Identification of dysregulated miRNA targets in human prostate cancer.
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MicroRNAs (miRNAs) are small, non-coding RNAs (18~24 nt) that regulate diverse cell functions in mammalian cells, including proliferation, differentiation, metabolism, stress resistance, and death. There is increasing evidence that altered expression and function of regulatory miRNAs contribute to the uncontrolled growth of human cancers. We examined the hypothesis that miRNA expression profiles can distinguish malignant from normal tissues. With the use of high throughput, liquid phase hybridization analyses (mirMASA) were carried out with 40 Stage 1c/2 prostate cancer specimens, using 114 miRNA-specific probes. We identified 5 miRNAs (miR -23b, -100, -145, -221, and -222) that were significantly downregulated in malignant prostate tissues as compared with their normal counterpart from the same patient. Decreased expression was confirmed by quantitative real-time PCR (qRT-PCR) analyses. The pathophysiological role of these downregulated miRNAs was further characterized in the prostate cancer LNCaP cell line, which exhibited similarly reduced expression of miR- 23b, -145, -221, and -222. Ectopic expression of mature miR-23b and miR-145 reduced LNCaP cell growth by >40%. In contrast, transfection with a miRNA (miR-141) that was upregulated did not markedly affect cancer cell growth. The altered expression of miR- 145 was of particular interest, in view of its similarly reduced expression in breast, cervical, colon, and lung carcinomas. In silico search (TargetScan) yielded 396 eligible targets for miR-145, of which 6 were markedly upregulated in LNCaP cells. These include the prostaglandin F receptor (PTGFR), transforming growth factor (TGFBR2), p21-activated kinase 7 (PAK7), SLIT-ROBO Rho GTPase activating protein 2 (SRGAP- 2), kinesin family member 3A (KIF3A), and Ras association (RalGDS/AF-6) domain family 2 (RASSF2). miR-145 was uniformly downregulated in all prostate caner lines tested (LNCaP, 22Rv1, PC-3, Du-145, as compared with the nonmalignant prostate line RWPE-1) This miRNA may be differentially expressed in androgen dependent (AD) and androgen independent (AI) prostate cancer cell lines according to qPCR analysis. Mean expression level of miR-145 was approximately 6-fold lower in AD (LNCaP, 22Rv1) than AI (PC3, Du-145) lines. qPCR analysis suggests that PTGFR and RASSF2 were upregulated in 3 of 4 prostate cancer cell lines, whereas MYCN was only upregulated in androgen independent cell lines. The expression of CCND2, MAP3K3 and MAP4K4 were not significantly upregulated by qPCR analysis. These findings suggest that altered miR-145 expression may impact prostate cancer growth through its loss of regulation of PTGFR and/or RASSF2 activities. qPCR analysis of miR-145 treated prostate cancer cells showed decreased expression of PTGFR and RASSF2 indicating these two genes are potential targets of miR-145 modulation. Direct binding only showed moderate reduction of RASSF2 and a more pronounced reduction of PTGFR. These studies suggest that miR-145 may modulate prostate cancer cell growth by modulating PTGFR activity.