KR101767605B1 - Methods of quantitative analysis for lactic acid content using NDRG3 protein - Google Patents

Abstract

The present invention relates to a method for quantifying lactic acid using an NDRG3 protein, and more particularly, to a method for quantifying lactic acid by measuring the amount of NDRG3 protein expression, thereby allowing prediction and analysis of cell proliferation and angiogenesis based on NDRG3 protein And can be usefully used for diagnosing, managing, treating, preventing, or related to the development and research of related diseases such as ischemic diseases in the future.

Description

Methods for quantitative analysis of lactic acid using NDRG3 protein (NDRG3 protein)

The present invention relates to a method for quantifying lactic acid using NDRG3 (N-myc downstream regulated gene 3) protein, and more specifically, to a method for quantifying the content of lactic acid in a cell by measuring the expression level of NDRG3 protein and its mutant .

The measurement of the content of intracellular metabolites is widely used in biology research and medicine industry because evaluation of metabolic activity through metabolism analysis is essential for evaluation of bio-condition and control and improvement of disease control. Lactic acid is produced in muscle cells, red blood cells, brain, and other tissues during hypoxic energy production, and is normally present in low concentrations in the blood. During the aerobic energy production process, mitochondria produce glucose and oxygen to produce adenosine triphosphate (ATP). When the oxygen concentration decreases or the mitochondria fail to function properly, the organism is converted into a hypoxic energy production process, Metabolism without function results in the production of ATP, the product of which is mainly lactic acid, which accumulates faster than the rate at which it can break down. Lactic acid is one of the most commonly observed metabolites in metabolic processes. Lactate in biological samples such as blood is a marker for detection of a variety of biological conditions and diseases, including ischemic diseases. It is strongly demanded from the standpoint.

Hypoxia-inducible factor-1 (HIF-1) is a nuclear transcription factor that induces a large amount of induction in a hypoxic state. In order to maintain the homeostasis of oxygen in the cells, erythropoiesis, angiogenesis, And to express genes involved in the glycolysis process. HIF-1 is divided into two groups, α and β. HIF-1α is a transcription factor, which is degraded at normal oxygen partial pressure but is known to stabilize protein under hypoxic condition. Stabilized HIF-1α binds to HIF-1β and translocates to the nucleus and expresses genes involved in angiogenesis and metabolism (Semenza et al., 1999; Wang et al., 1995; Wang and Semenza, 1995). The activity of HIF-1 has recently emerged as a major new drug target because it is closely related to pathological mechanisms of various chronic metabolic diseases including cancer development and metastasis, rheumatoid arthritis, ischemic stroke, and arteriosclerosis. However, it has been reported that the progress of hypoxic reaction can not be grasped completely by the promotion of HIF, and there is a HIF-independent pathway regulated by this. Therefore, in order to quantitate intracellular lactic acid under hypoxic response, it is necessary to combine and analyze not only HIF but also the promoter of factor involved in HIF independent-regulated pathway.

The gene for the NDRG (N-myc downstream regulated gene) gene was first identified as Ndr1, a gene whose expression is increased in N-Myc mutant mice. Since its human ortholog NDRG1 has been identified in human cell lines, it has been called by names such as Drg1, Cap43, RTP / rit42, and the like. Four different types of constitutive genes have been reported in the NDRG family of genes. These four types of NDRG1, NDRG2, NDRG3 and NDRG4 have high homology, but their expression pattern varies considerably depending on the development and growth of the individual (2002), 35-44, Deng et al., Int J Cancer, 2003 (106), 342-7). Therefore, although these genes are expected to function differently from these expression differences, there is no report on their definite function.

Conventional lactate determination methods include lactic acid analysis using TLC (thin layer chromatography), lactic acid analysis using HPLC (high performance liquid chromatography), and lactic acid analysis using lactate dehydrogenase. However, the method of quantifying intracellular lactic acid using NDRG3 protein There was a need for a quantitative method.

Accordingly, the present inventors have made efforts to find a method for quantifying lactic acid produced under hypoxic conditions. As a result, it has been confirmed that lactic acid produced in the hypoxic reaction binds to NDRG3 protein and accumulates, so that the NDRG3 protein can be used for lactic acid determination And completed the present invention.

It is an object of the present invention to provide a method for quantifying lactic acid using NDRG3 (N-myc downstream regulated gene 3) protein.

In order to accomplish the object of the present invention, the present invention provides a method for quantifying intracellular lactic acid, comprising the step of measuring the expression level of NDRG3 (N-myc downstream-regulated gene 3) protein in a cell.

In addition,

1) treating the cultured cells with the NDRG3 protein; And

2) measuring the expression level of NDRG3 protein in the cells of step 1).

The present invention provides a method for quantifying lactic acid produced under hypoxic conditions and a method for quantifying lactic acid by measuring the expression amount of NDRG3 (N-myc downstream-regulated gene 3) protein, thereby determining cell proliferation and angiogenesis And the like, thereby making it possible to diagnose related diseases such as cancer and ischemic diseases,

Treatment, prevention, or related drug development and research.

Figure 1a shows a pCAGGS plasmid encoding human NDRG3 for the production of NDRG3 (N-myc downstream regulated gene 3) over-expressing transgenic mice.
FIG. 1B shows that human NDRG3 gene is inserted into the genomic DNA of TG-2, TG-8 and TG-13 mice in the production process of NDRG3 overexpressing transgenic mouse.
FIG. 1C shows the expression of human NDRG3 gene in liver tissues of established NDRG3 overexpressing transgenic mice TG-2, TG-8 and TG-13.
FIG. 2 is a view showing the antigen-antibody reaction between the prepared rabbit anti-NDRG3 antibody and human NDRG3 (N66D) mutant.
FIG. 3A is a schematic diagram illustrating a process of selecting a candidate protein binding to PHD2 (prolyl hydroxylase domain 2).
3B is a diagram showing a protein binding to PHD2.
FIG. 3C shows the binding of PHD2 and NDRG3 proteins.
Figure 4a is a diagram showing the binding of hypoxia (hypoxia, 1% O ₂₎ under (Figure 4a, above) and (in-vitro) (Figure 4a, below) in vitro and PHD2 NDRG3.
FIG. 4b shows the induction of NDRG3 protein by inhibition of PHD2 in MCF-7 cells under normoxia (21% O ₂ ).
4C shows the induction of NDRG3 protein by inhibition of PHD2 in HeLa cells under normal oxygen conditions.
5A is a graph showing an increase in expression of NDRG3 protein in the PHD family and VHL deletion in a normal oxygen state.
FIG. 5B shows the binding of the PHD family group and the NDRG3 protein.
FIG. 6A shows a PHD2 docking site of NDRG3 through a protein-protein docking simulation.
FIG. 6B is a view for confirming the binding strength between PHD2 docking site and PHD2 of NDRG3 as confirmed by docking simulation.
FIG. 7A is a graph showing an increase in expression of NDRG3 protein after treatment with MG132, a proteasome inhibitor, in a normal oxygen state.
FIG. 7B shows the ubiquitination of NDRG3 protein under normal oxygen conditions.
FIG. 8A shows accumulation of NDRG3 protein according to oxygen concentration. FIG.
FIG. 8B shows the accumulation of NDRG3 protein according to a hypoxic state in a cell.
FIG. 8C shows the accumulation of NDRG3 protein in various kinds of cancer cells according to continuous hypoxic conditions.
FIG. 8D shows the inhibition of ubiquitination of NDRG3 protein in a hypoxic state.
FIG. 9A is a graph showing changes in NDRG3 protein expression according to a normal oxygen state and a continuous hypoxic state. FIG.
FIG. 9B is a graph showing a change in the expression of NDRG3 protein when recovering from a hypoxic state to a normal oxygen state.
FIG. 10A is a view showing the hydroxylation target site of the NDRG3 protein. FIG.
FIG. 10B is a view showing the hydroxylation target site mutation of NDRG3 protein and the binding of PHD2 / VHL.
11A is a graph showing changes in RNA expression of NDRG3 according to changes in oxygen state.
FIG. 11B is a graph showing changes in expression of NDRG3 protein due to inhibition of HIF protein.
FIG. 11C shows changes in the expression of NDRG3 protein in MCF-7 (HIF-1 ^{+ / +} and VHL ^{+ / +} ) and 786-O (HIF-1 ^{- / -} and VHL ^{- / -} ) cells under hypoxic conditions to be.
12A is an analysis of the functions of NDRG3 and HIF-1 associated with hypoxic response.
FIG. 12B is an analysis of the function of each gene up-regulated in cells overexpressing NDRG3 mutation (N66D) protein and cells under hypoxic state in normal oxygen state.
13A is a graph showing changes in angiogenic activity due to deletion of NDRG3 in a hypoxic state.
FIG. 13 (b) is a graph showing changes in expression of neovascularization markers IL8, IL1?, IL1? COX-2 and PAI-1 due to NDRG3 deletion in a hypoxic state.
13C is a diagram for confirming in vivo angiogenesis by NDRG3 deletion.
FIG. 14A is a graph showing changes in cell growth due to deletion of NDRG3 in a hypoxic state. FIG.
FIG. 14B is a chart showing changes in tumor formation due to deletion of NDRG3 and / or HIF.
FIG. 14C shows the change in tumor formation due to the expression of the ectopic mutant NDRG3 (N66D).
FIG. 14D shows the change in tumor volume in mice transplanted with NDRG3 and / or HIF deleted tumor cells.
FIG. 14E is a graph showing changes in tumor volume in a mouse transplanted with an ectopic mutant NDRG3 (N66D) -expressing tumor cells.
FIG. 14f is a graph showing changes in the expression of the cell proliferation marker Ki-67 and the neovascularization marker IL8 and CD31 proteins by NDRG3 or HIF deletion in tumor tissue.
FIG. 14g is a graph showing changes in the expression of neovascularization markers by NDRG3 or HIF deletion in tumor tissue.
FIG. 15A is a graph showing changes in protein expression and lactate production of NDRG3 and HIF-1.alpha. Depending on the oxygen state. FIG.
FIG. 15B is a graph showing changes in protein expression and lactate production of NDRG3 and HIF-1α according to hypoxic state after treatment with sodium oxamate, an LDHA (lactate dehydrogenase A) inhibitor.
FIG. 15C is a graph showing changes in expression of NDRG3 protein due to inhibition of lactic acid production in a hypoxic state.
FIG. 15D is a graph showing changes in expression of NDRG3 protein due to inhibition of glycolysis by 2-deoxyglucose (2-DG) in a hypoxic state.
FIG. 15E is a graph showing changes in the expression of NDRG3 protein due to excessive lactic acid production by pyruvate or LDHA.
FIG. 15f shows the change in ubiquitination of NDRG3 protein by lactic acid production.
16A shows a recombinant NDRG3 (G138W) mutant mutated at the lactic acid binding site of the NDRG3 protein and a recombinant NDRG3 wild type protein.
FIG. 16B shows the binding of recombinant NDRG3 wild type and recombinant NDRG3 (G138W) mutant protein to lactic acid.
16 (c) is a graph showing changes in the expression of mutants mutating the L-lactic acid binding site of NDRG3 in a hypoxic state.
16D shows the expression of NDRG3 protein by reoxygenation.
FIG. 17A is a graph showing cell growth inhibition by lactic acid production inhibition and ectopic mutant NDRG3 (N66D) expression in a hypoxic state (3% O ₂ ).
FIG. 17B is a graph showing changes in cell colony formation due to inhibition of lactic acid production and / or expression of the ectopic mutant NDRG3 (N66D).
FIG. 17C is a chart showing changes in cell growth due to LDHA deletion and / or expression of an ectopic mutant NDRG3 (N66D) in a hypoxic state (3% O ₂ ).
FIG. 17D shows changes in tumor formation in mice transplanted with tumor cells expressing LDHA deletion and / or heterologous variant NDRG3 (N66D).
FIG. 17E is a graph showing changes in NDRG3 protein expression due to inhibition of lactic acid production and / or expression of an ectopic mutant NDRG3 (N66D) in a continuous hypoxic state.
FIG. 17f shows the change in lactic acid production due to the expression of the heterologous variant NDRG3 (N66D) in a continuous hypoxic state.
FIG. 18 is a graph showing inhibition of lactic acid production and / or changes in angiogenesis due to the expression of an ectopic mutant NDRG3 (N66D) in a hypoxic state.
FIG. 19A is a diagram showing a change in protein phosphorylation due to deletion of NDRG3 in a hypoxic state. FIG.
FIG. 19B is a graph showing changes in the activity of ERK1 / 2 protein according to the expression of NDRG3 protein.
FIG. 19C is a graph showing changes in Raf-ERK1 / 2 activity due to NDRG3 deletion in a continuous hypoxic state.
Figure 19d shows the binding of NDRG3 and c-Raf proteins in vitro (left) and intracellular (right).
FIG. 19E shows the change of Raf-ERK1 / 2 activity by NDRG3 protein deletion or ectopic mutant NDRG3 (N66D) expression.
FIG. 19f shows the change in phosphorylation of c-Raf due to inhibition of PKC-beta activity by the ectopic mutant NDRG3 (N66D) and / or PKC-I.
FIG. 19g is a graph showing changes in NDRG3 protein expression and Raf-ERK1 / 2 activity upon inhibition of lactic acid production in a hypoxic state.
FIG. 20A shows the binding between NDRG3 and RACK-1 proteins. FIG.
20B is a graph showing changes in Raf-ERK1 / 2 activity due to the expression of NDRG3 (N66D), RACK-1 and / or Raf, which are NDRG3 deleted or heterologous mutants.
FIG. 20C shows the binding between the ectopic mutant NDRG3 (N66D) and PKC-beta due to RACK1 deletion in a hypoxic state.
FIG. 20D is a graph showing changes in ERK1 / 2 activity due to inhibition of PKC-beta activity in a hypoxic state.
FIG. 20E shows the formation of recombinant heterologous mutant NDRG3 (N66D) and c-Raf, RACK1 and PKC-beta complexes.
FIG. 20F shows the formation of NDRG3, c-Raf, RACK1 and PKC-beta complexes through simulation.
21A is a view showing the tumor formation of NDRG3-overexpressing transgenic mice and control mice.
FIG. 21B shows the results of confirming tumor formation in lung, intestine and hypogastrium of NDRG3-overexpressing transgenic mice and control mice.
FIG. 21C is a diagram showing B cells and T cells in mesenteric lymph node, spleen and liver tissues of NDRG3-overexpressing transgenic mice and control mice.
FIG. 21D is a graph showing the expression profiles of glutamine synthetase (GS) and heat shock protein 20 (HSP), which are hepatocellular carcinoma (HCC), in the liver tissues of NDRG3 overexpressing transgenic mice and control mice, The expression of PCNA and Ki-67 as markers.
FIG. 21E shows the expression of ERK1 / 2 activity and mRNA expression of neovascularization markers in liver tissues of NDRG3-overexpressing transgenic mice and control mice.
FIG. 22 shows the expression of NDRG3 and ERK1 / 2 activity in liver cancer patients.
Figure 23 is a schematic representation of the mechanism of NDRG3 as a mediator of the Raf-ERK pathway induced by lactic acid in continuous hypoxic response.
24 shows the expression of NDRG3 protein by lactic acid treated exogenously when LDHA expression is decreased in a hypoxic environment.
25 is a view showing the expression of NDRG3 protein by lactic acid treated with exogenous after oxamate treatment in a hypoxic environment.
26 is a graph showing the expression of NDRG3 protein when MCT (monocarboxylate transporter) 1 is inhibited in normal and hypoxic environments to block intracellular influx of exogenously treated lactic acid.
FIG. 27 is a graph showing the expression of NDRG3 protein and the dependence of MCT 1 on exogenously treated lactic acid after induction of oxamate treatment and glucose deficiency in a normal oxygen environment.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for quantifying intracellular lactic acid comprising the step of measuring the expression level of NDRG3 (N-myc downstream-regulated gene 3) protein in a cell.

