US20170130270A1 - Method for expecting and diagnosing uqcrb-related disease - Google Patents
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
Definitions
- Embodiments of the inventive concept described herein relate to a method for expecting and diagnosing UQCRB-related disease, and more particularly, relate to a method for diagnosing a UQCRB-related disease and a cholesterol biosynthesis related disease, as well as expecting risks of post-occurrence of the UQCRB-related disease and the cholesterol biosynthesis related disease, simply by measuring an expression level of miRNA, and a kit and a biomarker composition for the method.
- UQCRB Ubiquinol-cytochrome C Reductase Binding Protein
- UQCRB is encoded in a nuclear and is important in fabricating and maintaining mitochondria complex III. Additionally, UQCRB is known as being over-expressed in involved in a liver cancer and a stomach cancer, and involved in several diseases such as hypoclysemia, lactic acid acidosis, and muscle disease.
- the inventors as shown in FIG. 1 , has demonstrated that introduction of UQCRB into a cell induces Reactive Oxygen Species (ROS) to be generated in mitochondria and stabilizes HIF-1 ⁇ to induce angiogenesis (Jung, et al., Mol. Biosyst., 2013).
- ROS Reactive Oxygen Species
- complex III of mitochondria respiration chain generates ROS through sensing oxygen in a cell, performing an important function in regulating hypoxia-inducible angiogenesis.
- HIF Hypoxia-Inducible factor
- ROS generated by mitochondria complex III in a hypoxia condition stabilizes HIF-1 ⁇ protein which is a main regulator of angiogenesis.
- HIF is formed of a heterodimer complex of HIF-1 ⁇ and HIF-1 ⁇ , and especially HIF-la protein causes initiation of expression of an angiogenesis-friendly factor such as Vascular Endothelial Growth Factor (VEGF).
- VEGF Vascular Endothelial Growth Factor
- Micro-RNA is a small untranslated RNA formed of 18 ⁇ 25 nucleotides. Such miRNA is coupled with 3′-Untranslated Region (UTR) to regulate expression of gene (Bartel D P, et al., Cell 116: 281-297, 2004; Lewis B P, et al., Cell 120: 15-20, 2005) and processed from intron, exon, or intergenic region (Rodriguez A, et al., Genome Res14: 1902-1910, 2004).
- UTR 3′-Untranslated Region
- the miRNA is involved in various biological processes related to cancer development including proliferation and invasion of cancer cells. Expression of miRNA is known as being regulated bilaterally in many types of cancers (Esquela-Kerscher A, et al., Nat Rev Cancer 6: 259-269, 2006).
- Embodiments of the inventive concept provide a new method for diagnosing and expecting UQCRB and a UQCRB-related disease by measuring an expression level of a specific miRNA.
- Embodiments of inventive concept provide a biomarker composition for diagnosing and expecting UQCRB and a UQCRB-related disease, and a kit, including the biomarker composition, for diagnosing and expecting UQCRB and a UQCRB-related disease.
- a method for expecting and diagnosing a UQCRB-related disease includes measuring an expression level of at least one miRNA, which is selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p, in a sample, comparing a pattern of the measured expression level(s) with one of expression levels or several reference patterns; and diagnosing or expecting the UQCRB-related disease from the compared result of the sample and the reference patterns.
- miRNA which is selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p
- the sample may be selected from a group formed of a blood sample, a serum sample, a plasma sample, a urine sample, and sputum.
- the measuring of the expression level may include measuring an expression level of hsa-miR-1323.
- the measuring of the expression level may include measuring an expression level of hsa-miR-512-3p.
- the measuring of the expression level may include measuring an expression level of hsa-miR-10a-5p.
- an expression level of miRNA may be found by ascertaining a miRNA gene itself, or a miRNA level in which the gene is expressed, that is, an expression level of protein coded with the miRNA gene.
- a material capable for measuring the level may include a probe or primer specified peculiar to the miRNA gene.
- a probe or primer peculiar to the miRNA may be a probe or primer capable of peculiarly amplifying all or a specific region of each of the whole gene of the three miRNA, and the primer or probe may be designed through a method known in the art.
