JP2011062161A - Method for diagnosing colon cancer by using blood - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for rapidly and readily diagnosing colon cancer by using an antisense RNA containing sample having excellent treatability, and to provide a tool useful for the diagnosis. <P>SOLUTION: The examination method for diagnosing the colon cancer includes measuring the expression of an endogenous antisense RNA in the blood collected from a mammal. There are also provided the endogenous antisense RNA, and the diagnostic agent of the colon cancer containing a nucleic acid having a base sequence hybridizable therewith. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to an endogenous antisense RNA in blood exhibiting a colorectal cancer-specific expression pattern, and also relates to a test method for colorectal cancer diagnosis using the antisense RNA and a diagnostic agent for colorectal cancer.

  Until now, the central dogma in genetics is that RNA is transcribed from DNA and translated into protein. For RNA, messenger RNA (sense RNA) is produced using the genomic DNA encoding the protein as a template. The analysis has been proceeding with a focus on. However, in recent years, the results of genome / transcriptome analysis have reported that there are many antisense RNAs that are transcribed in opposite directions to sense RNA from both strands of DNA at the same locus (non-patent literature). 1).

  Antisense RNA is RNA having a base sequence complementary to messenger RNA, and specifically RNA read from the reverse strand of the DNA strand encoding the sense gene. Sense-antisense RNA has the ability to form double strands. For example, double-stranded RNA is required for RNA interference and is involved in protein translational control by small RNAs called microRNAs. Etc. are known.

  In mice, about 2,500 pairs of sense-antisense genes have been identified, and they contain many untranslated genes that do not encode proteins (Non-patent Document 2). About half of the sense-antisense gene pairs are considered to be translatable-untranslatable gene pairs. In humans, it is suggested that there are about 2,600 sense-antisense RNA pairs (Non-patent Document 3).

  One of the present inventors and their collaborators conducted research to elucidate the physiological role of this antisense RNA. As a result, specific endogenous antisense RNA (especially untranslated antisense RNA) was found. And the expression balance with the corresponding sense RNA was found to vary specifically in cancer tissues, and this has been reported (Patent Document 1). Another group reports that one antisense RNA epigenetically controls human canceration (Non-patent Document 4).

  Thus, it is becoming clear that the expression of antisense RNA is involved in the development of cancer, and analysis of this antisense RNA is considered to play an important role in cancer diagnosis and the like. However, no rapid and simple method for diagnosing such cancer has yet been developed, and in particular, comprehensive analysis of antisense RNA expression for samples that are easy to handle in the diagnosis, and cancer based on the results. No specific report has been made on the diagnostic method.

JP 2008-295392 A

Rosok O et al., Nat Biotechnol. 22, 104-108. (2004) Kiyosawa H et al., Genome Res. 13, 1324-1334. (2003) Yelin R et al., Nat Biotechnol. 21, 379-386. (2003) Yu W et al., Nature. 451, 202-206. (2008)

  The problem to be solved by the present invention is to provide a means for diagnosing colorectal cancer quickly and easily using an antisense RNA-containing sample excellent in handleability, and a useful tool used for the diagnosis.

  The inventors artificially assumed an antisense strand sequence deduced from the sequence of a known cDNA as a virtual antisense RNA, designed a probe for the antisense strand sequence (Artificial Antisense Sequence: AFAS), A microarray in which the probe (AFAS probe) was mounted together with a probe for a cDNA sequence (sense strand sequence) was constructed. Furthermore, the present inventors collected blood from cancer patients and healthy subjects as an antisense RNA-containing sample excellent in handleability, and prepared an RNA-containing sample from the blood.

  The present inventors label each sample by random priming, and as a result of comprehensive analysis of the expression of antisense RNA and sense RNA in each sample using the microarray, the specific antisense RNA is specific to colon cancer. It was found to have a typical expression pattern. As a result of further studies, the present inventors have demonstrated that the specific antisense RNA is useful for the diagnosis of colorectal cancer, and have completed the present invention.

