CN111748616A

CN111748616A - Annular RNA related to myocardial hypertrophy, expression vector and preparation method thereof

Info

Publication number: CN111748616A
Application number: CN202010615319.0A
Authority: CN
Inventors: 周祥; 王文静; 陈丽莉
Original assignee: Second Affiliated Hospital of Soochow University
Current assignee: Second Affiliated Hospital of Soochow University
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2020-10-09
Also published as: CN115961025B; CN115961025A

Abstract

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.

Description

Annular RNA related to myocardial hypertrophy, expression vector and preparation method thereof

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

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.

Examples

The specific implementation process of the invention is as follows:

first, construction of Chr153256629-53282092 over-expression adenovirus vector

The target gene is obtained by PCR method from plasmid cloning template containing the target gene, and by whole gene synthesis method if there is no template. And (3) carrying out enzyme digestion on the target gene and the target vector respectively. Purifying the enzyme digestion product, then connecting, converting the connecting product into a bacterial competent cell, carrying out enzyme digestion identification on the grown clone, and proving that the target gene is directionally connected into a target vector. And sequencing, analyzing and comparing the positive clones, wherein the correctly compared result is the successfully constructed target gene expression plasmid vector. The constructed recombinant vector is subjected to ultrapure extraction, and the flow is shown in figure 1. The method specifically comprises the following steps:

1. obtaining sequence fragments by PCR

1.1 design of oligo, upstream and downstream primers of Chr153256629-53282092 gene (SEQ ID NO: 1), EcoRI and BamHI and protective bases were added respectively for subcloning of vector, and the sequence of oligo is as follows:

TABLE 1Oligo primer sequence Listing

The primers were synthesized by Suzhou Jima Gen Ltd.

1.2 dissolving the oligo into 50 μ M, taking the same volume of oligo to a 1.5ml centrifuge tube, mixing uniformly to prepare oligo mix.

1.3 performing a first PCR reaction with the prepared oligo mix, wherein the PCR system is as follows:

TABLE 2 first round PCR reaction System

oligo mix	2μl
		10×Pfu Buffer(+Mg²⁺)	3μl
dNTP	0.6μl
		DMSO	1.2μl
ddH₂O	23μl
		Pfu DNA polymerase	0.2μl
Total volume	30μl

Circulation conditions are as follows:

TABLE 3 first round PCR reaction conditions

1.4 second round PCR reaction with oligo-1 and oligo-16, template is the product of the first round PCR reaction, and the PCR system is as follows:

TABLE 4 second round PCR reaction System

Circulation conditions are as follows:

TABLE 5 second round PCR reaction conditions

The first round of PCR can obtain a mixture of non-single-band PCR products mixed with the target gene band, and then the first round of PCR products is used as a template, and the second round of PCR can obtain a single target gene band.

1.5 after the PCR reaction is completed, the gene fragment is recovered by Agarose electrophoresis and gel cutting.

2. Cloning of Chr153256629-53282092 Gene into vector ADV-M12

2.1 the fragment of Chr153256629-53282092 gene was digested with EcoRI and BamHI at 37 ℃ for 2 hours as follows:

TABLE 6 digestion reaction system of fragment Chr153256629-53282092

10×Buffer	5μl
		Chr1 53256629-53282092	42μl
EcoRI	1.5μl
		BamHI	1.5μl
Total volume	50μl

2.2 vector ADV-M12 was digested with EcoRI and BamHI for 2 hours at 37 ℃ as follows:

TABLE 7 digestion reaction System of vector ADV-M12

2.3 electrophoresis, and recovering the Chr153256629-53282092 gene fragment and the vector ADV-M12 by using a DNA gel recovery kit.

2.4T 4 DNA ligase is used to connect the Chr153256629-53282092 gene fragment obtained by double digestion and the linearized vector, and the connection is carried out for 2 hours at 22 ℃, and the connection system is as follows:

TABLE 8 ligation reaction System of target Gene fragment and vector

T4 DNA ligase buffer	2μl
		ADV-M12	2μl
Chr1 53256629-53282092	5μl
		T4 DNA ligase	1μl
ddH₂O	10μl
		Total volume	20μl

2.5 preparation of competent cells (calcium chloride method)

2.5.1A single colony was picked from a fresh plate incubated at 37 ℃ for 16 hours and transferred to a 1L flask containing 100ml of LB medium. The cells were incubated at 37 ℃ for 3 hours with vigorous shaking (rotary shaker, 300 rpm).

