Annular RNA related to myocardial hypertrophy, expression vector and preparation method thereof
阅读说明：本技术 与心肌肥厚相关的环状rna、表达载体及其制备方法 (Annular RNA related to myocardial hypertrophy, expression vector and preparation method thereof ) 是由 周祥 王文静 陈丽莉 于 2020-06-30 设计创作，主要内容包括：本发明提供了与心肌肥厚相关的环状RNA、表达载体及其制备方法,该环状RNA是通过对心肌肥厚模型小鼠测序发现的,它在心肌肥厚发生发展过程中起着关键作用。(The invention provides a cyclic RNA related to myocardial hypertrophy, an expression vector and a preparation method thereof, wherein the cyclic RNA is discovered by sequencing a myocardial hypertrophy model mouse and plays a key role in the occurrence and development process of the myocardial hypertrophy.)
1. A circular RNA related to myocardial hypertrophy is characterized by being a fragment of Chr153256629-53282092 gene, and having a sequence shown as SEQ ID NO: 1 is shown.
2. A recombinant expression vector comprising the cyclic RNA associated with cardiac hypertrophy according to claim 1.
3. The recombinant expression vector of claim 2, wherein the recombinant expression vector is an adenovirus vector overexpressing Chr 153256629-53282092.
4. The recombinant expression vector of claim 3, wherein the adenoviral vector is ADV-M12.
5. The method of producing a recombinant expression vector according to any one of claims 2 to 4, comprising:
1) obtaining the target gene by using a PCR fishing method or a whole gene synthesis method, wherein the sequence of a primer used in PCR comprises SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16 and SEQ ID NO: 17;
2) respectively carrying out enzyme digestion and purification on a target gene and a target vector;
3) directionally connecting the purified enzyme cutting products;
4) transforming the connecting product into a bacterial competent cell, carrying out enzyme digestion identification on the grown clone firstly to prove that the target gene is directionally connected into a target vector, then sequencing, analyzing and comparing the positive clone, and obtaining the successfully constructed target gene recombinant expression vector if the comparison is correct.
6. The method for preparing a recombinant expression vector according to claim 5, wherein the step 1) comprises:
1-1) dissolving all primers respectively and mixing the primers in equal amount to prepare oligo mix;
1-2) carrying out a first round of PCR reaction by using prepared oligo mix to obtain a non-single-band PCR product mixture mixed with a target gene band;
1-3) using the PCR product of the first round as a template and the PCR product of SEQ ID NO: 2 and SEQ ID NO: and carrying out a second round of PCR reaction by using the primer with the sequence 17 to obtain a single target gene band.
7. The method for preparing the recombinant expression vector according to claim 6, wherein the reaction system of the first round of PCR reaction is:
；
The reaction conditions are as follows:
。
8. the method for preparing the recombinant expression vector according to claim 7, wherein the reaction system of the second round of PCR reaction is:
；
The reaction conditions are as follows:
9. the method of claim 5, wherein the step 2) comprises digesting the target gene and the target vector with EcoRI and BamHI, respectively.
10. The method for preparing a recombinant expression vector according to claim 5, wherein the purified enzyme cleavage products are directionally ligated using T4 DNA ligase in step 3).
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a circular RNA related to myocardial hypertrophy, an expression vector and a preparation method thereof.
Background
Cardiovascular disease is a major threat to human health, with an increasing incidence in recent years (Molkentin JD, LuJR, Antos CL, et al. A calcein-dependent transaction path for cardiovascular hypertension. cell 1998; 93: 215-28). Among them, myocardial hypertrophy is the most common pathological process in cardiovascular diseases, and it causes abnormal hypertrophy of myocardial cells, hypertrophy and asymmetry of ventricular septum, narrowing of ventricular cavity-space or limitation of ventricular filling (RockmanHA, Koch WJ, Lefkowwitz RJ. Seven-Transmembrane-plating receptors and Heart function. Nature. 2002; 415: 206-12; Evant AD, Tufro-McReddie A, Fisher A, et al.
CircRNAs are one of the non-coding RNAs (ncRNAs), originally discovered in the virus by electron microscopy in 1976 (Sanger HL, Klotz G, Riesner D, et al. viral are single-stranded coded with a generic approach circulation USA 1976; 73(11):3852-6.), have been ignored for decades. In recent years, interest in circRNA has increased dramatically with the progress of RNA sequencing and bioinformatic analysis, and the presence of circRNAs has been found in many organisms in abundance of about 2-4% of total cellular mRNA (Szabol, Salzman J. detection circular RNAs: bioinformatic and experimental transformations. Nat Rev Gene. 2016; 17(11): 679-92). It has also been recently discovered that circRNA has a non-negligible effect in aging, insulin secretion, atherosclerotic vascular disease, cancer and cardiac hypertrophy (Qu S, Zhong Y, Shang R, et al, the engineering landscapes of circular RNA in life processes, RNABiol.2017; 14(8): 992-9). Whole genome analysis and detailed characterization of Circular RNAs From cardiomyocyte ribosome failure RNAs From human, mouse and rat hearts, as well as From human embryonic stem cells (Khan MA, Reckman YJ, Aufield S, et al. RBM20 Regulation Circular RNA Production From the protein Gene. Circular Res.2016; 119(9): 996. D.1003; Werfel S, Notjunge S, Schwarzmayr T, et al. Characterification of Circular RNAs in man, mouse and rat J.Molcell Cardiol. 2016; 98: 103-7; Tan WL, Lim BT, Anene-Nzelu CG, Landscape expression of Circular ribosome failure RNA in man WL; found in heart RNA, heart failure RNA, 103. D.S.103. D.D.103. D.103. C. (found in heart failure RNA; heart failure RNA, human stem cell, DNA, et al. D.103. D.7; heart failure RNA, human stem cell, DNA, 103. D.D.103, 103. D.D.D.D.103, 103. D.D.D.103. D.3, 3, heart failure RNA, 7, heart failure RNA, 3, 7, heart failure RNA, heart failure, heart, lim BT, lene-Nzelu CG, et al.A landscapes of circular RNA expression in the human heart. Cardiovasc Res.2017; 113(3) 298 and 309), but the functions and mechanisms thereof are still not completely understood.