- the primer may mean single strand oligonucleotide capable of acting as an initiation point of template-directed DNA synthesis under a suitable condition (i.e., four different kinds of nucleoside triphosphate and polymerase) in suitable temperature and suitable buffer solution.
- a suitable length of a primer may be variable in accordance with diverse factors, for example, temperature and use of the primer.
- a primer is unnecessary to have a sequence fully complementary to a partial sequence of a template. It may be enough for a sequence of a primer if it has sufficient complementarity in a range capable of acting the unique function of the primer by hybridization with a template.
- a primer according to embodiments of the inventive concept is unnecessary to have a sequence fully complementary to a nucleotide sequence of a gene that is a template, and rather may be enough if it has sufficient complementarity in a range capable of acting its unique function by hybridization with the nucleotide sequence the gene. Additionally, it may be preferred for a primer according to embodiments of the inventive concept to be used in gene amplification.
- the measuring of the expression level may use any way for measuring an expression amount of miRNA, including selecting one from a group formed of reverse transcriptase polymerase chain reaction, real time polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.
- the probe may mean natural or modified monomer or linear oligomer of linkages, which may include deoxyribonucleotide and ribonucleotide and may be hybridized peculiarly with a target nucleotide sequence, and indicates a thing that naturally exists or is artificially synthesized.
- a probe according to embodiments of the inventive concept may be a single strand, preferably, oligodeoxyribonucleotide.
- a probe according to embodiments of the inventive concept may include natural dNMP (i.e., dAMP, dGMP, dCMP, and dTMP), a nucleotide analogue or derivative. Accordingly, a probe according to embodiments of the inventive concept may also include even ribonucleotide.
- protein-level measurement for measuring the expression level may use an antibody.
- the marker protein and an antibody peculiar thereto in a biological specimen may form a mixture, that is, an antigen-antibody complex, and an amount of the antigen-antibody complex may be quantitatively measured through a size of a signal of a detection label.
- This detection label may be selected from a group formed of enzymes, florescent materials, ligands, emitting materials, microparticles, redox molecules, and radioactive isotropes, but may not be restrictive hereto.
- the UQCRB-related disease may be related to cholesterol synthesis.
- UQCRB-related disease In a method for expecting and diagnosing UQCRB-related disease in accordance with embodiments of the inventive concept, it may be allowable to lower a serum cholesterol level in relation with cholesterol biosynthesis or to raise an LDL or HDL cholesterol.
- the UQCRB-related disease may be a dyslipidemia, hyperlipidemia, hypercholesterolemia, and microvascular disease and an acute nephritic syndrome which are generated in connection with cholesterol, as well as a kind of cancer such as breast cancer, colorectal cancer, liver cancer, or stroke.
- the UQCRB-related disease may arise from an increase of expression of cholesterol synthetase due to over-expression of UQCRB.
- the cholesterol synthetase may be HMG-CoA reductase (HMGCR), pyrophophosmevalnote decarboxylase (MVD), or lanosterol synthase (LSS).
- HMGCR HMG-CoA reductase
- MVD pyrophophosmevalnote decarboxylase
- LSS lanosterol synthase
- a biomarker composition for expecting and diagnosing UQCRB-related disease includes an agent detecting at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- an agent for detecting miRNA may mean molecules which can be used in detecting a marker by ascertaining an expression level of miRNA that is the marker whose expression is affected in relation with UQCRB as described above, preferably indicating a probe, primer, or antibody peculiar to the marker.
- the agent in the biomarker composition, may be a probe or primer having a sequence complementary to a nucleotide sequence of at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- a kit for expecting and diagnosing a UQCRB-related disease includes means configured to measure an expression level of at least one miRNA selected from a group, which is formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p, in a sample; and at least one reference pattern including an expression level that is to be compared with the measured expression level of the miRNA that is taken from the sample.
- the kit may selectively include a reagent necessary for PCR amplification, for example, buffer solution, DNA polymerase (e.g., thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Themis flavus, Thermococcus literalis , or Pyrococcus furiosus (Pfu)), DNA polymerase associated factor, and dNTPs.