The cells are preferably collected from any one selected from the group consisting of tissue, blood, plasma, serum, saliva, semen and urine, but are not limited thereto.

The NDRG3 protein But it is not limited thereto, but it is preferably composed of the amino acid sequence represented by SEQ ID NO: 1.

The NDRG3 protein is preferably decomposed when lactic acid is not bound, but accumulates when lactic acid is bound, but not limited thereto.

The lactic acid preferably binds to the 62nd aspartic acid, the 138th glycine, the 139th alanine or the 229th tyrosine in the amino acid sequence of the NDRG3 protein, but is not limited thereto.

In addition,

1) treating the cultured cells with the NDRG3 protein; And

2) measuring the expression level of NDRG3 protein in the cells of step 1).

The cultured cells in the step 1) are preferably cultured cells or animal cells collected from any one selected from the group consisting of tissue, blood, plasma, serum, saliva, semen and urine, but the present invention is not limited thereto.

The lactic acid in step 2) is preferably bound to the 62nd aspartic acid, the 138th glycine, the 139th alanine or the 229th tyrosine in the amino acid sequence of the NDRG3 protein, but is not limited thereto.

The method of quantifying lactic acid in step 2) is preferably, but not limited to, comparing the expression level of a normal NDRG3 protein with that of a NDRG3 protein having a mutated lactic acid binding site.

The NDRG3 protein is preferably decomposed when lactic acid is not bound, but accumulates when lactic acid is bound, but not limited thereto.

The NDRG3 protein is preferably an NDRG3 recombinant protein fused with a fluorescent protein, but is not limited thereto.

The fluorescent protein may be selected from the group consisting of Red Fluorescent Protein (RFP), Enhanced Red Fluorescent Protein (ERFP), Cyan Fluorescent Protein (CFP), Enhanced cyan Fluorescent Protein , ECFP), Yellow Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), mtFP (monomeric teal Fluorescent Protein), Green Fluorescent Protein (GFP) Green fluorescent protein (EGFP), but it is not limited thereto.

The expression level can be determined by in-vitro binding assay, enzyme immunoassay (ELISA), immunohistochemical staining, Western blotting, protein chip and real-time fluorescence imaging Method), but it is not limited thereto.

In a specific embodiment of the present invention, the present inventors have identified NDRG3 (NDRG3) as a candidate protein that binds to PHD2 (prolyl hydroxylase domain 2) protein involved in the activity of HIF in order to find a hypoxia-inducible factor (HIF) NDRG3 over-transformed C57 / BL6 mice TG-2, TG-8 and TG-13 were prepared to confirm the molecular biochemical function of NDRG3 protein in hypoxia (Figs. 1A to 1C , And 3A-3C), anti-NDRG3 polyclonal antiserum was obtained in rabbit as an antigen as a recombinant human NDRG3 protein (amino acid 32-315, SEQ ID NO: 2) and NDRG3 peptide (amino acid 244-255, No. 3) to prepare levit anti-human NDRG3 polyclonal antibody (see FIG. 2).

Further, in a specific embodiment of the present invention, the present inventors have found that in order to confirm the relationship between PHD2 and NDRG3 protein according to oxygen status, immunoprecipitation, western blotting, in-vitro pooling, Pull-down assay and RT-PCR showed that the PHD2 protein binds to the PHD2 protein among the four PHD family members, and the VHL suppression, the target protein of the PHD2 and E3 ubiquitin ligase complexes, Confirming that the NDRG3 protein is an intrinsic substrate of PHD2 and that the NDRG3 protein is a substrate for the post-PHD2 / VHL-mediated transcription process (Figs. 4A to 4C and Figs. 5A and 5B) 5b). Further, as a result of performing a protein-protein docking simulation, it was confirmed that the PHD2 docking site of NDRG3 was found and that the 47th or 66th amino acid position of NDRG3 in the PHD2 docking site was more important (FIGS. 6A and 6B) 6b). In vivo ubiquitination assay, PHD2 binds to the NDRG3 docking site in the normal oxygen state. Thus, NDRG3 is converted to ubiquitin (NAD) via the PHD2 / VHL-mediated proteasome pathway (See FIGS. 7A and 7B).

Further, in a specific embodiment of the present invention, the present inventors conducted immuno-precipitation, Western blotting, immunofluorescence staining, and in vivo ubiquitination assay to confirm the oxygen-dependency of the NDRG3 protein. As a result, The expression of NDRG3 protein is decreased and the expression and accumulation of NDRG3 protein are increased as the hypoxic state is continued in various cancer cells such as the neck, kidney and large intestine. On the contrary, when the oxygen state is restored to the normal oxygen state, the expression of NDRG3 protein is slowly removed (See Figs. 8a-8d and Figs. 9a and 9b), particularly when mass spectrometry is carried out to see that the 294th proline of NDRG3 is hydroxylated and the proline amino acid is alanine Alanine) increased expression, indicating that the 294th proline hydroxylation of NDRG3 was a hypoxic target site 10a and 10b). In addition, HIF decreases gradually after the increase in expression at the early stage of hypoxia, but NDRG3 protein plays an important role in HIF-independent, hypoxic response by confirming that the expression and accumulation of NDRG3 protein increases as the hypoxic state persists (See Figs. 11A to 11C).

Further, in a specific embodiment of the present invention, the present inventors performed gene expression profiling to confirm the biochemical function of the NDRG3 protein in the hypoxic reaction. As a result, unlike HIF, the NDRG3 protein exhibited cell proliferation, In vivo angiogenesis assay and in-vivo angiogenesis assay (Fig. 12A and Fig. 12B), which were found to be most related to angiogenesis, cell growth, As a result of transplanting tumor volume measurement and MTT assay, it was confirmed that NDRG3 promotes neovascularization, cell proliferation and tumor growth in a hypoxic reaction (FIGS. 13A and 13C, and FIGS. 14A to 14G).

Further, in a specific embodiment of the present invention, the inventors of the present invention found that in the hypoxic state, lactic acid production measurement, Western blotting, immunoprecipitation, in vitro ubiquitin As a result of the analysis, lactic acid was produced by the hypoxic reaction, and the lactic acid produced bound to the lactic acid binding site including the 62nd, 138th, 139th or 229th amino acids of NDRG3 inhibited the ubiquitination of the NDRG3 protein and inhibited the HIF- MTT assay, colony forming assay, in vivo implanted tumor volume measurement, and tube formation assay (see FIGS. 15A to 15F and FIGS. 16A to 16D) As a result, when the ectopic mutant NDRG3 (N66D) was expressed in the inhibition of lactic acid production, cell proliferation and angiogenesis were promoted, NDRG3 acts as an important mediator of lactic acid-induced cell proliferation and angiogenesis in persistent hypoxic conditions (see FIGS. 17A to 17F and FIG. 18).

Further, in a specific embodiment of the present invention, the inventors of the present invention conducted in vitro kinase assays, pull-down assays, immunoprecipitation assays and immunoassays to confirm the molecular regulatory mechanism of NDRG3- Western blotting showed that inhibition of NDRG3 expression by hypoxic inhibition of c-Raf and ERK1 / 2 phosphorylation inhibited NDRG3 in the c-Raf-ERK1 / 2 pathway activated by hypoxia-induced lactic acid lt; RTI ID = 0.0 > c-Raf-ERKl / 2 < / RTI > signal (see Figures 19a-19g). In addition, NDRG3 binds to RACK1 to induce PKC-β, forms NDRG3-RACK1-PKC-β-c-Raf complex with c-Raf, and induces PKC- -Raf protein was phosphorylated and c-Raf / ERK signal was activated, it was confirmed that NDRG3 regulated by lactic acid was a scaffold protein that regulates the activity of c-Raf (see Figs. 20A to 20F ).

Further, in a specific embodiment of the present invention, the present inventors conducted immunohistochemical anaylsis using the above-prepared NDRG3 transgenic mouse to confirm pathological changes caused by NDRG3, and found that NDRG3 and activated Western blotting and RT-PCR were performed to confirm expression of ERK1 / 2 protein. As a result, tumors were found in various organs such as lung, intestine and liver of NDRG3 transgenic mouse, and mesenteric lymph node And lymphocyte-expressing B-cells and T-cells were found in the secondary lymphoid organs such as the spleen, and the expression of the cell proliferation marker and the neovascularization marker was increased (see FIGS. 21A to 21E). Also, by confirming that the expression of NDRG3 protein and the activity of ERK1 / 2 protein are promoted in liver cancer tissue samples isolated from liver cancer patients (see FIG. 22), abnormal expression of NDRG3 activates c-Raf / ERK pathway, And promotes angiogenesis.

In addition, in a specific embodiment of the present invention, the present inventors treated L-lactic acid with LDHA-deficient Huh-1 cells to confirm the effect of exogenous lactic acid treatment on NDRG3 protein expression and hypoxic response, To investigate the relationship between exogenous lactate treatment and oxamate treatment, Huh-1 cells were treated with sodium oxamate, L-lactic acid, and Western blotting As a result, it was confirmed that exogenously treated lactic acid did not dose-dependently restore NDRG3 and did not affect the level of HIF-1α protein (FIG. 24), indicating that lactate production was inhibited by oxamate treatment (FIG. 25) The results of this study are as follows. In addition, in order to confirm the effect of the exogenous lactate treatment on the intracellular inflow of lactic acid, Huh-1 cells inhibited MCT (monocarboxylate transporter) 1 were treated with lactic acid and then subjected to Western blotting. Induced HH-1 cells to induce oxamate treatment or glucose deficiency, exogenously treat L-lactic acid, and inhibit MCT 1 and Western blotting. As a result, inhibition of MCT 1 inhibited NDRG3 expression by exogenous lactate (Fig. 26 and Fig. 27).

Therefore, the NDRG3 protein of the present invention is analyzed for the mechanism of inhibition of degradation by reacting with lactic acid, and it is confirmed that the lactic acid produced in the hypoxic reaction binds to and accumulates in the NDRG3 protein, so that the NDRG3 protein can be used for lactic acid determination He said.

The present invention provides a method for quantifying lactic acid by measuring the expression amount of NDRG3 protein, which is an important mediator such as cell proliferation and angiogenesis accumulation by HIF-independent accumulation of the NDRG3 protein by lactic acid, Cell proliferation and angiogenesis can be predicted and analyzed, so that it can be usefully used for diagnosis, management, treatment, prevention of related diseases such as ischemic diseases or research and development of related new drugs.

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> NDRG3  Production of Over-expressing Transgenic Mice

In order to examine the effect of NDRG3 expression on biochemical characteristics, NDRG3 transgenic mice were constructed.

Specifically, as shown in the schematic diagram of FIG. 1A, the human NDRG3 cDNA sequence (SEQ ID NO: 1) is inserted into a CAG-promoter (cytomegalovirus enhancer and chicken beta -actin promoter) and levitic- After linearization by cloning into a pCAGGS plasmid containing a polyadenylation sequence, the linearized construct was injected into the pronuclei of three C57 / BL6 mouse embryos. The tail of each of the three C57 / BL6 mice was cut into a mouse tail lysis buffer (60 mM Tris pH 8.0 / 100 mM EDTA / 0.5% SDS, 500 ug / ml Proteinase K), and then genotypes were confirmed by PCR using the primers shown in Table 1 (FIG. 1B). After confirming expression of the transgenic NDRG3 from each of the three C57 / BL6 mice, each mouse of the F1 generation was obtained by crossing with the normal mouse, and genotyping was confirmed in the same manner as above to obtain a transgenic mouse expressing NDRG3 Lines TG-2, TG-8 and TG-13 were established.