That is, the present invention is as follows.
[1] A test method for diagnosing colorectal cancer, comprising measuring endogenous antisense RNA selected from the following (1) to (30) in blood collected from a mammal.
(1) Endogenous antisense RNA for mammalian MAPK14 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 1
(2) Endogenous antisense RNA for mammalian RNF175 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 2.
(3) Endogenous antisense RNA for mammalian B4GALT5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 3
(4) Endogenous antisense RNA for mammalian LRRK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 4
(5) An endogenous antisense RNA against the mammalian LOC731208 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 5
(6) Endogenous antisense RNA for mammalian PSMF1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 6
(7) Endogenous antisense RNA for mammalian USP25 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 7
(8) Endogenous antisense RNA for mammalian RAB10 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 8
(9) Endogenous antisense RNA for mammalian LIN7A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 9
(10) Endogenous antisense RNA for mammalian PFN2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 10
(11) Endogenous antisense RNA for mammalian CREB5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 11
(12) Endogenous antisense RNA for mammalian DSC2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 12
(13) An endogenous antisense RNA against the mammalian LOC432369 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 13
(14) Endogenous antisense RNA for mammalian ANXA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 14
(15) An endogenous antisense RNA against the mammalian CENTB2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 15
(16) Endogenous antisense RNA for mammalian PFDN5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 16
(17) An endogenous antisense RNA against the mammalian KIAA0241 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 17
(18) Endogenous antisense RNA for mammalian HINT1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 18
(19) Endogenous antisense RNA for mammalian AYTL1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 19
(20) Endogenous antisense RNA for mammalian BAZ1A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 20
(21) Endogenous antisense RNA for mammalian PROK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 21
(22) Endogenous antisense RNA for mammalian TMEM80 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 22
(23) An endogenous antisense RNA against a mammalian C9orf102 gene, which comprises the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 23
(24) Endogenous antisense RNA for mammalian SETD6 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 24
(25) An endogenous antisense RNA against a mammalian LANCL1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 25
(26) Endogenous antisense RNA for mammalian WNT2B gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 26
(27) Endogenous antisense RNA for mammalian PNPLA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 27
(28) An endogenous antisense RNA against a mammalian ZNF533 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 28
(29) An endogenous antisense RNA against a mammalian ZBED4 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 29
(30) An endogenous antisense RNA against the mammalian LOC730966 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 30
[2] An endogenous antisense RNA selected from the following (1) to (30).
(1) Endogenous antisense RNA for mammalian MAPK14 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 1
(2) Endogenous antisense RNA for mammalian RNF175 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 2.
(3) Endogenous antisense RNA for mammalian B4GALT5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 3
(4) Endogenous antisense RNA for mammalian LRRK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 4
(5) An endogenous antisense RNA against the mammalian LOC731208 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 5
(6) Endogenous antisense RNA for mammalian PSMF1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 6
(7) Endogenous antisense RNA for mammalian USP25 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 7
(8) Endogenous antisense RNA for mammalian RAB10 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 8
(9) Endogenous antisense RNA for mammalian LIN7A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 9
(10) Endogenous antisense RNA for mammalian PFN2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 10
(11) Endogenous antisense RNA for mammalian CREB5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 11
(12) Endogenous antisense RNA for mammalian DSC2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 12
(13) An endogenous antisense RNA against the mammalian LOC432369 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 13
(14) Endogenous antisense RNA for mammalian ANXA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 14
(15) An endogenous antisense RNA against the mammalian CENTB2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 15
(16) Endogenous antisense RNA for mammalian PFDN5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 16
(17) An endogenous antisense RNA against the mammalian KIAA0241 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 17
(18) Endogenous antisense RNA for mammalian HINT1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 18
(19) Endogenous antisense RNA for mammalian AYTL1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 19
(20) Endogenous antisense RNA for mammalian BAZ1A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 20
(21) Endogenous antisense RNA for mammalian PROK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 21
(22) Endogenous antisense RNA for mammalian TMEM80 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 22
(23) An endogenous antisense RNA against a mammalian C9orf102 gene, which comprises the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 23
(24) Endogenous antisense RNA for mammalian SETD6 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 24
(25) An endogenous antisense RNA against a mammalian LANCL1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 25
(26) Endogenous antisense RNA for mammalian WNT2B gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 26
(27) Endogenous antisense RNA for mammalian PNPLA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 27
(28) An endogenous antisense RNA against a mammalian ZNF533 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 28
(29) An endogenous antisense RNA against a mammalian ZBED4 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 29
(30) An endogenous antisense RNA against the mammalian LOC730966 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 30
[3] A diagnostic agent for colorectal cancer comprising a nucleic acid comprising a base sequence capable of hybridizing under stringent conditions with the endogenous antisense RNA according to [2].

  Since the endogenous antisense RNA of the present invention has a cancer-specific expression pattern in colorectal cancer patients, colon cancer can be diagnosed efficiently by measuring the expression level of the antisense. Further, in the present invention, blood collected from a mammal is employed as an RNA-containing sample, and because it is excellent in handling in the diagnosis of colorectal cancer, it is possible to make a quick and simple diagnosis of colorectal cancer, It is possible to provide a test method for colorectal cancer that is also useful in clinical settings.

(A) MAPK14, (B) RNF175, (C) B4GALT5, (D) LRRK2, (E) LOC731208, (F) PSMF1, (G) USP25, (H) in blood samples collected from healthy individuals and colorectal cancer patients FIG. 4 is a graph showing the expression level of endogenous antisense RNA for RAB10 gene. In each graph, the vertical axis represents the RNA expression level. (A) LIN7A, (B) PFN2, (C) CREB5, (D) DSC2, (E) LOC432369, (F) ANXA3, (G) CENTB2, (H) in blood samples collected from healthy individuals and colorectal cancer patients FIG. 4 is a graph showing the expression level of endogenous antisense RNA for the PFDN5 gene. In each graph, the vertical axis represents the RNA expression level. In blood samples collected from healthy individuals and colorectal cancer patients, (A) KIAA0241, (B) HINT1, (C) AYTL1, (D) BAZ1A, (E) PROK2, (F) TMEM80, (G) C9orf102, (H FIG. 4 is a graph showing the expression level of endogenous antisense RNA for SETD6 gene. In each graph, the vertical axis represents the RNA expression level. Endogenous antisense RNA for (A) LANCL1, (B) WNT2B, (C) PNPLA3, (D) ZNF533, (E) ZBED4, (F) LOC730966 genes in blood samples collected from healthy individuals and colorectal cancer patients It is a figure which shows the expression level of. In each graph, the vertical axis represents the RNA expression level. It is a figure which shows the result of the clustering analysis about 12 sense RNA by which the statistically significant difference (p <0.001, Fold change> 2.0) was seen between the healthy subject and the colon cancer patient. It is a figure which shows the result of the clustering analysis about 30 antisense RNA by which the statistically significant difference (p <0.001, Fold change> 2.0) was seen between the healthy person and the colon cancer patient.