- DNA polymerase e.g., thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Themis flavus, Thermococcus literalis , or Pyrococcus furiosus (Pfu)
- DNA polymerase associated factor e.g., thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Themis flav
- kits according to embodiments of the inventive concept may selectively include a substrate of a label and a secondary antibody.
- a kit according to embodiments of the inventive concept may be fabricated to be compartments or a multiplicity of separate packages including the aforementioned specimen component.
- the kit may include a primer complementary to at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p and configured to amplify the miRNA.
- the kit may include an antibody, antisense oligonucleotide, or probe for at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- FIG. 1 shows a process that introduction of UQCRB induces generation of ROS in mitochondria and stabilizes HIF-1 ⁇ to induce angiogenesis
- FIG. 2 shows a process of selecting miRNA through comparison of expression patterns in UQCRB mutant cell strains
- FIG. 3 shows a result of measuring an expression amount of miRNA through qPCR in accordance with presence or absence of a process with UQCRB-1 (20M), which is a UQCRB inhibitor, in a UQCRB mutant cell strain;
- FIGS. 4 to 6 show a process of selecting a cholesterol metabolic process
- FIGS. 7 to 10 show results of measuring expression levels of three enzymes related to a cholesterol metabolic process
- FIGS. 11 to 13 show results of measuring effects that a cholesterol synthesis inhibitor affects growth of UQCRB mutant cell strains.
- FIGS. 14 to 18 show results of measuring expression levels of three miRNA and expression levels of enzymes related to a cholesterol metabolic process.
- HEK 293, UQCRB mutant cell strains MT1 and MT2, and HepG2 were cultivated in a DMEM medium to which FBS is added.
- the UQCRB mutant cell strains MT1 and MT2 were prepared according to the method disclosed in the article that had been previously published by the inventor (Chang, et al., Biochem. Biophys. Res. Commun., 2014).
- PC3, HCT116 were in a RPMI1640 medium to which FBS is added.
- Total RNA was separated from the HEK 293, the UQCRB mutant cell strains MT1 and MT2, by using a PureLink RNA isolation kit, and the separated total RNA is miRNA-sequenced in Macrogen Inc. to obtain sequencing data.
- FC a difference of expression levels
- CPM the own expression of the factor in each cells.
- Expression patterns of the selected 10 miRNAs were manufactured with RT-PCR and three miRNAs of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p were selected in comparison with expression patterns of UQCRB mutant cell strains.
- UQCRB-1 (20M) as a UQCRB inhibitor was processed in the UQCRB mutant cell strains MT1 and MT2 and an expression amount of miRNA was measured with qPCR.
- hsa-miR-1323 was restored in 18% of miRNA
- hsa-miR-512-3p was restored in 15% of miRNA
- hsa-miR-10a-5p was restored in 24% of miRNA in comparison with HEK293 that was used as a control group when processing a UQCRB inhibitor.
- This result means that reduction of expression amount of three miRNA is directly involved in over-expression of UQCRB in the UQCRB mutant cell strains MT1 and MT2.
- oncology processes (sterol metabolic process, cholesterol metabolic process, steroid metabolic process, sterol biosynthetic process, steroid biosynthetic process, terpenoid backbone biosynthesis, isoprenoid biosynthetic process, and cholesterol biosynthetic process) were over-expressed in UQCRB mutant cell strings MT1 and MT2.
- the cholesterol biosynthetic process which was the lowest oncology group, was selected from the eight processes.
- HMG-CoA reductase HMGCR
- MVD pyrophosphomevalnote decarboxylase
- LSS lanosterol synthase
- a cholesterol synthesis inhibitor was processed in UQCRB mutant cell strains to measure an effect that the cholesterol synthesis inhibitor affects growth of the UQCRB mutant cell strains, and the result thereof was shown in FIGS. 11 to 13 .
- Fatostatin sterol regulatory element-binding protein: SREBP inhibitor
- Mevastatin HMG-CoA reductase: HMGCR inhibitor
- YM-53601 squalene synthase: FDFT1 inhibitor
- the three miRNAs according to embodiments of the inventive concept may be used as a biomarker for diagnosing UQCRB and a disease related to a cholesterol metabolic process involved in UQCRB.
