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 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

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 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

Depletion of Zeb1 by siRNA mimics miR-23b overexpression.

<p>A) Zeb1 protein levels were significantly attenuated with 50 nM siRNA duplexes (Si) compared to a non-silencing siRNA duplex (Con) in T24 cells. B) Effect on cell proliferation. C–D) Effect on migration and invasion of T24 bladder cancer cells. E) Apoptosis assay showing induction of apoptosis after Zeb1 knockdown by siRNA in T24 cells. *p<0.05.</p

miR-23b induces cell cycle arrest and apoptosis in bladder cancer cells.

<p>A–B) Representative pictures of FACS analysis showing miR-23b over-expression induces G0/G1 cell cycle arrest in J82 and T24 cells with a corresponding decrease in S-phase cells. C–D) miR-23b over-expression induces apoptosis in J82 and T24 cells with a concomitant decrease in the viable number of cells. Data is shown from triplicate experiments ±SD.</p

miR-23b directly targets EMT regulator Zeb1.

<p>A) Complimentary miR-23b binding sequences in the Zeb1 3′UTR. B) Western blot analysis shows that miR-23b represses translation of Zeb1 protein in J82 and T24 bladder cancer cells. C) Luciferase assays showing decreased reporter activity after co-transfection of either the wild type or mutant Zeb1-3′UTR with miR-23b in J82 and T24 cells. Mut- Mutated Zeb1 3′UTR sequence.</p

Diagnostic and prognostic significance of miR-23b in bladder cancer.

<p>A) Clinicopathological characteristics of patient cohort. B) ROC curve analysis showing ability of miR-23b expression to discriminate between malignant and non-malignant tissue samples. C) Kaplan-Meier analysis for overall survival based on miR-23b expression.</p