2.5.2 transfer of bacteria under sterile conditions to a sterile, single-use, ice-pre-cooled 50ml polypropylene tube, and allow to stand on ice for 10 minutes, allowing the culture to cool to 0 ℃.

2.5.3 at 4 ℃, at 4000 rpm/separation for 10 minutes, recovering the cells.

2.5.4 pour out the culture and invert the tube for 1 minute to drain off the last traces of culture.

2.5.5 in 10ml of 0.1mol/L CaCl precooled with ice₂Resuspend each pellet and place on an ice bath.

2.5.6 was centrifuged at 4000 rpm for 10 minutes at 4 ℃ to recover the cells.

2.5.7 pour out the culture solution and invert the tube for 1 minute to drain the last remaining traces of culture solution.

2.5.8 Per 50ml of initial culture 2ml of 0.1mol/L CaCl precooled with ice₂Resuspend each cell pellet (containing 20% glycerol).

2.5.9 cells were divided into small aliquots (100. mu.l/piece) and stored frozen at-70 ℃.

(reference for preparation of competent cells: molecular cloning instructions second edition page 55)

2.6 transformation of the ligation products into competent cells

2.6.1 Take out the competent cells (E.coli DH 5. alpha.) from-70 deg.C, place the tube on ice for 4 minutes, after the competent cells have thawed, add 10. mu.l of ligation product, mix the contents gently and place on ice for 30 minutes.

2.6.2 Place centrifuge tubes on a test tube rack placed in a water bath preheated to 42 ℃ for 90 seconds without shaking the centrifuge tubes.

2.6.3 the tubes were quickly transferred to an ice bath and the cells were allowed to cool for 3 minutes.

2.6.4 Add 800. mu.l LB medium (without antibiotics) to each tube, then transfer the tubes to a 37 ℃ shaker, 250 rpm, incubate for 45 minutes to resuscitate the bacteria.

2.6.5 mu.l of the cultured cells were applied evenly to LB plates containing 50. mu.g/ml ampicillin.

2.6.6, etc., the liquid on the plate was absorbed, and the plate was placed upside down in an incubator at 37 ℃ and incubated for 16 hours.

2.7 picking clone colonies from the plate, miniextracting plasmids and identifying positive clones

2.7.1 from the cultured plate, 4 individual, filled colonies were picked and placed in a test tube containing 5ml (containing 50. mu.g/ml Ampicillin) of LB medium.

2.7.2 the tubes were incubated in a bacterial shaker at 37 ℃ and 250 rpm for 16 hours.

2.7.3 the plasmid was extracted from the cultured bacterial liquid with a plasmid miniprep kit (Tiangen Biochemical, DP 104-02).

3. Sequencing, verifying and massively extracting recombinant plasmid

3.1 taking 200 mul of bacterial liquid corresponding to the positive clone, sequencing, showing a gene synthesis report in figure 2, and preserving the residual bacterial liquid by using glycerol.

3.2 comparing the sequencing result with the target gene sequence, as shown in FIG. 3, inoculating the preserved glycerol bacterial liquid into LB culture medium, extracting a large amount of plasmid, and obtaining a sufficient amount of recombinant plasmid. At this point, the vector construction experiment was completed.

Second, primary cardiomyocyte culture and transfection

1. Collecting hearts

Take 1-2 d born SPF grade C57BL/6 male suckling mice. Placing a foam box sleeved with a preservative film in a biological safety cabinet, fixing a suckling mouse on the foam box by using a 1mL needle, spraying 75% alcohol to sterilize the surface skin of the mouse, pulling the upper abdominal skin of the mouse by using flat-headed tweezers, then using an ophthalmic scissors to cut the skin, pulling the skin backwards by using a left hand and fixing the skin, replacing a sterile head-cutting curved tweezers by a right hand to gradually pick up the chest wall, clamping the heart out and placing the heart in a DMEM culture medium, and repeating the steps.

2. Digestion of

The heart of the suckling rat is squeezed by sterile flat-headed tweezers to discharge blood in the heart cavity, the connective tissues at the bottom of the heart and epicardium are removed by the sterile sharp-pointed bent tweezers, the heart is placed in a 1.5mL sterile EP tube, the tube is repeatedly washed by PBS and then cut into pieces by ophthalmic scissors, 1mL digestive enzyme is added, and the mixture is incubated and digested in a 37 ℃ incubator for about 15 minutes. Under a microscope, after cardiomyocytes were seen to slough from the tissue, digestion was terminated by addition of complete medium after centrifugation at 1200rpm for 3min, followed by resuspension of the pellet. When larger pieces of tissue are not completely digested, the above steps can be repeated by using digestive enzyme digestion again.

