<i>MiR-125b</i> regulates the p53 network.

<p><i>A</i>) Western blot analysis of Mdm2 and p53 in miR-125bm-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were transfected with 50 nM of miR-125bm or miR-negative control (miR-NC) for 72 hrs. Equal amounts of protein (50 µg) were used to detect the expression levels of Mdm2, p53, p21 and Puma. <i>B</i>) Western blot analysis of p14^ARF, Mdm2 and p53 in <i>p14^ARF</i> siRNA (sip14)-treated LNCaP (<i>top</i>) and 22R<i>v</i>1 cells (<i>bottom</i>). Cells were treated with sip14 and the cellular levels of p14^ARF, p53 and Mdm2 were analyzed. β-actin was used as a loading control. <i>C</i>) Co-immunoprecipitation analysis of protein interaction between p14^ARF and Mdm2 in 22R<i>v</i>1 cells. Cells were transfected with miR-125bm and 1.0 mg protein was immunoprecipitated with anti-p14^ARF antibody or the rabbit IgG. The resultant immunecomplexes were used to detect the level of Mdm2 by Western blot analysis using anti-Mdm2 antibody. Input: 50 µg protein from total cell lysate. IP: immunoprecipitation. IB: immunoblotting.</p

<i>MiR-125b</i> down-regulates p14^ARF in CaP cells.

<p><i>A</i>) Western blot analysis of expression levels of p14^ARF in LNCaP (<i>top</i>) and 22Rv1 cells (<i>bottom</i>). Cells grown in 10% FBS media were transfected with 50 nM of miR-125bm or anti-<i>miR-125b</i> (anti-125b) for 72 hrs or treated with 5.0 nM of R1881 androgen for 48 hrs. Then, 50 µg of protein per sample was analyzed. Both miR-negative control (miR-NC) and anti-miR negative control (anti-NC) were used as controls, and β-actin was used as a loading control. <i>B</i>) Western blot analysis of expression levels of p14^ARF, mdm2 and p53 in lenti-<i>miR-125b</i>-overexpressed PC-346C xenograft tumor. Both untreated xenograft (untreat.) and lenti-miRNA control vector-infected PC-346C xenograft (vector) were used as controls. In both <i>A</i> and <i>B</i>, the numbers under the gels are the average fold changes of p14^ARF protein from three independent gels relative to the corresponding controls. Fold changes were calculated by scanning the p14^ARF bands and normalizing for β-actin bands. <i>C</i>) Luciferase assay of <i>miR-125b</i> binding to the 3′-UTR of <i>p14^ARF</i> mRNA in LNCaP cells. The assay was repeated three times with each assay being performed in three wells and similar results were obtained each time. The representative results are shown as a mean ±SD (n = 3).</p

Schematic model of <i>miR-125b</i>-controlled oncopathway in CaP cells.

<p>In CaP cancer cells, p14^ARF facilitates apoptosis in a p53-dependent (<i>left</i>) and p53-independent (<i>right</i>) manner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061064#pone.0061064-Muer1" target="_blank">[30]</a>. Since <i>miR-125b</i> directly targets p14^ARF and other pro-apoptotic molecules, deregulation of <i>miR-125b</i> can modulate proliferation and apoptosis in both p53-positive and p53-deficient CaP cells. Black arrows represent upregulated molecules and white arrows represent downregulated molecules. Broken arrow indicates undefined upregulation of Bak1 activity by p14^ARF.</p

WST-1 proliferation assay of LNCaP cells (<i>A</i>) and 22R<i>v</i>1 cells (<i>B</i>).

<p>Cells were transfected with 50 nM of miR-125bm or 50 nM of miRNA negative control (miR-NC) for 5 days. Cell proliferation was measured by WST-1 assay. <i>p14^ARF</i> siRNA (sip14) was used as a control. The results are expressed as proliferation relative to that of miR-NC-treated cells, and shown as mean ± SD (n = 4).</p

Inactivation of <i>miR-125b</i> induces apoptosis in p53-positive CaP cells.

