MicroRNA-940 as a Potential Serum Biomarker for Prostate Cancer

Abstract

Prostate cancer is one of the leading causes of death despite an astoundingly high survival rate for localized tumors. Though prostate specific antigen (PSA) test, performed in conjunction with digital rectal examinations, is reasonably accurate, there are major caveats requiring a thorough assessment of risks and benefits prior to conducting the test. MicroRNAs, a class of small non-coding RNAs, are stable molecules that can be detected in circulation by non-invasive methods and have gained importance in cancer prognosis and diagnosis in the recent years. Here, we investigate circulating miR-940, a miRNA known to play a role in prostate cancer progression, in both cell culture supernatants as well as patient serum and urine samples to determine the utility of miR-940 as a new molecular marker for prostate cancer detection. We found that miR-940 was significantly higher in serum from cancer patients, specifically those with clinically significant tumors (GS ≥ 7). Analysis of receiver operating characteristic curve demonstrated that miR-940 in combination with PSA had a higher area under curve value (AUC: 0.818) than the miR-940 alone (AUC: 0.75) for the diagnosis of prostate cancer. This study provides promising results suggesting the use of miR-940 for prostate cancer diagnosis.

Keywords: PSA; circulating miRNA; miR-940; prostate cancer; serum.

Figure 1

Expression of miR-940 in cell culture supernatant. (A) Higher levels of both precursor (Pre-miR-940) and mature (miR-940) miRNA are detected in the exosomes derived from the cell culture supernatant of the DU-145 cancer cells compared to the normal PWR-1E cells as measured by qPCR (normalized to RNU6-2). (B) Ratio of miR-940 from the secreted exosomes to miR-940 within the cells, as determined by qPCR, is plotted as a fold change in the normal and cancer cells. The experiment was repeated two independent times. ^#p-value ≤ 0.1; *p-value ≤ 0.05.

Figure 2

The expression of miR-940 and PSA in serum samples of prostate cancer patients and normal healthy males. (A) Scatter plot analysis of PSA expression in serum samples of normal healthy (n = 25) controls and prostate cancer (n = 32) patients. The data are expressed as the mean ± SEM (***p-value = 0.0005; two-tailed Student's t-test). (B) Scatter plot analysis of miR-940-fold expression in serum samples of healthy normal controls (n = 25) and prostate cancer patients (n = 32). The data are expressed as the mean ± SEM (***p-value = 0.0013; two-tailed Student's t-test). (C) ROC curve for (B) depicts the sensitivity (true positive: cancer in reality and according to test variable; i.e., fold change in miR-940) and specificity (100%—false positive: 100%—normal in reality with fold change in miR-940 predicting cancer) for the use of secreted miR-940 as an independent variable to determine cancer vs. normal states. The AUC values are shown on the graph.

Figure 3

Secreted miR-940 and PSA from serum is significantly higher in patients with clinically significant tumors. (A) Serum from patients who had GS 7 and above in their biopsy were grouped together as clinically significant tumor serum (GS ≥ 7, n = 25) and the ones with biopsy GS 6 as clinically insignificant tumor serum (GS = 6, n = 7). The fold change in the miR-940 was quantified using qPCR. The data are expressed as the mean ± SEM (*p-value = 0.0148; two-tailed Student's t-test). (B) Scatter plot analysis of PSA expression in serum samples of clinically insignificant (n = 25) and clinically significant (n = 32) prostate cancer patients. The data are expressed as the mean ± SEM (*p-value = 0.0134; two-tailed Student's t-test). (C) ROC curve for both miR-940, PSA, and combined (miR-940 and PSA) comparing clinically insignificant to clinically significant tumors. (D) Scatter plot analysis of miR-940 expression in serum samples of combined normal and clinically insignificant (Normal + GS 6, n = 32) tumors and clinically significant tumors (GS ≥ 7, n = 25) patients. The data are expressed as the mean ± SEM (***p-value = 0.0006; two-tailed Student's t-test). (E) PSA expression in serum samples of combined normal and clinically insignificant (Normal + GS 6, n = 32) tumors and clinically significant (GS ≥ 7, n = 25) tumors patients. (***p-value = 0.0003; two-tailed Student's t-test) (F) ROC curve for fold change in miR-940 to determine clinically significant cancers (GS ≥ 7) vs. normal and clinically insignificant cancers (healthy controls + GS = 6). (G) Scatter plot analysis of miR-940 expression in serum samples of normal (n = 25) healthy controls and clinically significant tumors (GS ≥ 7, n = 25) patients. The data are expressed as the mean ± SEM (***p-value = 0.0005; two-tailed Student's t-test). (H) PSA expression in serum samples of normal (n = 25) controls and clinically significant tumors (GS ≥ 7, n = 25) patients. (***p-value = 0.0002; two-tailed Student's t-test) (I) ROC curve for fold change in miR-940 to determine clinically significant cancers (GS ≥ 7) vs. normal (healthy controls).

Figure 4

The expression of miR-940 in urine of prostate cancer patients. Relative expression of Pre-miR-940 and miR-940 in the urine from clinically insignificant (GS ≥ 7) prostate cancer patients relative to clinically insignificant (GS = 6) patients (n = 3 and 5 respectively).