KR102293777B1 - A Novel UQCRB-related Circulating miRNA Biomarker and a Method for Diagnosing Colorectal Cancer Using the Same - Google Patents

Abstract

The present invention relates to a composition for diagnosis of gastrointestinal cancer, a composition for preventing or treating gastrointestinal cancer, and a screening method thereof. MiR-4435 discovered in the present invention can be usefully used as an excellent circulating biomarker that can simply collect accurate information on the onset and progression stage of gastrointestinal cancer, specifically UQCRB gene mutation-related colorectal cancer, only by analyzing body fluids. can In addition, the MiR-4435 inhibitor of the present invention can be applied as an effective anticancer composition by activating TIMP3, a tumor suppressor gene.

Description

A Novel UQCRB-related Circulating miRNA Biomarker and a Method for Diagnosing Colorectal Cancer Using the Same}

The present invention is based on the new discovery that miR-4435, a UQCRB-related circulating biomarker, is highly expressed in colorectal cancer and suppresses the formation and progression of colorectal cancer through its inhibition, measuring the expression level of miR-4435 and suppressing its expression It relates to a method for diagnosing and treating colorectal cancer, respectively.

Mitochondria play an important function in converting energy to produce ATP and contribute to various biosynthetic processes. The mitochondrial electron transport complex (ETC) generates an electrochemical proton gradient to produce energy by ATP synthesis ¹ . Mitochondrial dysfunction causes many mitochondrial-related diseases and is associated with various diseases such as metabolic diseases and cancer ^1-5 .

Ubiquinol-cytochrome c reductase binding protein (UQCRB) is located in the mitochondrial complex III and plays an important role in electron transport and complex III maintenance ⁶ . In addition, UQCRB is involved in the generation of mitochondrial reactive oxygen species (mROS) and hypoxia-inducing factor (HIF)-mediated angiogenesis, and regulates angiogenesis induced by signals of vascular endothelial growth factor receptor 2 (VEGFR2). ^7-8 . UQCRB is also known as a target protein of terpestacin, a natural anti-angiogenic compound, and in particular, it has been reported that terpestacin binds to UQCRB and has anti-angiogenic activity ⁹ . Furthermore, several studies have revealed that UQCRB plays a role in cancer pathogenesis by identifying genetic changes in UQCRB ^{in hepatocellular carcinoma 10} , ovarian cancer ¹¹ , pancreatic adenocarcinoma ¹² and colorectal cancer (CRC) ^{13 .}

Mutations in UQCRB can lead to mitochondrial defects and related diseases. For example, women with deletions in the UQCRB gene show hypoglycemia and lactic acidosis during the metabolic crisis in infancy ¹⁴ . Based on this, the present inventors have prepared two mutant UQCRB-expressing cell lines, MT1 and MT2. MT1 shows higher UQCRB mutation expression than MT2. We investigated the biological function of UQCRB in angiogenesis using these stable cell lines. The mutant UQCRB-expressing cell line showed greatly increased cell growth and angiogenic activity. In addition, the mitochondria of the mutant UQCRB-expressing cell line had an abnormal morphology and were more sensitive to UQCRB inhibitors ¹⁵ .

MicroRNAs (miRNAs) are small non-coding RNA molecules with a length of 21-23 nucleotides. The mature miRNA binds to the complementary region of the 3'UTR (untranslated region) of the target mRNA, causing mRNA silencing. Transcriptional regulation of ^{miRNA has been reported 16-17} , and miRNA can act as both a tumor suppressor and an oncogene ¹⁸ . Therefore, miRNAs are very important in the regulation of numerous physiological and pathological processes ^19-20 . The present inventors have recently reported that hsa-miR-10a-5p is associated with UQCRB. Reduction of miR-10a-5p in mutant UQCRB-expressing cells activates the cholesterol pathway by targeting cholesterol-synthesizing enzymes, suggesting the possibility that UQCRB-associated miRNAs may have a role in cancer cell proliferation ²¹ . Moreover, these miRNAs are stable in serum and plasma and their expression levels vary depending on the state of a disease such as cancer ²² . Recently, many studies have suggested that the miRNA expression profile can be used as a biomarker for various diseases. Recently, it has been reported that serum miR-155 is highly expressed in CRC patients compared to normal people, so miR-155 can be used as a CRC biomarker. ²³ was proposed. In addition, Lv, ZC et al. reported that four miRNAs were associated with overall survival in non-small cell lung cancer (NSCLC) ²⁴ . However, the mechanisms regulating these miRNAs in disease remain unclear.

CRC is the third most common cancer worldwide and the leading cause of death. Until recently, surgical resection was mainly performed in the early stages of cancer, but it is often not diagnosed until the disease has progressed significantly or even distant metastasis ²⁵ . Recently, the relationship between UQCRB and CRC has been studied, and it has been found that UQCRB genes and proteins are significantly higher expressed in colorectal cancer patients compared to adjacent non-tumor tissues ²⁶ . This suggests the possibility that UQCRB may be involved in tumorigenesis in CRC and may also be used as a diagnostic biomarker. The present inventors sought to identify novel CRC-related biomarkers by examining miRNA expression in mutant UQCRB-expressing cell lines. In addition, as a result of miRNA analysis on human CRC patient serum samples to investigate the clinical significance of UQCRB, miR-4435 was identified as a circulating miRNA biomarker for UQCRB-related cancers in CRC.

