Regulation of several androgen-induced genes through the repression of the miR-99a/let-7c/miR-125b-2 miRNA cluster in prostate cancer cells

Abstract

The androgen receptor (AR) stimulates and represses gene expression to promote the initiation and progression of prostate cancer. Here, we report that androgen represses the miR-99a/let7c/125b-2 cluster through AR and anti-androgen drugs block the androgen-repression of the miRNA cluster. AR directly binds to the host gene of the miR-99a/let7c/125b-2 cluster, LINC00478. Expression of the cluster is repressed or activated by chromatin remodelers EZH2 or JMJD3 in the presence or absence of androgen, respectively. Bioinformatics analysis reveals a significant enrichment of targets of miR-99a, let-7c and miR-125b in androgen-induced gene sets, suggesting that downregulation of the miR-99a/let7c/125b-2 cluster by androgen protects many of their target mRNAs from degradation and indirectly assists in the gene induction. We validated the hypothesis with 12 potential targets of the miR-99a/let7c/125b-2 cluster induced by androgen: 9 out of the 12 mRNAs are downregulated by the microRNA cluster. To ascertain the biological significance of this hypothesis, we focused on IGF1R, a known prostate cancer growth factor that is induced by androgen and directly targeted by the miR-99a/let7c/125b-2 cluster. The androgen-induced cell proliferation is ameliorated to a similar extent as anti-androgen drugs by preventing the repression of the microRNAs or induction of IGF1R in androgen-dependent prostate cancer cells. Expression of a microRNA-resistant form of IGF1R protects these cells from inhibition by the miR-99a/let7c/125b-2 cluster. These results indicate that a thorough understanding of how androgen stimulates prostate cancer growth requires not only an understanding of genes directly induced/repressed by AR, but also of genes indirectly induced by AR through the repression of key microRNAs.

Figure 1. miR-99a/let7c/125b-2 cluster and its host gene LINC00478 are repressed by androgen

A. qPCR was used to measure the expression level of miRNAs. The values were normalized to that of U6sn. Mean±s.d. n=3. The expression level under no androgen treatment is set as 1. LNCaP cells were treated with charcoal-stripped serum for 48 hrs followed by treatment with indicated concentrations of R1881. B. The expression of primary miRNAs was measured by qPCR and normalized to β-actin. Rest as in Fig. 1A. C. Six pairs of primers were used to measure the expression of the two variant transcripts of LINC00478 at different concentrations of androgen. Schematic at the top shows the two transcripts of LINC00478 with the exons indicated by black boxes and the microRNAs shown by vertical lines. Primer pairs are indicated by horizontal lines. F1R1 and F3R3 amplify across splice junctions of the mature RNA. qPCR value was normalized to GAPDH. Rest as in Fig. 1A

Figure 2. AR is required for androgen-repression of miR-99a/let7c/125b-2 cluster

A. The expression of primary miRNAs in C4-2 cells was measured by qPCR and normalized to β-actin. Rest as in Fig. 1A. B. The expression of primary miRNAs in PC-3 cells. Rest as in Fig. 2A. C. The expression of primary miRNAs in PC-3 cells expressing wild type AR. Rest as in Fig. 2A. D. The expression of primary miRNAs in LNCaP cells after knocking down AR by siRNA. LNCaP cells were treated with no androgen, 100pM or 1nM R1881. Rest as in Fig. 1A. E. The expression of primary miRNAs in LNCaP cells was measured by qPCR and normalized to β-actin. LNCaP cells were treated with no androgen, 1nM R1881, 1nM R1881 plus 30µM Bicalutamide or 1nM R1881 plus 30µM Flutamide.