Primer The sequence (5 '- &gt; 3') Human NDRG3
Forward AACCATAAATCCTGTTTCAATG (SEQ ID NO: 9) Reverse TCCACAACATTGGTTGTCAGG (SEQ ID NO: 10)

In order to confirm the overexpression of NDRG3 in the NDRG3 overexpressed transgenic mice TG-2, TG-8 and TG-13, PCR was performed using the primers shown in Table 1 as described above (FIG.

< Example  2> human NDRG3  Preparation of antibodies

In order to prepare an antibody against the human NDRG3 protein, the amino acid 32-315 sequence (SEQ ID NO: 2) of the recombinant human NDRG3 protein was cloned into the pET-28a vector and transformed into E. coli strain BL21, and the transformed E. coli strain BL21 The cells were cultured in an LB medium containing 100 mg / ampicillin at 37 until the OD value reached OD ₅₉₅ = 0.5. Then, 1 mM IPTG (isopropyl-β-D-thiogalactoside) Hr. &Lt; / RTI &gt; The strain was then centrifuged at 4,000 rpm for 4 to 15 minutes, resuspended in buffer [50 mM HEPES (pH 7.5) and 150 mM NaCl], and sonicated in an ice cube for 3 minutes with a pulse of 2 minutes Sonication, and centrifuged again at 12,000 rpm for 4 to 15 minutes. Then, the recombinant protein was purified according to the manufacturer's procedure through Ni-NTA agarose affinity chromatography using Ni-NTA agarose resin (Resin). The purified human NDRG3 recombinant protein (amino acid 32-315) was then immunized with rabbit (New Zealand White). Production of polyclonal antisera was commissioned by AbFrontier (Seoul, Korea). Antiserum was purified by affinity chromatography using NDRG3 peptide (QNDNKSKTLKCS; amino acids 244 to 255, SEQ ID NO: 3) to obtain anti-NDRG3 antibody. In order to confirm the specificity of the anti-NDRG3 antibody, Western blotting was performed.

Specifically, Myc-tagged NDRG1 expression vectors, Myc-tagged NDRG2 expression vectors, Myc-tagged NDRG4 expression vectors, and Myc-tagged NDRG3 expression vectors were cloned. In order to prepare the NDRG3 variant, the Myc-tagged NDRG3 expression vector was used as a template and site-directed mutagenesis was performed according to the manufacturer's procedure using the KOD-Plus-Mutagenesis Kit (Toyobo) as the primer shown in Table 2 to prepare NDRG3 Tagged NDRG3 (N66D) variant in which the 66th asparagine (Asn, N) in Aspergillus was replaced with aspartic acid. Then, each of the NDRG1-Myc, NDRG2-Myc, NDRG4-Myc and NDRG3 (N66D) -Myc was cultured in a DMEM medium containing 10% FBS (Gibco BRL) and 100 U / ml penicillin (Gibco BRL) One HEK293T cell (ATCC) was transfected using Lipofectamine (Invitrogen) according to the manufacturer's procedure. The transformed cells were then cultured and recovered in a 37 CO ₂ incubator (Sanyo) for 24 hours. Then, the recovered cells were dissolved in a lysis buffer (1% Triton X-100, 150 mM NaCl, 100 mM KCl, 20 mM HEPES (pH 7.9), 10 mM EDTA, protease inhibitor cocktail And the protein lysate (30) of the cells was electrophoresed on 9% SDS-PAGE, and then transferred to nitrocellulose membranes (PALL Life Sciences). Next, the obtained anti-NDRG3 antibody was reacted with the primary antibody and reacted, and HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane, which was confirmed by ECL (Pierce chemical co., USA) 2).

primer order NDRG3 (N66D)
Sense GACCATAAATCCTGTTTCAATGCATTCTTT (SEQ ID NO: 11) Antisense GAGGCCAATGTCATGATATGTTAGTATAAC (SEQ ID NO: 12)

As a result, as shown in Fig. 2, the anti-NDRG3 antibody produced was found to bind only to the NDRG3 (N66D) mutant except for NDRG1, NDRG2 and NDRG4, so that the anti-NDRG3 antibody binds to the NDRG3 antibody and the mutant Lt; RTI ID = 0.0 &gt; NDRG3 &lt; / RTI &gt; of the present invention (Figure 2).

< Example  3> PHD2 As a binding protein NDRG3  Confirm

In normal oxygen, PHI2 (Prolyl-hydroxylase domain 2) has been reported to modulate the activity of HIF-1α (Hypoxia-inducible Factor-1a), an important transcription factor for the expression of genes induced by hypoxia (Wenger, RH et al , Curr Pharm Des., 2009 (15), 3886-3894). Therefore, immunoprecipitation, immunostaining and micro-LC-MS / MS analysis were performed to find HIF-independent factors regulated by PHD2 in hypoxia responses.

Specifically, in order to identify the PHD2 binding protein, a construct for encoding Flag-tagged PHD2 was prepared as shown in the schematic diagram of FIG. 3A, and then 10% FBS (Gibco BRL) and 100 U / ml penicillin , The construct and control mock were transfected according to the manufacturer's procedure using Lipofectamine (Invitrogen) to MCF-7 cells (ATCC) cultured with DMEM medium transfection). Then, the in the transformed cell proteasome (proteasome) inhibitor, it processes the 10 μM MG132 contains a mixture consisting of _{92-94% N 2, 5% CO} 2 and 1% O ₂ O ₂ / CO using ₂ incubator (Sanyo) to maintain a low oxygen (hypoxia) for 24 hours, dissolved in buffer [1% Triton X-100, 150 mM NaCl, 100 mM KCl, 20 mM HEPES (pH 7.9), 10 mM EDTA, Protease inhibitor cocktail (Roche)]. The protein lysate (1 mg) of the cells was reacted with anti-FLAG M2 affinity gel (Sigma) for 4 days, centrifuged to immunoprecipitate, electrophoresed on 9% SDS-PAGE, (Coomassie Brilliant Blue, CBB). Protein bands exhibiting different immunoprecipitation patterns in the PHD2-Flag sample were then separated from the SDS-PAGE gel and digested with trypsin for micro-LC-MS / MS analysis. The digested protein was injected into a fused silica capillary column (100 mm inner diameter, 360 mm outer diameter) containing a 5-mm particle size Aqua C18 reversed phase column of 8 cm. The columns were transferred to an Agilent HP 1100 4th order LC pump and the peptides were separated using a separation system at a flow rate of 250 nl / min. In addition, buffer A (5% acetonitrile and 0.1% formic acid) and buffer B (80% acetonitrile and 0.1% formic acid) were used for 120 minute gradients. The eluted peptides were separated by electrospray method with a LTQ linear ion trap mass spectrometer (Thermo Finnigan) at 2.3 kV DC potential. A data dependent scan consisting of one full MS scan (400-1,400 m / z) and five data dependent MS / MS scans were used to generate the MS / MS spectra of the eluted peptides. The MS / MS spectra were then analyzed from the NCBI human protein sequence database using Bioworks version 3.1. DTASelect was used to filter the search results and Xcorr values were applied to different charge states of the peptide: 1.8 was applied as singly charged peptides, 2.5 was applied as double charge peptide, 3.5 was triple charged (Fig. 3B).

As a result, as shown in FIG. 3B, ten kinds of PHD2 binding proteins were identified by mass spectrometry among the proteins extracted from the PHD2-Flag sample. Among them, the PHD2 binding protein was identified, and the proliferation, migration ) And NDRG3 belonging to a gene family associated with invasion (Fig. 3B).

Immunoprecipitation and Western blotting were performed to further confirm that the NDRG3 was a PHD2 binding protein.

Specifically, MCF-7 cell lysate transformed with the control and Flag-tagged PHD2 constructs was immunoprecipitated using anti-FLAG M2 affinity gel (Sigma), and electrophoresis was performed by 9% SDS-PAGE And transferred to nitrocellulose membranes (PALL Life Sciences). Next, the primary anti-NDRG3 antibody and the anti-Flag antibody were treated and reacted with the primary antibody, HRP-conjugated secondary antibody was attached to the primary antibody attached to the membrane, and ECL (Pierce chemical co, USA) (Fig. 3C).

As a result, as shown in Fig. 3C, NDRG3 was confirmed to be a binding protein of PHD2 by confirming the protein of 42 KDa isolated from immunoprecipitated FLAG beads (Fig. 3C).

< Example  4> PHD2 As a unique substrate of NDRG3  Confirm

<4-1> PHD2  And NDRG3 Confirm the interaction of

In order to confirm the effect of interaction between NDRG3 and PHD2 as a binding protein of PHD2 and the interaction of PHD2 and NDRG3 in the expression of NDRG3 protein, immunoprecipitation, Western blotting, and in-vitro pull- (pull-down assay).

Specifically, constructs encoding Flag-tagged PHD2 as described in Example 3 were cultured in DMEM medium containing 10% FBS (Gibco BRL) and 100 U / ml penicillin (Gibco BRL) HeLa cells (ATCC) were transformed and maintained in a hypoxic condition with 1% oxygen for 24 hours and then dissolved. Cell lysates were then immunoprecipitated using anti-FLAG M2 beads and Western blotting was performed using anti-NDRG3 and anti-FLAG antibodies as primary antibodies (FIG. 4A, supra). Further, PHD2 was cloned into pET-28a recombinant plasmid by the method described in Example 1, transformed into E. coli strain BL21, and purified using Ni-NTA agarose resin. In addition, NDRG3 was cloned into pGEX-4T-2 recombinant plasmid, transformed into E. coli strain BL21, and purified using GST-binding agarose resin (ELPIS BIOTECH, Korea). The recombinant protein His-PHD2 10 and / or the recombinant protein GST-NDRG3 10 were then incubated with Ni-NTA agarose resin at a final concentration of 0.2 mg / 4 for 4 hours. Next, the NDRG3 protein bound to the resin was treated with an SDS buffer solution, subjected to SDS-PAGE electrophoresis as in Example 3, and subjected to Western blotting using an anti-NDRG3 antibody as a primary antibody 4a, below).

In order to confirm the functional relationship between PHD2 and NDRG3, MCF-7 cells (FIG. 4B) or HeLa cells (FIG. 4C) were cultured for 24 hours under normoxic condition (21% O ₂ ) with DFX (desferrioxamine) The cells were lysed as described in Example 3 above. The cell lysate was subjected to Western blotting using anti-NDRG3, anti-HIF-la and anti-beta-actin (Fig. 4B and Fig. 4C).

As a result, as shown in FIG. 4A, it was confirmed that PHD2 and NDRG3 directly interacted by confirming that PHD2 and NDRG3 immunoprecipitated under hypoxic condition bind and recombinant PHD2 and NDRG3 bind (FIG. 4A ).

4B and 4C, the expression level of NDRG3 protein was decreased when the activity of PHD2 was inhibited by DFX, which is a PHD2 inhibitor, in both MCF-7 and HeLa cells, although the basic expression level of NDRG3 protein was insufficient in HeLa cells Confirming that NDRG3 accumulates drug-dependently as PHD2 activity is suppressed, and NDRG3 is a unique substrate of PHD2 (FIGS. 4B and 4C).

<4-2> PHD family  Protein and NDRG3 Confirm the interaction of

The PHD family consists of PHD1, PHD2, PHD3, P4HTM and P4HA1, and these families have been reported to play an important role in the regulation of HIF proteins (Wenger, RH et al., Curr. Pharm. Des., 2009 15), 3886-3894). Therefore, in order to confirm the relationship between NDRG3 and VHL, the target proteins of the PHD family other than PHD2 and the E3 ubiquitin ligase complex, PHD was overexpressed in cells and knockdown due to RNA interference RT-PCR, immunoprecipitation and Western blotting were performed.

Specifically, siRNAs were prepared using the sequences shown in the following Table 3 with siVHL (siGENOME SMARTpool, Dharmacon) and Samchully (Korea), and then transformed into HeLa cells using lipofectamine 2000 according to the manufacturer's procedure , GFP, PHD1, PHD2, PHD3, P4HTM, P4HA1 or VHL were obtained and the cells in which the expression was inhibited were cultured for 48 hours under normoxic condition (21% O ₂ ) Respectively. Then, the recovered cells were subjected to total RNA isolation using Trizol Reagent (Invitrogen, Carlsbad, Calif.), And the separated RNA 5 was reacted with a reverse transcriptase to synthesize cDNA. And then visualized after electrophoresis on agarose gel (Fig. 5A).

siRNA order GFP
sense GUUCAGCGUGUCCGGCGAGTT (SEQ ID NO: 13) Antisense CUCGCCGGACACGCUGAACTT (SEQ ID NO: 14) PHD1
sense CAUCGAGCCACUCUUUGACTT (SEQ ID NO: 15) Antisense GUCAAAGAGUGGCUCGAUGTT (SEQ ID NO: 16) PHD2
sense AACGGGUUAUGUACGUCAUTT (SEQ ID NO: 17) Antisense AUGACGUACAUAACCCGUUTT (SEQ ID NO: 18) PHD3
sense CCAGAUAUGCUAUGACUGUTT (SEQ ID NO: 19) Antisense ACAGUCAUAGCAUAUCUGGTT (SEQ ID NO: 20) P4HTM
sense GAGUGUCGGCUCAUCAUCCTT (SEQ ID NO: 21) Antisense GGAUGAUGAGCCGACACUCTT (SEQ ID NO: 22) P4HA1
sense GAUCUGGUGACUUCUCUGATT (SEQ ID NO: 23) Antisense UCAGAGAAGUCACCAGAUCTT (SEQ ID NO: 24)

In order to confirm the interaction between the PHD family and the NDRG3 protein, PHD1, Flag-tagged PHD2, Flag-tagged PHD3, Flag-tagged P4HTM and Flag-tagged P4HA1 expression vectors and NDRG3 expression vector , NDRG3 and the Flag-tagged PHD family were transformed into HeLa cells according to the method described in Example 3, 20 μM MG132 was treated for 8 hours in a steady state, and cells were obtained Lt; / RTI &gt; The cell lysate was then immunoprecipitated with anti-FLAG M2 beads and Western blotting was performed using anti-NDRG3 antibody (Fig. 5B).

