CN113774017B

CN113774017B - Application of circRNA XPO4 in osteogenic differentiation of interstitial cells of human aortic valve

Info

Publication number: CN113774017B
Application number: CN202110782668.6A
Authority: CN
Inventors: 王勇军; 董念国; 韩东; 周廷文; 史嘉玮
Original assignee: Tongji Medical College of Huazhong University of Science and Technology
Current assignee: Tongji Medical College of Huazhong University of Science and Technology
Priority date: 2021-07-12
Filing date: 2021-07-12
Publication date: 2023-02-17
Anticipated expiration: 2041-07-12
Also published as: CN113774017A

Abstract

The invention relates to application of circRNA XPO4 in osteogenic differentiation of interstitial cells of human aortic valves. The differential expression of circRNA is identified and integrated and analyzed through a high-throughput sequencing and bioinformatics method, a complex competitive endogenous RNA (ceRNA) network exists in human calcified aortic valve tissues, then the differential circRNA expression in normal and calcified aortic valve tissues is screened through a high-throughput sequencing technology, and the circRNA XPO4 directly related to miRNA (miRNA-204-5 p) related to osteogenic differentiation of human aortic valve interstitial cells is screened by combining the bioinformatics method. Through cell function tests, the circRNA XPO4 is determined to play a regulating role in the osteogenic differentiation of human aortic valve interstitial cells, and the expression of osteogenic key genes in the human aortic valve interstitial cells can be promoted, so that the osteogenic differentiation of the human aortic valve interstitial cells is induced.

Description

Application of circRNA XPO4 in osteogenic differentiation of interstitial cells of human aortic valve

The technical field is as follows:

the invention relates to the field of biomedicine, in particular to application of circRNA XPO4 in osteogenic differentiation of interstitial cells of a human aortic valve.

Background art:

calcified Aortic Valve Disease (CAVD) is a progressive disease of high morbidity and mortality in the elderly, with major pathophysiological changes being fibroproliferative calcification of the aortic valve leaflets leading to valve stiffening and causing hemodynamic changes affecting cardiac function. From the past, CAVD is considered to be an age-related degenerative disease, and is a degenerative and hardening process of valve tissue with age. However, basic research in recent years indicates that CAVD is an active progress process involving complex pathological changes such as endothelial injury, inflammatory cell infiltration, extracellular matrix remodeling and valve interstitial cell osteogenic differentiation. Cells in valve tissue mainly include Valve Endothelial Cells (VECs), valve Interstitial Cells (VICs), valve precursor cells, and the like, and research has proved that calcium salt increase caused by osteogenic differentiation of valve interstitial cells may be an important initiation factor for valve calcification. Currently, CAVD lacks an effective clinically available drug treatment regimen, the primary treatment of which is aortic valve replacement surgery. However, the patient who receives the surgical operation inevitably entails a high medical operation risk and an economic burden. Therefore, exploring the specific pathogenesis of CAVD, the effective prevention and/or treatment of calcified aortic valve disorders by non-surgical methods is currently an urgent need for clinical treatment of CAVD.

Circular RNA (circRNA) is a type of non-coding RNA that is in a closed loop structure and is not affected by exoRNAs. Because the protein is widely expressed and stable in eukaryotes, has good conservation among species and is not easy to degrade, the protein becomes a hotspot in the research field of non-coding RNA in recent years. A great deal of research shows that the circular RNA plays an important role in cardiovascular diseases such as atherosclerosis, myocardial infarction, heart failure and the like, and the characteristics of the circular RNA enable the circular RNA to have very wide prospects in the development and application of novel disease diagnosis and treatment methods.

The currently disclosed research on osteogenic differentiation of valve mesenchymal cells mainly adopts a transcription and translation level regulation mechanism, and a plurality of miRNAs (micro ribonucleic acids) are proved to be capable of regulating the osteogenic differentiation of human valve mesenchymal cells, such as miR-204, miRNA-483-5p and miRNA-214. In the related studies of this project, through bioinformatics analysis, identification and integration analysis of the differential expression of lncRNA and circRNA, it was found that a complex competitive endogenous RNA (ceRNA) network exists in calcified aortic valve tissue. Therefore, specific IncRNA and circRNA can be used as cerRNA to regulate osteogenic differentiation of human aortic valve interstitial cells and influence the progression of aortic valve calcification diseases, and the research on the mechanism that the circRNA is used as the cerRNA to regulate the osteogenic differentiation of the valve interstitial cells proves that the IncRNA MALAT1 promotes the osteogenic differentiation of the valve interstitial cells through interaction with miR-204 through verification by searching for IncRNA which is functionally related to miR-204.

