MiRNA fingerprint in the diagnosis of prostate cancer
MicroRNAs (miRNA) are a recently discovered class of small non-coding RNAs (17-14 nucleotides). Due to their function as regulators of gene expression they play a critical role both in physiological and in pathological processes, such as cancer. The present invention provides novel methods for diagnosing prostate cancer based on the determination of specific miRNAs that have altered expression levels in different conditions, e.g. disease states compared to healthy controls.
1. A method for diagnosing prostate cancer, comprising:
(a) determining an expression profile of hsa-miR-144* (SEQ ID NO:714) and hsa-miR-148a (SEQ ID NO:701) in blood cells obtained from a blood sample from a subject suspected to be affected by prostate cancer, wherein said blood cells consist of erythrocytes, leukocytes and thrombocytes and are obtained by removing the acellular fraction from the blood sample, and wherein determining said expression profile comprises nucleic acid amplification performed using real-time polymerase chain reaction (RT-PCR),
(b) comparing said expression profile to a reference expression profile of healthy subjects,
(c) diagnosing the presence of prostate cancer in the subject suspected to be affected by prostate cancer when down-regulation of each of hsa-miR-144* and hsa-miR-148a is detected in comparison to the reference expression profile, and
(d) subjecting the subject diagnosed to be affected by prostate cancer to therapy for prostate cancer selected from the group consisting of surgery, radiation therapy, hormonal therapy, chemotherapy, cryosurgery, and high intensity focused ultrasound.
2. The method according to claim 1 , wherein determining the expression profile in step (a) comprises:
(i) extracting any RNA-fraction from said blood cells,
(ii) performing a reverse transcriptase reaction using miRNA specific primers to produce non naturally occurring cDNA-transcripts of the predetermined set of miRNAs,
(iii) amplifying the cDNA-transcripts using a set of primer pairs, and
(iv) quantifying the cDNA-transcripts to produce an expression profile.
3. The method according to claim 1 , wherein the reference expression profile is an averaged expression profile determined using blood cells from a plurality of healthy subjects.
4. The method according to claim 3 , wherein said plurality of healthy subjects are the same gender and of similar age as the subject to be diagnosed.
5. The method according to claim 1 , wherein the miRNA from blood cells is protected against degradation by collection tubes or by additives that stabilize the miRNA.
6. The method according to claim 1 , wherein the predetermined set of miRNAs from blood cells is protected against degradation by collection tubes.
7. The method of claim 1 , wherein the diagnosis comprises determining survival rate, responsiveness to drugs, or monitoring the course of the prostate cancer or the therapy, staging the prostate cancer, measuring the response of a patient to therapeutic intervention, segmentation of patients suffering from the prostate cancer estimating the severity of the prostate cancer, or predicting the response of a patient with the prostate cancer to therapeutic intervention.
8. The method of claim 7 , wherein said therapeutic intervention is chemotherapy.
9. The method according to claim 1 , wherein the reference expression profile is a mathematical average expression profile of the predetermined set of miRNAs.
10. The method according to claim 1 , wherein the expression profile of said subject and the reference expression profile and optionally the predetermined set of miRNAs are stored in a database.
11. The method according to claim 1 , further comprising determining the expression profile of at least 2, 7, 10, 15, 20, 25, 30, 35, 40, 50, 75, or 100 additional miRNAs.
12. The method according to claim 11 , further comprising determining the expression profile of hsa-miR-519b-5p, hsa-miR-1324, hsa-miR-137, hsa-miR-556-5p, and hsa-miR-330-3p.