The endogenous antisense RNA used for the diagnosis of colorectal cancer of the present invention is any one selected from the following (1) to (30).
(1) Endogenous antisense RNA for mammalian MAPK14 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 1
(2) Endogenous antisense RNA for mammalian RNF175 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 2.
(3) Endogenous antisense RNA for mammalian B4GALT5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 3
(4) Endogenous antisense RNA for mammalian LRRK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 4
(5) An endogenous antisense RNA against the mammalian LOC731208 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 5
(6) Endogenous antisense RNA for mammalian PSMF1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 6
(7) Endogenous antisense RNA for mammalian USP25 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 7
(8) Endogenous antisense RNA for mammalian RAB10 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 8
(9) Endogenous antisense RNA for mammalian LIN7A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 9
(10) Endogenous antisense RNA for mammalian PFN2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 10
(11) Endogenous antisense RNA for mammalian CREB5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 11
(12) Endogenous antisense RNA for mammalian DSC2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 12
(13) An endogenous antisense RNA against the mammalian LOC432369 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 13
(14) Endogenous antisense RNA for mammalian ANXA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 14
(15) An endogenous antisense RNA against the mammalian CENTB2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 15
(16) Endogenous antisense RNA for mammalian PFDN5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 16
(17) An endogenous antisense RNA against the mammalian KIAA0241 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 17
(18) Endogenous antisense RNA for mammalian HINT1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 18
(19) Endogenous antisense RNA for mammalian AYTL1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 19
(20) Endogenous antisense RNA for mammalian BAZ1A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 20
(21) Endogenous antisense RNA for mammalian PROK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 21
(22) Endogenous antisense RNA for mammalian TMEM80 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 22
(23) An endogenous antisense RNA against a mammalian C9orf102 gene, which comprises the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 23
(24) Endogenous antisense RNA for mammalian SETD6 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 24
(25) An endogenous antisense RNA against a mammalian LANCL1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 25
(26) Endogenous antisense RNA for mammalian WNT2B gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 26
(27) Endogenous antisense RNA for mammalian PNPLA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 27
(28) An endogenous antisense RNA against a mammalian ZNF533 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 28
(29) An endogenous antisense RNA against a mammalian ZBED4 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 29
(30) An endogenous antisense RNA against the mammalian LOC730966 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 30

  In the diagnosis of colorectal cancer of the present invention, the endogenous antisense RNA may be used alone or in combination of two or more. If two or more types are used in combination, the degree of false positive or false negative in the diagnosis or test can be suppressed, and colorectal cancer can be diagnosed with high accuracy.

  Animals capable of diagnosing colon cancer with the endogenous antisense RNA of the present invention are mammals (eg, humans, mice, rats, monkeys, dogs, cows, horses, pigs, sheep, goats, rabbits, hamsters, etc.). Although there is no particular limitation as long as it is, it is preferably a human, mouse, rat, monkey, dog or the like, more preferably a human or mouse.

  The nucleotide sequence shown in SEQ ID NO: 1 is the antisense strand sequence of the 691-750th nucleotide sequence of the cDNA sequence of the human MAPK14 (mitogen-activated protein kinase 14) gene registered as Accession No. NM_001315 in the GenBank database. is there. As shown in Examples below, the human antisense MAPK14 of the present invention (antisense RNA for MAPK14 gene; hereinafter, similar abbreviations may be used for antisense RNA for other genes) is shown in SEQ ID NO: 1. It can hybridize with a 60-mer base sequence complementary to the base sequence. Accordingly, the human antisense MAPK14 is an RNA having a base sequence containing at least the whole or a part of the base sequence shown in SEQ ID NO: 1.