Numerous papers and patent documents are referenced throughout this specification and their citations are indicated. The disclosure contents of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly describe the level of the technical field to which the present invention pertains and the content of the present invention.

Non-Patent Document 1. JE Kim et al., Hsa-miR-10a-5p downregulation in mutant UQCRB-expressing cells promotes the cholesterol biosynthesis pathway Scientific Reports (8): 12407 (2018)

The present inventors strive to discover circulating biomarkers that can be measured simply by examination of body fluids without tissue samples while providing comprehensive and reliable information for the diagnosis of gastrointestinal cancer, specifically gastric cancer or colorectal cancer. did. As a result, the three miRNAs are not only specifically expressed in gastrointestinal cancer, but are also highly expressed as the cancer progresses to a later stage, providing accurate information on whether or not cancer occurs and the stage of progression, and secreted via exosomes. By discovering that diagnosis is possible only by simple analysis of a sample, the present invention has been completed.

Accordingly, an object of the present invention is to provide a composition for diagnosis of gastrointestinal cancer.

Another object of the present invention is to provide a composition for preventing or treating gastrointestinal cancer.

Another object of the present invention is to provide a method for screening a composition for preventing or treating gastrointestinal cancer.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention relates to gastrointestinal cancer comprising, as an active ingredient, an agent for measuring the expression level of one or more miRNAs selected from the group consisting of miR-4435, miR-21-3p and miR-1226-3p. Provided is a composition for diagnosis of

The present inventors strive to discover circulating biomarkers that can be measured simply by examination of body fluids without tissue samples while providing comprehensive and reliable information for the diagnosis of gastrointestinal cancer, specifically gastric cancer or colorectal cancer. did. As a result, three miRNAs of miR-4435 (SEQ ID NO: 1), miR-21-3p (SEQ ID NO: 2) and miR-1226-3p (SEQ ID NO: 3) were specifically found in gastrointestinal cancer. Not only is it expressed, but it is also highly expressed as the cancer progresses to a later stage, providing accurate information on the onset of cancer and the stage of its progression. found that it can.

As used herein, the term “diagnosis” includes determination of an individual's susceptibility to a specific disease, determination of whether an individual currently has a specific disease, and determination of the prognosis of an individual with a specific disease. do. Since the expression level of the miRNA of the present invention is a genetic marker for the risk of onset as well as the current onset, by measuring it, it is determined whether gastrointestinal cancer has occurred in the current individual and the current individual has not suffered from gastrointestinal cancer, but it is likely to develop in the future. Provides information for the determination of prognosis related to likelihood. Accordingly, the term “diagnosis of gastrointestinal cancer” may be expressed as “prediction of the risk of developing gastrointestinal cancer”.

As used herein, the term “composition for diagnosis of gastrointestinal cancer” refers to an integrated mixture or device including an expression level measuring means such as miR-4435 to determine whether a subject develops gastrointestinal cancer, Accordingly, it may be expressed as “a kit for diagnosing gastrointestinal cancer”.

According to a specific embodiment of the present invention, the miRNA is miR-4435.

According to a specific embodiment of the present invention, the gastrointestinal cancer diagnosed by the composition of the present invention is gastric cancer or colorectal cancer, and more specifically, colorectal cancer having a mutation in the UQCRB gene.

According to a specific embodiment of the present invention, the colorectal cancer diagnosed with the composition of the present invention is stage 3 or 4 colorectal cancer. According to the present invention, miR-4435, a circulating biomarker of the present invention, is specifically and highly expressed in colorectal cancer, and as a result of analysis of serum samples from patients of various ages in CRC stages 1 to 4, as the CRC stage progresses to a later stage, the , in particular, it was confirmed that the expression was further increased in stage 3 and 4 patients (see Table 1 and Figure 2e). Therefore, miR-4435 of the present invention can provide information on the progress of colorectal cancer as well as the onset of colorectal cancer.

According to a specific embodiment of the present invention, the agent for measuring the expression level of the miRNA is a nucleic acid molecule that specifically binds to the miRNA.

As used herein, the term “nucleic acid molecule” has a meaning encompassing DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include natural nucleotides as well as analogs ( analogues) (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90:543-584 (1990)). “A nucleic acid molecule that specifically binds to miRNA” refers to a nucleic acid molecule that has a nucleotide sequence substantially complementary to the miRNA of the present invention and can specifically hybridize thereto, and includes, for example, primers and probes.

As used herein, the term “primer” refers to a condition in which the synthesis of a primer extension product complementary to a nucleic acid strand (template) is induced, that is, the presence of a polymerization agent such as nucleotides and DNA polymerase, and a suitable temperature and pH. It refers to an oligonucleotide that acts as a starting point. Specifically, the primer is a single-stranded deoxyribonucleotide. The primer used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP, and dTMP), modified nucleotides or non-natural nucleotides, and may also include ribonucleotides.

The primer of the present invention may be an extension primer that is annealed to a target nucleic acid to form a sequence complementary to the target nucleic acid by a template-dependent nucleic acid polymerase, which is extended to the position where the immobilized probe is annealed so that the probe is It occupies the annealed area.

The extension primer used in the present invention includes a hybridization nucleotide sequence complementary to a specific nucleotide sequence of a target nucleic acid, for example, miR-4435. The term “complementary” means that a primer or probe is sufficiently complementary to selectively hybridize to a target nucleic acid sequence under certain annealing or hybridization conditions, and when substantially complementary and perfectly complementary ), and specifically means a completely complementary case. As used herein, the term “substantially complementary sequence” includes not only a completely identical sequence, but also a sequence that is partially mismatched with a sequence to be compared within the range that can function as a primer by annealing to a specific sequence.

