miRNA targeting cJun signal channel as well as preparation method and application thereof
A signal pathway and targeting technology, which is applied in the field of biomedicine and can solve the problems of low bioavailability, metabolic inactivation, high bioinhibitory activity, etc.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] Example 1 Preparation of miRNA miR-4632
[0050] The mature sequence of miR-4632 is 5'-UGCCGCCCUCUCGCUGCUCUAG-3' (22bp, SEQ ID NO.1) which can be prepared in the following way:
[0051] 1) chemical synthesis
[0052] The analog of miR-4632 (miR-4632mimic) was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., which is a double-stranded RNA that can mimic the high-level expression of endogenous mature miR-4632; standard purification, stored at -20°C. Dissolve in RNase-free water to prepare a 20M stock solution, store at -80°C for later use, and dilute to the required concentration when used.
[0053] 2) Expression of miR-4632 mediated by viral vector
[0054] (1) Obtain the mature sequence and precursor sequence (pre-miRNA) information of miR-4632 in miRBase (http: / / www.mirbase.org / ) database. Find two flanking sequences of about 300bp in the mature sequence in the human genome database, design primers based on the flanking sequences, and add restriction sites an...
Embodiment 2
[0065] Example 2 miR-4632 targets and inhibits cJun molecules and their signaling pathways
[0066] (1) Cell culture: HEK293A cells (purchased from ATCC, Manassas, VA) were cultured with DMEN medium containing 10% fetal bovine serum, and the cells in good growth state were collected and divided into 6×10 4 Inoculate / well in a 24-well plate at 37°C, 5% CO 2 Cultivate for 24 hours;
[0067] (2) Human pulmonary artery smooth muscle cells were purchased from Lonza (Walkersville, MD), cultured in SMCM complete medium containing 5% serum, collected cells in good growth state, and centrifuged at 6×10 5 Inoculate in 60mm dishes, 37°C, 5% CO 2 Cultivate for 24 hours;
[0068] (3) Transfection: when the cell density reaches 70%, the cells are transfected by the calcium phosphate method, and the 3'-UTR dual-luciferase reporter vector or the mutated 3'-UTR dual-luciferase reporter vector of CCJUN is co-transfected, For the miR-4632 overexpression vector pLVX-CMV-miR-4632 or pLVX-CMV-c...
Embodiment 3
[0076] Example 3 miR-4632 inhibits the proliferation of human pulmonary artery smooth muscle cells induced by platelet-derived growth factor BB (PDGFBB).
[0077] To investigate the regulation of miR-4632 on the cJun signaling pathway activated by platelet-derived growth factor BB (PDGFBB), thereby causing the abnormal function of human pulmonary artery smooth muscle cells: Infection with miR-4632-expressing lentivirus to increase miR- 4632 level, and then determine the cell proliferation level by cell proliferation marker protein PCNA and EdU cell proliferation detection.
[0078] Use the EdU Cell Proliferation Detection Kit (Ribobio) for EdU labeling, and perform the experimental operation according to the kit instructions. The main steps are as follows: 1) Cell transfection: inoculate 1×10 4 Cells were cultured for 24 hours in a 48-well plate for transfection experiments. After 24 hours of culture the next day, they were starved with serum-free medium, and then added with p...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More