miR-34a inhibits cell growth.

<p>(A) Relative miR-34a expression in laser capture microdissected (LCM) prostate cancer tissues (C) and matched adjacent normal regions (N). (B) miR-34a overexpression suppresses soft agar colony formation of a stably transfected miR-34a PC-3 cell line. After 14 days incubation, colonies with over 50 cells were counted. The values are normalized to those of control. (C) Representative images of the colonies. (D) miR-34a inhibits <i>in vivo</i> tumor growth. Time course of tumor growth in nude mice after subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. *, P<0.05 compared with control. (E) Representative images of tumors in nude mice at 5 weeks after subcutaneous injection of a stably transfected miR-34a PC-3 cell line or control cell line. (F) miR-34a induces apoptosis in PC-3 cells. PC-3 cells were transfected with pre-miR negative control (NC) or pre-miR-34a for 3 days. PC-3 cells were stained with AnnexinV-FITC/7-AAD and apoptosis was analyzed by flow cytometry. The data shows the percentage of early apoptotic and apoptotic cells out of the total cell population of PC-3 cells. *, P<0.05 compared with control.</p

c-Myc overexpression partially rescues RhoA suppression by miR-34a.

<p>(A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Myc for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by Western blot. (B) c-Myc expression partially rescues inhibition of invasion induced by miR-34a. PC3 cells were transiently transfected with pCMV6-ENTRY only (control) or pCMV6-ENTRY-c-Myc for 8 h followed by transfection with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. *, P<0.05 compared with control. (C) Representative images of invaded cells.</p

miR-34a suppresses the c-Myc transcriptional complex of RhoA.

<p>PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre-miR-34a for 72 h. (A) RhoA mRNA level was analyzed by real-time PCR. (B) RhoA protein expression was analyzed by Western blot. (C) c-Myc pulls down endogenous Miz1, Skp2 and Max in PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and total cell lysates were immunoprecipitated with c-Myc antibody or IgG (control), followed by Western blot analysis. (D) miR-34a decreases the recruitment of c-Myc to the RhoA promoter. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and ChIP assays were performed. *, P<0.05 compared with control.</p

miR-34a inhibits invasion and migration of PC-3 cells.

<p>(A) miR-34a inhibits invasion of PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. The values are normalized to those of NC. *, P<0.05 compared with control. (B) Representative images of the invaded cells. (C) miR-34a inhibits wound-healing of PC-3 cells. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to wound-healing assay. The width of the remaining open wound calculated in relation to separation at time 0 h. *, P<0.05 compared with control. (D) Representative images of the wound healing assay.</p

miR-34a suppresses the c-Myc-P-TEFb complex.

<p>(A) miR-34a suppresses the formation of the endogenous c-Myc-P-TEFb complex. PC-3 cells were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 72 h and total cell lysates were immunoprecipitated with c-Myc antibody or IgG (control), followed by Western blot analysis. (B) miR-34a suppresses CAD and NUC mRNA expression and DRB revereses the suppression. PC-3 cells were treated with 20 µM of DRB for 12 h and were transiently transfected with pre-miR negative control (NC) or pre–miR-34a for 48 h. CAD and NUC mRNA level was analyzed by real-time PCR.</p

c-Met overexpression partially rescues suppression of cell invasion by miR-34a.

<p>(A) PC-3 cells were transiently transfected with pCMV6-ENTRY only or pCMV6-ENTRY-c-Met for 8 h followed by transient transfection with pre-miR negative control (NC) or pre–miR-34a for 72 h. Protein level was analyzed by Western blot. (B) c-Met expression partially rescues inhibition of invasion induced by miR-34a. PC3 cells were transiently transfected with pCMV6-ENTRY only (control) or pCMV6-ENTRY-c-Met for 8 h followed by transfection with pre-miR negative control (NC) or pre–miR-34a for 48 h. The cells were harvested and subjected to transwell invasion assay. *, P<0.05 compared with control. (C) Representative images of invaded cells.</p

Effect of miR-182-5p knock down on prostate cancer cells (PC-3, DU145).

<p>Two prostate cancer cell lines (PC-3 and DU145) were transiently transfected with either miR-182-5p inhibitor or miR-negative control (miR-NC-inhibitor). A. Relative miR-182-5p expression (miR-NC inhibitor or miR-182-5p inhibitor transfected PC cells), B. Cell viability assay (miR-NC inhibitor or miR-182-5p inhibitor transfected PC cells), C. Invasion assay, D. Wound healing assay (24 hours). Error bars represent±S.D. (standard deviation).</p

Inhibition of <i>in vivo</i> tumor growth by miR-182-5p knockdown in PC3 cells.

<p>A. Time record of tumor size in miR-NC and miR-182-5p inhibitor xenografts. B. Relative miR-182-5p expression at injection of cells and harvest of xenografts. Error bars represent ±S.D. (standard deviation). MiR-182-5p expression was significantly lower in the miR-182-5p inhibitor group. C. Typical gross appearance of nude mouse tumors in control and miR-182-5p inhibitor groups. D. Expression of FOXF2, RECK and MTSS1 protein in tumor xenografts.</p

Expression of miR-182-5p in cell lines and effect of miR-182-5p overexpression on normal prostate cells (RWPE-1).

<p>A. The expression of miR-182-5p was significantly higher in three prostate cancer cell lines compared to a normal prostate cell line (RWPE-1), B. Relative miR-182-5p expression (miR-NC or miR-182-5p precursor transfected RWPE-1 cells), B. cell viability assay (miR-NC or miR-182-5p precursor transfected RWPE-1 cells), C. Wound healing assay (miR-NC or miR-182-5p precursor transfected RWPE-1 cells). Error bars represent ±S.D. (standard deviation).</p

Prostate cancer patient information (n = 52).

<p>Prostate cancer patient information (n = 52).</p