Similarly, the base sequence shown in SEQ ID NO: 2 is a part of the antisense strand sequence for the cDNA sequence of the human RNF175 (ring finger protein 175) gene registered as Accession No. NM — 173662 in the GenBank database.
The nucleotide sequence shown in SEQ ID NO: 3 is an antisense strand sequence against the cDNA sequence of the human B4GALT5 (UDP-Gal: betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 5) gene registered as Accession No. NM_004776 in the GenBank database. It is a part.
The base sequence shown in SEQ ID NO: 4 is a part of the antisense strand sequence for the cDNA sequence of human LRRK2 (leucine-rich repeat kinase 2) gene registered as Accession No. NM_198578 in the GenBank database.
The base sequence shown in SEQ ID NO: 5 is a part of the antisense strand sequence for the cDNA sequence of the human LOC731208 gene registered as Accession No. XM — 001132091 in the GenBank database.
The base sequence shown in SEQ ID NO: 6 is a part of the antisense strand sequence for the cDNA sequence of the human PSMF1 (proteasome (prosome, macropain) inhibitor subunit 1) gene registered as Accession No. NM_006814 in the GenBank database.
The base sequence shown in SEQ ID NO: 7 is a part of the antisense strand sequence for the cDNA sequence of the human USP25 (ubiquitin specific peptidase 25) gene registered as Accession No. NM — 013396 in the GenBank database.
The base sequence shown in SEQ ID NO: 8 is a part of the antisense strand sequence for the cDNA sequence of the human RAB10 gene registered as Accession No. NM_016131 in the GenBank database.
The base sequence shown in SEQ ID NO: 9 is a part of the antisense strand sequence for the cDNA sequence of the human LIN7A (lin-7 homolog A) gene registered as Accession No. NM_004664 in the GenBank database.
The base sequence shown in SEQ ID NO: 10 is a part of the antisense strand sequence for the cDNA sequence of human PFN2 (profilin 2) gene registered as Accession No. NM_002628 in the GenBank database.
The base sequence shown in SEQ ID NO: 11 is a part of the antisense strand sequence for the cDNA sequence of the human CREB5 (cAMP responsive element binding protein 5) gene registered as Accession No. NM_004904 in the GenBank database.
The base sequence shown in SEQ ID NO: 12 is a part of the antisense strand sequence for the cDNA sequence of the human DSC2 (desmocollin 2) gene registered as Accession No. NM_024422 in the GenBank database.
The base sequence shown in SEQ ID NO: 13 is a part of the antisense strand sequence for the cDNA sequence of the human LOC432369 gene registered as Accession No. NR_002162 in the GenBank database.
The base sequence shown in SEQ ID NO: 14 is a part of the antisense strand sequence for the cDNA sequence of the human ANXA3 (annexin A3) gene registered as Accession No. NM_005139 in the GenBank database.
The base sequence shown in SEQ ID NO: 15 is a part of the antisense strand sequence for the cDNA sequence of the human CENTB2 (centaurin, beta 2) gene registered as Accession No. NM_012287 in the GenBank database.
The base sequence shown in SEQ ID NO: 16 is a part of the antisense strand sequence for the cDNA sequence of human PFDN5 (prefoldin subunit 5) gene registered as Accession No. NM — 145897 in the GenBank database.
The base sequence shown in SEQ ID NO: 17 is a part of the antisense strand sequence for the cDNA sequence of the human KIAA0241 gene registered as Accession No. NM_015060 in the GenBank database.
The base sequence shown in SEQ ID NO: 18 is a part of the antisense strand sequence for the cDNA sequence of the human HINT1 (histidine triad nucleotide binding protein 1) gene registered as Accession No. NM_005340 in the GenBank database.
The base sequence shown in SEQ ID NO: 19 is a part of the antisense strand sequence for the cDNA sequence of the human AYTL1 (acyltransferase like 1) gene registered as Accession No. NM_017839 in the GenBank database.
The base sequence shown in SEQ ID NO: 20 is a part of the antisense strand sequence for the cDNA sequence of human BAZ1A (bromodomain adjacent to zinc finger domain, 1A) gene registered as Accession No. NM_182648 in the GenBank database.
The base sequence shown in SEQ ID NO: 21 is a part of the antisense strand sequence for the cDNA sequence of the human PROK2 (prokineticin 2) gene registered as Accession No. NM — 021935 in the GenBank database.
The base sequence shown in SEQ ID NO: 22 is a part of the antisense strand sequence for the cDNA sequence of human TMEM80 (transmembrane protein 80) gene registered as Accession No. XM_001129808 in the GenBank database.
The base sequence shown in SEQ ID NO: 23 is a part of the antisense strand sequence for the cDNA sequence of the human C9orf102 (chromosome 9 open reading frame 102) gene registered as Accession No. NM_020207 in the GenBank database.
The base sequence shown in SEQ ID NO: 24 is a part of the antisense strand sequence for the cDNA sequence of the human SETD6 (SET domain containing 6) gene registered as Accession No. NM_024860 in the GenBank database.
The base sequence shown in SEQ ID NO: 25 is a part of the antisense strand sequence for the cDNA sequence of the human LANCL1 (LanC lantibiotic synthetase component C-like 1) gene registered as Accession No. NM_006055 in the GenBank database.
The nucleotide sequence shown in SEQ ID NO: 26 is a part of the antisense strand sequence for the cDNA sequence of the human WNT2B (wingless-type MMTV integration site family, member 2B) gene registered as Accession No. NM_024494 in the GenBank database. .
The base sequence shown in SEQ ID NO: 27 is a part of the antisense strand sequence for the cDNA sequence of the human PNPLA3 (patatin-like phospholipase domain containing 3) gene registered as Accession No. NM — 025225 in the GenBank database.
The base sequence shown in SEQ ID NO: 28 is a part of the antisense strand sequence for the cDNA sequence of the human ZNF533 (zinc finger protein 533) gene registered as Accession No. NM_152520 in the GenBank database.
The nucleotide sequence shown in SEQ ID NO: 29 is a part of the antisense strand sequence for the cDNA sequence of the human ZBED4 (zinc finger, BED-type containing 4) gene registered as Accession No. NM_014838 in the GenBank database.
The base sequence shown in SEQ ID NO: 30 is a part of the antisense strand sequence for the cDNA sequence of the human LOC730966 gene registered as Accession No. XM — 001127909 in the GenBank database.

