Circulating microRNAs as stable blood-based markers for cancer detection

Abstract

Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.

Fig. 1.

Identification of miRNAs in human plasma. (A) Cloning and sequencing of miRNAs from human plasma. The schematic diagram depicts the preparation of a small RNA library from human plasma. Briefly, the 18- to 24-nt fraction from ≈250 ng of plasma total RNA from a single donor (individual 006; described in Table S6) was isolated by PAGE. Purified miRNAs were then 3′ and 5′ligated to single-stranded oligonucleotides that contained universal primer sequences for reverse transcription and PCR. Reverse transcription and PCR generated a library of small RNA cDNA molecules that were ligated into a plasmid vector (pCR4-TOPO) and transformed into Escherichia coli. Inserts from a total of 125 individual colonies yielded high-quality sequence. Sequences were compared to a reference database of known miRNA sequences (miRBase Release v.10.1) (19) and to GenBank. Seventy-three percent of sequences corresponded to known miRNAs as shown. The next most abundant species were matches to the sequence of synthetic RNAs spiked in as radiolabeled 18- and 24-nt molecular size markers during gel isolation steps. When only endogenously derived RNA sequences are considered, miRNAs represent 93% (91 of 98) of the recovered sequences. The miRNA read designated as “let-7f G15A” denotes a sequence matching the known let-7f miRNA except for a G-to-A substitution at nucleotide position 15. (B) Quantification of representative miRNAs in normal human plasma by TaqMan qRT-PCR. The graph indicates the number of copies of each of three representative miRNAs measured in plasma obtained from three healthy individuals. In each case, values represent the average of two replicate reverse transcription reactions followed by real-time PCR. For each miRNA assay, a dilution series of chemically synthesized miRNA was used to generate a standard curve that permitted absolute quantification of molecules of miRNA/μl plasma as shown here (see Fig. S2 for standard curve plots). Values were median-normalized by using measurements of synthetic normalization controls spiked in immediately after addition of denaturing solution during RNA isolation (see SI Text for full details). The absence of amplification in reverse transcriptase-negative controls indicated that amplification was originating from an RNA template (real-time PCR plots corresponding to plasma RNA samples and negative controls are provided in Fig. S3).

Fig. 2.

Characterization of miRNA stability in human plasma. (A) miRNA levels remain stable when plasma is subjected to prolonged room temperature incubation or freeze-thawed multiple times. (Upper) The graphs show normalized Ct values for the indicated miRNAs measured in parallel aliquots of human plasma samples incubated at room temperature for the indicated times. The experiment was carried out by using plasma from the two different individuals noted. Normalization of raw Ct values across samples is based on the measurement of three nonhuman synthetic miRNAs spiked into each sample at known molar amounts after initial plasma denaturation for RNA isolation (described in detail in SI Text). (Lower) The graphs show normalized Ct values for the indicated miRNAs measured in parallel aliquots of human plasma samples subjected to the indicated number of cycles of freeze-thawing. Raw Ct values were normalized across samples by using the same approach as described above. (B) Exogenously added miRNAs are rapidly degraded in plasma, whereas endogenous miRNAs are stable. Three C. elegans miRNAs (chosen for the absence of sequence similarity to human miRNAs) were chemically synthesized and added either directly to human plasma (from individual 003; described in Table S6) or added after the addition of denaturing solution (containing RNase inhibitors) to the plasma (referred to as “denatured plasma”). RNA was isolated from both plasma samples, and the abundance of each of the three C. elegans miRNAs was measured by TaqMan qRT-PCR (Left), as was that of three endogenous plasma miRNAs (Right). Asterisks indicate that the abundance ratios of cel-miR-39, cel-miR-54, and cel-miR-238 added to human plasma directly, relative to addition to denatured plasma, were 1.7 × 10⁻⁵, 9.1 × 10⁻⁶, and 1.1 × 10⁻⁵, respectively and therefore too low to accurately display on the plot. (C) Abundance of miRNAs in serum and plasma collected from the same individual is highly correlated. Each plot depicts the average Ct values (average of two technical replicates) of the indicated miRNAs measured in serum and plasma samples collected from a given individual at the same blood draw. Results from three different individuals are shown. miRNA measurements were highly correlated in both sample types. Results shown for synthetic C. elegans miRNAs spiked into each plasma or serum sample (after addition of denaturing solution) demonstrate that experimental recovery of miRNAs and robustness of subsequent qRT-PCR is not affected by whether it is plasma or serum that is collected.

Fig. 3.

Tumor-derived miRNAs are detectable in plasma. (A) Schema for 22Rv1 human prostate cancer xenograft experiment. (B) MiRNAs are present in plasma of healthy control mice and their levels are not nonspecifically altered in cancer-bearing mice. Plasma levels of miR-15b, miR-16, and miR-24 were measured in 12 healthy control mice and 12 xenograft-bearing mice. The mature sequence of these miRNAs is perfectly conserved between mice and humans. Ct values were converted to absolute number of copies/μl plasma by using a dilution series of known input quantities of synthetic target miRNA run on the same plate as the experimental samples (dilution curves are provided in Fig. S2). Values shown have been normalized by using measurements of C. elegans synthetic miRNA controls spiked into plasma after denaturation for RNA isolation (details of the normalization method are provided in SI Text). (C) Tumor-derived miRNAs are detected in plasma of xenograft-bearing mice and can distinguish cancer-bearing mice from controls. Plasma levels of miR-629* and miR-660 (two human miRNAs that are expressed in 22Rv1 cells and do not have known murine homologs) were measured in all control and xenografted mice. Ct values were converted to absolute number of copies/μl plasma and normalized as described for B (see Table 5) threshold. Given that homologous miRNAs are not believed to exist in mice, the low level of signal detected for a few mice in the control group, particularly for the miR-660 assay, is likely to represent nonspecific background amplification. As expected, in the control (nontumor-bearing) mice group, qRT-PCR for miR-629* or miR-660 in plasma from most animals could not detect any appreciable signal. These points are therefore not shown on the graph, even though plasma samples from the entire group of 12 mice in the control group were studied.

Fig. 4.

Detection of human prostate cancer by serum levels of tumor-associated miRNA miR-141. (A) Serum levels of miR-141 discriminate patients with advanced prostate cancer from healthy controls. Serum levels of the prostate cancer-expressed miRNA miR-141 were measured in 25 healthy control men and 25 patients with metastatic prostate cancer (clinical data on subjects is provided in Table S3). Ct values were converted to absolute number of copies/μl serum by using a dilution series of known input quantities of synthetic target miRNA run simultaneously (on the same plate) as the experimental samples (dilution curves are provided in Fig. S2). Values shown have been normalized by using measurements of C. elegans synthetic miRNA controls spiked into plasma after denaturation for RNA isolation (details of normalization method are provided in SI Text). The dashed line indicates a 100% specificity threshold. (B) Receiver Operating Characteristic (ROC) plot. The data shown in A were used to draw the ROC plot shown. (C) Serum levels of nontumor-associated miRNAs are not substantially different between patients with prostate cancer and controls. Serum levels of miR-16, miR-24, and miR-19b were measured as negative controls as they are not expected to be cancer-associated in the serum. Absolute quantification of miRNAs and data normalization were carried out as described for A.