- a method according to embodiments of the inventive concept may expect and diagnose a UQCRB-related disease and a disease involved in cholesterol biosynthesis related to UQCRB simply by measuring a specific miRNA expression level.
Abstract
Description
- A claim for priority under 35 U.S.C. §119 is made to Korean Patent Application No. 10-2015-0155528 filed Nov. 6, 2015, in the Korean Intellectual Property Office, the entire contents of which are hereby incorporated by reference.
- Embodiments of the inventive concept described herein relate to a method for expecting and diagnosing UQCRB-related disease, and more particularly, relate to a method for diagnosing a UQCRB-related disease and a cholesterol biosynthesis related disease, as well as expecting risks of post-occurrence of the UQCRB-related disease and the cholesterol biosynthesis related disease, simply by measuring an expression level of miRNA, and a kit and a biomarker composition for the method.
- Ubiquinol-cytochrome C Reductase Binding Protein (UQCRB), which is one of elements of mitochondria complex III, is known as a target protein of terpestacin that is a low molecular compound inhibiting angiogenesis.
- In detail, UQCRB is encoded in a nuclear and is important in fabricating and maintaining mitochondria complex III. Additionally, UQCRB is known as being over-expressed in involved in a liver cancer and a stomach cancer, and involved in several diseases such as hypoclysemia, lactic acid acidosis, and muscle disease.
- The inventors, as shown in
FIG. 1 , has demonstrated that introduction of UQCRB into a cell induces Reactive Oxygen Species (ROS) to be generated in mitochondria and stabilizes HIF-1α to induce angiogenesis (Jung, et al., Mol. Biosyst., 2013). - According to other several reports, complex III of mitochondria respiration chain generates ROS through sensing oxygen in a cell, performing an important function in regulating hypoxia-inducible angiogenesis.
- A Hypoxia-Inducible factor (HIF) has been watched in the point that it importantly affects cell survival and initiation of angiogenesis in a hypoxia condition. ROS generated by mitochondria complex III in a hypoxia condition stabilizes HIF-1α protein which is a main regulator of angiogenesis. HIF is formed of a heterodimer complex of HIF-1α and HIF-1β, and especially HIF-la protein causes initiation of expression of an angiogenesis-friendly factor such as Vascular Endothelial Growth Factor (VEGF).
- Micro-RNA (miRNA) is a small untranslated RNA formed of 18˜25 nucleotides. Such miRNA is coupled with 3′-Untranslated Region (UTR) to regulate expression of gene (Bartel D P, et al., Cell 116: 281-297, 2004; Lewis B P, et al., Cell 120: 15-20, 2005) and processed from intron, exon, or intergenic region (Rodriguez A, et al., Genome Res14: 1902-1910, 2004).
- The miRNA is involved in various biological processes related to cancer development including proliferation and invasion of cancer cells. Expression of miRNA is known as being regulated bilaterally in many types of cancers (Esquela-Kerscher A, et al., Nat Rev Cancer 6: 259-269, 2006).
- However, it has been unknown that miRNA is involved in UQCRB and diseases related to UQCRB.
- Embodiments of the inventive concept provide a new method for diagnosing and expecting UQCRB and a UQCRB-related disease by measuring an expression level of a specific miRNA.
- Embodiments of inventive concept provide a biomarker composition for diagnosing and expecting UQCRB and a UQCRB-related disease, and a kit, including the biomarker composition, for diagnosing and expecting UQCRB and a UQCRB-related disease.
- According to an aspect of an embodiment, a method for expecting and diagnosing a UQCRB-related disease includes measuring an expression level of at least one miRNA, which is selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p, in a sample, comparing a pattern of the measured expression level(s) with one of expression levels or several reference patterns; and diagnosing or expecting the UQCRB-related disease from the compared result of the sample and the reference patterns.
- In the method, the sample may be selected from a group formed of a blood sample, a serum sample, a plasma sample, a urine sample, and sputum.
- In the method, the measuring of the expression level may include measuring an expression level of hsa-miR-1323.
- In the method, the measuring of the expression level may include measuring an expression level of hsa-miR-512-3p.
