Application of circRNA PRDM5 in development of diagnostic kit and therapeutic drug for calcified aortic valve diseases

An aortic valve and disease diagnosis technology, applied in the field of biomedicine, can solve problems such as lack of research on the function of calcified aortic valve disease, and achieve the effect of inhibiting osteogenic differentiation ability and promoting osteogenic differentiation

Active Publication Date: 2021-11-19
XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

PRDM5, member 5 of the PR domain protein family (PRDI-BF1 and RIZ domain containing, PRDM), belongs to the zinc finger protein family and is a type of tissue-specific transcription factor. It has been reported that it is closely related to bone development, but its role in Function is lacking in calcific aortic valve disease

Method used

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  • Application of circRNA PRDM5 in development of diagnostic kit and therapeutic drug for calcified aortic valve diseases
  • Application of circRNA PRDM5 in development of diagnostic kit and therapeutic drug for calcified aortic valve diseases
  • Application of circRNA PRDM5 in development of diagnostic kit and therapeutic drug for calcified aortic valve diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: 4 cases of normal and calcified aortic valve tissues were collected, and high-throughput sequencing was performed to screen the differential expression profile of circRNA in normal and diseased tissues.

[0031] In this example, the conserved circRNA PRDM5 gene in humans and mice was screened out based on the differentially expressed circRNAs up-regulated in calcified aortic valve tissue in the sequencing data, and then QRT-PCR technology was used to verify the differentially expressed circRNA PRDM5 gene in normal and calcified aortic valve tissues. Expression in arterial valve tissue.

[0032] The specific experimental plan is as follows:

[0033] 1. RNA extraction

[0034]1) Tissue processing: Take about 50 mg of aortic valve tissue, grind it to a satisfactory level with liquid nitrogen, add 1 ml Trizol reagent, oscillate and homogenate, fully lyse, centrifuge at 12,000 rpm, 4°C for 15 minutes, and take the supernatant.

[0035] 2) Add 200 ul of chlorofor...

Embodiment 2

[0054] Example 2: Construction of circRNA PRDM5 small interfering RNA and overexpression plasmid

[0055] 1. circRNA PRDM5 small interfering RNA design: The circRNA PRDM5 siRNA sequence was designed and synthesized by Guangzhou Jisai Biotechnology Co., Ltd. The siRNA sense strand sequence of circRNA PRDM5 is shown in SEQ ID NO:6, and the antisense strand sequence is shown in SEQ ID NO:7. The sense strand and antisense strand of the negative control group sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively.

[0056] 2. Overexpression of circRNA PRDM5 by pCD5-ciR vector and transient transfection of human valve mesenchymal cells: Synthesize the complete linear sequence of circRNAPRDM5, anneal the sequence into a double-stranded DNA fragment and insert it into the pCD5-ciR vector through multiple cloning sites, and identify the recombinant plasmid by sequencing , the control group was pCD5-ciR empty vector.

[0057] 3. siRNA and overexpression efficacy verificati...

Embodiment 3

[0065] Example 3: The effect of knocking down or overexpressing circRNA PRDM5 gene on the osteogenic differentiation ability of human valve mesenchymal cells.

[0066] 1. Experimental method

[0067] 1) Human valve interstitial cells were planted in a 6-well plate. After the confluence of the cells reached 80%, according to the method in Example 2, siRNA and overexpressed circRNA PRDM5 plasmids were respectively transfected. Cells were collected after 14 days of culture, and western blot was performed to detect the expression changes of calcification marker genes RUNX2 and Osterix protein. After 21 days of culture, alizarin red staining and calcium quantitative analysis were performed to detect the effects of different treatments on the osteogenic differentiation of valve interstitial cells.

[0068] 2) Method for induction of osteogenic differentiation of valve mesenchymal cells

[0069] After 24 hours of transfection of the cells in each group, observe the confluence of th...

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Abstract

The invention relates to application of circRNA PRDM5 in development of a diagnostic kit and a therapeutic drug for calcified aortic valve diseases. According to the invention, the circRNA PRDM5 related to osteogenic differentiation of human valvular interstitial cells is screened, the application of the circRNA PRDM5 is verified, the expression level of the circRNA PRDM5 in normal and calcified aortic valve tissues is detected, cell function study is carried out on the circRNA PRDM5 gene, and it is found that overexpression of the circRNA PRDM5 can remarkably promote the osteogenic differentiation capacity of the human valvular interstitial cells; and thus, it is proved that the circRNA PRDM5 and an expression product thereof can be used as markers for diagnosing the calcified aortic valve diseases and can also be used as target genes for preparing medicines used for treating the calcified aortic valve diseases, and a new non-surgical treatment scheme is provided for treating aortic valve calcification.

Description

Technical field: [0001] The invention relates to the field of biomedicine, in particular to the application of circRNA PRDM5 in the development of diagnostic kits and therapeutic drugs for calcific aortic valve disease. Background technique: [0002] Calcific aortic valve disease (CAVD) is an age-related progressive disease with high morbidity and mortality. Hemodynamic changes that affect cardiac function. In the past, it was believed that CAVD is an age-related degenerative disease, which is the process of degeneration and hardening of valve tissue with age. However, basic research in recent years has shown that CAVD is an active progressive process involving complex pathological changes such as endothelial injury, inflammatory cell infiltration, extracellular matrix remodeling, and osteoblastic differentiation of valvular interstitial cells. The cells in valve tissue mainly include valve endothelial cells (Valvular endothelial cells, VECs), valve interstitial cells (VIC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12N15/113
CPCC12Q1/6883C12Q1/686C12Q2600/178C12Q2600/158Y02A50/30
Inventor 王勇军董念国韩东周廷文史嘉玮
Owner XIEHE HOSPITAL ATTACHED TO TONGJI MEDICAL COLLEGE HUAZHONG SCI & TECH UNIV
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