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MiR-378a-3p is critical for Burkitt lymphoma cell growth

ABSTRACT: To gain further knowledge on the role of microRNAs (miRNAs) in the pathogenesis of Burkitt lymphoma (BL), we performed small RNA sequencing in BL cell lines and normal germinal center B (GC-B) cells. This revealed 26 miRNAs with significantly different expression levels. Altered levels were validated by qRT-PCR for six of eight selected miRNAs. For five miRNAs, the differential expression pattern was confirmed in BL primary tissues compared to GC-B cells. Inhibition of miR-378a-3p reduced the growth of multiple BL cell lines. RNA immunoprecipitation of Argonaute 2 followed by microarray analysis (Ago2-RIP-Chip) upon inhibition and ectopic overexpression of miR-378a-3p revealed 63 and 20 putative miR-378a-3p targets, respectively. These included 28 genes with a putative miR-378a-3p binding site. Effective targeting by miR-378a-3p was confirmed by luciferase reporter assays for four out of eight selected genes: MNT, FOXP1, IRAK4, and lncRNA JPX, all of which have been implicated in proliferation and cancer. In summary, our study identified several differentially expressed miRNAs in BL. We identified miR-378a-3p as a miRNA with an oncogenic role in BL and revealed 4 novel miR-378a-3p target genes, i.e. MNT, FOXP1, IRAK4, and JPX.

ORGANISM(S): Homo sapiens

PROVIDER: GSE141691 | GEO | 2020/06/30

REPOSITORIES: GEO

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Project description:We aimed at extending the repertoire of high-quality miRNA normalizers for reverse transcription-quantitative PCR (RT-qPCR) of human plasma with special emphasis on the extremely guanine-cytosine-rich portion of the miRNome. For high-throughput selection of stable candidates, microarray technology was preferred over small-RNA sequencing (sRNA-seq) since the latter underrepresented miRNAs with a guanine-cytosine (GC) content of at least 75% (p = 0.0002, n = 2). miRNA abundances measured on the microarray were ranked for consistency and uniformity using nine normalization approaches. The eleven most stable sequences included miRNAs of moderate, but also extreme GC content (45%–65%: miR-320d, miR-425-5p, miR-185-5p, miR-486-5p; 80%–95%: miR-1915-3p, miR-3656-5p, miR-3665-5p, miR-3960-5p, miR-4488-5p, miR-4497 and miR-4787-5p). In contrast, the seven extremely GC-rich miRNAs were not found in the two plasma miRNomes screened by sRNA-seq. Stem-loop RT-qPCR was employed for stability verification in 32 plasma samples of healthy male Caucasians (age range: 18–55 years). In general, inter-individual variance of miRNA abundance was low or very low as indicated by coefficient of variation (CV) values of 0.6%–8.2%. miR-3665 and miR-1915-3p outperformed in this analysis (CVs: 0.6 and 2.4%, respectively). The eight most stable sequences included four extremely GC-rich miRNAs (miR-1915-3p, miR-3665, miR-4787-5p and miR-4497). The best-performing duo normalization factor (NF) for the condition of human plasma, miR-320d and miR-4787-5p, also included a GC-extreme miRNA. In summary, the identification of extremely guanine-cytosine-rich plasma normalizers will help to increase accuracy of PCR-based miRNA quantification, thus raise the potential that miRNAs become markers for psychological stress reactions or early and precise diagnosis of clinical phenotypes. The novel miRNAs might also be useful for orthologous contexts considering their conservation in related animal genomes.

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MiR-378a-3p is critical for Burkitt lymphoma cell growth

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