阅读说明：本技术 circRNA PRDM5在钙化性主动脉瓣疾病的诊断试剂盒及治疗药物开发中的应用 (Application of circRNA PRDM5 in diagnosis kit and therapeutic drug development of calcified aortic valve diseases ) 是由 王勇军 董念国 韩东 周廷文 史嘉玮 于 2021-07-12 设计创作，主要内容包括：本发明涉及circRNA PRDM5在钙化性主动脉瓣疾病的诊断试剂盒及治疗药物开发中的应用。本发明通过筛选一种人瓣膜间质细胞成骨分化相关的circRNA PRDM5并验证其应用,通过检测正常与钙化主动脉瓣组织中的circRNA PRDM5的表达水平,并对circRNA PRDM5基因进行细胞功能学研究,发现过表达circRNA PRDM5,可以显著促进人瓣膜间质细胞成骨分化能力,证明circRNA PRDM5及其表达产物可作为诊断钙化性主动脉瓣疾病的标志物,也可作为制备治疗钙化性主动脉瓣疾病药物的靶基因,为治疗主动脉瓣钙化提供新的非手术治疗方案。(The invention relates to application of circRNA PRDM5 in the development of a diagnostic kit and a therapeutic drug for calcified aortic valve diseases. The invention screens circRNA PRDM5 related to osteogenic differentiation of human valve interstitial cells and verifies the application, the expression level of the circRNA PRDM5 in normal and calcified aortic valve tissues is detected, and the cell function research is carried out on the circRNA PRDM5 gene, so that the over-expression of the circRNA PRDM5 is found, the osteogenic differentiation capability of the human valve interstitial cells can be remarkably promoted, and the result proves that the circRNA PRDM5 and the expression product thereof can be used as a marker for diagnosing calcified aortic valve diseases, can also be used as a target gene for preparing medicaments for treating calcified aortic valve diseases, and provides a novel non-operative treatment scheme for treating aortic valve calcification.)

1. A calcified aortic valve disease diagnostic kit is characterized by comprising a reagent for detecting circRNA PRDM5, wherein the circbaseID of the circRNA PRDM5 is hsa _ circ _ 0005654.

2. The calcified aortic valve disease diagnostic kit of claim 1, wherein the kit comprises a primer for amplifying circRNA PRDM 5.

3. The diagnostic kit for calcified aortic valve diseases according to claim 2, wherein the primer comprises a primer pair consisting of DNA sequences shown by nucleotide sequences SEQ ID NO. 2 and SEQ ID NO. 3.

4. A pharmaceutical composition for treating calcified aortic valve diseases, which comprises an agent for inhibiting or preventing the expression of circRNA PRDM5 gene or an agent for inactivating the circRNA PRDM5 expression product, wherein the nucleotide sequence of the circRNA PRDM5 gene is shown as SEQ ID NO. 1.

5. The pharmaceutical combination for treating calcific aortic valve disease of claim 4, wherein the agent comprises shRNA or siRNA for silencing circRNA PRDM5 gene expression.

6. The pharmaceutical combination according to claim 5, wherein the sense strand nucleotide sequence of the siRNA is as shown in SEQ ID NO:4, the antisense strand nucleotide sequence of the siRNA is shown as SEQ ID NO:5, respectively.

7. The pharmaceutical combination for treating calcific aortic valve diseases according to claim 6, further comprising a negative control NC (negative control) sequence, wherein the sense strand nucleotide sequence of the negative control NC is as shown in SEQ ID NO:6, the antisense strand nucleotide sequence of the negative control NC is shown as SEQ ID NO: shown at 7.

The application of the circRNA PRDM5 gene and its expression product as target in preparing or screening medicine for treating calcified aortic valve diseases, wherein the nucleotide sequence of the circRNA PRDM5 is shown as SEQ ID NO. 1.

The technical field is as follows:

the invention relates to the field of biological medicines, in particular to application of circRNA PRDM5 in the development of a diagnostic kit and a therapeutic drug for calcified aortic valve diseases.

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, which 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 osteogenic differentiation of valve interstitial cells. 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 is subjected to the surgical operation inevitably bears 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.

circRNA PRDM5(circbaseID: hsa _ circ _0005654), which maps on the genome: chr4:121675708-121732604, the corresponding linear gene is PRDM5 (NM-018699), and the circularized sequence length is 758 bases. PRDM5, member 5 of the PR region protein family (PRDI-BF1 and RIZ domain conjugation, PRDM), belongs to the zinc finger protein family, is a tissue-specific transcription factor, and has been reported to have a close relationship with bone development, but its function in calcified aortic valve diseases is still lacking.