As a result, as shown in FIGS. 5A and 5B, when the expression of PHD2 and VHL was inhibited by RNA interference, it was confirmed that NDRG3 accumulates. As a result, PHD2 among PHD family members showed a significant NDRG3 is a target protein of ubiquitin in the normal oxygen state, thus confirming that NDRG3 is a substrate for the PHD2 / VHL-mediated transcription process (FIGS. 5A and 5B).

<4-3> NDRG3 in PHD2 The docking site

Docking simulation and docking simulation were performed to confirm the location of docking of PHD2 on NDRG3 as a substrate of PHD2 and the PHD2-docking site and the docking site of NDRG3 confirmed through the docking simulation Immunoprecipitation and Western blotting were performed using NDRG3 variants mutated at the docking site using a site-directed mutation in order to confirm the binding ability with PHD2.

Specifically, the protein-protein docking simulation of the predicted target protein was performed by HEX 6.3 (D. W. Ritchie. Et al., Genet, 2000 (39), 178-194). For the calculation options, shape and electrostatistics were chosen as the correlation type, and bumps and volumes were selected after the process. The remaining choices use the default configuration. For a single target docking (i.e., NDRG3-EGLN1), the simulation was performed using the options listed above once. Docking for multiple target structures (i.e., NDRG3 and PHD2) was performed through a two-step experiment. In the first step, NDRG3 was used as a receptor protein in a docking experiment for each target. In the second step, the resulting protein-protein interaction was used as a receptor protein for the docking of other proteins. The input order for the second experiment was PHD2. Docking calculations were performed by root-mean-square deviation (RMSD) and the results were filtered where the output from a single input matched the output from multiple inputs. Of the filtered results, the most stable one was selected using the HEX 6.3 total score (sum of shape scores and vocational school scores) (Figure 6a).

In order to confirm the binding strength of PHD2 with the docking site of NDRG3 confirmed by docking simulation, site-directed mutagenesis was performed according to the manufacturer's procedure using a KOD-Plus-Mutagenesis kit (Toyobo) with the NDRG3-Myc expression vector as a template A Myc-tagged NDRG3 (R47D) mutant in which the 47th arginine (Arg, R) of NDRG3 was substituted with an aspartic acid (Asp, D), a Myc-tag in which 66th asparagine (Asn, N) of NDRG3 was substituted with an aspartic acid Tagged NDRG3 (Q97E) mutant in which NDRG3 (N66D) mutant, NDRG3 97th glutamine (Gln, Q) was replaced with glutamic acid (Glu, E), NDRG3 296th valine To obtain Myc-tagged NDRG3 (V296D) mutants. Each of the Myc-tagged NDRG3 variants, Flag-tagged PHD2 and HA-tagged VHLs as in Example 3, The construct was simultaneously transformed with HEK293T cells (ATCC) and the transfected HEK293T cells were treated with 20 [mu] M MG132 for 8 hours and then lysed. The cell lysates were immunoprecipitated using anti-Myc affinity gel (Sigma) and Western blotting was performed using anti-Flag, anti-HA and anti-Myc antibodies as primary antibodies (FIG.

As a result, from the docking model between the disclosed PHD2 substrate (Structure. 2009 (7), 981-9) and the NDRG3 substrate, the 47th arginine to the PHD2-docking site on the putative NDRG3, Asparagine, 68th lysine, 69th serine, 72nd asparagine, 73rd alanine, 76th asparagine, 77th phenylalanine, 78th glutamic acid, (Glutamine), 97th glutamine, 98th glutamine, 99th glutamic acid, 100th glycine, 101st alanine, 102nd proline, 103rd serine, 203th leucine, 204 The second aspartic acid, the 205th leucine, the 208th threonine, the 209th tyrosine, the 211th methionine, the 212th histidine, the 214th alanine, the 215th glutamine, 216th aspartic acid, 217th isoleucine ( Isoleucine), 218th asparagine, 219th glutamine, 296th valine, 297th valine, 298th glutamine, 300th glycine and 301th lysine site were identified and the 47th, 66th, The 97th and 296th amino acid positions were more important (Fig. 6a).

As shown in FIG. 6B, the NDRG3 (V296D) mutant and the NDRG3 (Q97E) mutant bind PHD2, while the NDRG3 mutant that mutated at the 47th or 66th position of NDRG3 does not bind to PHD2, The NDRG3 variant exhibited various PHD2-binding activities (V296D>Q97E> R47DN66D), confirming that the NDRG3 mutant exhibiting high affinity with PHD2 was immunoprecipitated in large amounts by VHL, Th amino acid position is important for docking of PHD2 and is also related to VHL (Fig. 6B).

<4-4> In normal oxygen condition PHD2 / VHL -medium Proteasome  By path NDRG3 Confirmation of control of

To investigate the effect of VHL, a target element of the ubiquitin ligating enzyme complex of PHD2 and E3 under normal oxygen conditions, on protein expression and regulation of NDRG3, MG132 was used to inhibit proteasome activity and to inhibit Western blotting In-vivo ubiquitination assay was performed.

Specifically, a vector for transfection (MSCV retrovirus system) was constructed using full-length NDRG3 cDNA (SEQ ID NO: 1) and pMSCVneo retrovirus vector (Clontech). For virus production, the GP293 cell line was transfected using Lipofectamine (Invitrogen). After 48 hours, the cell supernatants containing NDRG3-retrovirus or control-retrovirus were treated with HeLa cells for 24 hours, such as 6 / ml polybrene, and then the cells overexpressing NDRG3 were incubated for 20 hours at 20 Cells were obtained after treatment or no treatment of μM MG132, and Western blotting was performed using anti-NDRG3 antibody and anti-beta actin antibody (Fig. 7A).

In vivo ubiquitination assay was also performed to confirm ubiquitination of NDRG3 under normal oxygen conditions. First, to inhibit the expression of NDRG3, transfection vectors were constructed using shNDRG3 (Sigma-Aldrich, SEQ ID NO: 4) and lentivirus vectors. For virus production, the cell line was transfected as described above. Subsequently, the cell supernatant containing the NDRG3 shRNA-expressing lentivirus was treated with 6 / ml polybrene in HeLa cells, maintained in DMEM medium containing 10% FBS, and then treated with control HeLa cells, HeLa cells in which NDRG3 expression was inhibited and HeLa cells in which NDRG3 was overexpressed were transformed with HA-tagged ubiquitin as described in Example 3, treated with 20 μM MG132 for 8 hours, and then cells were obtained and lysed . Subsequently, the cell lysate was pre-cleared by adding 30 protein G-agarose beads (Santa Cruz Biotechnology), followed by immunoprecipitation with an anti-NDRG3 antibody, followed by polyubiquitinated form Of NDRG3 was confirmed by Western blotting using an anti-HA antibody (Fig. 7B).

As a result, as shown in FIGS. 7A and 7B, when protease was inhibited by MG132 under normal oxygen conditions, it was confirmed that NDRG3 protein was actively ubiquitinated in NDRG3 overexpressed cells. Thus, NDRG3 It was confirmed to be ubiquitinated and degraded through the proteasome pathway (FIGS. 7A and 7B). Thus, the results of Example 4 demonstrate that NDRG3 is a unique PHD2-interacting protein and that expression of the NDRG3 protein is regulated post-transcriptionally by the PHD2 / VHL-mediated proteasome pathway.

< Example  5> Hypoxia  In state NDRG3 Confirmation of expression of

<5-1> NDRG3  Identification of oxygen-dependent expression of proteins

Since the activity of PHD2 is dependent on the O ₂ availability, the NDRG3 protein, which is an intrinsic substrate of PHD2, was also tested for its effect on the stability of oxygen by various cell types with different oxygen conditions, such as immunoprecipitation, Western blotting, Fluorescence staining and in vivo ubiquitination assay.

Specifically, 10% FBS and 100 U / ml penicillin, 1% of the MCF-7 cells cultured in the DMEM medium containing 3%, 5% and 21% O ₂ and 92-94% N ₂ and 5% CO ₂ . The cells were maintained in the O ₂ / CO ₂ incubator containing the mixed gas. Then, the cells were lysed and cell lysates were Western blotted using anti-NDRG3 and anti-beta actin to confirm the expression of NDRG3 (FIG. 8A).

MCF-7 cells cultured on cover slips with DMEM medium containing 10% FBS and 100 U / ml penicillin were mixed with 1% O ₂ , 94% N _2, and 5% CO ₂ The hypoxic state was maintained over time using the included O ₂ / CO ₂ incubator. The cells were then fixed with 4% paraformaldehyde / PBS for 20 minutes, infiltrated with 0.3% TritonX-100 / PBS for 5 minutes at room temperature, and then blocked with blocking solution (PBS with 1% BSA) And cultured for 30 minutes. Then, the cells were reacted with anti-NDRG3 antibody (1 / 1,000) at room temperature for 1 hour, washed, and then reacted with a secondary antibody (Alexa Flour 488-conjugated goat anti-rabbit IgG , Or Alexa Flour 594-conjugated goat anti-mouse IgG (1 / 1,000); Amersham] and DAPI (3 [mu] M, Sigma). It was then visualized using a Zeiss LSM 510 confocal microscope (Fig. 8B).

In addition, MCF-7 (breast), PLC / PRF / 5 (liver), Huh-1 (liver), HeLa (cervix), HEK293T (cervix) cultured with DMEM medium containing 10% FBS and 100 U / ml penicillin kidney) and MCF-10A (breast) cells, and 10% FBS and 100 U / ml penicillin, a cultured in RPMI 1640 medium supplemented with SW480 (colon) and IMR-90 (lung), as the O ₂ / CO ₂ incubator (1% O ₂ ), and cells were obtained. Then, the cells were lysed and the cell lysate was Western blotted with anti-NDRG3 and anti-beta actin to confirm the expression of NDRG3 and to graph (Fig. 8C).

In vivo ubiquitination assay was also performed to confirm ubiquitination of NDRG3 under hypoxic conditions. HA-tagged ubiquitin and Myc-tagged NDRG3 were transformed into HeLa cells as in Example 3, cultured in normal oxygen for 40 hours, and treated with 20 μM MG132 for 8 hours. The cells were then cultured in an additional hypoxic condition (1% O ₂ ) for 24 hours before cells were harvested and lysed. Then, the cell lysate was pre-cleared by adding 30 protein G-agarose beads (Santa Cruz Biotechnology), followed by immunoprecipitation with an anti-Myc antibody and Western blotting using an anti-HA antibody (Fig. 8D).

As a result, as shown in FIGS. 8A and 8B, it was confirmed that accumulation of NDRG3 protein increased as the O ₂ concentration decreased and the time elapsed in the cell, thereby confirming the accumulation of NDRG3 protein in inverse proportion to the O ₂ concentration (Figs. 8A and 8B).

Further, as shown in FIG. 8C, accumulation of NDRG3 protein according to the hypoxic state is confirmed not only in non-transformed cells but also in cancer cells derived from various tissues such as large intestine, liver, cervix, kidney, ₂ concentration and the inverse proportion of NDRG3 protein was a typical phenomenon (Fig. 8C).

Further, as shown in FIG. 8D, it was confirmed that the NDRG3 protein underwent a decrease in ubiquitination under hypoxic conditions (FIG. 8D). Therefore, it was confirmed through these results that accumulation of NDRG3 protein was increased and ubiquitination was decreased under hypoxic condition unlike the normal oxygen condition.

&Lt; 5-2 > NDRG3  Confirm protein stability

Western blotting was performed using cells with different oxygen conditions in order to examine the expression of NDRG3 protein according to hypoxic and normal oxygen conditions.

Specifically, in order to confirm the expression of NDRG3 protein in a continuous hypoxic state, MCF-7 cells cultured in the hypoxic state (1% O ₂ ) over time as shown in Example <5-1> O2), MCF-7 cells were cultured in the same manner as in Example 3, and then solubilized in the same manner as in Example 3, followed by Western blotting using anti-NDRG3, anti-HIF-1α and anti- (Fig. 9A).

In order to confirm the change in the expression of NDRG3 protein when restored to the normal oxygen state, MCF-7 cells cultured in a hypoxic condition (1% O ₂ ) for 24 hours and normal oxygen MCF-7 cells maintained and cultured at a time in a state (21% O ₂ ) were obtained and dissolved in the same manner as in Example 3, followed by the addition of anti-NDRG3, anti-HIF-1α and anti- Western blotting and graphing (Figure 9b).

As a result, as shown in FIGS. 9A and 9B, expression of HIF-1.alpha. Protein was induced at the early stage of hypoxia and decreased as the hypoxia persisted. In contrast, expression of NDRG3 protein at the late stage of hypoxia It is confirmed that it is maintained continuously. It was also confirmed that the NDRG3 expression induced by the hypoxic state was slowly removed until the cells returned to the normal oxygen state (FIGS. 9A and 9B).