In combination with previous studies, circRNA has been studied just before, but has many advantages over other non-coding RNAs (such as microrna and lncRNA), and plays an important role in various diseases. The research of the circRNAs related to the myocardial hypertrophy still stays at the primary stage, so the research of the circRNAs for finding a new treatment target point for the myocardial hypertrophy has important significance.
Disclosure of Invention
The invention provides a cyclic RNA related to myocardial hypertrophy, an expression vector and a preparation method thereof, wherein the cyclic RNA is discovered by sequencing a myocardial hypertrophy model mouse and plays a key role in the occurrence and development process of the myocardial hypertrophy.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a circular RNA related to myocardial hypertrophy, which is a Chr153256629-53282092 gene fragment, and has a sequence shown as SEQ ID NO: 1 is shown.
The invention also provides a recombinant expression vector containing the gene segment.
Preferably, the recombinant expression vector is an adenovirus vector overexpressing Chr 153256629-53282092.
More preferably, the adenoviral vector is ADV-M12.
The invention also provides a preparation method of the recombinant expression vector, which comprises the following steps:
1) obtaining the target gene by using a PCR fishing method or a whole gene synthesis method, wherein the sequence of a primer used in PCR comprises SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: 11. SEQ ID NO: 12. SEQ ID NO: 13. SEQ ID NO: 14. SEQ ID NO: 15. SEQ ID NO: 16 and SEQ ID NO: 17;
2) respectively carrying out enzyme digestion and purification on a target gene and a target vector;
3) directionally connecting the purified enzyme cutting products;
4) transforming the connecting product into a bacterial competent cell, carrying out enzyme digestion identification on the grown clone firstly to prove that the target gene is directionally connected into a target vector, then sequencing, analyzing and comparing the positive clone, and obtaining the successfully constructed target gene recombinant expression vector if the comparison is correct.
Preferably, step 1) comprises:
1-1) dissolving all primers respectively and mixing the primers in equal amount to prepare oligo mix;
1-2) carrying out a first round of PCR reaction by using prepared oligo mix to obtain a non-single-band PCR product mixture mixed with a target gene band;
1-3) taking the PCR product of the first round as a template and the sequence of the PCR product is SEQ ID NO: 2 and SEQ ID NO: 17, performing a second round of PCR reaction to obtain a single target gene band.
More preferably, the reaction system of the first round of PCR reaction is:
the reaction conditions are as follows:
more preferably, the reaction system of the second round of PCR reaction is:
the reaction conditions are as follows:
。
preferably, in step 2), the target gene and the target vector are digested with EcoRI and BamHI, respectively.
Preferably, in step 3), the purified enzyme-cleaved products are subjected to directional ligation using T4 DNA ligase.
The invention has the following beneficial effects:
a mouse myocardial hypertrophy model is established through Transverse Aortic Coarctation (TAC), a sham operation group is used as a control, circRNA high-flux sequencing is carried out on two groups of mice, the expression of the gene in the TAC group of mice is only 0.4 of that in the control group, the expression of the gene fragment in the TAC group of mice is found through tissue extraction and quantitative PCR, and the gene fragment is really down-regulated compared with that in the control group; then, after the cyclic RNA Chr153256629-53282092 is over-expressed and AngII induction is carried out, the myocardial hypertrophy marker and the myocardial hypertrophy surface area of the over-expressed Chr1 group are lower than those of the AngII group, so that the mice induced by AngII myocardial hypertrophy progression can be obviously reduced after the Chr1 is over-expressed.
Drawings
FIG. 1 is a flowchart of an experiment for constructing an overexpression adenovirus vector Chr153256629-53282092 according to an embodiment of the present invention.
FIG. 2 is a gene synthesis report form of recombinant plasmid sequencing according to an embodiment of the present invention.
FIG. 3 is a comparison map of the sequencing result of the recombinant plasmid and the target gene sequence in the embodiment of the present invention.
FIG. 4 shows the results of qPCR analysis of the expression of the cardiac hypertrophy marker in cardiomyocytes a. the expression of the hypertrophy marker SEARCA-2 a; b. expression of the hypertrophy marker β -MHC; c. expression of the hypertrophy marker BNP; d. expression of the hypertrophy marker ACTA-1; *: p is less than 0.05; **: p is less than 0.01.
FIG. 5 is a photograph of cardiomyocytes under a confocal laser confocal microscope, A, B, C, D is a cytoplasm stained with DyLight 59, E, F, G, H is a nucleus stained with DAPI, I, J, K, L is an image after Merged, wherein A, E, I is the control group, B, F, J is the CHACR + AngII 1. mu.M group, and C, G, K is the AngII 1. mu.M group; D. h, L is NC + AngII 1. mu.M group.
FIG. 6 is a statistical graph of the mean area of groups of cardiomyocytes as measured by image J analysis according to an embodiment of the present invention; *: p is less than 0.05.
Detailed Description
Exemplary embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. It should be noted that this example is provided for the purpose of enabling a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art, and the present invention may be implemented in various forms without being limited to the embodiments set forth herein.