Identification of miR-128 Target mRNAs That Are Expressed in B Cells Using a Modified Dual Luciferase Vector

Autor: Sandra Schreiber, Patrick Daum, Heike Danzer, Manuela Hauke, Hans-Martin Jäck, Jürgen Wittmann
Jazyk: angličtina
Rok vydání: 2023
Předmět:
Zdroj: Biomolecules, Vol 13, Iss 10, p 1517 (2023)
Druh dokumentu: article
ISSN: 2218-273X
34740546
DOI: 10.3390/biom13101517
Popis: MicroRNAs (miRNAs) are 21–25 nucleotide long non-coding ribonucleic acids that modulate gene expression by degrading transcripts or inhibiting translation. The miRNA miR-128, originally thought to be brain-specific, was later also found in immune cells. To identify a valuable immune cell model system to modulate endogenous miR-128 amounts and to validate predicted miR-128 target mRNAs in B cells, we first investigated miR-128 expression using Northern blot analysis in several cell lines representing different stages of B cell development. The results showed that only primary brain cells showed significant levels of mature miR-128. To study the function of miR-128 in immune cells, we modified dual luciferase vectors to allow easy transfer of 3′ UTR fragments with predicted miR-128 binding sites from widely used single to dual luciferase vectors. Comparison of in silico predicted miR-128-regulated mRNAs in single and dual luciferase constructs yielded similar results, validating the dual luciferase vector for miRNA target analysis. Furthermore, we confirmed miR-128-regulated mRNAs identified in silico and in vivo using the Ago HITS-CLIP technique and known to be expressed in B cells using the dual luciferase assay. In conclusion, this study provides new insights into the expression and function of miR-128 by validating novel target mRNAs expressed in B cells and identifying additional pathways likely controlled by this miRNA in the immune system.
Databáze: Directory of Open Access Journals
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