Construction of stable cell lines.

<p><i>A–B,</i> Ectopic expression of miRs-143 or -145 in PC-3 (A) and LNCaP (B) were verified by qRT-PCR (<i>t-test</i>, <i>p</i><0.01).</p

Detection of miRs-143 and -145 in primary PCa tissues by ISH.

<p>Sections at upper panel showed the identification of tumor cells and stromal cells by H&E-stainning. Sections at lower panel showed the location of miR-143 (left) and miR-145 (right) in PCa cells by LNA-ISH. Signals of miRs-143 and -145 were in purple blue. Pictures were taken under microscope 200×.</p

Validation of select miRNAs predicted to be downregulated in prostate cancer.

<p><i>A,</i> The certified result of microarray analysis. <i>B,</i> Real-time RT–PCR assays on miR-143 (left panel), miR-145 (right panel), and miR-125b (bottom panel) in 16 primary prostate cancer and 13 bone metastasis tissues. The order of the tissue samples is the same for all three plots. <i>p</i>-values by <i>t-test</i>.</p

Down-regulation of miRs-143 and -145 is associated with clinicopathological features of primary PCa.

<p><i>A and D,</i> The expression of miRs-143 or -145 in the patients with bone metastases was significantly lower than that without metastases (<i>t-test</i>, <i>p</i> = 0.039, <i>p</i> = 0.041, respectively). <i>B and E,</i> Tumor samples were divided into four groups with approximately equal sample sizes based on level of free PSA. The level of free PSA in patients with the primary tumor is presented on the x axis. The y axis is the mean of miRs-143 or -145 within each group. The bars represent the standard errors. There was a statistically significant Spearman correlation that characterized an inverse relationship between miRs-143 or -145 expression and free PSA (Spearman correlation = −0.501, <i>p</i> = 0.018; Spearman correlation = −0.536, <i>p</i> = 0.010). <i>F,</i> The level of total PSA is also correlated with miR-145 (Spearman correlation = −0.456, <i>p</i> = 0.033). <i>C and G,</i> The Gleason scores of primary tumor group are presented on the x axis. The y axis is the mean of miRs-143 or -145 within each group. The bars represent the standard errors. There was a statistically significant Spearman correlation that characterized an inverse relationship between miRs-143 or -145 expression and the Gleason scores (Spearman correlation = −0.574; <i>p</i> = 0.005; Spearman correlation = −0.546, <i>p</i> = 0.009).</p

Both of miRs-143 and -145 repressed the development and invasion of PC-3 cells in bone.

<p>Male SCID mice were inoculated with PC-3 cells through the intra-tibial route. Skeletal lesions in radiographs are demonstrated by arrows (upper panel), and histologic analysis was carried by H&E-staining in which tumors were lined out by dashed line and marked as “T” (middle panel, taken under microscope 40×). Lesion scores of control and experimental specimens were shown in lower panels, where results were showed by means ± SEM of each group, <i>p</i> = 0.035 and <i>p</i> = 0.014 respectively.</p

Differentially expressed miRNAs identified in bone metastasis of prostate cancer compared to primary prostate cancer by miRNA microarray.

<p>*Significantly differentially expressed miRNAs selected as following standards: |Score(d)|≥2, Fold Change≥2 or ≤0.5, q-value(%)≤5.</p

miRs-143 and -145 mediates bone metastasis of prostate cancer via regulating the EMT.

<p><i>A–B,</i> The levels of E-Cadherin, Fibronectin and Vimentin were shown in PC-3 (A) and LNCaP (B) cells. α-Tubulin was shown as loading control. <i>C,</i> The phenotype of PC-3 cell lines were photographed under microscope (200×).</p

miRs-143 and -145 showed similar expression tendencies in clinical specimens.

<p><i>A–B,</i> Expressions of miRs-143 and -145 and their relationships in primary prostate cancer samples (A) and bone metastasis samples (B).</p

Ectopic expression of miRs-143 or -145 repressed metastasis and increased adhesion of PCa cells.

<p><i>A,</i> The migration actions of over-expressing miR-143 were analyzed by wound healing assay. PC-3 cell lines transfected with special plasmids showed significantly different healing conditions. <i>B,</i> The invasive properties of indicated cells were tested in invasion assay in a Transwell insert coated with Matrigel. Penetrated cells were counted and analyzed in histogram. <i>C,</i> The ability of adhesion of indicated cells were tested in fibronectin-coated plate assay. Adhered cells were counted and analyzed in histogram.</p