Several studies have revealed that miRNAs are important regulators in cellular processes, such as cell proliferation, invasion and metastasis by repressing several oncogenes.11, 12, 13, 36, 37 To investigate the potential targets of miR-101, we used freely available web-based miR target prediction resources: TargetScan,38 miRanda39 and Diana-microT.40 We identified that miR-101 could potentially target SUB1. The binding site for miR-101 at 3′-UTR (untranslated region) of SUB1 is indicated (Figure 1a). MiR-101 is known to have tumor suppressor function by targeting several oncogenes including EZH2,11 proteasome assembly factor POMP,13 COX2,12 and others, therefore we sought to determine its role in SUB1 regulation. We treated prostate cancer cell line DU145 with precursor miRNA, miR-101 and tested some of the potential targets. We observed downregulation of QK1 and DDIT4 protein levels along with SUB1 (Figure 1b). Quantitative real-time PCR (qPCR) analyses also showed that miR-101 downregulates SUB1, DDIT4 and STC1 mRNA levels (Supplementary Figure S1). Here we show that miR-101 targets other known players in prostate cancer DDIT4, STC1 and QK1. To find the effect on SUB1 protein levels, we treated prostate cancer cells with precursor miRs, miR-101, -23a, -23b, -30a, -30b, -124 and -122 individually and measured SUB1 protein levels. As shown in Figure 1c, miR-101-treated cells showed significant reduction in SUB1 protein levels, whereas the control and other miR precursors did not alter SUB1 expression. Further, we measured the effect of miR-101 on cell growth using a colony growth assay using DU145, PC3 and LnCaP cells (Figure 1d). To determine whether miR-101 directly binds SUB1 3′-UTR and regulates it, HEK-293T cells were co-transfected with miR-101 and pMir-REPORT-SUB1 3′-UTR plasmids. MiR-101 showed substantial reduction in luciferase reporter activity compared with non-targeting (Non-T) control miR (Figure 1e). This effect is reversed by mutating miR-101 target site (Supplementary Figures S2a and b). These results indicate that SUB1 is a direct target of miR-101.