Figure 1.
Serum miRNA profiling and validation.
(A) Measurement of circulating miRNAs in sera pooled from patients with advanced prostate cancer as compared to healthy donors (comprising a Discovery Set) by TLDA profiling. Blue- and brown-filled circles represent serum miRNAs increased or decreased (with unadjusted p-value <0.05), respectively, in mCRPC patients compared to healthy controls. Inset: Nine miRNAs demonstrated >5-fold change (unadjusted P<0.05, Student’s t-test). FC, fold-change. (B) Confirmation of mCRPC-associated serum miRNAs in individual samples from the Discovery Set from the University of Washington samples. Upper: miRNA biomarker candidates were measured in individual samples by TaqMan miRNA qRT-PCR (P value assigned by Wilcoxon signed-rank test), where miRNA abundance is given in terms of miRNA copies/µl serum. Red bars, mean +/− SEM of miRNA copies/µl serum for each group. Lower: Receiver operating characteristic (ROC) curves plot sensitivity vs. (1 - specificity) to assess the ability of each miRNA biomarker to distinguish cases from controls. (C) Validation of mCRPC-associated serum miRNAs in an independent Validation Set. Upper: Serum concentration (copies/µl) of miR-141, miR-375, miR-200c, miR-200a and miR-210 was measured by TaqMan miRNA qRT-PCR. Dot-plot associated P values were assigned by Wilcoxon signed-rank test. Dot plots and ROC curves were generated as described for Fig. 1. Lower: Red, results from the validation sample set obtained from the University of Michigan. Black, results from the primary sample set obtained from the University of Washington reproduced from Fig. 1B, lower. AUC, area under the curve; mCRPC, prostate cancer patient sera; FC, fold-change; CTL, control sera (from age-matched male individuals with normal PSA and negative digital rectal exam).
Figure 2.
Exposure of prostate cancer cell lines to hypoxia induces production and release of miR-210 into the extracellular environment.
Left column, miR-210 copies/ng RNA in LNCaP and VCaP human prostate cancer cell lines cultured in normoxic (20% O2) (white bars) or hypoxic (1% O2) (blue bars) conditions for 24, 48 or 72 hours. Right column, miR-210 copies/µl in filtered conditioned media corresponding to cellular samples. *, P value <0.05; **, P value <0.01; ***, P value <0.001 (Student’s t-test).
Figure 3.
Relationship between serum miR-210 levels and PSA response in patients with metastatic castration resistant prostate cancer.
Upper: miR-210 copies/µl serum versus %PSA change/day. %PSA change/day represents a measure of response to treatment and was calculated using available clinical PSA values measured most recently prior to, and at the time of serum miR-210 draw. Mean time elapsed between the two blood draws was 30 days. Closed circles represent the subset of patients defined as "miRNA-high" based on higher abundance of mCRPC-associated serum miRNAs compared to all control individuals (as described in Results and Discussion). Open circles represent the patients with mCRPC who did not meet the definition for “miRNA-high”. Solid line represents trend line of the miRNA-high patient subset, dotted line represents trend line of all patients. Middle: miR-210 copies/µl serum in patients with either a PSA Response (R) or No PSA Response (NR). PSA Response is defined as a decreasing or stable PSA (any change less than a 25% increase), and No PSA Response is defined as a PSA increase of 25% or more, similar to the Prostate Cancer Working Group criteria. Lower: Copies/µl serum of miR-141, miR-200a, miR-200c and miR-375 in patients, R and NR.