<5-3> NDRG3  Confirming the mechanism of protein stability

To confirm the target site of the NDRG3 protein in the hypoxic state, micro-LC-MS / MS analysis was performed and Western blotting was performed using cells overexpressing the NDRG3 mutant produced by site-directed mutagenesis. Immunoprecipitation and Western blotting were also performed to confirm the relationship between hypoxic target sites of NDRG3 protein and PHD2 / VHL under normal oxygen conditions.

Specifically, in order to identify the target site of the NDRG3 protein under hypoxic conditions, MCF-7 cells overexpressing NDRG3 obtained by the method described in Example <4-2> were treated with 10 μM MG132, And immunoprecipitated using an anti-NDRG3 antibody as described in Example 3, and micro-LC-MS / MS analysis was performed (FIG. 10A).

In order to confirm the binding of PHD2 / VHL to the NDRG3 protein target region confirmed through the results of the micro-LC-MS / MS analysis, the 294th proline (Pro, P) expected as the NDRG3 protein target site was firstly labeled with alanine , A), a Myc-tagged NDRG3 P294A mutant was constructed by performing site-directed mutagenesis as described in Example < 4-3 &gt;. Then, the NDRG3 mutant or the wild type NDRG3 construct was transformed with HEK293T cells as in Example 3, cultured under normoxic condition (21% O ₂ ) for 40 hours, and cultured for 20 hours at 20 μM MG132 treated and untreated cells were obtained. After dissolution, the obtained cells were subjected to Western blotting using anti-Myc and anti-beta actin antibody (Fig. 10b, top). In addition, Flag-tagged PHD2, HA-tagged VHL, and Myc-tagged NDRG3P294A mutant or Myc-tagged NDRG3 were co-transfected into HEK293T cells as described in Example 3, Cells were cultured under oxygen conditions (21% O ₂ ) and treated with 20 μM MG132 for an additional 8 hours. The obtained cells were then lysed, immunoprecipitated with anti-Myc-affinity gel, and Western blotting was performed using anti-Flag, anti-HA and anti-Myc antibodies (FIG.

As a result, as shown in FIG. 10A, it was confirmed through mass analysis that the 294th proline of NDRG3 was the site of PHD2-mediated hypoxia target, and that the site was hydroxyl-dependent hydroxyl-dependent site (FIG. 10A).

In addition, as shown in Fig. 10B, in the cells overexpressing the NDRG3 mutant in which the NDRG3 294th amino acid region as the predicted hypoxic target site was replaced with alanine, accumulation of the NDRG3 protein mutant was increased in the normal oxygen state (Fig. 10 ), And confirming that the binding of the NDRG3 protein region to PHD2 / VHL in the normal oxygen state is controlled by PHD2 in an oxygen dependent manner, by confirming that the binding of the NDRG3 protein region with PHD2 and VHL decreases (Fig. 10B, below) (Fig. 10B).

&Lt; 5-4 > HIF - Independent NDRG3  Confirm expression control

Western blotting and RT-PCR were performed using cells with different oxygen conditions in order to determine whether the regulation of NDRG3 expression was related to HIF protein according to the oxygen status.

Specifically, in order to confirm the expression of HIF protein according to the oxygen state, MCF-7 cells cultured for 24 hours in a normal oxygen state (21% O ₂ ) as in Example <5-2, % O ₂₎ and kept in collecting the cultured MCF-7 cells over time, by using a half wherein -HIF-1α, 2α--HIF wherein -β- and anti-actin as the <example 3> Western The expression of the protein was confirmed by blotting, and half of the mRNA was confirmed by RT-PCR as in Example <4-2> (FIG. 11A).

Western blotting was also performed to confirm the expression of NDRG3 protein by inhibition of HIF and PHD2. First, shHIF-1α (Sigma-Aldrich), shHIF-2α (Sigma-Aldrich) or a control shGFP and a lentivirus vector were added to inhibit the expression of HIF-1α or HIF- To prepare a vector for transfection. Then, the cell supernatant containing the shHIF-1α or shHIF-2α-expressing lentivirus was treated with PLC / PRF / 5 cells with 6 / ml polybrene, and then cultured in DMEM medium containing 10% FBS PLC / PRF / 5 cells inhibited the expression of HIF-1α or shHIF-2α and were treated with 1 μM of a PHD2 inhibitor, DFX (desferrioxamine), under normoxic condition (21% O ₂ ) Cells were obtained and dissolved as in Example 3, and the lysate was treated with anti-NDRG3, anti-HIF-1 alpha, anti-HIF-2 alpha and anti-beta-actin Western blotting was performed (FIG. 11B).

In order to confirm the expression of NDRG3 protein by deletion of HIF and VHL, the expression levels of MCF-7 (HIF-1 ^{+ / +} and VHL ^{+ / +} ) and HIF-1α and VHL loci were 786 -O (HIF-1 ^{- / -} and VHL ^{- / -} ) cells were cultured for 24 hours in a hypoxic state (1% O ₂ ) as in Example <5-2> And the cell lysate was subjected to Western blotting using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin (FIG. 11C).

As a result, as shown in Fig. 11A, it was confirmed that the expression level of NDRG3 mRNA was maintained unchanged while the hypoxic state was maintained regardless of the remarkable increase in expression of HIF-1α protein in the early stage of hypoxia (Fig. 11A).

11B and 11C, it was confirmed that the accumulation of NDRG3 protein was increased as the inhibition of PHD2 activity was continued without being affected by the deletion of HIF under the normal oxygen state (FIG. 11B), and in the hypoxic state It was confirmed that expression of NDRG3 protein was conserved without being affected by HIF and VHL deletion (FIG. 11C). Thus, through the results of Example 5 above, the expression of NDRG3 protein is regulated by PHD2 in an oxygen dependent manner, and the expression of NDRG3 protein and mRNA is expressed by HIF activity as the hypoxic state continues. And thus confirmed that NDRG3 has a major function in HIF-independent persistently hypoxic reactions.

< Example  6> Continuous Hypoxia  As an adjustor of the reaction NDRG3  Confirm

<6-1> Hypoxia  In state NDRG3 Confirm the function of

In order to confirm the function of NDRG3 in the hypoxic response, gene expression profiling through GAzer (Gene Set Analyzer) of microarray-based transcriptome data using cells in which expression of NDRG3 was inhibited .

Specifically, Huh-7 cells inhibited the expression of NDRG3 or HIF-1α were prepared by the method described in Example <4-4> and Example <5-4> using shNDRG3 and shHIF-1α, The cells were maintained in a hypoxic state (1% O ₂ ) as in Example <5-2> over time and cells were recovered. Then, total RNA was isolated from the recovered cells using an RNA isolation kit (RNeasy midi-prep, Qiagen) according to the manufacturer's procedure. Next, for the microarray analysis, 200 ng of the separated total RNA was amplified using an Illumina TotalPrep ^™ RNA amplification kit, and then 700 ng of the amplified cRNA was amplified using a HumanHT-12 v3 / v4 expression bead chip ) For 16 hours at &lt; RTI ID = 0.0 &gt; 58 C. &lt; / RTI &gt; After washing and staining, the bead chips were scanned using an Illumina BeadArray Reader and Bead Scan software (Illumina). Expressed genes were classified by function using gene (gene ontology) analysis (Ashburner, M. et al., Nat. Genet., 2000 (25), 25-29). In this process, the Z score (standard value) transformation was used to calculate the standardized deviation score by each gene grouping. The Z score value indicates the activity of the GO biological process (Fig. 12A).

In addition, 136 genes expressing NDRG3 (N66D) mutant were transfected into HeLa cells 1.5 times or more as compared with the control group as in Example 3, and expression levels of 1,5-fold or more in hypoxic state (N66D) variants and hypoxic conditions were selected. To investigate the function of the genes selected above, genes were analyzed by Gene Set Analyzer (Standard value) of the biological ontology classified into the above-mentioned categories (FIG. 12B).

As a result, as shown in FIG. 12A, the NDRG3 deletion statistically confirmed significant expression changes in the functional gene group under hypoxic conditions for 24 hours, and the hypoxia function most affected by the NDRG3 deletion was angiogenesis ) And cell proliferation, while glycolysis was one of the least relevant functions. Conversely, in the case of HIF-1α, it was confirmed that the most relevant function of the hypoxic function is the corresponding process, while the angiogenesis and cell proliferation are less related (FIG. 12A).

As shown in FIG. 12B, when overexpression of the NDRG3 N66D variant mutated at the docking site of PHD2 in the normal oxygen state, the angiogenesis> cell apoptosis cell migration, cell migration> (Fig. 12B). Therefore, it was confirmed that NDRG3 has an important function in the hypoxic response.

<6-2> Hypoxia  In state NDRG3 Confirming Promotion of Angiogenesis by

In order to confirm the effect of NDRG3 in the production of angiogenesis, which is highly related to NDRG3 in hypoxia confirmed by the analysis of the above <6-1>, a tube forming assay using RT- -PCR, Western blotting and in vivo angiogenesis assay were performed.

Specifically, tube formation assay was performed to confirm the change of angiogenic activity by NDRG3 deletion. First, the expression of NDRG3 using shNDRG3 by the method described in Example <4-4> inhibition Huh-7 (2 × 10 ⁵ cells / ml) and control Huh-7 cells to hypoxia (1% O ₂ ) And cultured for 24 hours. Then, the culture solution (1 ml) was recovered and cultured in a 6-well dish coated with Matrigel together with HUVEC (human umbilical vein endothelial cells) (1 × 0 ⁵ cells / ml) The tube formation was observed by incubating for 6-12 hours (Fig. 13A).

In order to confirm the expression of IL8, IL1? And IL1? COX-2 and PAI-1, which are neovascularization markers due to NDRG3 deletion in a hypoxic state, NDRG3 deletion mutants obtained by the method described in Example <6-1> 7 cells and control Huh-7 cells were cultured under normoxic condition (21% O ₂ ) or in hypoxic condition (1% O ₂ ) for 24 hours and then the cells were recovered. The NDRG3 (N66D) mutant prepared by the method described in Example <4-3> was transformed into HeLa cells as described in Example 3, and cultured for 24 hours under normal oxygen conditions. Cells were recovered Respectively. Then, the total RNA was isolated and RT-PCR was performed by the method described in Example <4-4> above. Further, a portion of each of the above cells was subjected to Western blotting using an anti-NDRG3 antibody as in Example 3 to confirm the expression of NDRG3 (FIG. 13B).

Matrigel plug assay was also performed for in vivo angiogenesis analysis by NDRG3 deletion. First, NDRG3-deficient Huh-7 cells (1 × 10 ⁶ cells / ml) and control Huh-7 cells (1 × 10 ⁶ cells / ml) obtained by the method described in the above Example <6-1> and cold Matrigel (BD Biosciences) Cells / ml). The mixed Matrigel 500 was then subcutaneously injected into the abdominal region of a 6-week-old female BALB / c mouse (Japan SLC). Seven days later, the mice were sacrificed and matrigel plaques were obtained. Hemoglobin quantification was performed by homogenizing the cells in 500 ml water in ice cubes and centrifuging at 12,000 rpm for 4 to 15 minutes. Then, only the supernatant was separated, and the resultant was reacted according to the manufacturer's procedure using Drabkin's reagent (Sigma), and absorbance was measured at a wavelength of 570 nm using a spectrophotometer to graphically show (FIG.

By As a result, as shown in Figs. 13a and 13b, determine that the tube is formed lower than that in the case of the NDRG3 expression control cell control under hypoxic conditions, also NDRG3 deletion inhibit the angiogenic activity induced by hypoxic response (Fig. 13A). In addition, expression of IL8, IL1 [alpha] and IL1 [beta] COX-2 and PAI-1 mRNA, which are markers of neovascularization, is significantly decreased in the hypoxic NDRG3 deleted cells (Fig. 13b, left) By confirming that mRNA expression of the factor is increased in cells overexpressing the mutated NDRG3 (N66D) mutant (FIG. 13B, right), the expression of the angiogenic factor is increased by NDRG3 in the hypoxic response, (Figs. 13A and 13B).

Further, as shown in Fig. 13C, it was confirmed that the hemoglobin concentration was decreased in the mouse transplanted cells in which expression of NDRG3 was suppressed, and it was confirmed that NDRG3 stimulated angiogenesis in vivo similarly to the above results 13c).

<6-3> Hypoxia  In state NDRG3 Promoting cell proliferation by

In order to confirm the effect of NDRG3 on the cell proliferation of highly functional NDRG3 in hypoxia confirmed by the analysis of <6-1> described above, MTT assay was performed to analyze cell growth, and in vivo transplantation Tumor volume was measured, and immunofluorescence staining, Western blotting and RT-PCR were performed.

Specifically, MTT assay was performed to confirm cell growth by NDRG3 deletion. First, NDRG3-deficient Huh-1 cells and control Huh-1 cells obtained by the method described in Example <6-1> were dispensed into a 96-well plate in a number of 2,000 cells / well, , 5% CO2 incubator, and hypoxic condition (3% O ₂ ). Then, 1 mg / MTT solution diluted with PBS was treated and reacted for 2 hours. Then, the medium containing MTT was removed and treated with 100 DMSO to dissolve MTT formazan crystal. Absorbance was then measured at a wavelength of 570 nm with a spectrophotometer (Fig. 14A).

HIF-1α, HIF-2α, NDRG3, and HIF-1α, which were prepared by the methods described in Examples <4-4> and <5-4>, were used to confirm the degree of tumor formation due to deletion of NDRG3 and HIF Or Huh-7 cells (2 × 10 ⁶ cells / 100) in which NDRG3 and HIF-2α expression were inhibited were subcutaneously injected into the flank of six-week-old female BALB / c mice (FIG. Further, Huh-1 cells (2 × 10 ⁶ cells / 100) transformed with the NDRG3 (N66D) mutant prepared by the method described in Example <4-3> as described in <Example 3> And subcutaneously in the side of the mouse (Fig. 14C). Then, at 16 days and 20 days after administration, tumor sizes were imaged for comparison, respectively (Fig. 14B and Fig. 14C).

