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A sle-related circular RNA hsa_circ_0025843 and its application

A circular and DNA sequence technology, applied in the fields of molecular biology and immunity, can solve the problems of unknown key mechanisms and achieve the effect of promoting proliferation and secretion of IL-2

Active Publication Date: 2022-08-09
WENZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a large number of studies have shown that genetics, epigenetics, environmental factors and immunological mechanisms are involved in the occurrence and development of systemic lupus erythematosus, the key mechanism is still unknown.

Method used

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  • A sle-related circular RNA hsa_circ_0025843 and its application
  • A sle-related circular RNA hsa_circ_0025843 and its application
  • A sle-related circular RNA hsa_circ_0025843 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036]Example 1: Discovery and detection of circ_FGD4 in clinical samples

[0037] see Figure 1 to Figure 3 ,

[0038] (1) High-throughput sequencing technology screened the circular RNA circ-FGD4 in peripheral blood mononuclear cells of SLE patients

[0039] 1. Experimental grouping and collection of blood samples

[0040] A total of 30 SLE patients (age 39.5±12.1 years old) were outpatients or inpatients of the First Affiliated Hospital of Wenzhou Medical University and the Rheumatology and Immunology Department of the Second Affiliated Hospital of Wenzhou Medical University from November 2016 to May 2017. The selected cases all met the diagnostic criteria for SLE revised by the American Rheumatology Association (ARA) in 1997. Blood sample collection and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) data statistics were completed before the patients took glucocorticoids and immunosuppressive agents. The normal control group consisted of 30 healthy volunte...

Embodiment 2

[0069] Example 2: Stability experiment of circ_FGD4

[0070] see Figure 4 to Figure 5 ,

[0071] 1. To study the stability of circ_FGD4 to exonuclease (RNase R)

[0072] (1) RNase R experiment

[0073] Taking CD3+ T cells of SLE patients and healthy volunteers as research objects, RNA was extracted for RNAase R experiment, and they were divided into RNAase R+ and RNAase R- groups.

[0074] Reaction system: Storage Buffer: 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% X-100, 50% Glycerol 10X Reaction Buffer: 200mM Tris-HCl (pH 8.0), 1M KCl, 1mM MgCl2

[0075] Prepare the RNAase R+ reaction system (prepared on ice) with the following components, see Table 2.

[0076] Reagent name Usage amount RNA >5ug 10X Reaction Buffer 5ul RNAase R 3-4ul / ng RNase-free H2O To 50ul

[0077] Table 2

[0078] The above reaction solution was fully mixed and placed in a 37° water bath for 15 min.

[0079] (2) Reverse transcript...

Embodiment 3

[0099] Example 3: Positioning of circ_FGD4

[0100] see Image 6 ,

[0101] The Fish probe sequence of circ_FGD4 is as follows, see Table 5.

[0102]

[0103] table 5

[0104] 1) Probe dilution: nmole / OD260=4.17, then add 41.7 μl sterilized DEPC water to each OD probe dry powder product, and mix well to obtain a stock solution with a concentration of about 100 μM.

[0105] 2) Prepare the probe mixture: 70μl Buffer E, 2μl probe, 28μl DEPC water, the final volume is 100ul

[0106] 3) Smear: drop about 20ul of T cell suspension on a glass slide treated at -20 degrees, and take another slide as a pusher. When the cell droplets are evenly attached between the two slides, The angle between the two glass slides is 30 to 40 degrees, and the slide slide is slowly and evenly pushed from the left side of the cell drop to the right side of the slide glass, and the uniform cell membrane is pushed out, and then the slide glass is placed on the alcohol lamp. Baked into a white film. ...

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Abstract

The present invention discloses a circular RNA circ-FGD4 and use thereof, and the nucleotide sequence of the circular RNA circ-FGD4 gene is shown in SEQ ID NO: 1; in addition, the present invention provides a pharmaceutical composition , A systemic lupus erythematosus diagnostic kit and the use of a circ-FGD4 gene as a target gene in the preparation of a drug for the treatment of systemic lupus erythematosus. The circ-FGD4 gene and its expression products are used as markers for the diagnosis of systemic lupus erythematosus, making the diagnosis of systemic lupus erythematosus more accurate and rapid. Therapeutic targets and therapeutic pathways.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and immunity, in particular to an SLE-related circular RNA hsa_circ_0025843 and applications thereof. Background technique [0002] Systemic lupus erythematosus (SLE) has been recognized for many years as a classic hypersensitivity state mediated by inflammatory processes following organ-specific deposition of immune complexes, accompanied by activation of inflammatory pathways. In SLE patients, there are immune cell dysfunction, especially abnormally activated T / B lymphocytes, which lead to the production of a large number of autoantibodies, which attack their own tissues and organs and cause damage. In the pathogenesis of systemic lupus erythematosus, T and B lymphocytes play a crucial role, especially the recycling of T lymphocytes is widely exposed to antigenic substances in the body, strengthens the immune response, and further exerts cellular immunity and immune regulation. func...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12N15/113C12N15/11A61K48/00A61K31/7088A61P29/00A61P37/02
CPCC12Q1/6883A61K31/7088A61P29/00A61P37/02C12Q2600/178C12Q2600/158
Inventor 叶辉万乐予向敏王雪汪子晗褚晓颖胡煊煊李美琪杜邦张玉桑侃如
Owner WENZHOU MEDICAL UNIV
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