Reticuloendotheliosis virus strain T induces miR-155, which targets JARID2 and promotes cell survival

Abstract

The oncogenic microRNA miR-155 is upregulated by several oncogenic viruses. The precursor of miR-155, termed bic, was first observed to cooperate with myc in chicken B-cell lymphomas induced by avian leukosis proviral integrations. We identified another oncogenic retrovirus, reticuloendotheliosis virus strain T (REV-T), that upregulates miR-155 in chicken embryo fibroblasts. We also observed very high levels of miR-155 in REV-T-induced B-cell lymphomas. To study the role of miR-155 in these tumors, we identified JARID2/Jumonji, a cell cycle regulator and part of a histone methyltransferase complex, as a target of miR-155. The overexpression of miR-155 decreased levels of endogenous JARID2 mRNA. We confirmed that miR-155 directly targets both human and chicken JARID2 by assaying the repression of reporters containing the JARID2 3'-untranslated regions. Further, the overexpression of a sponge complementary to miR-155 in a tumor cell line increased endogenous JARID2 mRNA levels. The overexpression of JARID2 in chicken fibroblasts led to decreased cell numbers and an increase in apoptotic cells. The overexpression of miR-155 rescued cells undergoing cytopathic effect caused by infection with subgroup B avian retroviruses. Therefore, we propose that miR-155 has a prosurvival function that is mediated through the downregulation of targets including JARID2.

FIG. 1.

miR-155 is upregulated in v-rel- and c-myc-induced B-cell lymphomas and by REV-T. (A) Total RNA from cells and tissues was harvested, and miR-155 levels were assayed by RNase protection. KBMC and CM758 are v-rel-derived tumor cell lines. CEFs were infected with RCAS(A) and RCAS(A)miR-155. BK3A, S13, S234S4, and H1 are c-myc-induced cell lines. miR-155 levels were compared to those for RCAS(A) and normal liver and bursa controls. (B) CEFs were infected with REV-T virus harvested from CM758 cells. Total RNA was harvested after confirming positive infection by RT assay, and miR-155 levels were assayed by RNase protection.

FIG. 2.

JARID2 3′UTR has conserved (Cons) miR-155 target sites. The miR-155 target site was predicted using RNAhybrid (44) and miRBase (20). The miR-155 target site was mapped using the UCSC BLAT genome browser, and the corresponding Vista plot showing conservation across species is displayed. X_tropicalis, Xenopus tropicalis.

FIG. 3.

JARID2 mRNA is downregulated by miR-155 in CEFs. CEFs were infected with RCAS(A), RCAS(B), RCAS(A)miR-155, and RCAS(B)miR-155 and passaged for 10 days. Total RNA was harvested, and JARID2 mRNA levels were determined by qRT-PCR and normalized to results for GAPDH mRNA. Relative changes (n-fold) were calculated by the ΔΔCT method; data represent means ± standard deviations from five experiments.

FIG. 4.

JARID2 3′UTR is sensitive to miR-155 in a dose-dependent manner. (A) Splinted ligation to assay levels of miR-155 expressed from pBIC. Based on target prediction programs, the chicken JARID2 3′UTR contains one target sequence (B) and the human JARID2 3′UTR contains two target sequences (C). The entire 3′UTR of JARID2 mRNA was cloned downstream of firefly luciferase (WT). Cells (5 × 10⁵) were cotransfected with 80 ng of firefly luciferase plasmid, 40 ng of renilla luciferase plasmids, and increasing amounts of pBIC (80 to 4,000 ng). Ratios on the x axis indicate the amount of reporter vector to microRNA vector. pcDNA3.1(+) was used as a filler to keep the total amount of DNA per transfection constant. Relative luciferase units (RLU) is a ratio of firefly luciferase values normalized to renilla luciferase values; data represent means ± standard deviations from six experiments.

FIG. 5.

Inhibition of JARID2 and Tp53INP1 mRNA was abrogated in the presence of miR-155 sponge. (A) Eight bulged targets sites of miR-155 were cloned into the 3′UTR of MS2 coat protein. CEFs were transfected with 80 ng of firefly luciferase, 40 ng of renilla luciferase, and 500 ng of pBIC. Two different concentrations of MS2-sponge plasmid were transfected (400 and 800 ng). pcDNA3.1(+) was used as a filler to keep the total amount of DNA per transfection constant. Relative luciferase units (RLU) is a ratio of firefly luciferase values normalized to renilla luciferase values; data represent means ± standard deviations from three experiments. CMV, cytomegalovirus; SV40, simian virus 40. (B and C) KBMC cells (1 × 10⁵) were infected with either RCAS(A)eGFP or RCAS(A)eGFP-sponge virus. Cells then were passaged continuously and total RNA harvested on the indicated days. (B) JARID2 mRNA and (C) Tp53INP1 mRNA were assayed by qRT-PCR and normalized to GAPDH mRNA results. Differences (n-fold) were calculated as a ratio of mRNA levels in RCAS(A)eGFP-sponge-infected cells to RCAS(A)eGFP-infected control cells; data represent means ± standard deviations from five experiments.

FIG. 6.

Overexpression of JARID2 decreased cell number through apoptosis. CEFs were transfected with various amounts of JARID2 as indicated. The total amount of DNA was kept constant by using pCMV-SPORT as a control vector. (A) Upon transfection, cells were split equally into three 12-mm dishes. Cells were counted each day using a hemacytometer; data represent means ± standard deviations from six experiments. (B) Along with JARID2, cells were cotransfected with an eGFP expression vector. On the day of analysis, cells were fixed and treated with RNase A and PI. GFP and cells were analyzed for cell cycle profile by PI staining. The number of cells in sub-G₁, G₁, S, and G₂/M phase were counted. (C) Representation of GFP⁺ sub-G₁ cells with increasing amounts of JARID2. Data are represented as the percentage of GFP⁺ cells (transfected cells); data represent means ± standard deviations from four experiments.

FIG. 7.

RCAS(B) viral infection leads to decreased cell growth and a decrease in miR-155. (A) CEFs were infected with RCAS(A) and RCAS(B) retroviral vectors, plus retroviral vectors expressing miR-155, and passaged for 7 days. After confirming positive infection by an RT assay, cells were counted using a hemacytometer. Data represent means ± standard deviations from six experiments. (B) miR-155 levels in CEFs infected with corresponding viruses were assayed by splinted ligation and relative intensity determined by PhosphorImager analysis. Data represent means ± standard deviations from six experiments.