Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer

Abstract

MicroRNAs are small noncoding RNAs that regulate the expression of protein-coding genes. To evaluate the involvement of microRNAs in prostate cancer, we determined genome-wide expression of microRNAs and mRNAs in 60 primary prostate tumors and 16 nontumor prostate tissues. The mRNA analysis revealed that key components of microRNA processing and several microRNA host genes, e.g., MCM7 and C9orf5, were significantly up-regulated in prostate tumors. Consistent with these findings, tumors expressed the miR-106b-25 cluster, which maps to intron 13 of MCM7, and miR-32, which maps to intron 14 of C9orf5, at significantly higher levels than nontumor prostate. The expression levels of other microRNAs, including a number of miR-106b-25 cluster homologues, were also altered in prostate tumors. Additional differences in microRNA abundance were found between organ-confined tumors and those with extraprostatic disease extension. Lastly, we found evidence that some microRNAs are androgen-regulated and that tumor microRNAs influence transcript abundance of protein-coding target genes in the cancerous prostate. In cell culture, E2F1 and p21/WAF1 were identified as targets of miR-106b, Bim of miR-32, and exportin-6 and protein tyrosine kinase 9 of miR-1. In summary, microRNA expression becomes altered with the development and progression of prostate cancer. Some of these microRNAs regulate the expression of cancer-related genes in prostate cancer cells.

Figure 1

Analysis of the relationship between transcript levels of microRNAs and their respective target mRNAs in prostate tissue. Shown is the global distribution of the Pearson correlation coefficients between mRNAs and miR-106b (A) or miR-181a (B). The black-lined curves show the distribution of the correlation coefficients for all mRNAs. The red-lined curves show the correlation coefficient distribution for only those mRNAs that are a predicted target of either miR-106b or miR-181a. The red-lined curves have an additional shoulder (arrow) indicating an enrichment of target mRNAs, whose transcript levels are negatively correlated with the transcript levels of the microRNA.

Figure 2

Inhibition of protein expression by miR-1 (A) and miR-106b (B). LNCaP and PC-3 human prostate cancer cells were transfected with either microRNA precursor (miR-1 and miR-106b) or antisense microRNA (antisense miR-1 and antisense miR-106b), or their respective vector controls, scrambled precursor microRNA (Scrambled-P) and scrambled antisense microRNA (Scrambled-A). Protein extracts were prepared 48 hours after transfection and protein expression was examined by Western blot analysis. Loading: 50 µg protein per lane.

Figure 3

miR-106b and miR-32 inhibit expression of E2F1 and Bim, respectively, by a 3’UTR-mediated mechanism. (A,C) LNCaP and PC-3 human prostate cancer cells were transfected with either microRNA precursor (miR-106b or miR-32) or antisense microRNA (antisense miR-106b or antisense miR-32), or their respective vector controls, scrambled precursor microRNA (Scrambled-P) and scrambled antisense microRNA (Scrambled-A). Protein extracts were prepared 48 hours after transfection and protein expression was examined by Western blot analysis. To obtain relative intensity values, E2F1 and Bim expression were normalized to β-actin. (B) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3’UTR target sequence of miR-106b in the E2F1 gene were co-transfected into LNCaP cells with either precursor microRNA negative control or miR-106b precursor (each n = 3; mean ± standard deviation). For comparison, cells were also transfected with the pGL3 control vector that did not contain the 3’UTR. After 24 hours, luciferase activity was determined in the cell extracts. In the presence of the wild-type E2F1 3’UTR, transfection with precursor miR-106b lead to a significant inhibition of the luciferase reporter when compared with the vector control (P = 0.045, two-sided t-test). This inhibition was not observed if the reporter construct contained a mutant 3’UTR target sequence of miR-106b. (D) pGL3 luciferase reporter constructs containing either the wild-type or mutant 3’UTR target sequence of miR-32 in the BCL2L11 (Bim) gene were co-transfected into LNCaP cells with either precursor microRNA negative control or miR-32 precursor (each n = 3). In the presence of the wild-type BCL2L11 3’UTR, transfection with miR-32 lead to a significant inhibition of the luciferase reporter when compared with the vector control (P = 0.003, two-sided t-test). This inhibition was attenuated if the reporter construct contained a mutant 3’UTR target sequence of miR-32.