- In the method, the measuring of the expression level may include measuring an expression level of hsa-miR-10a-5p.
- In embodiments of the inventive concept, an expression level of miRNA may be found by ascertaining a miRNA gene itself, or a miRNA level in which the gene is expressed, that is, an expression level of protein coded with the miRNA gene. A material capable for measuring the level may include a probe or primer specified peculiar to the miRNA gene. In embodiments of the inventive concept, a probe or primer peculiar to the miRNA may be a probe or primer capable of peculiarly amplifying all or a specific region of each of the whole gene of the three miRNA, and the primer or probe may be designed through a method known in the art.
- In embodiments of the inventive concept, the primer may mean single strand oligonucleotide capable of acting as an initiation point of template-directed DNA synthesis under a suitable condition (i.e., four different kinds of nucleoside triphosphate and polymerase) in suitable temperature and suitable buffer solution. A suitable length of a primer may be variable in accordance with diverse factors, for example, temperature and use of the primer. Additionally, a primer is unnecessary to have a sequence fully complementary to a partial sequence of a template. It may be enough for a sequence of a primer if it has sufficient complementarity in a range capable of acting the unique function of the primer by hybridization with a template. Accordingly, a primer according to embodiments of the inventive concept is unnecessary to have a sequence fully complementary to a nucleotide sequence of a gene that is a template, and rather may be enough if it has sufficient complementarity in a range capable of acting its unique function by hybridization with the nucleotide sequence the gene. Additionally, it may be preferred for a primer according to embodiments of the inventive concept to be used in gene amplification.
- In the method, the measuring of the expression level may use any way for measuring an expression amount of miRNA, including selecting one from a group formed of reverse transcriptase polymerase chain reaction, real time polymerase chain reaction, Western blot, Northern blot, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, and immunoprecipitation assay.
- In embodiments of the inventive concept, the probe may mean natural or modified monomer or linear oligomer of linkages, which may include deoxyribonucleotide and ribonucleotide and may be hybridized peculiarly with a target nucleotide sequence, and indicates a thing that naturally exists or is artificially synthesized. A probe according to embodiments of the inventive concept may be a single strand, preferably, oligodeoxyribonucleotide. A probe according to embodiments of the inventive concept may include natural dNMP (i.e., dAMP, dGMP, dCMP, and dTMP), a nucleotide analogue or derivative. Accordingly, a probe according to embodiments of the inventive concept may also include even ribonucleotide.
- In a method for expecting and diagnosing UQCRB-related disease in accordance with embodiments of the inventive concept, protein-level measurement for measuring the expression level may use an antibody. In this case, the marker protein and an antibody peculiar thereto in a biological specimen may form a mixture, that is, an antigen-antibody complex, and an amount of the antigen-antibody complex may be quantitatively measured through a size of a signal of a detection label. This detection label may be selected from a group formed of enzymes, florescent materials, ligands, emitting materials, microparticles, redox molecules, and radioactive isotropes, but may not be restrictive hereto.
- In the method for expecting and diagnosing a UQCRB-related disease, the UQCRB-related disease may be related to cholesterol synthesis.
- In a method for expecting and diagnosing UQCRB-related disease in accordance with embodiments of the inventive concept, it may be allowable to lower a serum cholesterol level in relation with cholesterol biosynthesis or to raise an LDL or HDL cholesterol. The UQCRB-related disease may be a dyslipidemia, hyperlipidemia, hypercholesterolemia, and microvascular disease and an acute nephritic syndrome which are generated in connection with cholesterol, as well as a kind of cancer such as breast cancer, colorectal cancer, liver cancer, or stroke.
- In the method, the UQCRB-related disease may arise from an increase of expression of cholesterol synthetase due to over-expression of UQCRB.
- In the method, the cholesterol synthetase may be HMG-CoA reductase (HMGCR), pyrophophosmevalnote decarboxylase (MVD), or lanosterol synthase (LSS).