The primer should be long enough to prime the synthesis of extension products in the presence of a polymerizer. A suitable length of a primer depends on a number of factors, such as temperature, pH and source of the primer, but is typically 15-30 nucleotides. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template. The design of such a primer can be easily performed by those skilled in the art with reference to the target nucleotide sequence, for example, using a primer design program (eg, PRIMER 3 program).

As used herein, the term “probe” refers to a natural or modified monomer or a linear oligomer including deoxyribonucleotides and ribonucleotides capable of hybridizing to a specific nucleotide sequence. Specifically, the probe is single-stranded for maximum efficiency in hybridization, more specifically deoxyribonucleotides. As the probe used in the present invention, a sequence that is perfectly complementary to the specific nucleotide sequence of miR-4435 may be used, but a sequence that is substantially complementary within a range that does not interfere with specific hybridization is may be used. In general, since the stability of the duplex formed by hybridization tends to be determined by the match of the sequences at the ends, it is preferable to use a probe complementary to the 3'-end or 5'-end of the target sequence. do.

Suitable conditions for hybridization are Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory Press, NY (2001) and Haymes, BD, et al., Nucleic Acid Hybridization, A Practical Approach , IRL Press, Washington , can be determined with reference to the details disclosed in DC (1985).

According to a specific embodiment of the present invention, the diagnostic composition of the present invention is a body fluid diagnostic composition. The body fluid is urine, saliva, semen, tears, amniotic fluid, cerebrospinal fluid, synovial fluid, pericardial fluid, ascites (Peritoneal Fluid), blood, plasma and one or more bodily fluids selected from the group consisting of serum. Most specifically, the bodily fluid is blood, plasma or serum.

According to the present invention, the miR-4435 of the present invention circulates in the blood via exosomes secreted from cancer cells, thereby enabling diagnosis of cancer by measuring the concentration in the blood. Through this, the present invention achieves both the convenience of the patient and the simplicity and economy of the procedure by providing reliable clinical information without the need for biopsy.

According to another aspect of the present invention, the present invention provides a composition for preventing or treating gastrointestinal cancer comprising an inhibitor of miR-4435 expression as an active ingredient.

In the present invention, since gastrointestinal cancer, which is a target for diagnosis or treatment, has already been described above, description thereof will be omitted to avoid excessive duplication.

As used herein, the term “prevention” refers to inhibiting the occurrence of a disease or disease in a subject who has never been diagnosed with a disease or disease, but is likely to have the disease or disease.

As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or condition; or (c) eliminating the disease, condition or symptom. When the composition of the present invention is administered to a subject, TIMP3 , a tumor suppressor gene, is activated through suppression of expression of miR-4435, and blocks the formation and progression of a tumor, thereby inhibiting the development of symptoms according to the tumor, removing it, or alleviating the role do Accordingly, the composition of the present invention may be a therapeutic composition for gastrointestinal cancer itself, or may be administered together with other pharmacological components to be applied as a therapeutic adjuvant for the disease. Accordingly, as used herein, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic adjuvant” or “therapeutic adjuvant”.

As used herein, the term “administration” or “administering” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the subject's body.

In the present invention, the term “therapeutically effective amount” refers to a composition in which the pharmacological component (ie, miR-4435 expression inhibitor) in the composition is contained in an amount sufficient to provide a therapeutic or prophylactic effect to an individual to whom the composition of the present invention is to be administered. It means the content of, and is meant to include a “prophylactically effective amount”.

As used herein, the term “subject” includes, without limitation, humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons or rhesus monkeys. Specifically, the subject of the present invention is a human.

As used herein, the term “expression inhibitor” refers to a substance that causes a decrease in the activity or expression of a target gene, whereby the activity or expression of the target gene becomes undetectable or exists at an insignificant level, as well as the target gene. It refers to a substance that reduces the activity or expression to the extent that the biological function of the drug can be significantly reduced. The expression inhibitors include nucleic acid molecules, compounds, peptides and natural products.

According to a specific embodiment of the present invention, the miR-4435 expression inhibitor is a nucleic acid molecule that specifically binds to miR-4435. A nucleic acid molecule that specifically binds to a target gene for suppression of expression of the target gene is, for example, shRNA, siRNA, miRNA, ribozyme that inhibits the expression of miR-4435 whose sequence is known in the art. , PNA (peptide nucleic acids), antisense oligonucleotides, including, but not limited to, the CRISPR system including a guide RNA recognizing miR-4435, means of suppressing any gene known in the art may be used.

As used herein, the term “shRNA (small hairpin RNA)” is a single strand of 50-70 nucleotides forming a stem-loop structure in vivo. An RNA sequence that creates a tight hairpin structure. Usually, a long RNA of 19-29 nucleotides is base-paired on both sides of a loop of 5-10 nucleotides to form a double-stranded stem, and in order to always be expressed, it is introduced into a cell through a vector containing a U6 promoter. It is transduced and is usually passed on to daughter cells so that suppression of the expression of the target gene is inherited.

