miR-24-3p Is Overexpressed in Hodgkin Lymphoma and Protects Hodgkin and Reed-Sternberg Cells from Apoptosis

Abstract

miRNAs play important roles in biological processes, such as proliferation, metabolism, differentiation, and apoptosis, whereas altered expression levels contribute to diseases, such as cancers. We identified miRNAs with aberrant expression in Hodgkin lymphoma (HL) and investigated their role in pathogenesis. Small RNA sequencing revealed 84 significantly differentially expressed miRNAs in HL cell lines as compared to germinal center B cells. Three up-regulated miRNAs-miR-23a-3p, miR-24-3p, and miR-27a-3p-were derived from one primary miRNA transcript. Loss-of-function analyses for these miRNAs and their seed family members resulted in decreased growth on miR-24-3p inhibition in three HL cell lines and of miR-27a/b-3p inhibition in one HL cell line. Apoptosis analysis indicated that the effect of miR-24-3p on cell growth is at least in part caused by an increase of apoptotic cells. Argonaute 2 immunoprecipitation revealed 1142 genes consistently targeted by miRNAs in at least three of four HL cell lines. Furthermore, 52 of the 1142 genes were predicted targets of miR-24-3p. Functional annotation analysis revealed a function related to cell growth, cell death, and/or apoptosis for 15 of the 52 genes. Western blotting of the top five genes showed increased protein levels on miR-24-3p inhibition for CDKN1B/P27 and MYC. In summary, we showed that miR-24-3p is up-regulated in HL and its inhibition impairs cell growth possibly via targeting CDKN1B/P27 and MYC.

MicroRNA High Throughput Loss-of-Function Screening Reveals an Oncogenic Role for miR-21-5p in Hodgkin Lymphoma

“…Statistical analysis of GFP competition assays was performed as described previously [16]. Briefly, decrease in percentages of GFP + cells over time was compared with the control miRZIP-NT1 using a mixed model with time and the interaction of time and miRNA inhibitor type as fixed effect and measurement repeat within miRNA inhibitor type as random effect in SPSS (22.0.0.0 version, IBM, Armonk, New York, USA).…”

“…We included a total of 63 constructs in the pool, these were partly selected based on being highly abundant or differentially expressed in cHL compared to GC B cells (n=17) [16], and partly included based on availability of constructs (n=41; 34 miRNAs with similar expression levels in cHL cell and GC-B cells, 1 miRNA with decreased expression and 6 miRNAs not expressed in cHL) ( Table S1) Table S2. In addition to the miRNA inhibition constructs the library also included 186 shRNA inserts targeting genes Fig.…”

“…This list was used to identify the miR-21-5p target genes consistently Ago2-IP enriched in at least 2 out of 3 cHL cell lines using our previously published Ago2-IP data in cHL cell lines [16].…”

“…Lentivirus production, infection and sorting Lentiviral particles were produced in HEK-293T cells by calcium phosphate precipitation transfection procedure as described previously [16]. CHL cells were transduced with virus in the presence of 4μg/ ml polybrene.…”

“…This resulted in the identification of miR-21-5p as a miRNA critical to cHL cell growth by inhibiting apoptosis. Insights into the potential mechanism of miR-21-5p were obtained by identifying target genes in our previously published Ago2-IP data [16].…”