KR20170021580A - A biomarker for lung cancer - Google Patents

Abstract

The present invention relates to a biomarker for lung cancer and, more specifically, to a micro RNA biomarker for non-small cell lung cancer. According to the present invention, miRNA, which is distinguished and expressed from a lung cancer patient, can be used as a biomarker. Provided is a micro RNA marker for diagnosing lung cancer which is selected from the group consisting of hsa-miR-144-5p, hsa-miR-182-5p, hsa-miR-205-5p, and hsa-miR-891a-5p.

Description

A biomarker for lung cancer < RTI ID = 0.0 >

The present invention relates to a biomarker of lung cancer, and more particularly to a microRNA biomarker of non-small cell lung cancer.

Lung cancer, mainly non-small cell lung cancer (NSCLC), is the most common cause of cancer deaths worldwide. CA Cancer J. Clin., 56: 106-130 (2006) (Jamal et al.). Early stage NSCLC patients have a recurrence rate of 40% within 5 years after treatment. This suggests that the stage of the disease is a factor associated with clinical prognosis. Am. J. Respir. Cell Mol. Biol. 33: 216-223 (2005) (Miller). However, one of these factors is insufficient to predict the clinical prognosis.

Gene expression profiling, particularly the microRNA profiling technique, has been suggested to be useful in both cancer diagnosis and prognosis. J. Clin. Oncol., 22: 811-819 (2004) (Endoh et al.) And N. Engl. J. Med., 355: 570-580 (2006) (Potti et al.). For example, in determining the subtype of cancer, the expression pattern of a specific microRNA is known to be more accurate than the expression pattern of a protein-coding gene. Nat. Rev. Cancer, 6: 857-866 (2006) (Calin et al.) And Proc. Natl. Acad. Sci. U.S.A., 103: 2257-2261 (2006) (Volinia et al.).

MicroRNAs are small nonprotein-encoded RNAs that regulate a wide range of biological pathways, such as cell proliferation, differentiation and cell apoptosis, by controlling the expression of hundreds of genes through RNA interference in the post-transcriptional stage of the gene. See Calin et al. (2006). It has been reported that certain microRNA markers - i.e., one or more microRNAs - showing a particular pattern of expression in a patient group are associated with the clinical prognosis of chronic lymphocytic leukemia, lung adenocarcinoma, breast cancer, and pancreatic cancer. See Calin et al. (2006). Since these microRNA markers are effective in predicting the clinical prognosis of various cancers, particularly NSCLC, it is of great interest to find novel microRNA markers.

[Prior Patent Literature]

Korean Patent Publication No. 1020080099666

The present invention has been made in view of the above needs, and it is an object of the present invention to provide novel microRNA markers for diagnosis of lung cancer.

In order to accomplish the above object, the present invention provides a method of treating a lung cancer selected from the group consisting of hsa-miR-144-5p, hsa-miR-182-5p, hsa-miR-205-5p, Diagnostic microRNA markers.

In one embodiment of the present invention, the hsa-miR-144-5p is preferably composed of the nucleotide sequence shown in SEQ ID NO: 1,

In another embodiment of the present invention, the hsa-miR-182-5p is preferably composed of the nucleotide sequence of SEQ ID NO: 2,

In another embodiment of the present invention, the hsa-miR-205-5p is preferably composed of the nucleotide sequence shown in SEQ ID NO: 3,

In another embodiment of the present invention, the hsa-miR-891a-5p comprises the nucleotide sequence shown in SEQ ID NO: 4, but is not limited thereto.

In a preferred embodiment of the present invention, the lung cancer is preferably non-small cell lung cancer (NSCLC), but is not limited thereto.

The present invention also provides a composition for diagnosing lung cancer comprising the microRNA marker of the present invention as an active ingredient.

The present invention also provides a microarray for diagnosing lung cancer in which the microRNAs of the present invention are immobilized.

The present invention also provides a method for detecting the expression of hsa-miR-205-5p or hsa-miR-182-5p, which is a microRNA of a subject, And

And judging the lung cancer to be lung cancer when the expression amount of the subject is up-regulated based on the expression amount of the microRNAs.

The present invention also provides a method for detecting hsa-miR-144-5p, comprising the steps of: detecting the expression level of hsa-miR-144-5p, And

And judging the lung cancer to be lung cancer if the expression level of the subject is down-regulated based on the expression level of the microRNAs.