3. Purification of

Culturing the obtained cell plate in a 60mm dish for 1.5h, removing fibroblasts through differential adherence when the cardiac fibroblasts are basically adhered to the wall and the cardiac cells are not adhered to the wall, taking the culture medium to place in a 15mL centrifuge tube, cleaning the culture dish by PBS, washing out residual partial cardiac cells, placing in the centrifuge tube, centrifuging again at 1200rpm for 5min, and discarding the supernatant.

4. Adherent culture

And adding complete culture medium (containing 0.1 mu mol/mL Brdu) into the precipitate to resuspend the cells, uniformly seeding the cells in a culture dish, removing the culture medium after culturing for 24h, washing the cells twice by PBS (primary cells, a lot of residual tissue fragments and washing for one time), adding the complete culture medium, and starting to use in experiments when the cell morphology and pulsation are good.

5. Grouping of cells

The main grouping cases can be seen in the following table:

TABLE 9 cell grouping Table

Note: "CHACR" is the provisional name for Chr153256629-53282092 in the present application.

Control group (control group):

normal cultured cardiomyocytes, without intervention;

and (3) thickening group:

i AngII 1. mu.M group: after culturing the myocardial cells for 48h, adding AngII1 mu M to induce for 48 h;

ii CHACR + AngII 1. mu.M group: adding 0.01 mu l of CHACR overexpression adenovirus, changing the solution after 24h, adding AngII1 mu M after 24h, and culturing for 48 h;

iii NC + AngII 1. mu.M group: adding 0.01 mul of unloaded adenovirus, changing the solution after 24h, adding AngII1 mul for culturing for 48h after 24 h.

6. Detection of

6.1 the grouped treated myocardial cells are directly added with trizol for cracking, total RNA of the cells is extracted, then the concentration of the RNA is measured, the RNA is quantitatively and reversely transcribed into cDNA, and qPCR is used for detecting the expression of myocardial hypertrophy markers such as ANP, BNP, beta-MHC, SERCA-2a and the like in the myocardial cells by taking GAPDH as an internal reference.

6.2 after immunofluorescence staining (antibodies expressed on the membrane of the cardiomyocytes), pictures were taken under confocal laser, pictures were analyzed by image j, ten fields per group were selected, and the mean surface area of the cardiomyocytes was calculated.

Third, results and analysis

1. The qPCR detection shows that the cardiac hypertrophy marker (SEARCA-2 a/beta-MHC/BNP/ACTA-1) of the AngII-induced hypertrophy model cell is obviously higher than that of the control group, which indicates that the modeling of the hypertrophic cardiac muscle cell model is successful, but the increase of the AngII-induced hypertrophy marker after the CHACR is over-expressed is reduced, as shown in FIG. 4. The results demonstrate that CHACR inhibits AngII-induced cardiomyocyte hypertrophy.

2. In addition to the detection of changes in markers of cellular hypertrophy by qPCR, we next validated by immunofluorescence analysis of changes in surface area after cardiac myocyte hypertrophy. Experimental groups are as above, the average surface area of the cardiomyocytes is measured by laser confocal microscope image collection analysis, and the result shows that the surface area of the cardiomyocytes is obviously reduced after CHACR overexpression compared with the control group, which indicates that CHACR can obviously inhibit AngII-induced cardiomyocyte hypertrophy. As shown in fig. 5 and 6.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or additions or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are also included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Suzhou university affiliated second hospital

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ctgatgactt tagcactcag tatgttttag atggcagtgg ccacatactt tctcagaagc 420

cttcacatct tggtcaaggt acaactgtaa ctgctctaaa gttgtttaag aatctgcctg 480

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Claims

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:

3) directionally connecting the purified enzyme cutting products;

6. The method for preparing a recombinant expression vector according to claim 5, wherein the step 1) comprises:

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:

first round PCR product 1μl 10×Pfu Buffer(+Mg²⁺) 5μl dNTP 1μl DMSO 2μl SEQ ID NO: 2 sequence of the sequence II 1μl SEQ ID NO: 17 sequence primer 1μl ddH₂O 39μl Pfu DNA polymerase 0.3μl Total volume 50.3μl

；

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).