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

ABSTRACT: 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.

SUBMITTER: Schreiber S

PROVIDER: S-EPMC10605364 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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

Schreiber Sandra S Daum Patrick P Danzer Heike H Hauke Manuela M Jäck Hans-Martin HM Wittmann Jürgen J

Biomolecules 20231013 10

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 represen ...[more]

PMID: 37892199

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Project description:ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3'-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3'-UTR and pGL3 Control LDLR mutant-3'-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3'-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3'-UTR-driven (p<0.01), but not mutant LDLR-3'-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = -0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3'-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

Dataset Information

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

Publications

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

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