The invention content is as follows:

technical problem to be solved

Aiming at the background, the invention provides a calcified aortic valve disease diagnosis kit based on the circRNA PRDM5 gene by researching the expression of the circRNA PRDM5 in calcified aortic valve and the regulating effect on the osteoblastic differentiation of interstitial cells of the human aortic valve, and provides an application of the circRNA PRDM5 gene and an expression product thereof as targets in preparing or screening medicines for treating calcified aortic valve diseases, thereby providing a new non-operative treatment scheme for treating aortic valve calcification.

(II) technical scheme

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention discloses a first aspect and provides a calcified aortic valve disease diagnostic kit, which comprises a reagent for detecting circRNA PRDM5, wherein the circbaseID of the circRNA PRDM5 is hsa _ circ _ 0005654.

Further, the kit comprises primers for amplifying circRNA PRDM 5.

Further, the primer includes a primer pair composed of DNA sequences shown by nucleotide sequences SEQ ID NO. 2 and SEQ ID NO. 3.

The invention discloses a second aspect, provides a pharmaceutical composition for treating calcified aortic valve diseases, which comprises an agent for inhibiting or preventing the expression of circRNA PRDM5 gene or an agent for inactivating the circRNA PRDM5 expression product by binding, wherein the nucleotide sequence of the circRNA PRDM5 gene is shown as SEQ ID NO. 1.

Further, the agent comprises shRNA or siRNA for silencing circRNA PRDM5 gene expression.

Further, the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO:4, the antisense strand nucleotide sequence of the siRNA is shown as SEQ ID NO:5, respectively.

Further, the pharmaceutical composition for treating calcified aortic valve diseases further comprises a negative control NC (negative control) sequence, wherein the sense strand nucleotide sequence of the negative control NC is shown as SEQ ID NO:6, the antisense strand nucleotide sequence of the negative control NC is shown as SEQ ID NO:7 is shown in

The invention discloses a third aspect, and provides an application of a circRNA PRDM5 gene and an expression product thereof as targets in preparation or screening of medicines for treating calcified aortic valve diseases, wherein the nucleotide sequence of the circRNA PRDM5 is shown as SEQ ID NO. 1.

(III) advantageous effects

The invention has the following beneficial effects: (1) the invention carries out RT-PCR by designing a specific circular RNA primer, and confirms the real existence of the circRNA PRDM5 gene; (2) according to the invention, by detecting the circRNA PRDM5 gene expression in human calcified aortic valve tissues, the expression level is proved to be obviously increased compared with that of normal aortic valve tissues, and the circRNA PRDM5 can be used as a diagnostic marker of calcified aortic valve diseases; (3) according to the invention, by researching the influence of circRNA PRDM5 on the osteogenic differentiation of human aortic valve interstitial cells, the over-expression of circRNA PRDM5 is proved to remarkably promote the osteogenic differentiation of human aortic valve interstitial cells, and the down-regulation of circRNA PRDM5 can obviously inhibit the osteogenic differentiation capacity of human aortic valve interstitial cells, so that a basis is provided for the preparation of an anti-aortic valve calcification therapeutic drug taking circRNA PRDM5 as a target spot; (4) the invention provides a circRNA PRDM5 and an expression product thereof, which can be used as a marker for diagnosing calcified aortic valve diseases, can also be used as a target gene for preparing medicaments for treating calcified aortic valve diseases, and provides a novel non-operative treatment scheme for treating aortic valve calcification.

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 verification of the inter-species (human, murine) conservation of the circRNA PRDM5 gene;

FIG. 2 is a graph of QRT-PCR detection of the expression level of circRNA PRDM5 in normal and calcified aortic valve tissue in example one. (NC: normal aortic valve, CAVs: calcified aortic valve);

FIG. 3 is a graph of the results of validation of the overexpression plasmid and siRNA knockdown efficiency of circRNA PRDM5 in example two;

FIG. 4 is a graph showing the results of calcium-related marker protein (RUNX2, ALP) detection (FIG. 4A) after culturing for 14 days in osteogenic differentiation induction medium, followed by alizarin red staining (FIG. 4B) and calcium salt quantitative analysis (FIG. 4C) after culturing for 21 days in osteogenic differentiation induction medium, in the third example, after transfecting CircRNA PRDM 5-specific siRNA to interfere with CircRNA PRDM5 gene in human primary valvular stromal cells;

FIG. 5 is a graph showing the results of transfection of a circRNA PRDM 5-specific overexpression plasmid into human primary valvular stromal cells, overexpression of the circRNA PRDM5 gene, detection of a calcification-associated marker protein (RUNX2, ALP) after 14 days in osteogenic differentiation induction medium (FIG. 5A), followed by alizarin red staining after 21 days in osteogenic differentiation induction medium (FIG. 5B), and quantitative calcium salt analysis (FIG. 5C).