  The antisense RNA of the present invention is capable of hybridizing to a 60-mer base sequence complementary to the base sequence shown in each SEQ ID NO. Therefore, the antisense RNA is RNA having a base sequence including at least a part or all of the base sequence shown in each SEQ ID NO.

  The “base sequence substantially identical to the base sequence shown in SEQ ID NO: n (n = 1 to 30; hereinafter the same)” is a fragment of the base sequence shown in SEQ ID NO: n, or a non-human mammal It means the nucleotide sequence of the region corresponding to SEQ ID NO: n, or their natural allelic variant or polymorphism in the antisense strand sequence for the cDNA sequence of each gene ortholog in an animal.

  The antisense RNA of the present invention can be detected by a known RNA detection method using a nucleic acid containing a base sequence that can hybridize with them as a probe or primer. Therefore, the antisense RNA can be used as a marker for diagnosis or detection of colorectal cancer, and the present invention also includes a colorectal cancer containing a nucleic acid containing a base sequence capable of hybridizing with the antisense RNA. Provide diagnostics.

  The nucleic acid for detecting an antisense RNA of the present invention is not particularly limited as long as it contains a nucleotide sequence that can hybridize with a target antisense RNA sequence under hybridization conditions that can be used in normal gene expression analysis. . Preferably, the probe is a nucleic acid comprising a base sequence capable of hybridizing under stringent conditions with a target antisense RNA sequence. “Stringent conditions” refers to 95% or more, preferably 96% or more, more preferably 97% or more, particularly preferably 98% or more, most preferably a base sequence that is completely complementary to the target nucleic acid sequence. Means a condition under which only a base sequence having 99% or more identity can hybridize. Those skilled in the art can appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Can be easily adjusted to the desired stringency.

  The nucleic acid for detecting antisense RNA of the present invention includes a nucleic acid probe that can specifically hybridize with the antisense RNA to be detected, and a pair that can function as a primer that amplifies part or all of the antisense RNA. Oligonucleotides (primers) and the like. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used.

The nucleic acid used as the probe may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid. The length of the nucleic acid is not particularly limited as long as it can specifically hybridize with the target antisense RNA, and is, for example, about 15 bases or more, preferably about 30 bases or more. The nucleic acid is preferably labeled with a labeling agent in order to enable detection or quantification of the target nucleic acid. As the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance or the like is used. As the radioisotope, for example, [ ³² P], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used. Furthermore, biotin- (strept) avidin can also be used for the binding between the probe and the labeling agent. On the other hand, when the nucleic acid to be a probe is immobilized on a solid phase, the nucleic acid in the sample can be labeled using the same labeling agent as described above.

  As a set of oligonucleotides used as primers, the sense strand and the antisense strand of double-stranded DNA synthesized by a method known per se from the antisense RNA containing the base sequence shown in SEQ ID NO: n are specific. There are no particular limitations as long as they can be hybridized and can amplify the DNA fragments sandwiched between them, for example, about 15 to about 100 bases each, preferably about 15 to about 50 bases in length. And a set of oligo DNAs designed to amplify DNA fragments of about 100 bp to several kbp.

  The nucleic acid functioning as a probe capable of detecting the antisense RNA of the present invention uses a primer set capable of amplifying a part or all of the antisense RNA, and uses any mammalian cell (for example, hepatocyte, spleen cell, Nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (Eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts , Osteoclasts, mammary cells, hepatocytes or stromal cells, or precursor cells of these cells, stem cells, cancer cells, etc.) or any tissue in which those cells are present (eg, brain, brain regions) (Eg, olfactory bulb, amygdaloid nucleus, basal cerebral sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, Skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, blood (eg, peripheral blood, umbilical cord blood, etc.), prostate, testis, ovary, placenta, uterus, bone, It can be obtained by amplifying a nucleic acid having a desired length using RT-PCR method using total RNA derived from joints, adipose tissue, skeletal muscle, etc.) as a template. Alternatively, the nucleic acid is based on each base sequence information of a sense RNA (cDNA) corresponding to the antisense RNA of the present invention (for example, in the case of human MAPK14, each is registered as Accession No. NM_001315 in the GenBank database). It can also be obtained by chemically synthesizing part or all of the base sequence and / or its complementary strand sequence using a commercially available DNA / RNA automatic synthesizer or the like. In addition, a chip (array) in which the nucleic acid is solid-phased can be produced by directly synthesizing the nucleic acid in situ (on chip) on a solid phase such as silicon or glass.

