Down-Regulation of eIF4GII by miR-520c-3p Represses Diffuse Large B Cell Lymphoma Development

<div><p>Deregulation of the translational machinery is emerging as a critical contributor to cancer development. The contribution of microRNAs in translational gene control has been established however; the role of microRNAs in disrupting the cap-dependent translation regulation complex has not been previously described. Here, we established that elevated miR-520c-3p represses global translation, cell proliferation and initiates premature senescence in HeLa and DLBCL cells. Moreover, we demonstrate that miR-520c-3p directly targets translation initiation factor, eIF4GII mRNA and negatively regulates eIF4GII protein synthesis. miR-520c-3p overexpression diminishes cells colony formation and reduces tumor growth in a human xenograft mouse model. Consequently, downregulation of eIF4GII by siRNA decreases translation, cell proliferation and ability to form colonies, as well as induces cellular senescence. <i>In vitro</i> and <i>in vivo</i> findings were further validated in patient samples; DLBCL primary cells demonstrated low miR-520c-3p levels with reciprocally up-regulated eIF4GII protein expression. Our results provide evidence that the tumor suppressor effect of miR-520c-3p is mediated through repression of translation while inducing senescence and that eIF4GII is a key effector of this anti-tumor activity.</p></div

Influence of miR-520c-3p on the senescent phenotype.

<p>Cells were transfected/transduced as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004105#s4" target="_blank">Materials and Methods</a>. (A) Six days after transfection the percentage of apoptotic cells was determined by flow cytometry. (B) 72 h after transfection cells were stained with PI and subjected to cell cycle analysis. (C) HeLa cells were tested for β-galactosidase activity six days after transfection. Representative pictures are shown. (D) Six days after transfection the levels of p16, p53 and HuR were assessed by Western blot analysis. GAPDH served as a loading control. The data are representative of at least three independent experiments. In the graphs (A) and (B) the means and SEM are shown. * p<0.05.</p

DLBCL cells express high levels of eIF4GII protein and low levels of miR-520c-3p.

<p>(A) eIF4GII protein levels in normal B-cells and DLBCL cells were analyzed by Western blot. β-Actin was used as a loading control. Data are representative of three independent experiments. (B) Pictured are representative fields (20× and 400× magnifications) of germinal center B-cell centroblasts of reactive lymph nodes and DLBCL tissue microarrays immunohistochemically stained with anti-eIF4GII antibody. HE staining was used for morphologic examination. (C) RNA from tissue microarrays used in (B) was purified and expression of miR-520c-3p was measured by RT-qPCR. Graphs represent the means and SEM from repeats of three independent assays.</p

Influence of eIF4GII on gene translation, cell proliferation and the senescent phenotype.

<p>(A) 72 h after transfecting HeLa cells with either siRNA Ctrl or siRNA targeting eIF4GII, cell lysates were fractionated through 10–50% linear sucrose gradients, and the polysome distribution profiles were analyzed. (B) HeLa cells were transfected as described in (A) and nascent protein synthesis was assessed by incorporation of ³⁵S-labeled amino acids. The ³⁵S-amino acid incorporation was quantified and presented as percentage of signal intensity relative to control transfection (graph.). (C) Protein levels of miR-520c-3p target genes were measured by Western blotting 72 h after transfection of HeLa cells with either control siRNA or eIF4GII siRNA. GAPDH was used as a loading control. (D) Cells were transfected as described in (A) and the mRNA levels of selected miR-520c-3p target mRNAs and housekeeping GAPDH mRNA in each gradient fraction were measured by RT-qPCR and plotted as a percentage of the total mRNA levels in each sample. Data represent the average of three independent experiments showing similar results. (E) 72 h after transfection HeLa and Farage cells with either siRNA Ctrl or targeting eIF4GII siRNA, cell numbers were counted with a hemocytometer. (F) Six days after transfection as described in (E) cells were tested for apoptosis. (G) 72 h after transfection as described in (E) HeLa cells were stained with PI and subjected to cell cycle analysis. (H) β-galactosidase activity was measured in HeLa cells six days after transfection as described in (F) (I) Six days after transfections as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004105#s4" target="_blank">Materials and Methods</a>, the levels of p16, p53, and HuR in HeLa and Farage cells were assessed by Western blot analysis. GAPDH served as a loading control. Data are representative of at least three independent experiments. In the graphs (B), (E), (F) and (G) the means and SEM are shown. * p<0.05.</p

miR-520c-3p is downregulated in DLBCL cells and affects global gene translation and cell proliferation.

<p>(A) Total RNA from normal B-cells, DLBCL, BL, and HeLa cell lines were used to measure miR-520c-3p abundance by RT-qPCR. Graphs represent the means and SEM from repeats of three independent assays. (B) 48 h after transfection with Pre-miR-Ctrl or Pre-miR-520c-3p HeLa cells were fractionated through sucrose gradients. Absorbance at 254 nm was used to identify the fractions containing ribosomal subunits 40S and 60S, monosomes 80S, and polysomes LMW and HMW (low- and high-molecular weight). (C) 48 h after transfection as described in (B) HeLa cells incubated for 20 min with ³⁵S-labeled amino acids. Lysate aliquots corresponding to the same cell numbers were size fractionated by SDS-PAGE, transferred onto PVDF membranes, and visualized using a PhosphorImager. The ³⁵S-amino acid incorporation was quantified (graph) and is presented as percent signal intensity relative to control transfection group. (D) Polysome profiles obtained 48 h after transduction of SUDHL4 cells with either empty vector (pCDH-Vector; V) or vector overexpressing miR-520c-3p (pCDH-520c-3p). (E) SUDHL4 cells were transduced as described in (D) and radiolabeled 48 h later as described in (C). (F) 72 h after transfection of HeLa cells as described in (B) cell numbers were counted using a hemocytometer. (G) 72 h after transfection cells as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004105#s4" target="_blank">Materials and Methods</a> cell numbers were counted using a hemocytometer. (H) After transfection as in (B), HeLa cell proliferation in real-time was monitored by the xCELLigence System. The data are representative of at least three independent experiments. In the graphs (A), (C), (E), (F) and (G) the means and SEM are shown. * indicates p<0.05.</p