- According to an aspect of an embodiment, a biomarker composition for expecting and diagnosing UQCRB-related disease includes an agent detecting at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- In embodiments of the inventive concept, an agent for detecting miRNA may mean molecules which can be used in detecting a marker by ascertaining an expression level of miRNA that is the marker whose expression is affected in relation with UQCRB as described above, preferably indicating a probe, primer, or antibody peculiar to the marker. In other words, in the biomarker composition, the agent may be a probe or primer having a sequence complementary to a nucleotide sequence of at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- According to still another embodiment of the inventive concept, a kit for expecting and diagnosing a UQCRB-related disease includes means configured to measure an expression level of at least one miRNA selected from a group, which is formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p, in a sample; and at least one reference pattern including an expression level that is to be compared with the measured expression level of the miRNA that is taken from the sample.
- In the case that a kit for expecting and diagnosing UQCRB-related disease in accordance with embodiments of the inventive concept is applied to a PCR amplification process, the kit may selectively include a reagent necessary for PCR amplification, for example, buffer solution, DNA polymerase (e.g., thermostable DNA polymerase obtained from Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Themis flavus, Thermococcus literalis, or Pyrococcus furiosus (Pfu)), DNA polymerase associated factor, and dNTPs. In the case that a kit according to embodiments of the inventive concept is applied to immunity analysis, the kit may selectively include a substrate of a label and a secondary antibody. Besides, a kit according to embodiments of the inventive concept may be fabricated to be compartments or a multiplicity of separate packages including the aforementioned specimen component.
- The kit may include a primer complementary to at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p and configured to amplify the miRNA.
- The kit may include an antibody, antisense oligonucleotide, or probe for at least one miRNA selected from a group formed of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p.
- The above and other objects and features will become apparent from the following description with reference to the following figures, wherein like reference numerals refer to like parts throughout the various figures unless otherwise specified, and wherein:
-
FIG. 1 shows a process that introduction of UQCRB induces generation of ROS in mitochondria and stabilizes HIF-1α to induce angiogenesis; -
FIG. 2 shows a process of selecting miRNA through comparison of expression patterns in UQCRB mutant cell strains; -
FIG. 3 shows a result of measuring an expression amount of miRNA through qPCR in accordance with presence or absence of a process with UQCRB-1 (20M), which is a UQCRB inhibitor, in a UQCRB mutant cell strain; -
FIGS. 4 to 6 show a process of selecting a cholesterol metabolic process; -
FIGS. 7 to 10 show results of measuring expression levels of three enzymes related to a cholesterol metabolic process; -
FIGS. 11 to 13 show results of measuring effects that a cholesterol synthesis inhibitor affects growth of UQCRB mutant cell strains; and -
FIGS. 14 to 18 show results of measuring expression levels of three miRNA and expression levels of enzymes related to a cholesterol metabolic process. - Hereafter, embodiments of the inventive concept will be described in detail. However, the inventive concept may not be restrictive embodiments described below.
- HEK 293, UQCRB mutant cell strains MT1 and MT2, and HepG2 were cultivated in a DMEM medium to which FBS is added. The UQCRB mutant cell strains MT1 and MT2 were prepared according to the method disclosed in the article that had been previously published by the inventor (Chang, et al., Biochem. Biophys. Res. Commun., 2014). PC3, HCT116 were in a RPMI1640 medium to which FBS is added.
- For the purpose of processing miRNA, which has different expression patterns in the UQCRB mutant cell strains MT1 and MT2, in comparison with HEK 293 which is used as a control group, total RNA was separated from the UQCRB mutant cell strains MT1 and MT2 and thereafter sequenced.
- Total RNA was separated from the HEK 293, the UQCRB mutant cell strains MT1 and MT2, by using a PureLink RNA isolation kit, and the separated total RNA is miRNA-sequenced in Macrogen Inc. to obtain sequencing data.
- As a result of separating total RNA from the UQCRB mutant cell strains MT1 and MT2, 1255 miRNAs were separated and, as shown in
FIG. 2, 10 miRNAs satisfying the following three conditions were selected. - |log2FC|>1, |log2CPM|>2, FDR<0.05
- FC: a difference of expression levels
- CPM: the own expression of the factor in each cells.
- Expression patterns of the selected 10 miRNAs were manufactured with RT-PCR and three miRNAs of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p were selected in comparison with expression patterns of UQCRB mutant cell strains.
