MiR-23b/

<p>-<b>27b significantly increases E-cadherin expression in castration –resistant cell lines.</b> ALVA31 or PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control and LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR were subjected to western blotting for E-cadherin and actin. Representative blots are shown of a total of 2–6 experiments.</p

MiR-23b/

<p>-<b>27b expression decreases the invasiveness of castration-resistant prostate cancer cells while inhibition of miR-23b/</b>-<b>27b in androgen-dependent prostate cancer cells increases invasiveness.</b> A, Matrigel invasion assays were performed on ALVA31 cells (A) and PC3-ML cells (B) expressing miR-23b/-27b or scrambled control-transduced cells. Upper panels show representative regions of the chamber filters with crystal violet-stained cells. The fold change (±SEM) represents the number of invaded cells per chamber divided by controls from three independent experiments performed in triplicate for A and the means (±SD) of one independent experiment done in triplicate for B (***P<0.001). C, LNCaP cells were transfected with 50 nM control antagomiR or antagomiR-23b/-27b. Matrigel invasion assays were performed 72 hours post transfection as explained above. The means (±SD) of two independent experiments performed in triplicate are shown (***P<0.001).</p

MiR-23b/

<p>-<b>27b expression decreases castration-resistant prostate cancer cell migration while inhibition of miR-23b/</b>-<b>27b increases migration of androgen-dependent prostate cancer cells.</b> Scratch assays were performed on ALVA31 cells expressing miR-23b/-27b or scrambled control (A) and LNCaP cells transfected with 50 nM antagomiR-23b-27b or control antagomiR (B). Images of the cleared zones (representative images shown on the right) were taken before and after a 4 hr incubation and the cleared areas measured using Image J software. The mean percentage of gap closure (±SD) (due to cell migration) is shown for two independent experiments from 7 scratches (A) and 6 scratches (B) (***P<0.001).</p

MiR-23b/

<p>-<b>27b significantly decreases Rac1 activity but not total Rac1 levels in castration-resistant prostate cancer cell lines.</b> Rac1 activity assays were performed on ALVA31 (A) and PC3-ML (B) cells expressing miR-23b/-27b or transduced with scrambled control. GTP-bound Rac1 was separated from GDP-Rac1 using a pull-down assay as described in Materials and Methods. Complexes containing GTP-Rac1 (active Rac1) were collected, denatured and resolved by SDS-PAGE followed by Western blotting with anti-Rac1 antibodies. Total Rac1 (GDP-and GTP-bound) represents 5% of the original cell lysate. Actin was used as a loading control. Quantification of three independent experiments is shown with error bars representing SEM (*P<0.05), (**P<0.01).</p

MiR-23b/

<p>-<b>27b does not regulate prostate cancer cell proliferation.</b> A and B, Proliferation of cells expressing miR-23b/-27b was compared to that of scrambled control-transduced cells. The mean cell number (±SEM) of a total of three independent experiments performed in triplicate is shown for ALVA31 cells and the mean cell number (±SD) of a representative experiment performed in triplicate is shown for PC3-ML cells. C, Proliferation of LNCaP cells transfected with antagomiR-23b/-27b or control antagomiR was assessed. The mean cell number (±SD) from two independent experiments performed in triplicate is shown.</p

MiR-23b/

<p>-<b>27b decreases anchorage independent growth of castration-resistant prostate cancer cell lines.</b> Soft agar assays were performed on ALVA31 and PC3-ML cells expressing miR-23b/-27b or transduced with scrambled control (**P<0.01). Mean colony number (±SEM) per plate from two independent experiments performed in triplicate for each cell line is shown (**P<0.01).</p

Systemic Gene Silencing in Primary T Lymphocytes Using Targeted Lipid Nanoparticles

Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4⁺ T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4⁺ T lymphocytes. <i>Ex vivo</i>, tLNPs demonstrated specificity by targeting only primary CD4⁺ T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4⁺ T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4⁺ T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs’ efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases

Systemic Gene Silencing in Primary T Lymphocytes Using Targeted Lipid Nanoparticles

Modulating T cell function by down-regulating specific genes using RNA interference (RNAi) holds tremendous potential in advancing targeted therapies in many immune-related disorders including cancer, inflammation, autoimmunity, and viral infections. Hematopoietic cells, in general, and primary T lymphocytes, in particular, are notoriously hard to transfect with small interfering RNAs (siRNAs). Herein, we describe a novel strategy to specifically deliver siRNAs to murine CD4⁺ T cells using targeted lipid nanoparticles (tLNPs). To increase the efficacy of siRNA delivery, these tLNPs have been formulated with several lipids designed to improve the stability and efficacy of siRNA delivery. The tLNPs were surface-functionalized with anti-CD4 monoclonal antibody to permit delivery of the siRNAs specifically to CD4⁺ T lymphocytes. <i>Ex vivo</i>, tLNPs demonstrated specificity by targeting only primary CD4⁺ T lymphocytes and no other cell types. Systemic intravenous administration of these particles led to efficient binding and uptake into CD4⁺ T lymphocytes in several anatomical sites including the spleen, inguinal lymph nodes, blood, and the bone marrow. Silencing by tLNPs occurs in a subset of circulating and resting CD4⁺ T lymphocytes. Interestingly, we show that tLNP internalization and not endosome escape is a fundamental event that takes place as early as 1 h after systemic administration and determines tLNPs’ efficacy. Taken together, these results suggest that tLNPs may open new avenues for the manipulation of T cell functionality and may help to establish RNAi as a therapeutic modality in leukocyte-associated diseases