14d) and the NDRG3 (N66D) mutant were transfected with Huh-7 cells transfected with the NDRG3, HIF-1α, HIF-2α, NDRG3 and HIF-1α or NDRG3 and HIF- The tumor volume was measured using a caliper at a given time after transplantation in a mouse transplanted with Huh-1 cells (Figure 14e). The volume of the tumor was measured by measuring the length (a), the width (b), and the height (c) as shown in the following Equation 1 (FIGS. 14D and 14E).

Tumor tissues of mice transplanted with Huh-7 cells (2 × 10 ⁶ cells / 100) in which the expression of NDRG3, HIF-1α and HIF-2α were inhibited were extracted and fixed in 10% formalin at room temperature And fixed for one day. The parinffin was then treated and quadrifected. Then, the cells were infiltrated with 0.3% Triton X-100 / PBS for 5 minutes at room temperature, and incubated with a blocking solution (PBS containing 1% BSA) for 30 minutes. The cells were incubated with secondary antibody (Alexa Flour 488-conjugated goat anti-rabbit IgG (1 / 1,000), or Alexa Flour 594-conjugated goat anti-mouse IgG (1 / 1,000)] and DAPI and visualized using a Zeiss LSM 510 confocal microscope (Fig. 14f).

Tumor tissues of mice transplanted with Huh-7 cells (2 × 10 ⁶ cells / 100) in which NDRG3, HIF-1α and HIF-2α expression were inhibited were extracted and frozen with liquid nitrogen. Then, RT-PCR was performed by the method described in the above Example <4-2> to confirm the expression of mRNA, and the expression of anti-NDRG3 and anti-beta actin antibody was measured by the method described in <Example 2> And the protein expression was confirmed by conducting the ligation (Fig. 14 (g)).

As a result, as shown in FIG. 14A, it was confirmed that cell growth of cells lacking NDRG3 in a mild hypoxic state was decreased over time as compared with the control group (FIG. 14A).

In addition, as shown in Figs. 14B to 14E, the tumor size of a mouse transplanted with NDRG3 expression-suppressed cells was inhibited over time as compared with mice transplanted with control and HIF-expressing cells, It was confirmed that no tumor was generated in mice transplanted with the cells of NDRG3 and HIF-la or -2a simultaneously (Figs. 14B and 14D). On the other hand, tumor size of mice transplanted with cells overexpressing the NDRG3 (N66D) mutant in which the docking site of PHD2 was mutated was significantly increased compared to the control, and tumor was not formed in the control group until 20 days after transplantation, whereas NDRG3 N66D) mutant over-expressing cell transplanted mice showed that the tumor size was increased to about 900 mm ³ , thereby confirming that tumor growth was promoted by the NDRG3 (N66D) mutant (FIG. 14B to FIG. 14E).

As shown in FIGS. 14F and 14G, it was confirmed that the expression of Ki-67, a cell growth marker, was suppressed in tumor tissues of NDRG3-deficient cell transplanted mice (FIG. 14F). In addition, it was confirmed that the expression of proteins and mRNAs of IL-8 and CD31, which are tumor angiogenesis markers, in tumor tissues of NDRG3 deletion cell transplantation mice was inhibited (FIGS. 14F and 14G). Therefore, it was confirmed that NDRG3 plays an important role in promoting angiogenesis and cell proliferation in the hypoxic state.

< Example  7> NDRG3 - Mediated Hypoxia  L-lactate production and exogenous L-lactic acid treatment in the reaction, NDRG3  Determination of L-lactic acid by measurement

<7-1> Hypoxia  In state NDRG3  Identification of protein accumulation and lactic acid production

The accumulation and degradation of NDRG3 protein in long lag periods identified through Example <5-2> indicates that several steps are involved in the regulation of hypoxic expression of NDRG3. Therefore, in order to confirm the biochemical characteristics related to hypoxia and the relationship of NDRG3, lactic acid production measurement, Western blotting and RT-PCR were performed in a hypoxic condition, and in-vitro was performed to confirm ubiquitination of NDRG3. Ubiquitin assay was performed.

Specifically, MCF-7 cells were cultured for 24 hours in a normal oxygen state (21% O ₂ ) or maintained in a hypoxic state (1% O ₂ ) for a time period as in Example <5-2> , Cells were collected and dissolved as in Example 3 above. Then, the cell lysate was subjected to Western blotting using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin, and subjected to the manufacturer's procedure using EnzyChrom TM L-lactic acid assay kit (BioAssay Systems) The production of L-lactate (L-lactate) was measured and plotted. The values were normalized by L-lactic acid standard curve, and it was confirmed that the content of lactic acid according to the expression amount of NDRG3 protein in the cells could be calculated (FIG. 15A).

To confirm the effect of inhibition of L-lactic acid production on NDRG3 protein expression, MCF-7 cells were treated with sodium lactate dehydrogenase (LDHA) inhibitor sodium oxamate in a concentration-dependent manner, And maintained in a hypoxic state (1% O ₂ ) for 24 hours. Then, the cells were recovered and dissolved as in Example 3 above. Western blotting was then performed using anti-NDR-3, anti-HIF-1 alpha and anti-beta-actin as described above and L-lactate production was measured and plotted. The values were normalized by the L-lactic acid standard curve, and it was confirmed that the lactic acid content could be calculated according to the expression amount of NDRG3 protein in the cells (FIG. 15B).

In order to confirm the effect of inhibition of L-lactic acid production on NDRG3 protein expression, siLDHA (siGENOME SMARTpool, Dharmacon) or siMCT4 (siGENOME SMARTpool, Dharmacon) were exposed to LDHA or lactic acid by the method described in Example <4-2> MCF-7 cells lacking MCF4, which is a monocarboxylate transproter that is involved in the export of MCF4, were prepared and cultured in a hypoxic state (1% O ₂ ) for 24 hours , And the cells were collected and dissolved as in Example 3 above. Western blotting was then carried out using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin as described above and L-lactate production was measured and plotted (Figure 15c) . In order to confirm the deletion of LDHA and MCT4, RT-PCR was performed as in Example <4-2> (FIG. 15C).

2-deoxyglucose (2-DG), which inhibits the process, was treated with MCF-7 cells in a concentration-dependent manner to examine the effect of the inhibition of NDRG3 on the expression of the NDRG3 protein, -2> and as hypoxia (1% O ₂₎ maintained for 24 hours in a culture and then, the <example 3> the cells recovered as follows, and dissolved and wherein -NDRG3 and wherein using the actin -β Western blotting (Fig. 15D).

Further, in order to confirm the expression of NDRG3 protein by excessive lactic acid production, a Flag-tagged LDHA expression vector was constructed and transformed into HeLa cells as in Example 3 and incubated with normal oxygen (21% O ₂ ) Lt; / RTI &gt; for 24 hours. Then, additional pyruvate (pyruvate) then treated with 50 mM and kept for 24 hours under mild hypoxia (3% O ₂₎ the culture, the cells are recovered, and dissolved, and wherein -NDRG3, wherein -Flag and anti-actin -β (Fig. 15 (e)).

In order to confirm the expression of NDRG3 protein by exogenously treated lactic acid, Huh-1 cells lacking LDHA were prepared using siLDHA (siGENOME SMARTpool, Dharmacon) according to the method described in Example <4-2> L-lactic acid was treated for each concentration and then cultured for 24 hours in a normal oxygen state (21% O ₂ ) and a hypoxic state (1% O ₂ ) as in Example <5-2. Cells were collected and lysed. Western blotting was then carried out using anti-NDRG3, anti-HIF-1.alpha., Anti-LDHA and anti-beta-actin as described above to confirm the expression of NDRG3 protein and HIF-1.alpha. Protein ). In order to confirm the effect on NDRG3 mRNA, RT-PCR was performed as in Example <4-2 (FIG. 24).

In addition, in order to examine the expression of NDRG3 protein by treatment with oxamate and removal of intracellular lactic acid, Huh-1 cells were treated with sodium oxamate and treated with L-lactic acid by concentration, The cells were cultured for 24 hours under a normal oxygen condition (21% O ₂ ) and a hypoxic state (1% O ₂ ) as in Example 5-2, and the cells were recovered and dissolved as in Example 3 above. Then, Western blotting was performed using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin as described above to confirm the expression of NDRG3 protein and HIF-1 alpha protein (FIG. 25).

In addition, in order to confirm that the action by the exogenous lactic acid treatment is maintained even when the intracellular inflow of lactic acid is blocked, the method described in the above Example <4-2> is performed using siMCT (siGENOME SMARTpool, Dharmacon) Huh-1 cells inhibiting MCT (monocarboxylate transporter) 1 were prepared and treated with L-lactic acid. Then, L-lactic acid was treated with a normal oxygen condition (21% O ₂ ) ₂ ) for 24 hours, and cells were collected and lysed as in Example 3 above. Then, Western blotting was performed using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin as described above to confirm expression of NDRG3 protein and HIF-1 alpha protein (FIG. 26).

In addition, in order to clarify the effect of inhibiting MCT (monocarboxylate transporter) 1, Huh-1 cells inhibiting MCT 1 were prepared as described above. Then, glucose-deficient or oxamate treatment was performed to remove intracellular lactic acid, Lactic acid was treated and cultured for 24 hours under normal oxygen condition (21% O ₂ ) as in Example <5-2, and the cells were recovered and dissolved as in Example 3 above. Then, Western blotting was performed using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin as described above, and expression of NDRG3 protein and HIF-1 alpha protein was confirmed (FIG.

In addition, in vitro ubiquitin assay was performed to confirm the effect of lactic acid production on ubiquitination of NDRG3. First, Flag-tagged PHD2 and HA-tagged VHL were transformed into HEK293T cells as described in Example 3, and cells were recovered and lysed. Protein lysate 500 was then reacted with anti-HA affinity gel (Sigma) or anti-Flag affinity gel (Sigma) for 4 days and centrifuged to obtain recombinant PHD2 proteins or VHL proteins. Subsequently, the recombinant human NDRG3-GST (SEQ ID NO: 2) obtained by the method described in the above Example < 4-1 > and the recombinant PHD2 / VHL- 4), 500 ng ubiquitin-activating enzyme (E1, Upstate), 1 ubiquitin-conjugating enzyme (E2 UbcH5a), Upstate, 2.5 Ubiquitin- (pH 7.3), 5 mM MgCl2, 1 mM DTT, and 2 mM ATP at 37 for 1 hour. The mixture was then incubated with GST-conjugated agarose resin for 4 to 4 hours, then the precipitate was washed and Western blotting was performed using anti-Flag as in Example 3 (Fig. 15F ).

As a result, as shown in FIGS. 15A and 15B, it was confirmed that the expression of HIF-1.alpha. Protein was remarkably increased and decreased in the early stage of hypoxic state, but accumulation of lactic acid production and NDRG3 protein increased remarkably as the hypoxic state continued (Fig. 15A). Conversely, by confirming that NDRG3 protein accumulation is inhibited proportionally to the expression of lactic acid (Fig. 15B) when sodium oxamate is treated to inhibit lactate production, expression of NDRG3 protein in the hypoxic state is associated with lactic acid production (Figs. 15A and 15B).

In addition, as shown in FIGS. 15C to 15E, when the production is suppressed by inhibiting the process by LDHA deletion or 2-deoxyglucose treatment, the expression of NDRG3 protein decreases in the hypoxic state, while the expression of MCT4 deletion, Or LDH overexpression and / or pyruvate-containing medium, the accumulation of NDRG3 protein in the hypoxic state is increased. As a result, unlike the hypoxic induction of HIF protein, oxygen deficiency in NDRG3 protein accumulation It was further confirmed that glycolytic lactate production was further required (Figs. 15C to 15E).

In addition, as shown in Figures 24-27, exogenously treated lactic acid in Huh-1 cells restored dose-dependent NDRG3 expression, which was reduced in expression by LDHA deletion, and did not affect the level of HIF-1α protein (Fig. 24), confirming that similar results were obtained when lactate production was inhibited by oxamate treatment (Fig. 25). However, siRNAs targeting MCT1 (a monocarboxylate transporter that transports extracellular lactate into cells) did not have the effect of restoring the expression of NDRG3 by lactic acid mediated, either normal or hypoxic conditions (Fig. 26 ), And a similar effect was observed when Huh-1 cells knockdown MCT1 with glucose deficiency or oxamate treatment (FIG. 27).

Further, as shown in FIG. 15F, it was confirmed that the ubiquitination of the recombinant NDRG3 protein by the PHD2 / VHL complex was reduced when the lactic acid was treated. Thus, lactic acid production under hypoxic condition inhibited the ubiquitination of NDRG3 protein and accumulated in the cells (Fig. 15F).

<7-2> Hypoxia  In state NDRG3  And lactate binding

In order to confirm the interaction of lactic acid and NDRG3 protein according to the oxygen state, NDRG3 recombinant protein and site-directed mutagenesis were used to prepare an NDRG3 mutant recombinant protein and used for in-vitro binding assay. , Immunostaining and Western blotting were performed.