MicroRNAs, also called miRNAs or miRs, are short RNA strands that are approximately 21-23 nucleotides in length. MiRNAs are transcribed from DNA but not decoded into proteins, and thus encoded by genes containing non-coding RNAs.

miR is processed from a known primary transcript pri-miRNA into a short stem-loop structure termed pre-miRNA and finally processed into a single stranded miRNA that is finally produced. Pre-miRNAs typically form a self-folding structure within the self-complementary region. These structures are then processed by nuclease in the next animal or by DCL1 in plants. Mature miRNA molecules are partially complementary to one or more messenger RNA (mRNA) molecules and can function to regulate the transcription of proteins. Identified sequences of miRNAs can be evaluated in publicly available databases, such as www.microRNA.org, www.mirbase.org, or www.mirz.unibas.ch/cgi/miRNA.cgi.

miRNAs are generally numbered according to a naming convention such as "mir- [number]". The number of miRNAs is assigned according to the sequence found for miRNA species that have already been identified. For example, if the most recently revealed miRNA is mir-121, then the miRNA found will be named mir-122. If the miRNA is found to be similar to a known miRNA of a different organism, the name is provided in the form of [organism identifier] - mir- [number], the name of the selector organism identifier. The verifier includes hsa for Homo sapiens and mmu for Mus Musculus. For example, the human homolog at mir-121 will be named hsa-mir-121, the mouse homologue will be named mmu-mir-121, and the rat homologue will be named rno-mir-121.

Mature microRNAs are usually prefixed with "miR", and gene or precursor miRNAs are prefixed with "mir". For example, mir-121 is a precursor of miR-121. When different miRNA genes or precursors are processed with the same mature miRNA, this gene / precursor can be represented by a numbered suffix. For example, mir-121-1 and mir-121-2 may refer to distinct genes or precursors that are processed with miR-121. Character suffixes are used to represent closely related maturation sequences. For example, mir-121a and mir-121b may be processed with closely related miRNAs miR-121a and miR-121b, respectively. In the context of the present invention, any microRNA (miRNA or miR) with the prefix mir- * or miR- * should be understood to include both precursors and / or mature species unless explicitly stated otherwise.

In some cases, it is observed that both mature miRNA sequences originate from the same precursor. When one of the sequences is more common than the other, the "*" suffix is used to indicate a less common variant. For example, miR-121 is the predominant product, whereas miR-121 * represents a less common variant seen on the opposite side of the precursor. If dominant variants are not identified, miR can be distinguished by appending the suffix "5p" to the mutants from the 5 'branch of the precursor and the suffix "3p" to the mutants from the 3' branch. For example, miR-121-5p originated from the 5 'branch of the precursor, while miR-121-3p originated from the 3' branch. Although not common, 5p and 3p variants can be referred to as sense ("s") and antisense ("as"). For example, miR-121-5p may be referred to as miR-121-s, whereas miR-121-3p may be referred to as miR-121-as.

The naming convention has evolved over time and is a general guide rather than an absolute rule. For example, the let- and lin- families of miRNAs are constantly being referred to as generic names. The mir / miR for precursor / mature types should also be considered as guidelines and content to determine what form to call. For more information on miR nomenclature, see www.mirbase.org or Ambros et al., A uniform system for microRNA annotation, RNA 9: 277-279 (2003).

Many miRNAs are involved in gene regulation, and miRNAs are part of a growing class of non-coding RNAs that are currently recognized as a key step in gene regulation. In some cases, miRNAs can interfere with detoxification by inducing inhibition of detoxification by binding to regulatory sites embedded in 3-UTRs of their target mRNA. Target cognition involves the complementary base pairing of the target site with the miRNA's seed region (positions 2-8 at the 5'-end of the miRNA), the exact level of seed complementarity is not precisely determined, . join. In other cases, miRNAs function similarly to small interfering RNAs (siRNAs) and bind to perfectly complementary mRNA sequences to destroy the target transcript.

The characteristics of multiple miRNAs indicate that they affect various processes including early development, cell proliferation and apoptosis, apoptosis (self-destruction) and fat metabolism. For example, some miRNAs such as lin-4, let-7, mir-14, mir-23, and bantam have been shown to play a major role in cell differentiation and tissue development. Others are assumed to have a similar important role due to their differential spatial and temporal pattern of expression.

Hereinafter, the present invention will be described in detail.

Clinical characteristics

Nine non-small cell lung cancer patients have the following clinical characteristics.