As used herein, the term “siRNA” refers to a short double-stranded RNA capable of inducing an RNAi (RNA interference) phenomenon through cleavage of a specific mRNA. It is composed of a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. The total length is 10 to 100 bases, preferably 15 to 80 bases, most preferably 20 to 70 bases, and if the expression of the target gene can be inhibited by the RNAi effect, blunt ends or cohesive ends Both ends are possible. The structure of the adhesive end can be both a structure in which the three-terminal protrudes and a structure in which the five-terminal side protrudes.

As used herein, the term “miRNA (microRNA)” refers to a single-stranded RNA molecule that is not expressed in cells, has a short stem-loop structure, and inhibits the expression of a target gene through complementary binding to the mRNA of the target gene. do.

As used herein, the term “ribozyme” is a type of RNA and refers to an RNA molecule having the same function as an enzyme that recognizes the base sequence of a specific RNA and cuts it by itself. A ribozyme is a complementary nucleotide sequence of a target mRNA strand and consists of a region that binds with specificity and a region that cuts the target RNA.

As used herein, the term “peptide nucleic acid (PNA)” refers to a molecule capable of complementary binding to DNA or RNA while having both nucleic acid and protein properties. PNA is not found in nature but is artificially synthesized by a chemical method, and forms a double strand through hybridization with a natural nucleic acid of a complementary base sequence to control the expression of a target gene.

As used herein, the term “antisense oligonucleotide” refers to a nucleotide sequence complementary to a sequence of a specific mRNA, which binds to a complementary sequence in a target mRNA and translates its protein into a protein, translocation into the cytoplasm, maturation, or any other It refers to a nucleic acid molecule that inhibits an essential activity for an overall biological function. Antisense oligonucleotides may be modified at one or more bases, sugars or backbone positions to enhance efficacy (De Mesmaeker et al., Curr Opin Struct Biol. , 5(3):343-55, 1995). . The oligonucleotide backbone can be modified with phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyls, cycloalkyls, short chain heteroatomics, heterocyclic sugarscholphonates, and the like.

As used herein, the term “express” means that a gene is introduced into a cell of a subject by artificially introducing it using a gene delivery system to allow a subject to express an exogenous gene or to increase the natural expression level of an endogenous gene. It means becoming capable of replication as an extrachromosomal factor or by the completion of chromosomal integration. Thus, the term “expression” is synonymous with “transformation”, “transfection” or “transduction”.

As used herein, the term “gene carrier” refers to any means of transporting a gene into a cell, and gene delivery has the same meaning as transduction of a gene. At the tissue level, the term gene transfer has the same meaning as the spread of a gene. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.

The gene delivery system of the present invention may include in the form of an expression cassette, which is a polynucleotide construct including all elements necessary for self-expression of a gene to be introduced. The expression cassette usually includes a promoter operably linked to the gene, a transcription termination signal, a ribosome binding site, and a translation termination signal. The expression cassette may be in the form of an expression vector capable of self-replication.

The gene delivery system used in the present invention can be applied to any gene delivery system used for conventional gene insertion, for example, plasmids, adenoviruses, adeno-associated viruses (AAV), retroviruses, and lentiviruses. , herpes simplex virus, basinia virus, liposomes and niosomes.

Methods for delivering the gene carrier of the present invention into a host cell of a subject include, for example, microinjection (Harland and Weintraub, J. Cell Biol. 101:1094-1099 (1985)), calcium phosphate precipitation (Chen and Okayama, Mol). Cell. Biol. 7:2745-2752 (1987)), electroporation (Tur-Kaspa et al., Mol. Cell Biol. , 6:716-718 (1986)), liposome-mediated transfection (Nicolau) et al., Methods Enzymol. , 149:157-176 (1987)), DEAE-dextran treatment (Gopal, Mol. Cell Biol. , 5:1188-1190 (1985)), and gene bambadment (Yang et al.) al., Proc. Natl. Acad. Sci. , 87:9568-9572 (1990)).

According to a specific embodiment of the present invention, the composition for preventing or treating gastrointestinal cancer of the present invention increases the expression or activity of tissue inhibitor of metalloproteinases 3 (TIMP3). As described above, when the composition of the present invention is administered to a subject, TIMP3 , a tumor suppressor gene, is activated through suppression of miR-4435 expression, thereby effectively inhibiting tumor formation and progression.

According to another aspect of the present invention, the present invention provides a method for screening a composition for preventing or treating gastrointestinal cancer comprising the following steps:

(a) contacting a test substance with a biological sample comprising cells expressing miR-4435; and

(b) measuring the expression level of miR-4435 in the sample,

When the expression level of miR-4435 is reduced, the test substance is determined as a composition for preventing or treating gastrointestinal cancer.

Since the gastrointestinal cancer to be prevented or treated in the present invention has already been described above, the description thereof will be omitted to avoid excessive duplication.

As used herein, the term “biological sample” refers to any sample containing miR-4435-expressing cells obtained from mammals, including humans, and includes, but is not limited to, tissues, organs, cells, or cell cultures. More specifically, the cells expressing miR-4435 are cancer cells, and most specifically, colorectal cancer cells.

The term “test substance” used while referring to the screening method of the present invention is added to a sample containing miR-4435-expressing cells and used in screening to test whether the activity or expression level of miR-4435 is affected. It means an unknown substance. The test substance includes, but is not limited to, compounds, nucleotides, peptides and natural extracts. The step of measuring the expression level or activity of miR-4435 in the biological sample treated with the test substance may be performed by various methods for measuring the expression level and activity known in the art. As a result of the measurement, when the expression level or activity of miR-4435 is reduced, the test substance may be determined as a composition for preventing or treating gastrointestinal cancer.