Analysis of microarray data in HeLa cells.

<p>(A) Functional categories of total and polysome associated mRNAs in Pre-miR-520c-3p compared to Pre-miR-Ctrl transfected cells. Heat map represents GO annotations with the most altered values in sucrose gradient fractions. Top 100 categories are illustrated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004105#pgen.1004105.s004" target="_blank">Figure S4</a>. T indicates total RNA; lanes 1 through 11 represent RNA from sucrose fractions of increasing molecular weight. (B and C) The levels of total mRNAs of validated genes were measured by RT-qPCR in cells transfected with Pre-miR-520c-3p compared to Pre-miR-Ctrl. Graphs represent the means and SEM from three repeats of three independent assays. (D) Protein levels of miR-520c-3p target genes were measured by Western blotting. GAPDH was used as a loading control. (E) Cells were fractionated through sucrose gradients and the relative distribution of selected miR-520c-3p target mRNA (and housekeeping GAPDH mRNA) was studied by RT-qPCR analysis of RNA in each of 11 gradient fractions. Data are representative of three independent experiments.</p

Immunohistological analysis of eIF4GII expression in DLBCL and normal GCB.

<p>Immunohistological analysis of eIF4GII expression in DLBCL and normal GCB.</p

Overexpression of miR-520c-3p in DLBCL diminished colony formation in clonogenic assay and decreased tumor growth in xenograft model.

<p>(A) Farage cells after transfection with either Pre-miR-Ctrl or Pre-miR-520c-3p were cultured in agarose/medium for two weeks. Representative pictures (10×) are shown. Graphs represent the means and SD of three independent experiments. (B) Farage cells were transfected with Ctrl siRNA or eIF4GII siRNA, assay was performed and analyzed as described in (A). * p<0.05. (C and D) SCID Beige mice (n = 5) received a subcutaneous injection of SUDHL4 cells either expressing empty pCDH-Vector (on left sites; red arrow) or overexpressing miR-520c-3p, pCDH-520c-3p (on right sites; green arrow). The average tumor volume of each group with SEM is shown as a function of time. The repeated measure ANOVA showed a significant effect of time on tumors growth F(8,64) = 40.23, p<0.001, and significant repression of growth by miR-520c-3p as revealed by significant effect of treatment F(1,8) = 49.98, p<0.001 and significant treatment x time interaction F(8,64) = 5.93, p<0.001. (E) Lysates from SUDHL4 xenograft tumors obtained in C and D were fractionated by centrifugation through 10–50% linear sucrose gradients, and the polysome profiles were studied. (F) Protein extracts from xenograft tumors from (C) and (D) were subjected to Western blot analysis using indicated senescence markers antibodies.</p

Image-guided drug delivery with magnetic resonance guided high intensity focused ultrasound and temperature sensitive liposomes in a rabbit Vx2 tumor model

Clinical-grade Doxorubicin encapsulated low temperature sensitive liposomes (LTSLs) were combined with a clinical magnetic resonance-guided high intensity focused ultrasound (MR-HIFU) platform to investigate in-vivo image-guided drug delivery. Plasma pharmacokinetics were determined in 3 rabbits. Fifteen rabbits with Vx2 tumors within superficial thigh muscle were randomly assigned into three treatment groups: 1) free doxorubicin, 2) LTSL and 3) LTSL+MR-HIFU. For the LTSL+MR-HIFU group, mild hyperthermia (40–41°C) was applied to the tumors using an MR-HIFU system. Image-guided non-invasive hyperthermia was applied for a total of 30 min, completed within 1 hour after LTSL infusion. High-pressure liquid chromatography (HPLC) analysis of the harvested tumor and organ/tissue homogenates was performed to determine doxorubicin concentration. Fluorescence microscopy was performed to determine doxorubicin spatial distribution in the tumors. Sonication of Vx2 tumors resulted in accurate (mean=40.5±0.1°C) and spatially homogenous (SD=1.0°C) temperature control in the target region. LTSL+MR-HIFU resulted in significantly higher tumor doxorubicin concentrations (7.6- and 3.4-fold greater compared to free doxorubicin and LTSL respectively, p<0.05, Newman-Keuls). This improved tumor concentration was achieved despite heating <25% of the tumor volume. Free doxorubicin and LTSL treatments appeared to deliver more drug in the tumor periphery as compared to the tumor core. In contrast, LTSL+MR-HIFU treatment suggested an improved distribution with doxorubicin found in both the tumor periphery and core. Doxorubicin bio-distribution in non-tumor organs/tissues was fairly similar between treatment groups. This technique has potential for clinical translation as an image-guided method to deliver drug to a solid tumor