Figure 3. AR directly binds to host gene of miR-99a/let7c/125b-2 cluster and the repression is attenuated when EZH2 is knocked down

A. The AR binding sites at the LINC00478 locus from published AR ChIP-seq data are shown. Seven sites shown as vertical lines are labeled as BS1–7. The two primary transcripts and the microRNAs are shown. B. AR ChIP was performed and followed by qPCR. Primers were designed to detect AR ChIP-seq peaks ARBS1–5. A PSA enhancer containing an ARE was used as a positive control. ChIP grade IgG antibody was used as negative control. The mean and standard deviation from three independent experiments are indicated. The value was expressed as percentage of input DNA. The dashed line (---) marks the background threshold at 0.05% of input DNA. The statistical difference between “−”and “+” R1881 for AR ChIP was assessed by t-test. * indicated p-value less than 10⁻⁶. C. The expression of primary miRNAs in LNCaP cells was measured by qPCR and normalized to GAPDH. LNCaP cells were transfected with siRNA against EZH2 or control si-GL2, and treated without or with 1nM R1881. Mean±s.d. n=3. The expression in no androgen is set as 1. * indicates p-value of difference from si-GL2 <0.05 and ** indicates p-value <0.01. D. The expression of primary miRNAs in LNCaP cells was measured by RT-qPCR and normalized to GAPDH. LNCaP cells were transfected with siRNA against JMJD3 or control si-GL2, and treated without or with 1nM R1881. Mean±s.d. n=3. The expression in 1nM R1881 is set as 1. * indicates p-value of difference from si-GL2 <0.05 and ** indicates p-value <0.01.

Figure 4. Validation of androgen-induced targets of miR-99a/let7c/125b-2 cluster

A. The expression of four predicted targets each of miR-99a, let-7c or miR-125b respectively was measured by RT-qPCR and normalized to GAPDH. LNCaP cells grown in charcoal stripped serum were treated with 0pM, 100pM or 1nM R1881. Mean±s.d. n=3. The expression in no androgen is set as 1. B. The expression of targets of miR-99a/let7c/125b-2 cluster after transfecting LNCaP cells with miR-99a, let-7c, miR-125b miRNA duplex or control siRNA duplex si-GL2. Expression was measured by RT-qPCR and normalized to GAPDH. Rest as in Fig. 4A. * indicates p-value of difference from si-GL2 <0.01.

Figure 5. IGF1R is a key target molecule regulated by miR-99a/let7c/125b-2 cluster

A. Top: The 3’UTR of IGF1R, the relative positions of each miRNA binding site and the fragments inserted in the luciferase vectors. * indicates miRNA binding sites validated by luciferase assays. Bottom: Luciferase assay was performed with control luciferase vector, vector with indicated 3’UTR fragment of IGF1R, or 3’UTR fragments with mutation in the predicted target sites (indicated by MUT). The ratio of the renilla luciferase to firefly luciferase (transfection control) was normalized to that in the si-GL2 transfection. Mean±s.d. n=3 * indicates p-value of difference from si-GL2 <0.005 and ** indicates p-value <0.001. B. The level of IGF1R mRNA after transfecting miRNA duplex mixture of miR-99a and let-7c (marked as miRs) or control si-GL2 was measured by RT-qPCR and normalized to β-actin. Mean±s.d. n=3. The value in si-GL2 treated cells was set as 1. C. Western blot was used to detect IGF1R protein after transfecting miR-99a, let-7c, miRNA duplex mixture of miR-99a and let-7c (miRs) or control si-GL2. β-actin was used as a loading control. D. LNCaP cells were treated with no androgen or 1nM R1881 during the experiment. 30µM Bicalutamide and Flutamide were added to LNCaP cells for 2 hours before addition of 1nM R1881. miR-99a, let-7c, miR-125b, siRNA against 3’UTR of IGF1R or si-GL2 was transfected twice at 0 hr and 48 hrs. At 96 hrs, cell number was counted using an automated cell counter. The mean and standard deviation from triplicate samples are shown. Cell numbers were normalized to that of cells in 1nM R1881 and transfected with si-GL2. * indicates p-value<0.01 and ** indicates p-value <0.001. E. miR-99a, let-7c, siRNA against 3’UTR of IGF1R or si-GL2 was transfected twice at 0 hr and 48 hrs. LNCaP cells were treated with 1nM R1881 during the experiment. At 96 hrs, cell number was counted in an automated cell counter. The mean and standard deviation from triplicate samples are shown. Cell numbers were normalized to that of cells transfected with si-GL2. * indicates p-value<0.05. F. Schematic of how the repression of miR-99a/let-7c/miR-125b-2 by androgen stimulates prostate epithelial cell proliferation.