The invention content is as follows:

technical problem to be solved

Aiming at the background, the invention carries out research on the relation between circRNA and aortic valve calcification and human valve interstitial cell osteogenic differentiation, detects the differentially expressed circRNA in normal and calcified aortic valve tissues based on a high-throughput sequencing technology, and predicts and screens the circRNA XPO4 with a binding site for miRNA-204-5p related to valve interstitial cell osteogenic differentiation by combining TOP10 circRNA gene in the differentially expressed gene with a starbase database. The fact that the expressions of bone formation key expression genes ALP (alk-phosphate) and Runx2 (run-related transcription factor 2) in human aortic valve interstitial cells after the CircRNA XPO4 is silenced is confirmed to be reduced, and the deposits of alizarin red positive nodules and calcium salts are obviously reduced, proves that the CircRNA XPO4 has a promoting effect on the bone formation differentiation of the human aortic valve interstitial cells, and opens up a wider prospect for the non-operative treatment of aortic valve calcification.

(II) technical scheme

In order to solve the technical problems, the invention adopts the following technical scheme:

the invention discloses a first aspect and provides application of circRNA XPO4 as an osteogenic differentiation inducer for interstitial cells of a human aortic valve.

Further, the application comprises the application of circRNA XPO4 as ALP or Runx2 agonist in osteogenic differentiation of interstitial cells of human aortic valves.

Further, the circRNA XPO4 is a sequence with a registration number Hsa _ circ _0100041 in circBase.

The invention discloses a second aspect, and provides an application of a circRNA XPO4siRNA silencing reagent as an inhibitor for osteoblastic differentiation of interstitial cells of human aortic valves.

Further, the circRNA XPO4 silencing agent comprises a circRNA XPO4siRNA sequence. The nucleotide sequence of the sense strand is shown as SEQ ID NO:6 is shown in the specification; the nucleotide sequence of the antisense strand is shown as SEQ ID NO: shown at 7.

The invention discloses a third aspect, and provides application of a circRNA XPO4 detection reagent in preparation of a kit for detecting the osteogenic differentiation level of interstitial cells of human aortic valves.

Further, the circRNA XPO4 detection reagent is a PCR detection reagent, the PCR detection reagent comprises a group of primer sequences, and the primer sequences comprise a sequence consisting of SEQ ID NO:2 and SEQ ID NO:3, and (b) a primer pair consisting of the DNA sequences shown in the specification.

Further, the primer sequence also comprises an internal reference primer, the internal reference primer is beta-actin, and the internal reference primer sequence comprises a primer consisting of SEQ ID NO:4 and SEQ ID NO:5, and (b) a primer pair consisting of the sequences shown in the specification.

The invention discloses a fourth aspect, and provides an application of circRNA XPO4 as a marker for evaluating osteogenic differentiation capacity of interstitial cells of a human aortic valve.

The invention discloses a fifth aspect, which provides a method for treating aortic valve calcification by inhibiting osteogenic differentiation of human aortic valve interstitial cells, wherein the method is used for treating aortic valve calcification by transferring circRNA XPO4siRNA into human aortic valve interstitial cells.

(III) advantageous effects

The invention has the following beneficial effects:

(1) The invention proves that the expression level of the circRNA XPO4 gene in human calcified aortic valve tissue is obviously higher than that of normal aortic valve tissue by detecting the expression of the circRNA XPO4 gene in human calcified aortic valve tissue, and the invention prompts that the circRNA XPO4 can be used as a diagnostic marker of calcified aortic valve diseases;

(2) According to the invention, by researching the influence of the circRNA XPO4 on the osteogenic differentiation of the interstitial cells of the human aortic valve, the over-expression of the circRNA XPO4 is proved to remarkably promote the osteogenic differentiation of the interstitial cells of the human aortic valve, and the down-regulation of the circRNA XPO4 can remarkably inhibit the osteogenic differentiation capacity of the interstitial cells of the human aortic valve, so that the osteogenic differentiation mechanism of the interstitial cells of the human aortic valve is further defined, and a new thought is provided for the treatment of the calcification of the aortic valve by taking the circRNA XPO4 as a target spot.

