Departments of Biochemistry and Molecular Genetics and Pathology, University of Virginia, Charlottesville, Virginia, USA.
Cancer Res. 2011 Feb 15;71(4):1313-24. doi: 10.1158/0008-5472.CAN-10-1031. Epub 2011 Jan 6.
MicroRNAs (miRNA) have been globally profiled in cancers but there tends to be poor agreement between studies including in the same cancers. In addition, few putative miRNA targets have been validated. To overcome the lack of reproducibility, we profiled miRNAs by next generation sequencing and locked nucleic acid miRNA microarrays and verified concordant changes by quantitative RT-PCR. Notably, miR-125b and the miR-99 family members miR-99a, -99b, and -100 were downregulated in all assays in advanced prostate cancer cell lines relative to the parental cell lines from which they were derived. All four miRNAs were also downregulated in human prostate tumor tissue compared with normal prostate. Transfection of miR-99a, -99b, or -100 inhibited the growth of prostate cancer cells and decreased the expression of prostate-specific antigen (PSA), suggesting potential roles as tumor suppressors in this setting. To identify targets of these miRNAs, we combined computational prediction of potential targets with experimental validation by microarray and polyribosomal loading analysis. Three direct targets of the miR-99 family that were validated in this manner were the chromatin-remodeling factors SMARCA5 and SMARCD1 and the growth regulatory kinase mTOR. We determined that PSA is posttranscriptionally regulated by the miR-99 family members, at least partially, by repression of SMARCA5. Together, our findings suggest key functions and targets of miR-99 family members in prostate cancer suppression and prognosis.
微小 RNA（miRNA）在癌症中进行了全面分析，但包括在相同癌症中的研究之间往往存在较差的一致性。此外，很少有假定的 miRNA 靶标得到验证。为了克服缺乏可重复性的问题，我们通过下一代测序和锁核酸 miRNA 微阵列对 miRNA 进行了分析，并通过定量 RT-PCR 验证了一致的变化。值得注意的是，miR-125b 和 miR-99 家族成员 miR-99a、-99b 和 -100 在所有检测中在高级前列腺癌细胞系中相对于它们衍生的亲本细胞系均下调。与正常前列腺组织相比，所有四种 miRNA 在人前列腺肿瘤组织中也下调。miR-99a、-99b 或 -100 的转染抑制了前列腺癌细胞的生长，并降低了前列腺特异性抗原（PSA）的表达，表明在这种情况下可能作为肿瘤抑制因子发挥作用。为了鉴定这些 miRNA 的靶标，我们将潜在靶标的计算预测与通过微阵列和多核糖体加载分析进行的实验验证相结合。以这种方式验证的 miR-99 家族的三个直接靶标是染色质重塑因子 SMARCA5 和 SMARCD1 以及生长调节激酶 mTOR。我们确定 PSA 是由 miR-99 家族成员通过抑制 SMARCA5 进行转录后调控的，至少部分是这样。总之，我们的研究结果表明 miR-99 家族成员在前列腺癌抑制和预后中的关键功能和靶标。