These nucleic acids can be provided as a solid in a dry state or in an alcohol-precipitated state, or can be provided in a state dissolved in water or a suitable buffer (eg, TE buffer). When used as a labeled probe, the nucleic acid can be provided in a state of being previously labeled with any of the above-mentioned labeling substances, or can be provided separately from the labeling substance and can be used after labeling. Alternatively, the nucleic acid can be provided in a state immobilized on an appropriate solid phase. Examples of the solid phase include, but are not limited to, glass, silicon, plastic, nitrocellulose, nylon, polyvinylidene difluoride, and the like. As immobilization means, a functional group such as an amino group, an aldehyde group, an SH group, or biotin is introduced into a nucleic acid in advance, and a functional group that can react with the nucleic acid on a solid phase (eg, aldehyde) Group, amino group, SH group, streptavidin, etc.), and the solid phase and nucleic acid are cross-linked by covalent bond between both functional groups, or the solid phase is polycation-coated for polyanionic nucleic acid. Examples of the method include immobilization of nucleic acid using electric coupling, but are not limited thereto.
A preferred example of the nucleic acid probe provided in a state immobilized on a solid phase is a DNA microarray. DNA microarray fabrication methods include Affymetrix method, which synthesizes nucleic acid probes one by one on a substrate (glass, silicon, etc.) using photolithography method, micro spotting method, inkjet method, bubble jet (registered trademark) method, etc. And a Stanford method in which a nucleic acid probe prepared in advance is spotted on a substrate. When using a probe of 30 mer or more, it is preferable to use the Stanford method or a combination of both methods.

  The present invention also includes a test for diagnosing colorectal cancer, comprising measuring the amount of the antisense RNA in an RNA-containing sample collected from a test mammal using a nucleic acid capable of detecting the antisense RNA of the present invention. Provide a method.

  Blood is adopted as the RNA-containing sample collected from the test mammal. Although it will not be restrict | limited especially if it is blood, Peripheral blood is preferably employ | adopted from points, such as being able to extract | collect rapidly and simply and being less invasive to the animal to extract | collect. There is no restriction | limiting in particular as a collection method of peripheral blood, It can extract | collect by a conventional method, For example, venous blood collection and arterial blood collection are mentioned. Moreover, there is no restriction | limiting in particular as a site | part from which peripheral blood is extract | collected, Peripheral blood extract | collected from any part of the body can be utilized suitably. In addition, the RNA-containing sample in the present invention includes plasma, serum, or cells constituting blood, and examples of the cells include red blood cells, white blood cells, and platelets. The leukocytes include neutrophils, eosinophils, basophils, lymphocytes, monocytes and the like.

  The expression of antisense RNA in a blood sample collected from a test mammal can be examined by preparing a total RNA fraction from the sample and detecting the antisense RNA contained in the fraction. The RNA fraction can be prepared using a known method such as guanidine-CsCl ultracentrifugation or AGPC, but a commercially available RNA extraction kit (eg, RNeasy Mini Kit, PAXgene Blood RNA Kit; manufactured by QIAGEN) Etc.) can be used to quickly and easily prepare high-purity total RNA from a small amount of sample. As a means for detecting gene transcripts in the RNA fraction, for example, a method using hybridization (Northern blot, dot blot, DNA chip (microarray) analysis, etc.) or PCR (RT-PCR, competitive RT-PCR, And a method using real-time RT-PCR).

  In the case of Northern blot or dot blot hybridization, detection of antisense RNA expression can be performed using the diagnostic reagent of the present invention containing a nucleic acid used as a labeled probe. That is, in the case of Northern hybridization, the RNA fraction prepared as described above is separated by gel electrophoresis, then transferred to a membrane such as nitrocellulose, nylon, polyvinylidene difluoride, etc., and the reagent of the present invention The amount of expression of each gene is determined by measuring the amount of label bound to the membrane by an appropriate method after hybridization in a hybridization buffer containing, preferably “under stringent conditions”. Can be measured. In the case of dot blots, the membrane on which the RNA fraction is spotted is similarly subjected to a hybridization reaction (respectively for each antisense RNA), and the amount of expression of each antisense RNA is determined by measuring the amount of label on the spot. Can be measured.

  In the case of DNA chip (microarray) analysis, for example, from the RNA fraction prepared as described above, a cDNA in which an appropriate promoter such as T7 promoter is introduced is synthesized by reverse transcription reaction, and further, cRNA is synthesized using RNA polymerase. (At this time, a labeled cRNA can be obtained by using a mononucleotide labeled with biotin or the like as a substrate). The amount of expression of each antisense RNA can be measured by bringing this labeled cRNA into contact with the above-mentioned immobilized probe, causing a hybridization reaction, and measuring the amount of label bound to each probe on the solid phase. DNA chip (microarray) analysis is preferable in that expression fluctuations of a plurality of marker genes can be collectively detected, and quantitativeness can be improved by selecting a detection method.

When quantitatively analyzing the expression of the antisense RNA of the present invention using a small amount of RNA sample, it is preferable to use competitive RT-PCR or real-time RT-PCR. Competitive RT-PCR is a method in which a known amount of another template nucleic acid that can be amplified by a set of primers that can amplify the target DNA is used as a competitor in the reaction solution to cause an amplification reaction competitively. A method for calculating the amount of target DNA by comparing the amounts. Therefore, when using competitive RT-PCR, the reagent of the present invention is amplified by the primer set in addition to the primer set, and can be distinguished from the target DNA (for example, the size of the target DNA is different from that of the target DNA). Nucleic acids that produce different amplification products, amplification products exhibiting different migration patterns by restriction enzyme treatment, and the like can be further contained. The competitor nucleic acid may be DNA or RNA. In the case of DNA, PCR may be performed by adding a competitor after synthesizing cDNA from an RNA sample by a reverse transcription reaction. In the case of RNA, RT-PCR can be performed by adding it to the RNA sample from the beginning. In the latter case, the efficiency of the reverse transcription reaction is taken into consideration, so that the absolute amount of the original mRNA can be estimated.
On the other hand, since real-time RT-PCR can monitor the amount of PCR amplification in real time, electrophoresis is unnecessary, and the expression of the antisense RNA of the present invention can be analyzed more quickly. Usually, monitoring is performed using various fluorescent reagents. Among these, in addition to reagents (intercalators) that emit fluorescence by binding to double-stranded DNA such as SYBR Green I and ethidium bromide, nucleic acids that can be used as the above probes (however, the nucleic acids are amplification regions) In which both ends of the nucleic acid are hybridized with a fluorescent substance (eg, FAM, HEX, TET, FITC, etc.) and a quencher (eg, TAMRA, DABCYL, etc.).

