Androgen-dependent expression of Dsc1 in Sertoli cells.

<p>(<b>A</b>) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Dsc1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Dsc1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (<b>B</b>) Real-time PCR analysis of <i>Dsc1</i> transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (<b>C</b>) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Dsc1 antibody (1∶250). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Dsc1 protein level for the control was set to 1. All images were taken at magnification of 600×.</p

MiR-471 regulates expression of Foxs1 in Sertoli cells.

<p>(<b>A</b>) Putative miR-471 binding sequence in the <i>Foxd1</i> 3′ UTR. (<b>B</b>) We co-transfected 15P1 Sertoli cells with <i>Renilla</i> luciferase expression construct pRL-CMV and firefly luciferase construct containing pMIR-<i>Foxd1</i> 3′ UTR in the absence and presence of miR-471 mimic. We normalized firefly luciferase activity of each sample to <i>Renilla</i> luciferase activity. Graphs show mean ± SEM of three independent experiments (performed in duplicate for each experiment). * <i>p</i><0.01; *** <i>p</i><0.001. (<b>C</b>) Real-time RT-PCR analysis of miR-471-overexpressing cells by using <i>Foxd1</i>-specific primers. (<b>D</b>) Western blot analysis of 15P1 cells transfected with miR-471 mimic by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Gel photographs represent three independent experiments. Values below the gel were quantified using Image J software (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>). Foxd1 protein level for the control was set to 1.</p

Dsc1 is a bona fide target of miR-471.

<p>(<b>A</b>) Putative miR-471 binding sequence in the <i>Dsc1</i> 3′ UTR. (<b>B</b>) We co-transfected 15P1 Sertoli cells with <i>Renilla</i> luciferase expression construct pRL-CMV and firefly luciferase construct containing pMIR-<i>Dsc1</i> 3′ UTR in the absence and presence of miR-471 mimic. We normalized firefly luciferase activity of each sample to <i>Renilla</i> luciferase activity. Graphs show mean ± SEM of three independent experiments (performed in duplicate for each experiment). * <i>p</i><0.01; *** <i>p</i><0.001. (<b>C</b>) Real-time RT-PCR analysis of miR-471-overexpressing cells by using <i>Dsc1</i>-specific primers. (<b>D</b>) Western blot analysis of 15P1 cells transfected with miR-471 mimic by using anti-Dsc1 antibody (1∶250). Actin was used as a loading control. Gel photographs represent three independent experiments. Values below the gel were quantified using the Image J software. Dsc1 protein level for the control was set to 1.</p

Androgen-dependent expression of FoxD1 in Sertoli cells.

<p>(<b>A</b>) Immunohistochemical analysis on testis sections from Sham, flutamide-acyline (Flut+Acy) and flutamide-acyline testosterone-replacement (Flut+Acy+T) mice, using antibodies against Foxd1 (1∶50) and Sox9 (1∶500). Insets show Sertoli cell-specific expression of Foxd1 in magnified testicular section from Sham, Flut+Acy, and Flut+Acy+T mice. (<b>B</b>) Real-time PCR analysis of <i>Foxd1</i> transcript levels in total cellular RNA isolated from the purified Sertoli cells from Sham, Flut+Acy, and Flut+Acy+T mice testes. All values are normalized against RNU19 levels. (<b>C</b>) Western blot analysis on total testicular lysates from Sham, Flut+Acy, and Flut+Acy+T mice by using anti-Foxd1 antibody (1∶1000). Tubulin was used as a loading control. Values below the gel were quantified using the Image J software. Foxd1 protein level for the control was set to 1. Arrowheads indicate Sertoli cells. All images were taken at magnification of 600×.</p

Expression pattern of testosterone-responsive miRNAs.

<p>Real-time RT-PCR analysis of selected miRNA expression in total cellular RNA prepared from the adult mouse tissues. All values are normalized against RNU19 levels. Bar graphs represent the mean fold increase ± SEM of miRNA expression over background for at least two RT reactions assayed in duplicate from three separate mice. Several miRNAs negatively regulated by the androgen were specifically expressed in the testis.</p

Androgen-Responsive MicroRNAs in Mouse Sertoli Cells

<div><p>Although decades of research have established that androgen is essential for spermatogenesis, androgen's mechanism of action remains elusive. This is in part because only a few androgen-responsive genes have been definitively identified in the testis. Here, we propose that microRNAs – small, non-coding RNAs – are one class of androgen-regulated <em>trans</em>-acting factors in the testis. Specifically, by using androgen suppression and androgen replacement in mice, we show that androgen regulates the expression of several microRNAs in Sertoli cells. Our results reveal that several of these microRNAs are preferentially expressed in the testis and regulate genes that are highly expressed in Sertoli cells. Because androgen receptor-mediated signaling is essential for the pre- and post-meiotic germ cell development, we propose that androgen controls these events by regulating Sertoli/germ cell-specific gene expression in a microRNA-dependent manner.</p> </div

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

<div><p>Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including <em>brain-derived neurotrophic factor</em> (<em>BDNF</em>), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.</p> </div

Genomic loss of miR-204 in cancers.