The specific implementation mode is as follows:

the technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

The invention firstly carries out high-throughput sequencing by collecting 4 cases of normal and calcified aortic valve tissues and screens the circRNA differential expression profiles of normal and disease group tissues. And screening out the circRNA PRDM5 gene with high conservation among species (human and mouse). Then, a specific primer capable of amplifying the circular RNA is designed, and a large sample is verified through a QRT-PCR technology to verify the circRNA PRDM5 gene expression in human calcified aortic valve tissues, so that the expression level is proved to be remarkably increased compared with that of normal aortic valve tissues, and the circRNA PRDM5 can be used as a diagnostic marker of calcified aortic valve diseases.

Furthermore, by researching the influence of circRNA PRDM5 on the osteogenic differentiation of human aortic valve interstitial cells, the invention proves that the overexpression of circRNA PRDM5 can obviously promote the osteogenic differentiation of the human aortic valve interstitial cells, and the reduction of circRNA PRDM5 can obviously inhibit the osteogenic differentiation capacity of the human aortic valve interstitial cells, thereby providing a basis for preparing the aortic valve calcification resisting treatment medicine taking the circRNA PRDM5 as a target spot.

The related technologies of the invention are all conventional technologies of molecular cloning, the related reagents and consumables belong to common products sold in the market, and the related detection means and instruments are well known and skilled by those skilled in the art.

The technical solution of the present invention is further illustrated by the following examples:

the first embodiment is as follows: 4 cases of normal and calcified aortic valve tissues are collected, high-throughput sequencing is carried out, and the circRNA differential expression profiles of normal and disease group tissues are screened.

In this example, human and mouse conserved circRNA PRDM5 gene was screened based on differentially expressed circRNA whose expression was up-regulated in calcified aortic valve tissues in sequencing data, and then large samples were used to verify the expression of differentially expressed circRNA PRDM5 gene in normal and calcified aortic valve tissues using QRT-PCR technique.

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 tube bottom 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 (10ul) by the company TAKARA, Japan:

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 detects the expression of circRNA PRDM5 in normal and calcified aortic valve tissues, and the primer pair consisting of the DNA sequences shown as SEQ ID NO. 2 and SEQ ID NO. 3 is used as the primer of circRNA PRDM 5.

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) And (3) performing amplification on the machine, confirming an amplification curve and a dissolution curve after the reaction is finished, and calculating a P value according to the CT value (threshold cycle values) and the T test of the expression intensity of each gene. The specific real-time fluorescent quantitative PCR primer sequence of the internal reference housekeeping gene beta-actin is shown as a primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5.

4) QRT-PCR results

The results are shown in FIG. 2. The expression of circRNA PRDM5 was detected in 30 normal aortic valve tissue and 30 calcified aortic valve tissue samples, showing that circRNA PRDM5 was significantly upregulated in calcified aortic valve tissue compared to normal aortic valve tissue.

Example two: construction of circRNA PRDM5 Small interfering RNA and overexpression plasmid

1. Design of circRNA PRDM5 Small interfering RNA: the circRNA PRDM5 siRNA sequence was designed and synthesized by Gisela Guangzhou Biotech. The siRNA sense strand sequence of the circRNA PRDM5 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 circRNA PRDM5 and transient transfection of human valve stromal cells: synthesizing a circular RNA PRDM5 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 valve interstitial cells are inoculated to a 24-well plate and cultured at 37 ℃ with 5% CO 2.

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% CO 2.

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. 3. The results show that siRNA can effectively reduce circRNA PRDM5 expression, while overexpression plasmid can significantly up-regulate circRNA PRDM5 expression level.

Example three: effect of knocking down or over expressing circRNA PRDM5 gene on human valve stromal cell osteogenic differentiation capacity.

1. Experimental methods

1) Human valve stromal cells are transfected into 6-well plates, and after the cell confluence reaches 80%, siRNA and over-expression circRNA PRDM5 plasmids are transfected 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 calcification marker genes RUNX2 and Osterix. 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) Osteogenic differentiation induction method for valve interstitial cells

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, and performing osteogenic differentiation induction on the human valve interstitial cells by newly configuring an osteogenic induction culture medium (50mg/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, changing the liquid every three days, and inducing for 14 days.

2. Analysis of results

The osteogenesis induction culture medium is induced for 14 days, and western blot detects the protein expression changes of calcification marker genes RUNX2 and Osterix. 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. 4, compared with si-NC group, the circRNA PRDM5 siRNA group has significantly reduced calcification marker genes RUNX2, Osterix protein expression (a); alizarin red staining positive calcium nodules are obviously reduced (B), and the deposition amount of calcium salt is also obviously reduced (C); in contrast, as shown in fig. 5, compared with the control group, the circRNA PRDM5 overexpression group had significantly increased expression of the calcification marker genes RUNX2 and Osterix protein (a); alizarin red staining positive calcium nodules (B) and salt deposition amount are also significantly increased (C).

Conclusion

The circRNA PRDM5 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 medicaments for treating calcified aortic valve diseases so as 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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