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 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

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

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-182-5p binds to the 3′ UTR of RECK and Smad4 mRNAs and down-regulates expression.

<p>A. RECK and Smad4 3′UTR position and complementary miR-182-5p sequences. B. 3′UTR Luciferase assay (miR-NC and miR-182-5p precursor), C. RECK, Smad4 and beta-tubulin protein expression in miR-NC inhibitor or miR-182-5p inhibitor transfected bladder cancer cells (T24, UM-UC-3).</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-182-5p expression and association with clinical parameters in bladder cancer tissues.

<p>A. miR-182-5p expression in clinical samples and bladder cancer cell lines, B. Association of miR-182-5p with clinic-pathological parameters, C. Kaplan Meier plots of overall survival.</p

Primer sequences used for plasmid construction.

<p>Primer sequences used for plasmid construction.</p

Effect of miR-182-5p over-expression on bladder cancer cell function (T24, UM-UC-3).

<p> Two bladder cancer cell lines (T24 and UM-UC-3) were transiently transfected with either miR-182-5p precursor or control (miR-NC). A. Relative miR-182-5p expression, B. Cell viability assay, C. Invasion assay, D. Wound healing assay (24 hours), E. Flow cytometric analysis of apoptosis in miR-NC or miR-182-5p transfected BC cells.</p