As used herein, the term “reduction in expression or activity” means that the degree of inhibition of the tumor suppressor gene TIMP3 by miR-4435 is significantly reduced, so that the intrinsic tumor suppressive action of TIMP3 is restored to a measurable level. It means that the expression level or intrinsic function in vivo decreases. Specifically, it may refer to a state in which the activity or expression level is reduced by 20% or more, more specifically, a state in which the activity or expression is reduced by more than 40%, more specifically, a state in which the activity or expression level is reduced by more than 60% compared to the control.

According to another aspect of the present invention, the present invention provides a UQCRB comprising an agent for measuring the expression level of one or more miRNAs selected from the group consisting of miR-4435, miR-21-3p and miR-1226-3p as an active ingredient. A composition for diagnosis of related diseases is provided.

According to a specific embodiment of the present invention, the miRNA is miR-4435.

As used herein, the term “UQCRB-related disease” refers to any disease in which a defect in mitochondrial function is induced due to a dysfunctional mutation or overexpression thereof in UQCRB. UQCRB-related diseases are mainly associated with complex III deficiency due to a deficiency of an enzyme that catalyzes electron transport, for example, cholesterol biosynthesis disease, hypoglycemia, lactic acidosis , metabolic acidosis, cardiomyopathy, myoglobinuria, retinitis pigmentosa, mitochondrial myopathy, hepatocellular carcinoma, ovarian cancer, pancreatic adenocarcinoma. it's not going to be

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a composition for diagnosis of gastrointestinal cancer, a composition for preventing or treating gastrointestinal cancer, and a screening method thereof.

(b) MiR-4435 of the present invention is usefully used as an excellent circulating biomarker that can simply collect accurate information on the onset and progression stage of gastrointestinal cancer, specifically UQCRB gene mutation-related colorectal cancer, simply by analyzing body fluids can be

(c) The present invention can also be applied as an effective anticancer composition by activating TIMP3 , a tumor suppressor gene, through a MiR-4435 inhibitor.

1 is selected in the present invention This figure shows that miRNAs are highly expressed in mutant UQCRB-expressing cells and colon cancer cells. 1a is a schematic diagram showing the miRNA selection process associated with mutant UQCRB-expressing cells and colorectal cancer. FC shows different expression between control and MT cells. CPM represents normalized expression of each miRNA. Figure 1b is a diagram showing the result of confirming the miRNA selected from the mutant UQCRB-expressing cell line using qRT-PCR. 1c is a diagram showing the result of confirming the miRNA selected in mutant colorectal cancer cells using qRT-PCR. All quantitative data were expressed as mean ± standard error (SEM) relative to controls (* p < 0.05, ** p < 0.01, *** p < 0.0001).
2 is Figure showing that MiR-4435 is secreted by exosomes. Figure 2a shows the result of qRT-PCR analysis of the expression level of miR-4435 in exosomes secreted from a mutant UQCRB-expressing cell line. Figure 2b shows the exosome marker racnf results in mutant UQCRB-expressing cell culture medium. CD63 was used as a positive marker of exosomes, and Calnexin was used as a negative marker, respectively. Figure 2c is a diagram showing the results of qRT-PCR analysis of the expression levels of miR-4435 (left) and miR-21 (right) in exosomes secreted from colon cancer cells. Figure 2d is a diagram showing the exosome marker detection results in the colorectal cancer cell culture medium. Figure 2e is a result of measuring miR-4435 in the serum of colorectal cancer patients using qRT-PCR, the miR-4435 expression level of each patient (left) and the average expression level of miR-4435 at each stage (right) show each All quantitative data were expressed as mean ± standard error (SEM) relative to controls (* p <0.05, ** p <0.01, *** p < 0.0001). All images are representative images of 3 independent replicates.
3 is This figure shows that TIMP3 , a tumor suppressor gene, is a potent target of MiR-4435. 3A shows the miR-4435 and TIMP3 seed sequences identified using the miRmap database. 3b and 3c are diagrams showing protein expression levels in mutant UQCRB-expressing cell lines and colorectal cancer cells. β-actin was used as an internal control. 3D and 3E are diagrams showing the expression level of miR-4435 after transfection of miR-4435 inhibitor into mutant UQCRB-expressing cell lines and colon cancer cells. 3f and 3g are diagrams showing the expression level of TIMP3 after transfection of miR-4435 inhibitor into mutant UQCRB-expressing cell lines and colorectal cancer cells. β-actin was used as an internal control. All quantitative data were expressed as mean ± standard error (SEM) relative to controls (* p < 0.05, ** p < 0.01, *** p < 0.0001). All images are representative images of 3 independent replicates.
4 is Figure showing that MiR-4435 is regulated by A1938, a UQCRB inhibitor. Figures 4a and 4b are diagrams showing the effect of A1938 on miR-4435 expression level in mutant UQCRB-expressing cell lines and colorectal cancer cells. 4c and 4d are diagrams showing the effect of A1938 on TIMP3 expression level in mutant UQCRB-expressing cell lines and colorectal cancer cells. β-actin was used as an internal control. 4e and 4f are diagrams showing cell proliferation after miR-4435 inhibitor transfection. 4g and 4h are diagrams showing the effect of A1938 on cell proliferation. Cells were treated with A1938 (30 μM) for 36 hours. All quantitative data were expressed as mean±standard error (SEM) relative to the control group (* p <0.05, ** p <0.01, *** p <0.0001). All images are representative images of 3 independent replicates.
5 is a diagram showing that miR-4435 is highly expressed in colorectal cancer cell lines. To evaluate the effectiveness of selected miRNAs in various cancer cell lines, miR-4435 expression was measured in gastric cancer (YCC16), liver cancer (HepG2), lung cancer (H1299) and colon cancer cells (HCT116) using qRT-PCR. . All quantitative data were expressed as mean ± standard error (SEM) for controls (*p<0.05, **p<0.01, ***p<0.0001).
6 is miR-21 in the serum of colorectal cancer patients using qRT-PCR. It is a figure showing the result of investigation of expression level. Each patient's miR-21 expression level (left) and the average miR-21 expression level at each stage (right) are shown, respectively. All quantitative data were expressed as mean ± standard error (SEM) for controls (*p<0.05, **p<0.01, ***p<0.0001).