Description of the drawings:

in order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings required to be used in the embodiments will be briefly described below.

FIG. 1 is a heat map of high throughput sequencing of differentially expressed circRNA genes from normal and calcified human aortic valve tissue.

FIG. 2 is a database of starbase (http:// www.starbase.sysu.edu.cn) to screen TOP10 circRNA for the presence of binding sites for circRNA XPO4 and miRNA-204-5p, predicted to be up-regulated in aortic valve calcified tissue.

FIG. 3 is a graph of the expression levels of circRNA XPO4 and β -actin in normal and calcified aortic valve tissue measured by QRT-PCR in accordance with one embodiment; (NC: normal aortic valve, CAVs: calcified aortic valve);

FIG. 4 shows the performance of circRNA XPO4 over-expression plasmid (B) and siRNA knockdown (A) in example two;

FIG. 5 is a graph showing the results of the third example, in which a calcium-related marker protein (RUNX 2, ALP) was detected after transfecting a CircRNA XPO 4-specific siRNA to interfere with a CircRNA XPO4 gene in human primary valvular stromal cells, followed by 14 days of culture in an osteogenic differentiation induction medium (FIG. 5A), followed by alizarin red staining after 21 days of culture in an osteogenic differentiation induction medium (FIG. 5B), and calcium salt quantitative analysis (FIG. 5C);

FIG. 6 is a graph showing the results of the transfection of a circRNA XPO 4-specific overexpression plasmid into human primary valvular stromal cells, overexpression of the circRNA XPO4 gene, subsequent detection of calcification-associated marker protein (RUNX 2, ALP) after 14 days of culture in osteogenic differentiation-inducing medium (FIG. 6A), subsequent alizarin red staining after 21 days of culture in osteogenic differentiation-inducing medium (FIG. 6B), and calcium salt quantitative analysis (FIG. 6C) in example III;

the specific implementation mode is as follows:

in order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example one

4 cases of normal and calcified aortic valve tissues were collected, high throughput sequencing was performed, and differential expression profiles of circRNA from normal and diseased tissues were screened, as shown in FIG. 1.

In this example, 10 circrnas as shown in table 1 were obtained based on the expression of upregulated circrnas in calcified aortic valve tissue in the sequencing data.

TABLE 1 high throughput circRNA sequencing screening for expression of Up-regulated Top10 circRNA in calcified valvular tissues

circBase database numbering	Fold change in differential expression	NCBI transcripts	Name of Gene
				hsa_circ_0100041	18.56	NM_022459	XPO4
hsa_circ_0002268	12.55	NM_030948	PHACTR1
				hsa_circ_0017995	10.50	NM_020200	PRTFDC1
hsa_circ_0002688	10.29	NM_007331	WHSC1
				hsa_circ_0093546	9.66	NM_145023	CCDC7
hsa_circ_0032029	9.03	NM_004986	KTN1
				hsa_circ_0114158	8.65	NM_015534	ZZZ3
hsa_circ_0016909	8.58	NM_020808	SIPA1L2
				hsa_circ_0119637	8.52	NM_000183	HADHB
hsa_circ_0016443	8.42	NM_005401	PTPN14

And screening target circRNA which has the highest expression difference fold and has a binding site with miRNA-204-5p related to aortic valve calcification in expression up-regulated TOP10 circRNA by combining a starbase database with human valve tissue sequencing data.

circRNA associated with miRNA-204-5p was predicted based on the startbase database (http:// www.starbase.sysu.edu.cn).

As can be seen from FIG. 2, the TOP10 circRNA with up-regulated expression in the calcified aortic valve tissue was screened for the presence of a binding site for circRNA XPO4 and miRNA-204-5p, and was sequenced at position 1 in the up-regulated gene.

To further validate circRNA XPO4, large sample qRT-PCR validation of circRNA XPO4 expression in human aortic valve tissue was performed by PCR technique by designing specific primers that amplify circular RNA. The qRT-PCR result shows that the circRNA XPO4 is highly expressed in calcified aortic valve tissues, so that a kit for detecting the expression change of the gene can be prepared for diagnosing aortic valve calcification.

The specific experimental scheme is as follows:

1. RNA extraction

1) Tissue treatment: taking about 50mg of aortic valve tissue, grinding the aortic valve tissue with liquid nitrogen to a satisfactory degree, adding 1ml of Trizol reagent, oscillating for homogenization, fully cracking, centrifuging at 12000rpm at 4 ℃ for 15 minutes, and taking supernatant.

