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Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro
Published on: November 15, 2024
LNCRNA XIST Inhibits miR-377-3p to Hinder Th17 Cell Differentiation through Upregulating ETS1
Chen Yao1, Chao Li2, Zhijia Liu2
1Beijing IROT Key Laboratory, The 8th Medical Center of Chinese PLA General Hospital, 100091 Beijing City, China.
Background:
Th17 cell differentiation is involved in the development and progression of many diseases, such as rheumatoid arthritis and systemic lupus erythematosus. Present study mainly focused on the role of LINC-XIST in Th17 cell differentiation.
Methods:
The naïve CD4+ T cells were isolated from human whole blood. Cells were cultured under Th17 cell-polarizing condition for 6 days. The expression of LINC-XIST and miR-153-3p was measured by qPCR. The relationship between LINC-XIST, miR-153-3p, and ETS1 was predicted by TargetScan website and authenticated by luciferase reporter assay. ELISA assays were conducted to evaluate the IL-17 concentration. Western blot was utilized to measure the protein expression of ETS1. Th17 cell frequency was examined by flow cytometry.
Results:
The expression of XIST markedly decreased and miR-153-3p expression markedly increased with Th17 cell differentiation. The mRNA expression of IL-17, IL-17 concentration, and Th17 cell frequency were observably decreased in overexpressed LINC-XIST group. Luciferase reporter assay authenticated that miR-153-5p was directly regulated by LINC-XIST. miR-153-3p inhibitor observably decreased IL-17 concentration, mRNA expression of IL-17, and Th17 cell frequency while si-XIST reversed this impact. ETS1 was confirmed to be regulated by miR-153-5p via luciferase reporter assay. In addition, ETS1 markedly decreased IL-17 mRNA expression, IL-17 concentration, and Th17 cell frequency while miR-153-5p mimic reversed this impact.
Conclusion:
LNCRNA XIST inhibited miR-377-3p to hinder Th17 cell differentiation through upregulating ETS1.
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