  In the method of the present invention, whether or not the test mammal has developed colorectal cancer (or is likely to develop in the future) is determined by, for example, the present method in an RNA-containing sample collected from a healthy control mammal. The expression level of the antisense RNA of the invention can be measured in the same manner, and the expression of the antisense RNA can be compared between the test animal and the control animal. As a result, when the expression of the antisense RNA is significantly fluctuated (increased or decreased) between the test animal and the control animal, the test animal develops colon cancer (or may develop in the future). It is possible to determine that the

  EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the scope of the present invention.

[Example 1] Production of microarray For transcription sequences of human genes, work was carried out to obtain probe polynucleotides (120 mer) from all genes. In addition, a gene having a transcript variant was designed so that the variant could be identified. Furthermore, a probe region was set so as not to have homology with each other even for a region having no possibility of transcription. Among the polynucleotides selected in this way (120 mer), a 60-mer polynucleotide was selected as an array probe and used as a sense probe. As the antisense probe, a sequence complementary to the sense probe was used. Various probes were mounted on the microarray using an Agilent system to produce a microarray chip.

[Example 2] RNA extraction and measurement of RIN (RNA Integrity Number) Targeting 17 patients with colorectal cancer and 6 healthy subjects before surgery, 5 mL of peripheral blood was collected after obtaining consent from each subject. did. Total RNA in whole blood was extracted from the collected blood using a QIAGEN RNA extraction kit (PAXgene Blood RNA Kit and PAXgene Blood RNA Tubes) according to the protocol described in the product manual. For the extracted RNA, RIN was measured using an Agilent 2100 bioanalyzer (manufactured by Agilent Technologies) in order to confirm whether degradation such as RNA degradation from the time of blood collection had occurred. RIN was measured according to the protocol specified for the instrument.

  As a result, all the test samples (RNA-containing samples) showed numerical values in the range of 6.70 to 9.20 as RIN. As a result, it was found that almost no degradation of RNA occurred in all test samples, and the quality of RNA was maintained at a high level, confirming that it could be used sufficiently for microarray experiments. It was.

[Example 3] Expression analysis of RNA-containing sample Using 5 µg of total RNA extracted in Example 2, a target cDNA labeled with a Cy3 fluorescent dye was prepared. The target cDNA was prepared using LabelStar Array kit (QIAGEN) and Cy3 fluorescent dye (GE Healthcare) according to the protocol described in the product manual. The obtained target cDNA was hybridized to the microarray chip prepared in Example 1, and the fluorescence intensity was measured using an Agilent DNA microarray scanner (manufactured by Agilent Technologies). Hybridization and washing and measurement of fluorescence intensity were performed according to protocols recommended by Agilent Technologies. About various measured values, data was corrected by Feature Extraction software (made by Agilent Technologies), and the signal value (processed signal) was computed.

  Based on the above data, we examined the expression level of sense RNA and antisense RNA in the test sample, compared between colorectal cancer patients and healthy individuals, and identified RNA that showed statistically significant differences did. As a result, when the condition of statistical significance was p <0.05 (T-test) and Fold change> 2.0, 121 sense RNAs and 197 antisense RNAs were identified. Among them, the number of patients with colorectal cancer that showed higher expression levels than that of healthy individuals was 57 for sense RNA and 162 for antisense RNA. In order to further improve the accuracy in comparison between colorectal cancer patients and healthy individuals, the conditions for statistical significance were p <0.01 (T-test) and Fold change> 2.0. Antisense RNAs have been identified. Of these, 27 sense RNAs and 111 antisense RNAs were expressed in colorectal cancer patients more than healthy individuals. To further improve the accuracy, statistical sense difference conditions were set to p <0.001 (T-test) and Fold change> 2.0, and 12 sense RNAs and 30 antisense RNAs were identified. It was. Similarly, when RNAs of colon cancer patients showed higher expression levels than healthy individuals, the number of sense RNAs was 7, and the number of antisense RNAs was 24.

[Example 4] Clustering analysis Clustering analysis was performed on the 12 sense RNAs and 30 antisense RNAs identified in Example 3 under the most accurate conditions using Gene Spring GX10 software (Agilent Technologies). Went. As a result, as shown in FIG. 5, 12 sense RNAs could not be classified into clusters between colorectal cancer patients and healthy persons based on the color distribution. In contrast, the 30 antisense RNAs had a clear color distribution as shown in FIG. 6, and could be classified into clusters. From the above results, it was considered that these 30 antisense RNAs in blood can be used for diagnosis or examination of colorectal cancer and may be a biomarker of a novel colorectal cancer diagnostic method.