2) Adding chloroform 200ul, shaking and mixing evenly, and standing for 15 minutes at room temperature.

3) Centrifuging at 12000rpm for 15 min at 4 deg.C, separating into three layers, collecting the upper water phase to new enzyme-free EP tube, adding equal volume of isopropanol, mixing, standing at room temperature for 10 min, and precipitating RNA.

4) Centrifugation was carried out at 12000rpm for 15 minutes at 4 ℃ and the supernatant, RNA pellet and bottom of the tube were carefully removed.

5) 1ml of 75% ethanol was added to each 1ml of Trizol, and the mixture was mixed by inversion.

6) Centrifugation was carried out at 8000g for 5 minutes at 4 ℃ and the supernatant was discarded and dried in the sun at room temperature (5-10 minutes).

7) Adding appropriate amount of DEPC water to dissolve RNA, and measuring the concentration of RNA. Reverse transcription was performed based on the quantitative results.

8)RNA A260/A280＝1.8-2.1

2. Reverse transcription of cDNA

9) Kit reverse transcription System (10 ul) by the company TAKARA, japan:

the reaction conditions are as follows: reverse transcription reaction at 37 deg.c for 15 min; 5s reverse transcriptase inactivation reaction at 85 ℃; the reaction was completed at 4 ℃ and the product was cDNA.

3. QRT-PCR on-machine amplification

1) The experimental system is as follows:

2) Reaction conditions are as follows:

at 95 ℃ for 2 minutes; 40 cycles (95 ℃,10 seconds; 60 ℃,60 seconds); dissolution curve at 60-95 ℃.

3) Performing amplification on the machine, confirming an amplification curve and a dissolution curve after the reaction is finished, and calculating a P value by T test after the expression intensity of each gene is normalized according to a CT value (threshold cycle values) and an internal reference gene (beta-actin).

4) QRT-PCR results

The results are shown in FIG. 3. The expression of circRNA XPO4 was detected in 30 normal aortic valve tissue samples and 30 calcified aortic valve tissue samples, and the results showed that circRNA XPO4 was significantly upregulated in calcified aortic valve tissue compared to normal aortic valve tissue.

Example two: construction of circRNA XPO4 small interfering RNA and overexpression plasmid

1. Design of circRNA XPO4 small interfering RNA: the circRNA XPO4siRNA sequence was designed and synthesized by Gisey Biotech, guangzhou. The siRNA sense strand sequence of the circRNA XPO4 is shown as SEQ ID NO. 6, and the antisense strand sequence is shown as SEQ ID NO. 7. The positive strand and the antisense strand of the negative control group are respectively shown as SEQ ID NO. 8 and SEQ ID NO. 9.

2. pCD5-ciR vector overexpression of circRNA XPO4 and transient transfection of human valvular stromal cells: synthesizing a circRNA XPO4 linear complete sequence, annealing the sequence to form a double-stranded DNA fragment, inserting the double-stranded DNA fragment into a pCD5-ciR vector through a multiple cloning site, identifying the recombinant plasmid through sequencing, and using a pCD5-ciR empty vector as a control group.

3. siRNA and overexpression potency validation:

1) The 2-5 generation human valvular stromal cells were inoculated into 24-well plates and cultured at 37 ℃ in 5% CO2.

2) After the cell confluence density reached 80%, transfection was started, 0.5ug of overexpression plasmid or 5ul siRNA and 0.5ug of internal reference plasmid were dissolved in 250ul opti-MEM and mixed well.

3) 1.5ul Lipofectamine 2000 was dissolved in 250ul opti-MEM and mixed well.

4) The two tubes of reagents were mixed, allowed to stand at room temperature for 20 minutes, and then added to a 24-well plate, and incubated at 37 ℃ with 5% by volume of CO2.

5) After 6-8 hours, the complete medium is replaced and the culture is continued.

6) Cells were harvested after 48 hours and quantified by QRT-PCR.

The results are shown in FIG. 4. The results show that siRNA can effectively reduce the expression of circRNA XPO4, and overexpression plasmids can obviously up-regulate the expression level of circRNA XPO4.

Example three: the effect of knocking down or over expressing circRNA XPO4 gene on the osteogenic differentiation capacity of human valve interstitial cells.