<p><i>A</i>) Detection of SMAC and activated caspase 3 (Cas-3) in LNCaP (<i>left</i>) and 22R<i>v</i>1 (<i>right</i>) cells. Cells were transfected with 50 nM miR-125bm or 50 nM anti-<i>miR-125b</i> (anti-125b) for 5 days, and the levels of SMAC and Cas-3 were measured by Western blot analysis. β-actin was used as loading control. The numbers under the gels are the average fold changes of SMAC and Cas-3 from three independent gels relative to the corresponding controls. <i>B</i>) Detection of anti-<i>miR-125b</i>-induced apoptosis in 22R<i>v</i>1 cells. Cells were transfected using 50 nM anti-<i>miR-125b</i> for 72 hrs and apoptotic cell death was detected using TUNEL assay. The green nuclear fluorescence indicates the apoptotic cleavage of nuclear DNA (<i>left</i>). For quantitation of apoptotic cell death, 400 cells were counted and apoptosis is expressed as % apoptosis (apoptotic cells/400×100%). Quantitative analysis was performed three times and result was expressed as mean ± SE (n = 3) (<i>right</i>). Cells treated with irradiation (IR, 6 Gy) were used as a positive control. <i>C</i>) TUNEL assay of apoptotic death of 22R<i>v</i>1 cells that were treated with anti-<i>miR-125b</i> followed by <i>p14^ARF</i> antisense (sip14). Result was expressed as mean ± SE (n = 3). <i>D</i>) Western blot analyses of p14^ARF, p53 and Bak1 levels in 22R<i>v</i>1 cells. <i>Left</i>: 22R<i>v</i>1 cells were transfected with anti-<i>miR-125</i>; <i>right</i>: anti-<i>miR-125</i>-transfected 22R<i>v</i>1 cells were treated with sip14. Both anti-miR-NC (anti-NC) and scramble siRNA were used as controls.</p

Let-7c is expressed in PCa cells.

<p><b>A</b>) Total RNAs from LNCaP, PC-3, DU145, LNCaP-S17 and LN-IL6+ cells were analyzed by qRT-PCR. Results are presented as relative fold change compared to expression levels in LNCaP. Data points represent mean ± SD of triplicate samples from two independent experiments. <b>B</b>) 20 µg each of the above RNAs were also analyzed by northern blotting. U6 snRNA was used as the loading control. <b>C</b>) qRT-PCR showing the decrease in let-7c expression in LNCaP cells expressing Lin28 compared to LNCaP cells transfected with the empty vector (Con). Inset Western blot shows expression of Lin28. <b>D</b>) qRT-PCR showing the increase in let-7c expression in C4-2B cells transfected with shRNA against Lin28 compared to C4-2B cells transfected with control GFP shRNA. Inset Western blot shows downregulation of Lin28. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*<i>p</i> < 0.05). <b>E</b>) Western blot showing the expression levels of Lin28 in PCa cells. Actin is used as a loading control.</p

Let-7c inhibits colony forming abilities of human PCa cells.

<p>Clonogenic (<b>A</b>) and soft agar colony forming (<b>B</b>) abilities of LNCaP-S17 and C4-2B cells transfected with let-7c or empty vector (Con) were assayed. Let-7c inhibited cell growth and survival in anchorage-dependent and –independent conditions. <b>C</b>) Clonogenic assay-Upper and lower panels represent colony sizes of C4-2B and LNCaP-S17 cells expressing control (empty vector) or let-7c respectively. <b>D</b>) Soft agar assay-Upper and lower panels represent colony sizes of C4-2B and LNCaP-S17 cells expressing control (empty vector) or let-7c respectively. <b>E</b>) Number of colonies formed in clonogenic assay by C4-2B cells stably expressing let-7c. Let-7c decreased the number of colonies formed by the PCa cells. Data points represent mean ± SD of triplicate samples from two independent experiments. Error bars denote ± SD (*<i>p</i> < 0.05).</p

Let-7c expression is downregulated in human PCa.

<p><b>A</b>) Relative expression levels of let-7c were measured by qRT-PCR in total RNAs extracted from 10 paired benign and tumor human prostate samples. <b>B</b>) Let-7c levels were measured using <i>in situ</i> hybridization in TMAs containing 160 cores each from unmatched benign and cancerous prostate biopsies. Representative images are shown for benign and cancer cores. Expression of let-7c was higher in benign prostates compared to cancerous prostates. <b>C</b>) Relative expression levels of Lin28 in the 10 paired benign and tumor human prostate samples. Expression levels of Lin28 were correlated inversely with those of let-7c. Error bars denote ± SD (*<i>p</i> < 0.05).</p

Let-7c suppresses tumor growth of human PCa xenografts <i>in vivo</i>.

<p>C4-2B (<b>A</b>), PC346C (<b>B</b>) and DU145 (<b>C</b>) cells were injected into both flanks of nude mice and the tumors received a single intratumoral injection of lentiviruses expressing either GFP (control) or let-7c. Tumor growth was monitored twice weekly over 3 weeks. Data points represent mean ± SD of tumor volume (mm³) of all mice at the indicated time points. <b>D</b>) Secretion of PSA by C4-2B and PC346C xenografts was measured in mouse sera by ELISA. Reconstitution of let-7c in the tumors reduced secretion of PSA by the xenografts. At the end of the experiments, tumor tissues were excised, total RNAs prepared and subjected to qRT-PCR to assess mRNA levels of let-7c (<b>E</b>) and Lin28 (<b>F</b>). Error bars denote ± SD (*<i>p</i> < 0.05).</p