Specifically, to confirm the interaction between L-lactic acid and NDRG3 protein, pGEX-4T-2-NDRG3 cloned by the method described in Example <4-1> was used as a template, (Glycine, G), which is the 138th amino acid in the L-lactic acid binding site of NDRG3, was transfected with tryptophan (Tryptophan, W) pGEX-4T-2-NDRG3 (G138W) variant construct was obtained. Subsequently, the recombinant plasmids pGEX-4T-2-NDRG3 wild-type and -NDRG3 (G138W) were transformed into E. coli strain BL21 for expression of the recombinant protein, and GST-binding And purified using agarose resin. Then, the cells were electrophoresed on 9% SDS-PAGE as described in Example 3, stained with Coomassie Brilliant Blue to confirm the detection. To confirm the expression of recombinant proteins, anti-GST and anti-NDRG3 antibodies (Fig. 16A).

primer order NDRG3 (G138W)
sense GTTTGGGCTGGAGCTTACATCCTCAGC (SEQ ID NO: 25) Antisense TCCAATTCCAATGATGCTTTTCAGGCT (SEQ ID NO: 26)

In addition, in vitro binding assay was performed to confirm the interaction of L-lactic acid and NDRG3 protein. GST or the GST-NDRG3 recombinant protein (50 μg) was reacted with unlabeled L-lactic acid (pH 7.0, Sigma) and 0.5 μCi of L- [ ¹⁴ C] -lactic acid (PerkinElmer) for 1 hour at 30 ° C. Then, GST-conjugated agarose resin was added and reacted for 4 hours. Then, a resin bound with NDRG3-GST protein was obtained in the reaction mixture, impurities were removed with PBS, and the NDRG3-bound agarose The resin was transferred to a scintillation solution containing 2 LSC-cocktail (PerkinElmer), and the value of ¹⁴ C was measured and plotted (Fig. 16B, left). The GST-NDRG3 recombinant protein (50) or the GST-NDRG3 (G138W) mutant recombinant protein (50 μg) was reacted with 0.5 μCi of L- [ ¹⁴ C] -lactic acid (PerkinElmer) at 30 ° C. for 1 hour Thereafter, the value of ¹⁴ C was measured in the same manner as above to graph (Fig. 16B, right).

NDRG3 variants were also constructed to confirm the interaction of L-lactate and NDRG3 protein binding sites in the hypoxic state. First, site-directed mutagenesis was carried out using the Myc-tagged NDRG3 expression vector as a template in Example <4-3>, and aspartic acid (Asp, D), which is the 62nd amino acid in the L-lactic acid binding site of NDRG3, NDRG3 (G138W) mutant in which Glycine (G), which is the 138th amino acid in the L-lactic acid binding site of NDRG3, is substituted with arginine (Arg, R), Myc- NDRG3 (A139E) mutant in which alanine (Ala, A) at position 139 of L-lactic acid binding site of NDRG3 was replaced with glutamic acid (Glu, E) and tyrosine NDRG3 (Y229P) mutant in which proline (Pro, P) was substituted for Tyr and Yyr. Then, each of the Myc-NDRG3 and Myc-NDRG3 mutants was transformed into HEK293T cells as described in Example 3, and then normal oxygen (21% O ₂ ) Cells were harvested and lysed in a hypoxic state (1% O ₂ ) or 20 μM MG132 treated and hypoxic (1% O ₂ ) for 24 hours. The cell lysates were then subjected to Western blotting using anti-Myc and anti-beta actin antibodies (Figure 16c).

MCF-7 cells were maintained in a hypoxic condition (1% O ₂ ) for 24 hours, then replaced with fresh medium, and further supplemented with normal oxygen (21% O ₂ ), and the cells were collected and lysed as in Example 3 above. Then, the cell lysate was subjected to Western blotting using anti-NDRG3, anti-HIF-1 alpha and anti-beta-actin antibody (FIG.

As a result, the expression of the recombinant protein of the NDRG3 (G138W) mutant in which the lactic acid binding site of NDRG3 and NDRG3 was mutated was confirmed (Fig. 16A), as shown in Figs. 16A to 16C. In the case of NDRG3 recombinant protein in vitro, (Fig. 16B, left), and the lactate-binding ability of the NDRG3 mutant was decreased (Fig. 16B, right). In addition, while normal NDRG3 increased protein accumulation in the hypoxic state, while mutants that mutated the lactate-binding site of NDRG3 showed reduced protein accumulation in the hypoxic state (Fig. 16c), indicating that NDRG3 binds to lactic acid , Which was confirmed to be associated with accumulation of NDRG3 protein (Figs. 16A to 16C).

In addition, as shown in FIG. 16D, the HIF-1α protein is rapidly eliminated by reoxygenation, whereas the NDRG3 protein accumulated in the hypoxic state and accumulated in the NDRG3 lactic acid complex is stably maintained in the cell even when the NDRG3 protein is restored to the normal oxygen state (Fig. 16D). Thus, the results of Example 7 demonstrate that hypoxia-induced lactic acid acts as a sensor of NDRG3 in sustained hypoxic reactions and promotes HIF-independent biological response by NDRG3.

< Example  8> Hypoxia  Lactic acid-dependent NDRG3 Confirm the function of

<8-1> Hypoxia  In the reaction NDRG3 Confirmation of Lactic Acid-Dependent Cell Growth Promotion by

In order to confirm the effect of NDRG3 on lactic acid-dependent cell growth under hypoxic conditions, MTT assay, colony forming assay and in vivo transplantation using NDRG3 mutant and lactic acid production inhibited cells were performed. The volume of one tumor was measured.

Specifically, to confirm the effect of the ectopic mutant NDRG3 (N66D) on cell growth after inhibition of lactic acid production, the expression vector of heterologous mutant NDRG3 (N66D) was transformed into Huh-1 cells as in Example <4-3> after transfection, a 96 well plate Huh-1 cells and the NDRG3 (N66D) mutant over-expressing Huh-1 cells, 1000 cells / well cultured, treated with sodium formate oxa by concentration and light hypoxia (3% O ₂₎ . &Lt; / RTI > Then, MTT assay was performed to confirm the cell growth as shown in the above Example <6-3> (FIG. 17A).

In addition, colony formation assay was performed to confirm the effect of ectopic mutant NDRG3 (N66D) on cell growth after inhibition of lactic acid production. After inoculation with Huh-1 cells and 0.5 × 10 ⁴ cells / 2 of Huh-1 cells overexpressing the NDRG3 (N66D) mutant in a 6-well plate, 40 mM sodium oxamate was treated or not treated, O ₂ ) for 10 days with dilution with fresh medium every 3 days. The colonies formed after 10 days of culture were identified and fixed with 100% methyl alcohol, and then stained with 30% giemsa stain solution (Sigma) and counted (Fig. 17B).

MTT assay was also performed to confirm the effect of ectopic mutant NDRG3 (N66D) on cell growth after inhibition of lactate production by LDHA deletion in hypoxic condition. First, Huh-1 cells lacking LDHA were prepared by the method described in Example <4-4>, and then transformed with an expression vector of NDRG3 (N66D) mutant as in <Example 3> And NDRG3 (N66D) mutant over-expressing Huh-1 cells. Then, the control GFP-deficient Huh-1 cells, the LDHA-deficient Huh-1 cells and the LDHA deletion / NDRG3 (N66D) -expressing Huh-1 cells were cultured in a 96-well plate at 1,000 cells / % O ₂ ). Then, MTT assay was performed to confirm the cell growth as shown in the above Example <6-3> (FIG. 17C).

In order to confirm the effect of the ectopic mutant NDRG3 (N66D) on the cell growth after inhibition of lactate production by LDHA deletion in vivo, the tumor volume was measured after transplantation of the tumor cells into the mouse. The control GFP-deficient Huh-1 cells, the LDHA-deficient Huh-1 cells and the LDHA deletion / NDRG3 (N66D) -expressing Huh-1 cells were transplanted into BALB / c mice by the method described in Example <6-3> After that, the tumor volume was measured using a caliper at a given time and plotted (Fig. 17D).

In order to confirm the effect of lactic acid production inhibition on the expression of ectopic mutant NDRG3 (N66D) protein in continuous hypoxic state, Huh-1 cells and the NDRG3 (N66D) mutant overexpressing Huh-1 cells were treated with 40 mM sodium oxamate The cells were maintained and cultured for a period of time under a light and hypoxic condition, and the cells were recovered and dissolved as in Example 3 above. Then, the cell lysate was subjected to Western blotting using anti-NDRG3 and anti-beta actin antibody (Fig. 17E).

In order to confirm the effect of the ectopic mutant NDRG3 (N66D) in the production of lactic acid under the continuous hypoxic state, Huh-1 cells treated with or without sodium oxamate as described above and cultured for a time period under mild hypoxic condition and NDRG3 (N66D) mutant over-expressing Huh-1 cells were recovered and the production of L-lactate (L-lactate) was measured by the method described in Example <7-1>. Values were normalized with L-lactic acid standard curve (Fig. 17f).

As a result, as shown in Figs. 17A to 17D, when sodium oxamate was treated in a low-oxygen state, cell growth was inhibited in a concentration-dependent manner, whereas when an ectopic mutant of NDRG3 was overexpressed, It was confirmed that the cell growth was increased (Fig. 17A). Even when lactate production is inhibited under normal oxygen conditions, tumor growth is promoted in the same manner as hypoxic conditions when the NDRG3 heterologous mutant is overexpressed (FIG. 17B). In addition, inhibition of lactate production by LDHA deletion inhibited tumor growth in vitro and in vivo, whereas tumor overgrowth was significantly increased when NDRG3 heterologous mutants were overexpressed even when LDHA was deleted (Fig. 17c and 17d).

17E and 17F, the accumulation of NDRG3 was inhibited when sodium oxamate was treated in the hypoxic state to inhibit the production of lactic acid. On the other hand, when the NDRG3 heterologous mutant was overexpressed, the NDRG3 mutant (Fig. 17E). In addition, it was confirmed that NDRG3 ectopic mutant had no direct effect on the production of lactic acid by confirming that sodium oxamate treatment in a low-oxygen state did not change lactic acid production even when NDRG3 heterologous mutant was overexpressed in the case of no treatment ). Thus, the above results confirm that NDRG3 is an important mediator in lactic acid-induced cell growth under hypoxic conditions.

<8-2> Hypoxia  In the reaction NDRG3 Confirmation of lactate-dependent angiogenesis promotion by

To confirm the effect of NDRG3 on lactate - dependent angiogenesis in hypoxic conditions, tube formation assay was performed using NDRG3 mutant overexpressing mutant overexpressing cells.

Specifically, Huh-1 cells overexpressing the NDRG3 (N66D) mutant were prepared and treated with or without sodium oxamate in control cells and Huh-1 cells overexpressing NDRG3 (N66D) ₂ ) for 24 hours, and the cells were recovered as in Example <6-2> to perform tube formation assay (FIG. 18).

As a result, as shown in FIG. 18, when sodium oxamate was treated to suppress lactic acid production, angiogenesis was suppressed in a hypoxic condition, whereas when NDRG3 isomeric mutant was overexpressed, Confirming that NDRG3 is an important mediator of lactate-induced angiogenesis in a sustained hypoxic state (Fig. 18). Therefore, lactic acid is an important signal of hypoxic cell proliferation and angiogenesis through the results of Example 8, and it was confirmed that NDRG3 acts as an important mediator of lactic acid-induced cell proliferation and angiogenesis in a continuous hypoxic condition .

< Example  9> NDRG3 - Mediated Hypoxia  Reaction Molecular  Confirm adjustment

<9-1> Hypoxia  In state NDRG3 C- Shelf - ERK  Confirming Active Regulation

In-vitro kinase assays, pull-down assays, immunoprecipitation and Western blotting were performed to confirm the role of NDRG3 in lactate-induced molecular responses in hypoxic conditions.

Specifically, under the above Example <4-4> The method used to control or shGFP shNDRG3 as described in GFP or deletion NDRG3 PLC / PRF / 5] After the fabrication of the cells, the cells to hypoxia (1% O ₂₎ 24 The cells were harvested as described in Example 3 above. Then, the phosphorylated protein was identified according to the manufacturer's procedure using a human phosphoryl-kinase array kit (FIG. 19A).

Huh-1, Huh-7 and 786-O cells were cultured for 24 hours under normal oxygen condition (21% O ₂ ) in order to confirm the molecular regulation function according to the expression level of NDRG3. Cells were recovered and dissolved as in Example 3, and Western blotting was performed using anti-NDRG3, anti-phosphorylated-ERK1 / 2, anti-ERK1 / 2 and anti-beta actin antibody (FIG.

In order to confirm the molecular function of NDRG3 in a hypoxic state, GFP or NDRG3-deficient SK-HEP-1 cells were prepared using the control shGFP or shNDRG3 by the method described in Example < 4-4 > Were maintained and cultured under hypoxic conditions over time, and cells were recovered and dissolved as in Example 3, and Western blotting was performed using anti-phosphorylated-ERK1 / 2 and anti-ERK1 / 2 antibodies , Above). The prepared cells were cultured under hypoxic conditions for 24 hours, and the cells were recovered and dissolved as in Example 3, and anti-NDRG3, anti-phosphorylated-c-Raf (S338), anti- Western blotting was performed using Raf, anti-phosphorylated-B-RAF1 (S445), anti-B-RAF1, anti-phosphorylated-A-RAF (S299), anti-A- RAF and anti- (Fig. 19C, bottom).