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Experimental method

cell culture

Among control (HEK293, human normal kidney cells) and HEK293 cells, mutant UQCRB-expressing cell lines, MT1 and MT2, were treated with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 1% antibiotic (Invitrogen); Invitrogen, Grand Island, NY). HCT116 (human colon cancer cells) were cultured in RPMI1640 (Invitrogen) under the same conditions. Human normal colon cells, CCD18Co, were cultured in DMEM supplemented with 20% fetal bovine serum (Gibco BRL) and 1× non-essential amino acids (Sigma-Aldrich, St Louis, MO, USA). All cells were cultured in a humidified incubator of pH 7.4 medium at 37° C., 5% CO ₂ . To maintain a stable mutant cell line, 1 mg/mL G418 was applied to DMEM medium.

RNA isolation

Cells were harvested using Trizol (Invitrogen, Carlsbad, CA) and total RNA was ^{extracted using the PureLink™} RNA Isolation Kit (Ambion) according to the manufacturer's instructions. Isolation of RNA from exosomes was performed ^{using SeraMir™} (System Biosciences) according to the manufacturer's instructions.

miRNA sequencing and miRNA expression

The isolated total RNA was subjected to miRNA-sequencing (Illumina HiSeq 2000), and a raw data set was obtained from Macrogen (Seoul, South Korea). In summary, small RNA samples were prepared according to the Illumina protocol for miRNA-silencing. 5' and 3' adapters were serially bound to small RNAs of 18-30 bases purified from 5-10 μg of total RNA. The adapter was excised from the adapter-bound small RNA, reverse transcribed, amplified and removed, and sequenced using a HiSeq 2000 (Illumina) according to the manufacturer's instructions. Next, the adapter was removed and mapped to a reference gene using the Bowtie tool ²⁷ . Data were standardized by the quartile method using edgeR. Finally, read values were normalized to counts per million (CPM) and fold change (FC) was calculated. Candidate miRNAs were identified by adjusting the false discovery rate (FDR) by adjusting the p -value using the Benjamini-Hochberg algorithm. Transcriptome analysis data of mutant UQCRB-expressing cells are available from the National Center for Life Research and Resource Information (KOBIC) (KBRS20171018-0000001 ~ KBRS20171018-0000336)

mRNA sequencing

We performed mRNA-sequencing (Illumina HiSeq 2000) on total RNA and obtained raw data sets from Macrogen (Seoul, South Korea). Briefly, a cDNA library was constructed with the TruSeq RNA library kit using 1 μg of total RNA. The protocol consists of poly A-selective RNA extraction, RNA fragmentation, random hexamer priming reverse transcription, and 100 nt paired-end sequencing using Illumina HiSeq 2000. Libraries were quantified according to the qRT-PCR quantification protocol guide using qRT-PCR and quantified using an Agilent Technologies 2100 Bioanalyzer. Next, the adapters of these mRNA reads were removed and mapped using TopHat2. Data on the reference genome sequence (hg19, Genome Reference Consortium GPCh37) and its annotation were downloaded from the UCSC website (http://genome.ucsc.edu).

Quantitative RT-PCR (qRT-PCR)

The isolated RNA was reverse transcribed using the TaqMan miRNA reverse transcription kit (Applied Biosystems, Waltham, MA) and Taqman primers (Applied Biosystems). On HT Fast Real Time PCR System (Applied Biosystems) using Taqman Fast Universal PCR master mix Applied Biosystems with TaqMan primers (Applied Biosystems), or LightCycler 96 system (Roche) using FastStart Essential DNA Probes Master (Roche) Real-time PCR was performed according to the manufacturer's instructions. Data were calculated using the 2- ^ΔΔCT me method and normalized to RNU48, an internal control small RNA.

Western blot analysis

Cell lysates were analyzed by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). A blot was performed with the primary antibody of anti-UQCRB(1:500) (A78559, Sigma-Aldrich, Saint Louis, MO), anti-TIMP3(1:3000) (ab39184, Abcam, Cambridge, MA) and β-actin (1). :3000) (ab6276, Abcam, Cambridge, MA) overnight at 4°C. Immunolabeling was performed using Clarity Western ECL substrate (Bio-Rad, Hercules, CA). Images were quantified using LabTM software (Bio-Rad).

