![Figure 5. miR-217 regulates a DNA damage response and repair gene network and dampens Bcl-6 degradation in GC B cells. (A-B) RNAseq analysis of genome-wide gene expression in control and miR-217TG GC B cells isolated from Peyer’s patches. Pools of 4 to 6 mice per genotype were used in each experiment. Data from 2 independent experiments are shown. (A) Enrichment in miRNA prediction targets within the 1236 mRNAs whose expression was down-regulated in miR-217TG vs control GC B cells. The GEO accession number for the RNAseq data is GSE47877. (B) Ingenuity pathway analysis of the genes differentially expressed in control vs miR-217TG GC B cells, showing enrichment for a DDR and repair gene network among the genes regulated by miR-217 (P = 10−31). Downregulated genes are shown in green; upregulated genes are shown in red. (C) Quantification of Nbs1, Xrcc2, Lig4, and Pds5b expression in Ramos cells transduced with control (open bars) or miR-217 (filled bars) retrovirus analyzed by qRT-PCR. Two-tailed Student t test with P values: *P < .05. (D) Cell cycle analysis of etoposide-treated control- and miR-217-transduced Ramos cells. Representative cell cycle plots of untreated (−Etop) or etoposide-treated (6 hours, 10 μM) (+Etop) Ramos cells. Fraction of cells in S cycle ± standard deviation is shown. The lower graph depicts the change in the proportion of cells at each phase of cell cycle in response to etoposide treatment (open bars, control-transduced cells; black bars, miR-217-transduced cells). Bars represent means ± standard deviations from 3 independent experiments (P = .004 for S phase cells, 2-tailed Student t test). (E) (Upper) Immunoblot of Bcl-6 in Ramos cells transduced with control or miR-217 retrovirus treated for 6 hours with the indicated doses of etoposide. (Lower) Immunoblot of Bcl-6 in miR-217-transduced cells left untreated (−Etoposide) or treated with 10 μM Etposide for 6 hours (+Etoposide) in the presence of increases doses of caffeine (0 mM [−], 2 mM [+], and 5 mM [++]. (F) Analysis of Bcl-6 expression in GC cells by flow cytometry. (Upper) Representative dot plots of Fas, GL7, and Bcl-6 staining of Peyer’s patch B cells. Quantification of Bcl-6 expression levels in B220+Fas+GL7+ GC B cells from Peyer’s patches of littermate controls (white bar) and miR-217KI mice (black bar) (MFI, mean fluorescence intensity; n = 3 mice/genotype, P = .0006, 2-tailed Student t test). (G) Bcl-6 expression in spleens of control and miR-217TG mice analyzed by immunohistochemical staining. A representative picture is shown. The proportion of Bcl-6+ stained in follicle areas was quantified in 205 and 201 spleen follicles of control and 10 miR-217TG mice, respectively. Statistical significance was determined by 2-tailed Student t test (P = .0008).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/124/2/10.1182_blood-2013-12-543611/4/229f5.jpeg?Expires=1722568034&Signature=uErBizDFSPArKEo5nNksjgGAZy5KsvjUtdjUCa-PE0qHH9KpqnsPKdRe75m6i~zqthfX-88rX4dil4OCD1YYBnT9uMNfZ45riQYP3YoJN4tSi52q1PlUkABofogVjriLuwMCZ5VTsKYhk~L7QATzoeQ5x9qzE8NPM8vgLfbXNaKWKd4XiipyGUAKV4YJUIy6uP-J7HIyYzskHlXX1pHd5sv~c4uigYrMkB2JSzWY8M0SWAzzExahQOKo8yGDot8SNjooAINERhZ8OtjuaUDQjwcYz2ta1baC~JFVb2w5m~cDfW3TsVyqE9g9FL4Otyn6TBuR-AuNw4gCaF34rREeNg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
miR-217 regulates a DNA damage response and repair gene network and dampens Bcl-6 degradation in GC B cells. (A-B) RNAseq analysis of genome-wide gene expression in control and miR-217TG GC B cells isolated from Peyer’s patches. Pools of 4 to 6 mice per genotype were used in each experiment. Data from 2 independent experiments are shown. (A) Enrichment in miRNA prediction targets within the 1236 mRNAs whose expression was down-regulated in miR-217TG vs control GC B cells. The GEO accession number for the RNAseq data is GSE47877. (B) Ingenuity pathway analysis of the genes differentially expressed in control vs miR-217TG GC B cells, showing enrichment for a DDR and repair gene network among the genes regulated by miR-217 (P = 10−31). Downregulated genes are shown in green; upregulated genes are shown in red. (C) Quantification of Nbs1, Xrcc2, Lig4, and Pds5b expression in Ramos cells transduced with control (open bars) or miR-217 (filled bars) retrovirus analyzed by qRT-PCR. Two-tailed Student t test with P values: *P < .05. (D) Cell cycle analysis of etoposide-treated control- and miR-217-transduced Ramos cells. Representative cell cycle plots of untreated (−Etop) or etoposide-treated (6 hours, 10 μM) (+Etop) Ramos cells. Fraction of cells in S cycle ± standard deviation is shown. The lower graph depicts the change in the proportion of cells at each phase of cell cycle in response to etoposide treatment (open bars, control-transduced cells; black bars, miR-217-transduced cells). Bars represent means ± standard deviations from 3 independent experiments (P = .004 for S phase cells, 2-tailed Student t test). (E) (Upper) Immunoblot of Bcl-6 in Ramos cells transduced with control or miR-217 retrovirus treated for 6 hours with the indicated doses of etoposide. (Lower) Immunoblot of Bcl-6 in miR-217-transduced cells left untreated (−Etoposide) or treated with 10 μM Etposide for 6 hours (+Etoposide) in the presence of increases doses of caffeine (0 mM [−], 2 mM [+], and 5 mM [++]. (F) Analysis of Bcl-6 expression in GC cells by flow cytometry. (Upper) Representative dot plots of Fas, GL7, and Bcl-6 staining of Peyer’s patch B cells. Quantification of Bcl-6 expression levels in B220+Fas+GL7+ GC B cells from Peyer’s patches of littermate controls (white bar) and miR-217KI mice (black bar) (MFI, mean fluorescence intensity; n = 3 mice/genotype, P = .0006, 2-tailed Student t test). (G) Bcl-6 expression in spleens of control and miR-217TG mice analyzed by immunohistochemical staining. A representative picture is shown. The proportion of Bcl-6+ stained in follicle areas was quantified in 205 and 201 spleen follicles of control and 10 miR-217TG mice, respectively. Statistical significance was determined by 2-tailed Student t test (P = .0008).