1. Experimental methods

1) After the confluency of the cells reaches 80%, siRNA and plasmid for over-expressing circRNA XPO4 are transfected into the 6-well plate respectively according to the method in the second embodiment. After 14 days of culture, cells are collected, and western blot is carried out to detect the protein expression changes of the calcification marker genes ALP and RUNX 2. After 21 days of culture, alizarin red staining and calcium quantitative analysis are carried out to detect the influence of different treatments on the osteogenic differentiation of valve interstitial cells.

2) The osteogenic differentiation induction method of the valve interstitial cells comprises the following steps:

after each group of cells are transfected for 24 hours, observing the confluence degree of the cells, taking human valve interstitial cells with the culture density of about 90%, starving the human valve interstitial cells by a DMEM high-sugar culture medium containing 2% fetal calf serum overnight, performing osteogenic differentiation induction on the human valve interstitial cells by newly configuring an osteogenic induction culture medium (50 mg/mL vitamin C,5mmol/L beta-glycerophosphate, 100nmol/L dexamethasone and a high-sugar DMEM culture medium containing 2% fetal calf serum in a solvent) on the second day, and changing the liquid every three days to respectively induce for 14 days and 21 days.

2. Analysis of results

The osteogenesis induction medium is induced for 14 days, and western blot detects the protein expression changes of calcification marker genes ALP and RUNX 2. And (5) inducing the osteogenesis induction culture medium for 21 days, and carrying out alizarin red staining and calcium quantitative analysis and detection. The results are shown in FIG. 5, compared with si-NC group, the protein expressions of the calcification marker genes ALP and RUNX2 in the circRNA XPO4siRNA group are obviously reduced; alizarin red staining positive calcium nodules are obviously reduced, and the deposition amount of calcium salt is also obviously reduced; in contrast, compared with the control group, the circRNA XPO4 overexpression group has obviously increased protein expression of calcification marker genes ALP and RUNX 2; alizarin red-staining positive calcium nodules and salt deposition were also significantly increased, and the results are shown in fig. 6.

In conclusion, the circRNA XPO4 and the expression product thereof can be used as a marker for diagnosing calcified aortic valve diseases and can also be used as a target gene for preparing the medicament for treating calcified aortic valve diseases to provide a new non-operative treatment scheme for treating aortic valve calcification.

Finally, it should be noted that the above examples are only used for illustrating the present invention and do not limit the protection scope of the present invention. In addition, after reading the technical content of the invention, the skilled person can make various changes, modifications or variations to the invention, and all the equivalents thereof also belong to the protection scope defined by the claims of the present application.

Sequence listing

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Claims

Application of circRNA XPO4 in preparing human aortic valve interstitial cell osteogenic differentiation inducer, wherein the nucleotide sequence of the circRNA XPO4 gene is shown as SEQ ID NO:1 is shown.
Application of circRNA XPO4siRNA silencing reagent in preparing human aortic valve interstitial cell osteogenic differentiation inhibitor.
3. The use of a circRNA XPO4siRNA silencing agent of claim 2 in the preparation of an inhibitor of human aortic valve interstitial cell osteogenic differentiation, wherein the circRNA XPO4 silencing agent comprises a circRNA XPO4siRNA with a sense strand nucleotide sequence as set forth in SEQ ID NO:6 is shown in the specification; the nucleotide sequence of the antisense strand is shown as SEQ ID NO: shown at 7.
4, application of the circRNA XPO4 detection reagent in preparation of a kit for detecting the osteogenic differentiation level of interstitial cells of human aortic valves.
5. The use of the circRNA XPO4 detection reagent as claimed in claim 4 in the preparation of a kit for detecting the level of osteogenic differentiation of human aortic valve stromal cells, wherein the circRNA XPO4 detection reagent is a PCR detection reagent comprising a set of primers consisting of the nucleotide sequence set forth in SEQ ID NO:2 and SEQ ID NO:3, and 3, or a DNA fragment thereof.
6. The use of the circRNA XPO4 detection reagent as claimed in claim 5, in the preparation of a kit for detecting the level of osteoblastic differentiation of stromal cells of human aortic valve, wherein the primers further comprise an internal reference primer, the internal reference primer is β -actin, and the internal reference primer comprises the sequence given by SEQ ID NO:4 and SEQ ID NO:5, and (b) a primer pair consisting of the DNAs shown in the figure.
And 7, application of the circRNA XPO4 as a marker for judging osteogenic differentiation capacity of interstitial cells of the human aortic valve.