<p>A, high resolution miRNA-CGH on selected pediatric renal tumors with (left) or without (right) miR-204 deletions. Deletion of genomic loci containing the miRNA is indicated by dotted perpendicular lines. Red and green dots indicate position and value of each probe reflecting copy number change, represented in triplicate on the CGH array. The grey trend line represents the average value of the triplicate probe for each tumor. B, graphs obtained from meta-analysis of high-resolution CGH of ovarian cancers (<i>n</i> = 354; obtained from TCGA) representing a subset of tumors with or without deletion. The deletion of genomic loci containing miR-204 is indicated by dotted perpendicular lines. C and D, allelic PCR of miR-204 genomic locus in pediatric renal tumors (C) and ovarian cancers (D). The y-axis shows log₂ transformed relative quantification values. Dotted lines show the loss of copy threshold. E–G, graphical representation of qRT-PCR analysis showing levels of miR-204 in pediatric renal tumors (<i>n</i> = 38; E), in advanced stage ovarian cancers (<i>n</i> = 11; F) and, in breast cancers (<i>n</i> = 10; G), when compared to normal matched control kidney (<i>n</i> = 38), normal ovarian tissues (<i>n</i> = 5) and normal matched breast tissues (<i>n</i> = 10).</p

MiR-204 regulates expression of BDNF in cancers.

<p>A–C, increased BDNF expression correlates strongly with lower miR-204 expression in multiple cancers. Graphical representation of qRT-PCR analysis showing the inverse correlation between miR-204 and <i>BDNF</i> in pediatric renal tumors (<i>n</i> = 38; A), advanced stage ovarian cancers (<i>n</i> = 11; B) and breast cancers (<i>n</i> = 10; C), compared to normal matched control kidney (<i>n</i> = 38), normal ovarian tissues (<i>n</i> = 5) and normal matched breast tissues (<i>n</i> = 10). D–H, <i>BDNF</i> is a bonafide target of miR-204. D, schematic of the putative miR-204 binding sequence in the <i>BDNF</i> 3′ UTR. E, HEK-293 cells were co-transfected with Renilla luciferase expression construct pRL-TK and firefly luciferase constructs containing either pMIR-<i>BDNF</i> 3′ UTR in the absence and presence of miR-204 mimic or pMIR-<i>BDNF</i> 3′ UTR mutant. Firefly luciferase activity of each sample was normalized to Renilla luciferase activity. Mean±SEM of three independent experiments (performed in duplicate for each experiment). (**) <i>P</i><0.01; (***) <i>P</i><0.001. F, qRT-PCR analysis of miR-204 overexpressing cells and cells transfected with miR-204 inhibitors using <i>BDNF</i>-specific primers. G, western blot analysis of HEK-293 cells transfected with miR-204 mimic using anti-BDNF antibody (1∶1000). β-actin was used as a loading control. Gel photographs are representative of three independent experiments. H, graphical representation of band intensities quantified using the Total Labs TL100 1D gel analysis software (<i>n</i> = 3; Nonlinear). BDNF protein level for the control was set to 100.</p

Systemic delivery of miR-204 suppresses tumor metastasis.

<p>A, injection of miR-204 oligonucleotide into tail vein suppressed lung metastasis. Live bioluminescence images of mice injected with miR-204 (<i>n</i> = 6) or miR-204 mutant (neg. control; <i>n</i> = 6) oligonucleotides using the Xenogen In Vivo Imaging System (IVIS) (Xenogen). Images were taken after subcutaneously injecting 150 mg/kg D-luciferin substrate in PBS to anesthetized mice. B, tumor metastasis volume was assessed starting from day 10 until animals were sacrificed at day 60. Using ROI analysis, tumor light intensity was calculated in photon/s, which corresponds with the number of live cells in vivo. C, representative lung images showing GFP^+ve foci (red circle) in neg. control groups. D, representative lung sections showing metastatic foci in neg. control groups. E, no hepatotoxicity in miR-204 injected mice. Sections of liver from miR-204 injected mice show no signs of hepatotoxicity. The presence of multifocal periportal lymphocytes is not unusual and is a common finding in young animals.</p