Isolation of exosomes

ExoQuick-TC ^TM was used to extract the exosomes from the cell culture medium (CCM). Cells (3×10 ⁵ ) were seeded in 100 mm cell culture plates for 72 hours and cultured in a humidified incubator at _{37° C., 5% CO 2 , pH7.4.} After 72 hours, the exosomes were isolated from the colorectal cancer patient sample using the ^{ExoQuick TM kit according to the manufacturer's instructions.} Serum samples from CRC patients were donated from the Severance Hospital Gene Bank (IRB approval number: 7001988-201803-BR-141-01E).

miRNA inhibitor transfection

Mutant UQCRB-expressing cells and colon cancer cells were transfected with miR-4435 inhibitor purchased from Bioneer Co. (Daejeon, Korea). Cells (1.5 x 10 ⁵ ) were seeded in 6-well plates 48 hours before transfection. Transfection was performed using Lipofectamine RNAiMAX (Invitrogen) reagent according to the manufacturer's instructions.

cell proliferation assay

Cells (3 x 10 ³ ) were seeded in 96-well plates for 24-72 hours and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma- Aldrich, SaintLouis, MO) cell proliferation was measured by analysis. Cells were treated with UQCRB inhibitor (A1938) and transfected with miR-4435 inhibitor.

statistical analysis

Result values are expressed as mean ± standard error (SEM) and all statistical analyzes were calculated using GraphPad Prism (ver. 5.00, GraphPad Software, San Diego, CA, www.graphpad.com). Student's t -test was used to determine the statistical significance between the control group and the test group. A p -value less than 0.05 was considered statistically significant (* p < 0.05, ** p < 0.01, *** p < 0.001).

Experiment result

Selected miRNAs had increased expression in mutant UQCRB-expressing cell lines and CRC cells.

The present inventors previously performed miRNA-sequencing in HEK293 and mutant UQCRB-expressing HEK293 cell lines (MT1 and MT2) to analyze the miRNA expressed differently in mutant cells compared to control cells (HEK293) ²¹ . First, 1,338 and 1,195 miRNAs differently expressed compared to the control were identified in MT1 and MT2 cell lines, respectively. Of these, 6 miRNAs (hsa-miR-4485, -4745-5p, -1908-3p, -1226-3p, -4435, -21-3p) were selected as major candidates according to the following three criteria: control Contrast |log ₂ FC|>1, |log ₂ CPM|>2, FDR<0.05. A schematic diagram of the miRNA candidate selection process is shown in Figure 1a. First, three candidates (miR-21-3p, miR-1226-3p, miR-4435) confirmed to increase in CRC among the six candidates were first selected, and exosomes were produced in CRC cells among these three candidates. miR-4435, which was confirmed to be secreted via the translocation, was selected as a final candidate. Next, as a result of confirming whether the miR-4435 level was increased in the mutant UQCRB-expressing cell line compared to the control cell line (HEK293) using quantitative RT-PCR, the miR-4435 level was the mutant UQCRB-expressing cell line compared to the control cell line (HEK293). was increased in (Fig. 1b). The CRC biomarker, miR-21, was also increased in CRC and was used as a positive control. Both the expression levels of miR-21 and miR-4435 were increased two-fold in the CRC cell line, HCT116, compared to the normal colon cell line (FIG. 1c). In addition, the present inventors investigated the expression level of miR-4435 in gastric cancer (YCC16), liver cancer (HepG2), lung cancer (H1299) and colorectal cancer (HCT116), which are known to have high prevalence of cancer. It was found that the expression was specifically increased. The place with the next highest expression was gastric cancer, which seems to be the effect of the gastrointestinal tract (FIG. 5). Taken together, these results show that miR-4435 expression is increased in mutant UQCRB-expressing cell lines and colon cancer cells.

Selected miRNAs increase in exosomes

An increase in the expression of miR-4435, a selected miRNA, was observed in mutant UQCRB-expression and CRC cells, and it was investigated whether miR-4435 was secreted through exosomes. Mutant UQCRB-expressing cells and CRC cells were cultured for 72 hours, exosomes were isolated from cell culture medium (CCM), and the expression level of candidate miRNAs in the exosomes was measured. The expression level of miR-4435 in the isolated exosomes was higher in the exosomes derived from the mutant UQCRB-expressing cell line than in the control (HEK293) (Fig. 2a). For further confirmation, exosomes were isolated and western blotting was performed for exosome-specific markers (CD63 and calnexin). As a result, exosomes were successfully isolated from CCM ( FIG. 2b ), and CRC compared to normal cells. The amount secreted from cells through exosomes was higher in miR-4435 than in miR-21 ( FIG. 2c ). It was also confirmed that the exosomes were well separated through the western blotting results for the exosome-specific markers (FIG. 2d). Taken together, miR-4435 was highly expressed in mutant UQCRB-expressing cell lines and CRC cells, as well as high levels of miR-4435 in exosomes secreted from these cells.

Next, we investigated the expression of miR-4435 in the serum of CRC patients. Exosomes were isolated from serum samples of CRC patients, and the expression level of miR-4435 in the exosomes was measured by qRT-PCR. As a result of analyzing serum samples from patients of various ages of CRC stages 1 to 4 (Table 1), the expression of miR-4435 increased as the CRC stage progressed to a later stage ( FIG. 2E ). On the other hand, the miR-21 expression level of the patient's serum samples did not show a correlation with the progression of the CRC stage (FIG. 6). These results show that miR-4435 can function as a circulating miRNA biomarker for CRC at specific stages. In particular, miR-4435 appears to be closely related to the degree of CRC progression.