- It can be seen from
FIG. 2 that the three miRNAs of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p are less expressed in the UQCRB mutant cell strains than in HEK 293 that is a normal cell. - For the purpose of ascertaining whether reduction of expression of three miRNAs of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p in the UQCRB mutant cell strains MT1 and MT2 was directly involved in over-expression of UQCRB, UQCRB-1 (20M) as a UQCRB inhibitor was processed in the UQCRB mutant cell strains MT1 and MT2 and an expression amount of miRNA was measured with qPCR.
- It could be monitored from
FIG. 3 that hsa-miR-1323 was restored in 18% of miRNA, hsa-miR-512-3p was restored in 15% of miRNA, and hsa-miR-10a-5p was restored in 24% of miRNA in comparison with HEK293 that was used as a control group when processing a UQCRB inhibitor. This result means that reduction of expression amount of three miRNA is directly involved in over-expression of UQCRB in the UQCRB mutant cell strains MT1 and MT2. - For the purpose of selecting a process in which UQCRB was participated, a process of separating and sequencing mRNA was performed in UQCRB mutant cell strains MT1 and MT2.
- As shown in
FIGS. 4 to 6 , eight oncology processes (sterol metabolic process, cholesterol metabolic process, steroid metabolic process, sterol biosynthetic process, steroid biosynthetic process, terpenoid backbone biosynthesis, isoprenoid biosynthetic process, and cholesterol biosynthetic process) were over-expressed in UQCRB mutant cell strings MT1 and MT2. The cholesterol biosynthetic process, which was the lowest oncology group, was selected from the eight processes. - For the purpose of verifying validity for selection of cholesterol metabolic process as a pathway in which UQCRB was participated, three enzymes (HMG-CoA reductase (HMGCR), pyrophosphomevalnote decarboxylase (MVD), and lanosterol synthase (LSS)) related to a cholesterol metabolic process were measured in UQCRB mutant cell strains MT1 and MT2 and the result thereof were shown in
FIGS. 6 to 9 . - For the purpose of verifying validity for selection of cholesterol metabolic process as a pathway in which UQCRB was participated, a cholesterol synthesis inhibitor was processed in UQCRB mutant cell strains to measure an effect that the cholesterol synthesis inhibitor affects growth of the UQCRB mutant cell strains, and the result thereof was shown in
FIGS. 11 to 13 . - After processing Fatostatin (sterol regulatory element-binding protein: SREBP inhibitor), Mevastatin (HMG-CoA reductase: HMGCR inhibitor), and YM-53601 (squalene synthase: FDFT1 inhibitor), as cholesterol synthesis inhibitors, in UQCRB mutant cell strains MT1 and MT2, a result of measuring growth rates of the UQCRB mutant cell strains MT1 and MT2 shows that the growth of the UQCRB mutant cell strains MT1 and MT2 are inhibited when processing Fatostatin, Mevastatin, YM-53601 that are cholesterol synthesis inhibitor.
- Expression levels of three miRNAs of hsa-miR-1323, hsa-miR-512-3p, and hsa-miR-10a-5p, which are selected by
Embodiment 1, were measured at the same time with cholesterol metabolic process enzymes in three cancer cell strains which are known as there was over-expressed UQCRB. The results of measuring expression levels are shown inFIGS. 14 to 18 . - It can be seen from
FIGS. 14 to 18 that as expression amounts of the cholesterol metabolic process enzymes increase in the three cancer cell strains which are known as there is over-expressed UQCRB, whereas expression amounts of the three miRNAs decrease in the three cancer cell strains, the three miRNAs according to embodiments of the inventive concept may be used as a biomarker for diagnosing UQCRB and a disease related to a cholesterol metabolic process involved in UQCRB. - A method according to embodiments of the inventive concept may expect and diagnose a UQCRB-related disease and a disease involved in cholesterol biosynthesis related to UQCRB simply by measuring a specific miRNA expression level.
- While the inventive concept has been described with reference to exemplary embodiments, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the inventive concept. Therefore, it should be understood that the above embodiments are not limiting, but illustrative.
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