NSCLC patients (n = 9) Age, year (mean ± SD)   5 9 (45 ~ 68) Male, n (%)      9 (100) Smoking, (mean ± SD) ^a    33 ± 15.6 Histological cell type, n (%) Squamous cell carcinoma   5 (55.6%)   Adenocarcinoma  4 (44.4%) TNM stage, n (%)       I  4 (44.4%)       II 5 (55.6%)       III 0 (0)

Table 1 shows the clinical characteristics of patients with non-small cell lung cancer

Expressed miRNAs  And mRNA  Profiling

| fold change | (Table 2). Among the miRNAs, miR-205-5p and miR-196a-5p were elevated and miR-133a and 5p were elevated. miR-139-5p was down.

miRNA fold change (LC / NOR) p value up-regulated hsa-miR-205-5p 18.8595 0.0306 hsa-miR-196a-5p 11.0968 0.0102 hsa-miR-1246 9.2112 0.0111 hsa-miR-577 8.4114 0.0006 hsa-miR-301b 7.6426 0.0004 hsa-miR-182-5p 7.4388 0.0015 hsa-miR-196b-5p 7.2126 0.0280 down-regulated hsa-miR-133a -7.8252 0.0004 hsa-miR-139-5p -7.1165 0.0002 hsa-miR-144-5p -6.5923 1.41E-06 hsa-miR-338-3p -6.5742 0.0002 hsa-miR-1 -6.1193 0.0002 hsa-miR-490-3p -6.1072 0.0001 hsa-miR-30a-3p -5.5812 0.0001

Table 2 shows the top seven up-regulated or downregulated microRNAs in NSCLC

A list of microRNAs in Table 2 was obtained in lung cancer tissues, and microRNAs that differed significantly in the blood of another lung cancer patient for validation were described in the claims of the present invention.

miRNA - mRNA  integrative genome analysis

MRNA pairs were found that negatively correlated with significantly elevated or depleted miRNAs. 49 putative putative negative regulatory miRNA-mRNA pairs were identified (see Table 3 and Table 4). Of these, 19 miRNAs were upregulated and 425 mRNAs were downregulated. Conversely, 30 miRNAs were downregulated and 425 mRNAs were upregulated.

miRNA miR.FC (LC / NOR) miRNA_rawP P value on Hypergeometric test hsa-miR-577 8.411 0.0006 0.0461 hsa-miR-301b 7.643 0.0004 0.0360 hsa-miR-944 5.765 0.0422 0.0416 hsa-miR-891a 5.683 0.0229 0.0020 hsa-miR-615-3p 5.211 0.0064 0.0299 hsa-miR-671-5p 2.606 0.0119 3.83E-06 hsa-miR-429 2.599 0.0429 0.0007 hsa-miR-210 2.246 0.0334 9.27E-06 hsa-miR-137 2.002 0.0412 0.0143 hsa-let-7c -2.044 0.0105 0.0046 hsa-miR-338-3p -6.574 0.0002 0.0442

Table 3 shows that miRNAs having a putative target gene

Table 4 shows the expression of miRNAs differentially expressed in NSCLC and their predicted target gene

Physical interaction analysis

2 is a cross-

As can be seen from the present invention, the present invention extracts miRNA and mRNA differentially expressed from lung cancer tissues and normal tissues using a next generation sequencing method in a patient who has undergone surgery with non-small cell lung cancer, pair and can be used as a biomarker by obtaining differentially expressed miRNA and target gene data.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.
FIG. 2 is a diagram showing Enrichment analysis for GO terms in the domain of biological processes,
Figure 3 shows seven miRNAs with a significant negative correlation with the cell cycle related genes

The present invention will now be described in more detail by way of non-limiting examples. The following examples are intended to illustrate the invention and the scope of the invention is not to be construed as being limited by the following examples.

In the present invention, normal lung tissues of 9 patients who were operated on from non-small cell lung cancer between March 2008 and March 2011 were distributed in pairs, and the mean age of patients was 59 years. All were men. 5 were squamous cell carcinoma and 4 were adenocarcinoma. The clinical characteristics of the patients were described in Table 1 above.