  According to the present invention, it is possible to provide an endogenous antisense RNA whose expression level is significantly different in colorectal cancer patients compared to healthy individuals. This makes it possible to provide a colorectal cancer marker useful in diagnosis or examination. Further, in the present invention, blood that is easy to handle in diagnosis or examination is adopted as an RNA-containing sample, so that a practical method for diagnosing or examining colorectal cancer in a clinical setting can be provided.

Claims (3)

A test method for diagnosing colorectal cancer, comprising measuring endogenous antisense RNA selected from the following (1) to (30) in blood collected from a mammal.
(1) Endogenous antisense RNA for mammalian MAPK14 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 1
(2) Endogenous antisense RNA for mammalian RNF175 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 2.
(3) Endogenous antisense RNA for mammalian B4GALT5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 3
(4) Endogenous antisense RNA for mammalian LRRK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 4
(5) An endogenous antisense RNA against the mammalian LOC731208 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 5
(6) Endogenous antisense RNA for mammalian PSMF1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 6
(7) Endogenous antisense RNA for mammalian USP25 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 7
(8) Endogenous antisense RNA for mammalian RAB10 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 8
(9) Endogenous antisense RNA for mammalian LIN7A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 9
(10) Endogenous antisense RNA for mammalian PFN2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 10
(11) Endogenous antisense RNA for mammalian CREB5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 11
(12) Endogenous antisense RNA for mammalian DSC2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 12
(13) An endogenous antisense RNA against the mammalian LOC432369 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 13
(14) Endogenous antisense RNA for mammalian ANXA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 14
(15) An endogenous antisense RNA against the mammalian CENTB2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 15
(16) Endogenous antisense RNA for mammalian PFDN5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 16
(17) An endogenous antisense RNA against the mammalian KIAA0241 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 17
(18) Endogenous antisense RNA for mammalian HINT1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 18
(19) Endogenous antisense RNA for mammalian AYTL1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 19
(20) Endogenous antisense RNA for mammalian BAZ1A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 20
(21) Endogenous antisense RNA for mammalian PROK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 21
(22) Endogenous antisense RNA for mammalian TMEM80 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 22
(23) An endogenous antisense RNA against a mammalian C9orf102 gene, which comprises the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 23
(24) Endogenous antisense RNA for mammalian SETD6 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 24
(25) An endogenous antisense RNA against a mammalian LANCL1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 25
(26) Endogenous antisense RNA for mammalian WNT2B gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 26
(27) Endogenous antisense RNA for mammalian PNPLA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 27
(28) An endogenous antisense RNA against a mammalian ZNF533 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 28
(29) An endogenous antisense RNA against a mammalian ZBED4 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 29
(30) An endogenous antisense RNA against the mammalian LOC730966 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 30 An endogenous antisense RNA selected from the following (1) to (30).
(1) Endogenous antisense RNA for mammalian MAPK14 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 1
(2) Endogenous antisense RNA for mammalian RNF175 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 2.
(3) Endogenous antisense RNA for mammalian B4GALT5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 3
(4) Endogenous antisense RNA for mammalian LRRK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 4
(5) An endogenous antisense RNA against the mammalian LOC731208 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 5
(6) Endogenous antisense RNA for mammalian PSMF1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 6
(7) Endogenous antisense RNA for mammalian USP25 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 7
(8) Endogenous antisense RNA for mammalian RAB10 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 8
(9) Endogenous antisense RNA for mammalian LIN7A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 9
(10) Endogenous antisense RNA for mammalian PFN2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 10
(11) Endogenous antisense RNA for mammalian CREB5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 11
(12) Endogenous antisense RNA for mammalian DSC2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 12
(13) An endogenous antisense RNA against the mammalian LOC432369 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 13
(14) Endogenous antisense RNA for mammalian ANXA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 14
(15) An endogenous antisense RNA against the mammalian CENTB2 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 15
(16) Endogenous antisense RNA for mammalian PFDN5 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 16
(17) An endogenous antisense RNA against the mammalian KIAA0241 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 17
(18) Endogenous antisense RNA for mammalian HINT1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 18
(19) Endogenous antisense RNA for mammalian AYTL1 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 19
(20) Endogenous antisense RNA for mammalian BAZ1A gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 20
(21) Endogenous antisense RNA for mammalian PROK2 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 21
(22) Endogenous antisense RNA for mammalian TMEM80 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 22
(23) An endogenous antisense RNA against a mammalian C9orf102 gene, which comprises the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 23
(24) Endogenous antisense RNA for mammalian SETD6 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 24
(25) An endogenous antisense RNA against a mammalian LANCL1 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 25
(26) Endogenous antisense RNA for mammalian WNT2B gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 26
(27) Endogenous antisense RNA for mammalian PNPLA3 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 27
(28) An endogenous antisense RNA against a mammalian ZNF533 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 28
(29) An endogenous antisense RNA against a mammalian ZBED4 gene comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 29
(30) An endogenous antisense RNA against the mammalian LOC730966 gene, comprising the same or substantially the same nucleotide sequence as shown in SEQ ID NO: 30   A diagnostic agent for colorectal cancer comprising a nucleic acid comprising a base sequence capable of hybridizing under stringent conditions with the endogenous antisense RNA according to claim 2.