UQCRB-associated key miRNAs are tumor suppressor genes TIMP3 to target

To investigate which gene miR-4435 targets, mRNA-sequencing results of mutant UQCRB-expressing cell lines (MT1 and MT2) and control were compared. Among them, 133 mRNAs with |FC|>2 compared to the control group were selected as a candidate group with a significant decrease. Then, as a result of identifying the target mRNA of miR-4435 using miRDB, a target prediction program, a total of 474 target genes were predicted. Compare the results with sequencing mRNA- miRDB result in mutant cell lines expressing UQCRB- go out the common gene, the TIMP3 received the highest score, 53 of which were selected as the final candidate target gene. The seed sequences of miR-4435 and TIMP3 are shown in FIG. 3A . TIMP3 is low expressed in CRC and is known as a tumor suppressor gene ²⁸ . Accordingly, as a result of examining the protein expression level, it was found that the TIMP3 protein was expressed lower in the mutant UQCRB-expressing cell line than in HEK293 ( FIG. 3b ). Furthermore, the intrinsic UQCRB level was higher in CRC cells than in normal control cells, whereas the protein level of TIMP3, predicted as a target of miR-4435, was lower than in the control cells (Fig. 3c). To further confirm whether TIMP3 is a target of miR-4435, a miR-4435 inhibitor was transfected into a mutant UQCRB-expressing cell line and colorectal cancer cells. As a result of measuring the expression level of miR-4435 using qRT-PCR in a mutant UQCRB-expressing cell line transfected with 50 nM of miR-4435 inhibitor, it was confirmed that the expression was reduced by about 50% ( FIG. 3D ). In CRC cells, miR-4435 expression was decreased in a concentration-dependent manner by miR-4435 inhibitor treatment (Fig. 3e). Next, the protein level of TIMP3 after inhibition of miR-4435 was investigated. As a result, the TIMP3 protein level was increased in the mutant UQCRB-expressing cells transfected with the miR-4435 inhibitor (FIG. 3f), and also in colorectal cancer cells, the protein level of TIMP3 was increased in the untreated group by treatment with the miR-4435 inhibitor. increased compared to that (Fig. 3g). These results show that by adjusting the relationship between miR-4435, and the target TIMP3 UQCRB TIMP3 and, ultimately, in part, contribute to the CRC tumor formation.

MiR-4435 is regulated by the UQCRB inhibitor A1938

Next, as a ^{result of investigating whether UQCRB-related miRNAs can be regulated by A1938 29} , a known UQCRB inhibitor, it was confirmed that miR-4435 levels were reduced after treatment with A1938 in a mutant UQCRB-expressing cell line ( FIG. 4a ). In particular, miR-4435 levels were decreased in a concentration-dependent manner in A1938-treated CRC cells (Fig. 4b). Since A1938 regulated miR-4435 expression level, the present inventors investigated the effect of A1938 on TIMP3, a target of miR-4435. As a result, the protein level of TIMP3 was increased in the mutant UQCRB-expressing cell line (Fig. 4c) and CRC cells (Fig. 4d) by A1938 treatment. Through this, it can be seen that a series of expression of miR-4435 and TIMP3 is regulated when UQCRB is inhibited by treatment with A1938.

In addition, the present inventors investigated how cell proliferation is affected after restoration of TIMP3 expression through treatment with miR-4435 inhibitor and A1938. First, mutant UQCRB-expressing cells and CRC cells were treated with miR-4435 inhibitor and A1938. MiR-4435 inhibitor treatment at 20 nM and 50 nM inhibited proliferation of mutant UQCRB-expressing cells ( FIG. 4E ), and colon cancer cell proliferation was also inhibited by treatment with miR-4435 inhibitor ( FIG. 4F ). Proliferation of mutant UQCRB-expressing cells (FIG. 4G) and colon cancer cells (FIG. 4H) was inhibited in a dose-dependent manner even when cells were treated with A1938, a UQCRB inhibitor. Through these results, it can be seen that the restoration of TIMP3 expression level by treatment with A1938 and miR-4435 inhibitors affects cell proliferation.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (17)

A composition for blood diagnosis of colorectal cancer, comprising as an active ingredient a formulation for measuring the expression level of miR-4435.
delete delete The composition of claim 1, wherein the colorectal cancer is colorectal cancer having a mutation in the UQCRB gene.
The composition of claim 1, wherein the colorectal cancer is stage 3 or stage 4 colorectal cancer.
The composition of claim 1, wherein the agent for measuring the expression level of miR-4435 is a primer or probe that specifically binds to the miR-4435.
delete delete A composition for preventing or treating colorectal cancer comprising an inhibitor of miR-4435 expression as an active ingredient.
delete The composition of claim 9, wherein the colorectal cancer is colorectal cancer having a mutation in the UQCRB gene.
The composition according to claim 9, wherein the miR-4435 expression inhibitor is a nucleic acid molecule that specifically binds to miR-4435.
The composition according to claim 9, wherein the composition increases the expression or activity of TIMP3 (tissue inhibitor of metalloproteinases 3).
A method for screening a composition for preventing or treating colorectal cancer, comprising the steps of:
(a) contacting a test substance with a biological sample comprising cells expressing miR-4435; and
(b) measuring the expression level of miR-4435 in the sample,
When the expression level of miR-4435 is reduced, the test substance is determined as a composition for preventing or treating colon cancer. delete delete delete

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