm38-OE group # p<0.05， ## p<0.01)。
Hearts were taken 12 weeks after molding, paraffin sections were cut, masson trichromatic staining was performed and photographed, and the results are shown in fig. 6B, in which the myocardial fibrosis was more severe in the myocardial tissue of the Vector mice than in the GRCm38-OE group.
The application provides a novel heart failure treatment target point: the application discloses the position, the size, the sequence information, the coding capacity and the nuclear mass distribution of the lncRNA-GRCm38, aiming at improving the curative effect of heart failure treatment, and performing functional verification on the regulation and control of myocardial fibrosis of the lncRNA-GRCm 38.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application, and not for limiting the same; although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the application.
Claims (9)
1. An application of lncRNA-GRCm38 in screening or preparing a drug target for detecting, preventing and/or treating heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
2. The use according to claim 1, wherein the medicament is a medicament for improving heart failure heart function.
3. The use according to claim 1, wherein the medicament is a medicament for inhibiting myocardial fibrosis.
4. The use of claim 1, wherein the treatment of heart failure is achieved by increasing expression of lncRNA-GRCm 38.
5. An application of lncRNA-GRCm38 in preparing a medicament for detecting, preventing and/or treating heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
6. The use according to claim 5, wherein the medicament is a medicament for improving heart failure heart function.
7. The use according to claim 5, wherein the medicament is a medicament for inhibiting myocardial fibrosis.
8. The use of claim 5, wherein the treatment of heart failure is achieved by increasing expression of lncRNA-GRCm 38.
9. The application of the lncRNA-GRCm38 in preparing a kit, a reagent and/or a chip for detecting heart failure, wherein the lncRNA-GRCm38 nucleotide sequence comprises 5'RACE and 3' RACE, the sequence of the 5'RACE is shown as SEQ ID NO.1, and the sequence of the 3' RACE is shown as SEQ ID NO. 2.
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