miRNA fingerprint in the diagnosis of COPD
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 a state of health 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 of diagnosing COPD in a subject, the method comprising:
providing a blood sample comprised of a blood cell fraction from a subject,
determining an expression profile of a predetermined set of at least two miRNAs in the blood sample, wherein the predetermined set of two miRNAs comprises:
(i) miR-26a, and
(ii) at least one additional miRNA selected from the group consisting of: hsa-miR720, hsa-miR-328, hsa-miR-675, hsa-miR-182, hsa-miR-877, hsa-miR-1275, hsamiR-363, miR-324-3p and hsa-miR-664;
wherein the expression profile is determined by measuring levels of the at least two miRNAs in the predetermined set of at least two miRNAs;
comparing the expression profile to at least one reference expression profile obtained from at least one control known to have lung cancer;
detecting an up-regulation relative to the reference expression profile of:
hsa-miR-26a and
and at least one of the following deregulations relative to the reference expression profile:
a down-regulation of hsa-miR-720,
a down-regulation of hsa-miR-328,
a down-regulation of hsa-miR-675,
an up-regulation of hsa-miR-182,
a down-regulation of hsa-miR-877,
a down-regulation of hsa-miR-1275,
an up-regulation of hsa-miR-363,
a down-regulation of hsa-miR-324-3p, and
a down-regulation of hsa-miR-664;
determining the presence of COPD in the subject; and
further comprising:
subjecting the subject to therapeutic treatment for COPD; and
monitoring the therapeutic treatment on the basis of the predetermined set of at least two miRNAs.
2. The method of claim 1 , wherein total RNA is isolated from the blood cell fraction;
cDNA transcripts are obtained from the total RNA isolated by reverse transcription reaction; and
the cDNA transcripts of the predetermined set of at least two miRNAs are amplified to determine the expression profile of the predetermined set of at least two miRNAs.
3. The method of claim 1 , wherein the at least one additional miRNA comprises miR-720, and the predetermined set of at least two miRNAs comprises least one set of miRNAs selected from the group consisting of:
SLC-465 set consisting of: hsa-miR-328, hsa-miR-26a, and hsa-miR-720;
SLC-694 set consisting of: hsa-miR-675, hsa-miR-26a, and hsa-miR-720;
SLC-130 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-877;
SLC-79 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-720;
SLC-621 set consisting of: hsa-miR-26a, hsa-miR-1275, and hsa-miR-720;
SLC-719 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-182;
SLC-646 set consisting of: hsa-miR-26a, hsa-miR-363, and hsa-miR-720;
SLC-28 set consisting of: hsa-miR-26a and hsa-miR-720;
SLC-155 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-324-3p;
SLC-358 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-664; and,
SLC-492 set consisting of: hsa-miR-1275, hsa-miR-26a, and hsa-miR-720.
4. The method of claim 1 , wherein the at least one additional miRNA in the predetermined set of at least two miRNAs comprises hsa-miR-324-3p having SEQ ID NO: 466.
5. The method of claim 1 , wherein the measuring comprises nucleic acid hybridization, nucleic acid amplification, polymerase extension sequencing, mass spectroscopy, flow cytometry, or any combination thereof.
6. The method of claim 1 , wherein the measuring comprises using specific or non-specific primer pairs to determine the expression profile.
7. The method of claim 6 , wherein the primer pairs are used to amplify cDNA transcripts of the predetermined set of at least two miRNAs.
8. The method of claim 6 , wherein the primer pairs are complemented by buffers, enzymes, dye, or labeled probes suitable for conducting RT-PCR.
9. The method of claim 1 , wherein the measuring comprises using primers to perform reverse transcriptase reaction and real-time polymerase chain reaction (RT-PCR).
10. The method of claim 1 , wherein the blood cellular fraction consists of erythrocytes, leukocytes, and thrombocytes.
11. The method of claim 1 , wherein the predetermined set of at least two miRNAs comprises at least one set of miRNAs selected from the group consisting of
SLC-1 set consisting of: hsa-miR-26a and hsa-miR-675;
SLC-28 set consisting of: hsa-miR-26a and hsa-miR-720;
SLC-54 set consisting of: hsa-miR-26a and hsa-miR-877;
SLC-79 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-720;
SLC-105 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-877;
SLC-130 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-877;
SLC-155 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-324-3p;
SLC-179 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-324-3p;
SLC-203 set consisting of: hsa-miR-26a, hsa-miR-877, and hsa-miR-324-3p;
SLC-266 set consisting of: hsa-miR-26a and hsa-miR-324-3p;
SLC-358 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-664;
SLC-382 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-664;
SLC-406 set consisting of: hsa-miR-26a, hsa-miR-324-3p, and hsa-miR-664;
SLC-430 set consisting of: hsa-miR-328 and hsa-miR-26a;
SLC-436 set consisting of: hsa-miR-1275 and hsa-miR-26a;
SLC-455 set consisting of: hsa-miR-1275, hsa-miR-328, and hsa-miR-26a;
SLC-456 set consisting of: hsa-miR-328, has-miR-26a, and hsa-miR-675;
SLC-465 set consisting of: hsa-miR-328, hsa-miR-26a, and hsa-miR-720;
SLC-478 set consisting of: hsa-miR-1275, hsa-miR-26a, and hsa-miR-675;
SLC-492 set consisting of: hsa-miR-1275, hsa-miR-26a, and hsa-miR-720;
SLC-506 set consisting of: hsa-miR-328, hsa-miR-26a, and hsa-miR-877;
SLC-533 set consisting of: hsa-miR-675 and hsa-miR-26a;
SLC-534 set consisting of: hsa-miR-26a and hsa-miR-1275;
SLC-555 set consisting of: hsa-miR-26a and hsa-miR-363;
SLC-596 set consisting of: hsa-miR-675, hsa-miR-26a, and hsa-miR-1275;
SLC-597 set consisting of: hsa-miR-26a, hsa-miR-1275, and hsa-miR-363;
SLC-620 set consisting of: hsa-miR-675, hsa-miR-26a, and hsa-miR-363;
SLV-621 set consisting of: hsa-miR-26a, hsa-miR-1275, and hsa-miR-720;
SLC-646 set consisting of: hsa-miR-26a, hsa-miR-363, and hsa-miR-720;
SLC-671 set consisting of: hsa-miR-26a, hsa-miR-363, and hsa-miR-182;
SLC-694 set consisting of: hsa-miR-675, hsa-miR-26a, and hsa-miR-720;
SLC-695 set consisting of: hsa-miR-26a, hsa-miR-1275, and hsa-miR-182;
SLC-719 set consisting of: hsa-miR-26a, hsa-miR-720, and hsa-miR-182;
SLC-781 set consisting of: hsa-miR-26a, hsa-miR-675, and hsa-miR-1275.