Example  1: RNA Extraction and RNA Sequencing

Lung cancer tissues and normal lung tissues were categorized from cryopreserved lung tissues by pathologists. RNA was extracted from these tissues using the RNeasy 96 Universal Tissue Kit (Qiagen, Gaithersburg, USA) and analyzed using a NanoCrop 1000 spectrometer (Thermo Scientific, Wilmington, DE, USA) and Bioanalyzer 2100 (Agilent Technologies, Palo Alto CA, USA) And RNA was identified using this method. RNA-Seq reads were mapped using TopHat (version 1.3.3) and determined with Cufflinks software (version 1.2.1). The reference genome sequence was downloaded from the UCSC website ( http://genome.uscs.edu), and the RNA transcript was measured by FPKM (Fragments Per Kilobase of exon per Million fragments mapped). By comparing fold expression of gene expression with the FPKM extracted, we found differentially expressed genes (DEGs) in cancer tissues and normal lung tissues of non-small cell lung cancer patients. All data were analyzed using R 2.15.1 ( www.r-project.org) and FDR (false discovery rate) was corrected for p value using the Benjamini-Hochberg algorithm.

Example  2: miRNA  analysis

Immediately after surgery, mRNA and miRNAs were obtained from frozen tissues in a liquid nitrogen tank at -80 ° C.

Example  3: integrative analysis

1) A probe list showing differential expression was extracted from miRNA data set and mRNA data set, respectively.

: Significant cut-off: | fold change | ≥ 1.5 & raw.p <0.05

2) We extracted candidates that would have a real regulatory relationship among mRNA and miRNA list with differential expression. The putative microRNA - mRNA target pair was derived using miRBase Targets.

3) Negative correlation was confirmed and the final microRNA-mRNA regulatory association list was derived. A summary of the data analysis procedure is shown in FIG.

<110> KNU-Industry Cooperation Foundation <120> A biomarker for lung cancer <130> HY150857 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 22 <212> DNA <213> Homo sapiens <400> 1 ggatatcatc atatactgta ag 22 <210> 2 <211> 24 <212> DNA <213> Homo sapiens <400> 2 tttggcaatg gtagaactca cact 24 <210> 3 <211> 22 <212> DNA <213> Homo sapiens <400> 3 tccttcattc caccggagtc tg 22 <210> 4 <211> 79 <212> DNA <213> Homo sapiens <400> 4 ccttaatcct tgcaacgaac ctgagccact gattcagtaa aatactcagt ggcacatgtt 60 tgttgtgagg gtcaaaaga 79

Claims (15)

a lung cancer diagnostic microRNA marker selected from the group consisting of hsa-miR-144-5p, hsa-miR-182-5p, hsa-miR-205-5p, and hsa-miR-891a-5p. The microRNA marker for lung cancer diagnosis according to claim 1, wherein the hsa-miR-144-5p comprises the nucleotide sequence as set forth in SEQ ID NO: The microRNA marker for lung cancer diagnosis according to claim 1, wherein the hsa-miR-182-5p comprises the nucleotide sequence of SEQ ID NO: 2. 2. The microRNA marker for lung cancer diagnosis according to claim 1, wherein the hsa-miR-205-5p comprises the nucleotide sequence as set forth in SEQ ID NO: 3. The microRNA marker for lung cancer diagnosis according to claim 1, wherein the hsa-miR-891a-5p comprises the nucleotide sequence as set forth in SEQ ID NO: 4. The microRNA marker for lung cancer diagnosis according to claim 1, wherein the lung cancer is non-small cell lung cancer (NSCLC). A composition for diagnosing lung cancer comprising the microRNA marker of any one of claims 1 to 5 as an active ingredient. 8. The composition for diagnosing lung cancer according to claim 7, wherein the lung cancer is non-small cell lung cancer (NSCLC). A microarray for diagnosing lung cancer, wherein the microRNA is immobilized according to any one of claims 1 to 5. 10. The microarray for diagnosing lung cancer according to claim 9, wherein the lung cancer is non-small cell lung cancer (NSCLC). Detecting the expression levels of hsa-miR-205-5p, or hsa-miR-182-5p, microRNAs of the subject; And
Determining whether the amount of expression of the micro-RNA is lung cancer when the amount of expression of the micro-RNA is up-regulated relative to the normal amount. 12. The method according to claim 11, wherein the hsa-miR-182-5p comprises the nucleotide sequence of SEQ ID NO: 2. 12. The method according to claim 11, wherein the hsa-miR-205-5p comprises the nucleotide sequence of SEQ ID NO: 3. Detecting the expression level of hsa-miR-144-5p, which is a microRNA of the subject; And
And judging the lung cancer to be lung cancer when the expression level of the subject is down-regulated based on the expression level of the microRNAs. 15. The method according to claim 14, wherein the hsa-miR-144-5p comprises the nucleotide sequence of SEQ ID NO: 1.

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