Method for expecting and diagnosing UQCRB-related disease

Abstract

Disclosed is a method for expecting and diagnosing UQCRB-related disease, and more particularly, related 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.

Claims

1. A method for treating a Ubiquinol-cytochrome C Reductase Binding Protein (UQCRB)-related liver cancer, the method comprising: (i) detecting the UQCRB-related disease by determining that a sample from a subject over-expresses UQCRB protein comprising measuring an expression level of all of hsa-miR-1323, hsa-miR-512-3p and hsa-miR-10a-5p, in a sample from a subject; comparing the measured expression levels with an expression level for said miRs of a HEK293 cell; (ii) diagnosing the subject as having a UQCRB-related disease when the sample overexpresses UQCRB protein as indicated by a decrease in the level of all of hsa-miR-1323, hsa-miR-512-3p and hsa-miR-10a-5p relative to expression levels for said miRs of a HEK293 cell determined in step (i); and (iii) administering a cholesterol synthesis inhibitor YM-53601 to the subject wherein the UQCRB-related disease has been detected in step (ii).

2. The method of claim 1, wherein the measuring of the expression level of all of hsa-miR-1323, hsa-miR-512-3p and hsa-miR-10a-5p is carried out by reverse transcriptase polymerase chain reaction or real time polymerase chain reaction.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a process that introduction of UQCRB induces generation of ROS in mitochondria and stabilizes HIF-1.alpha. to induce angiogenesis.

(2) FIG. 2 shows a process of selecting miRNA through comparison of expression patterns in UQCRB mutant cell strains.

(3) FIGS. 3A-3C show 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.

(4) FIG. 4 shows a process of selecting a cholesterol metabolic process.

(5) FIG. 5 shows mRNAs significantly upregulated in mutant UQCRB-expressing cells compared to the control.

(6) FIG. 6 shows a list of genes upregulated in both of MT1 and MT2 cell lines.

(7) FIG. 7 shows the mRNA expression levels of HMG-CoA reductase (HMGCR), pyrophosphomevalonate decarboxylase (MVD), lanosterol synthase (LSS), mutant UQCRB, and endogenous UQCRB, measured by RT-PCR.

(8) FIG. 8 shows western blot analysis for measuring the protein levels of HMGCR, MVD, LSS and endogenous UQCRB.

(9) FIG. 9 shows the relative mRNA expression level of 5 genes of FIG. 7.

(10) FIG. 11 represents MTT colorimetric assay showing the cell growth of HEK293, MT1, and MT2 cells treated with a cholesterol inhibitor.

(11) FIG. 12 represents MTT colorimetric assay showing the cell growth of HEK293, MT1, and MT2 cells treated with a cholesterol inhibitor.

(12) FIG. 13 represents MTT colorimetric assay showing the cell growth of HEK293, MT1, and MT2 cells treated with a cholesterol inhibitor.

(13) FIG. 14 shows the expression level of miR-1323, miR-512-3p and hsa-miR-10a-5p in control MT1, MT2, HepG2, PC3 and HCT116 cell lines.

(14) FIG. 10 shows the relative protein expression level of 4 proteins of FIG. 8.

(15) FIG. 15 shows the mRNA expression levels of HMGCR, MVD, LSS, endogenous UQCRB and mutant UQCRB measured by RT-PCR in HEK293, MT1, MT2, HepG2, PC3 and HCT116 cell lines.

(16) FIG. 16 shows the relative mRNA expression level of 5 genes of FIG. 15.

(17) FIG. 17 shows the protein expression levels of HMGCR, MVD, LSS and endogenous UQCRB measured by western blotting.

(18) FIG. 18 shows the relative protein expression level of 4 proteins of FIG. 17.

DETAILED DESCRIPTION

(19) Hereafter, embodiments of the inventive concept will be described in detail. However, the inventive concept may not be restrictive embodiments described below.

(20) <Manufacturing example> Manufacturing Cell Strain

(21) 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.

<Embodiment 1> Selecting UQCRB-Related miRNA

<Embodiment 1-1> Separating and Sequencing miRNA

(22) 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.

(23) 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.

<Embodiment 1-2> Selecting miRNA Through Comparison of Expression Patterns

(24) 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.

(25) |log.sub.2 FC|>1, |log.sub.2 CPM|>2, FDR<0.05

(26) FC: a difference of expression levels

(27) CPM: the own expression of the factor in each cells.

(28) 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.

(29) 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.

<Embodiment 1-3> Expression Amount of miRNA According Presence or Absence of Processing UQCRB-1 (20M)

(30) 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.

(31) 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.

<Embodiment 2> Selecting UQCRB-Related Pathway

<Embodiment 2-1> Separating mRNA and Selecting Cholesterol Metabolic Process

(32) 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.

(33) 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.

<Embodiment 2-2> Verifying Validity for Selection of Cholesterol Metabolic Process

(34) 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.

<Embodiment 2-3> Verifying Validity for Selection of Cholesterol Metabolic Process

(35) 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.

(36) 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.

<Embodiment 3> Examining the Relativity of miRNA and Cholesterol Metabolic Process Enzymes

(37) 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 in FIGS. 14 to 18.

(38) 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.

(39) 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.

(40) 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.