In order to confirm the interaction of NDRG3 and c-Raf by hypoxic reaction in vitro, a recombinant plasmid pET-28a-c-Raf construct encoding c-Raf was first cloned. Then, the c-Raf expression vector was transformed into a BL21 Escherichia coli strain to obtain a recombinant protein, and the c-Raf recombinant protein was transformed with Ni-NTA agarose resin as described in Example <7-1> And purified according to the manufacturer's procedure. Next, the purified recombinant proteins c-Raf-His and NDRG3-GST were reacted with Ni-NTA agarose resin to perform His pull-down as in Example <4-1> Western blotting was performed using anti-NDRG3 antibody as in Example 3 (Fig. 19D, left). In addition, the Flag-tagged c-Raf expression vector was cloned and then the Flag-tagged c-Raf vector was transformed into HeLa cells as in Example 3 above. Then, the Flag-c-Raf-overexpressed HeLa cells were cultured in a hypoxic condition (1% O ₂ ) for 24 hours, immunoprecipitated using anti-FLAG M2 beads, Blotting was performed (Fig. 19D, right).

In order to confirm the molecular function of NDRG3 in a hypoxic state, the expression of NDRG3 was determined using four kinds of siNDRG3 (siGENOME SMARTpool, Dharmacon) (SEQ ID NOS: 5 to 8) as in Example <4-2> Inhibited SK-HEP-1 cells were prepared and transformed with the Flag-tagged c-Raf expression vector as described in Example 3 to obtain NDRG3 deletion / c-Raf overexpressing cells. Also, NDRG3 (N66D) and c-Raf-overexpressing HEK293T cells were obtained using Myc-tagged NDRG3 (N66D) and Flag-tagged c-Raf expression vectors as described in Example 3 above. Then, the cells were cultured for 24 hours in a normal oxygen state (21% O ₂ ), and the cells were recovered and dissolved as in Example 3, and then anti-NDRG3, anti-phosphorylated-c-Raf B-RAF1, anti-phosphorylated-ERK1 / 2, anti-ERK1 / 2 and anti-beta actin antibodies, (Fig. 19E).

In addition, in vitro kinase assays were performed using the [-32P] -ATP (PerkinElmer) label to identify phosphorylation by NDRG3 in hypoxic conditions. First, HEK293T cells transformed with the Myc-tagged NDRG3 (N66D) expression vector were prepared as described in Example 3, and the NDRG3 protein was immunoprecipitated using anti-Myc-affinity gel. The immunoprecipitated NDRG3 complex was then subjected to treatment with or without the PKC inhibitor PKC-1 5 [mu] M for radiation labeling and incubated with 40 reaction buffer (PKC active buffer and PKC contained in the SignaTECT protein kinase C assay kit (Promega) C-Raf-GST recombinant protein), for 30 min at room temperature for 30 min. The reaction mixture was then electrophoresed on 8% SDS-PAGE and ³² P-labeled c-Raf was detected by autoradiography (Fig. 19f).

MCF-7 cells were cultured under steady state (21% O ₂ ) for 24 hours in order to confirm lactate dependence of NDRG3-mediated molecular pathway activity in the hypoxic state, and in Example <7-1> Cells treated with LDFA-inhibited MCF-7 cells and sodium oxamate obtained by the method described in Example 1 were cultured in a hypoxic condition (1% O ₂ ) for 24 hours. Then, the cells were recovered and dissolved as in Example 3, and then treated with anti-NDRG3, anti-phosphorylated-c-Raf (S338), anti-c-Raf, anti- / 2 and an anti-beta actin antibody (Fig. 19G).

As a result, as shown in FIGS. 19A to 19D, when NDRG3 is deleted, the phosphorylation of ERK1 / 2 is decreased in a continuous hypoxic state (FIGS. 19A and 19C, By confirming that the phosphorylation level of ERK1 / 2 is different in proportion to the expression level of NDRG3 protein in different kinds of cells (Fig. 19B), it was confirmed that NDRG3 activates ERK1 / 2 in continuous hypoxic response. In addition, the phosphorylation of c-Raf and B-RAF1 was inhibited as well as ERK1 / 2 phosphorylation as the hypoxic state persisted in the absence of NDRG3 (Fig. 19c, By confirming the interaction of NDRG3 and c-Raf with the hypoxic response (19d, right), it was confirmed that NDRG3 is involved in activation of ERK and c-Raf in the hypoxic state (Figs. 19A to 19D).

19E and 19F, phosphorylation of ERK1 / 2 and c-Raf is induced even in a normal oxygen state when NDRG3 (N66D), which is an ectopic mutant, is overexpressed (Fig. 19 (e)) and NDRG3 isomeric mutant molecule Lt; RTI ID = 0.0 &gt; c-Raf &lt; / RTI &gt; protein (Fig. 19f).

In addition, as shown in Fig. 19G, it was confirmed that the expression of NDRG3 protein was inhibited and the activity of the c-Raf-ERK pathway was inhibited when lactic acid production was suppressed in a hypoxic state (Fig. 19G). Thus, the above results confirm that NDRG3 acts as an essential mediator of c-Raf-ERK signaling activity induced by lactic acid in the hypoxic response.

<9-2> Hypoxia  In state NDRG3 On by Kinase  Check path adjustment

RACK1 is well known to maintain active conformation as a scaffold protein for PKC (Lendahl, U. et al., Nat. Rev. Genet., 2009 (10), 821- 832), PKC has been reported to phosphorylate and activate c-Raf (Epstein, AC et al., Cell, 2001 (107), 43-54, Mahon, P. c. Et al., Genes Dev, 2001 (15), 2675-2686). Therefore, in order to confirm the association of RACK1 in the kinase pathway activity by NDRG3 in the hypoxic state, immunoprecipitation and Western blotting were carried out. To confirm the complex formation between NDRG3 and kinase pathway proteins, Modeling was performed.

Specifically, in order to confirm the interaction between NDRG3 and RACK1, NDRG3 and / or Flag-tagged RACK1 expression vector was transformed into HeLa cells as in the above Example 3 and cultured in normal oxygen (21% O ₂ ) After culturing for 8 hours in the presence of μM MG132, the cells were recovered and lysed and immunoprecipitated using anti-FLAG M2 beads. Western blotting was then performed using anti-NDRG3 antibody to identify NDRG3 associated with RACK-1 (Fig. 20A).

In order to confirm the relationship between the proteins involved in the kinase pathway by NDRG3, C-Raf, RACK1-overexpressing and / or NDRG3-deleted HEK293T cells and c-Raf, RACK1 and / (N66D) overexpressing HEK293T cells were treated or not treated with 5 [mu] M of PKC-I (LY333531) and cultured for 24 hours under normal oxygen conditions. Cells were recovered and dissolved as described in Example 3, Western blotting was performed using NDRG3, anti-phosphorylated-c-Raf (S338), anti-c-Raf, anti-phosphorylated-ERK1 / 2, anti-ERK1 / 2, anti- (Fig. 20B).

NDRG3 (N66D) overexpression and / or RACK1 (N66D) overexpression using the Myc-NDRG3 (N66D) expression vector and / or siREN1 (siGENOME SMARTpool, Dharmacon) were used to confirm the relationship between proteins involved in the kinase pathway by NDRG3 in the hypoxic state. Deficient HeLa cells were cultured under hypoxic conditions (1% O ₂ ) for 24 hours, immunoprecipitated using an anti-Myc-affinity gel as described in Example 3, and anti-PKC- (Fig. 20C).

In addition, under the settlement HeLa cells oxygen conditions (21% O ₂₎ in order to determine the effect of PKC on the ERK activation in hypoxic, or PKC inhibitors of PKC-I (GO; GO 6976 ) 1 μM or PKC-I ( LY; LY333531) and maintained in a hypoxic state (1% O2) for 24 hours. Cells were recovered and lysed as described in Example 3, and anti-phosphorylated-ERK1 / 2 and anti-ERK1 / 2 < / RTI > antibody (Fig. 20d).

In order to confirm the formation of the NDRG3-cRaf-RACK1-PKC-? Complex in the hypoxic state, Flag-c-Raf and Flag-RACK1 expression vectors and / or Myc-NDRG3 (N66D) HEK293T cells were transfected and cultured for 24 hours under normoxic conditions. Cells were harvested and lysed. Immunoprecipitation was then performed using anti-Myc-affinity gel, followed by SDS-PAGE electrophoresis followed by Western blotting using anti-Flag, anti-Myc and anti-PKC-beta antibody (Figure 20e) .

In order to confirm the formation of the NDRG3-cRaf-RACK1-PKC-beta complex in the hypoxic state, a protein docking simulation was performed as in Example <4-3>. Docking for multiple target structures (ie, NDRG3, RAF1, RACK1 (GNB2L1) and PKC-β) was performed through a two-step experiment. In the first step, NDRG3 was used as a receptor protein in a docking experiment for each target. In the second step, the resulting protein-protein interaction was used as a receptor protein for the docking of other proteins. The input order for the second experiment was c-Raf, RACK1 and PKC-β, in order. Docking calculations were performed by root-mean-square deviation (RMSD) and the results were filtered where the output from a single input matched the output from multiple inputs. Of the filtered results, the most stable one was selected using a total score of HEX 6.3 (sum of shape scores and points of vestibular function) (Fig. 20F, supra) Respectively. In the first phase, PDB (protein data bank) proteins in order to find a candidate template used for homology modeling from a well-known structure in the sequence database, - performing a protein BLAST (BLASTp) search, and having a small p- value less than 10 ^-3 One of the highest identity scores was identified. In the second step, BLAST was performed to perform the alignment process and restrained spaces were identified in the structure. In a third step, prediction of protein structure is performed using the well-known homology modeling program Modeller 9v10 (N. Eswar, MA et al., Current Protocols in Bioinformatics, John Wiley & Sons, Inc., Supplement 15, 5.6.1-5.6. Finally, the best protein model was evaluated using the DOPE (Discrete Optimized Protein Energy) evaluation method (Shen MY, et al., Protein Sci. 2006 Nov; 15 (11): 2507-24) (Fig. 20 (f), below).

As a result, as shown in Figs. 20A to 20D, it was confirmed that RACK1 interacts with NDRG3 (Fig. 20A). In addition, c-Raf and ERK1 / 2 phosphorylation was inhibited when NDRG3 was overexpressed while RACK1 was overexpressed, whereas c-Raf and ERK1 / 2 phosphorylation were increased when NDRG3 (N66D) mutant was overexpressed, while RACK1 and NDRG3 It was confirmed that phosphorylation of c-Raf and ERK1 / 2 was inhibited when PKC activity was inhibited even when the protein was overexpressed (Fig. 20B). In particular, when RACK1 is deleted under hypoxic conditions, the interaction of NDRG3 with PKC is inhibited (FIG. 20c), confirming that inhibition of PKC activity by PKC inhibitors inhibits phosphorylation of ERK1 / 2 at hypoxic conditions 20d), it was confirmed that NDRG3 interacts with RACK1 and PKC-beta in the hypoxic state to promote c-Raf-ERK phosphorylation (Figs. 20a to 20d).

In addition, as shown in FIGS. 20E and 20F, NDRG3 exists in a complex with c-Raf, RACK1 and PKC-β in the cell (FIG. 20E), and docking smulation and protein structure modeling are performed to form NDRG3-c -Raf-RACK1-PKC- < / RTI > quaternary complex (Figure 20F). Therefore, NDRG3 interacts with RACK1 through the results of Example 10, whereby c-Raf is phosphorylated by PKC? Induced by the NDRG3-RACK1 complex, and thus lactic acid-regulated NDRG3 binds to c-Raf and RACK1 as a scaffold protein.

< Example  10> NDRG3  Confirm pathological changes by expression

<10-1> NDRG3  Promoting tumor formation and angiogenesis in overexpressing transgenic mice

To confirm pathologically the effect of NDRG3 expression, immunohistochemical analysis using NDRG3 transgenic mice was performed to confirm tumor formation, and expression of NDRG3 and the expression of activated ERK1 / 2 protein Western blotting and RT-PCR were performed to confirm gene expression changes.

Specifically, to examine the effect of NDRG3 overexpression on tumor formation, NDRG3-overexpressed transformed C57 / BL6 mice (40 rats) and control mice (32 mice), which were prepared by overexpression of the NDRG3 gene as described in Example 1, Tumor formation was determined from time to time until the 24th month, and tumor-free Kaplan-Meier analysis was performed (FIG. 21A).

In order to examine the effect of NDRG3 overexpression on the tumor formation of various tissues, the lungs of the above-mentioned NDRG3 overexpressing transgenic mice and control mice, the 20-month old NDRG3-overexpressing transgenic mice and the control mice, And tumor formation was observed after the hypogastrium tissues of the NDRG3-overexpressing transgenic mice and the control mice were extracted (FIG. 21B).

In order to confirm the effect of NDRG3 overexpression on lymphoma expression, immunohistochemical analysis was performed using the mesenteric lymph node, spleen, and liver tissues in the NDRG3 transgenic and control mice, Respectively. First, lymph nodes, spleen, and liver tissues were extracted from the NDRG3 over-expressing transgenic mice and control mice, and fixed with 10% formalin at room temperature for one day. The fixed tissue was then treated with paraffin and sectioned to 4 μm and transferred to a silanylated slide (Histoserv). The sectioned tissue slides were treated with 0.01 M citrate buffer (pH 6.0) for antigen detection and heated at 100 for 2 minutes. The slides were then cooled and treated with 3% hydrogen peroxide / PBS for 5 minutes to inactivate peroxidase in the tissues, followed by 30% non-immunized mouse or goat serum Min. CD45R and anti-CD3 antibodies for the detection of CD45R and T cell marker CD3, which are B cell markers expressed in lymphomas, and the label is DAB (3, 3'-diaminobenzidine) substrate chromogen chromogen) solution and detected by avidin-biotin complex (ABC) method. Further, the slide was stained with hematoxylin-eosin (H & E) and observed and photographed under a microscope (Fig. 21C).

In order to examine the effect of NDRG3 overexpression on hepatic tumors, liver tissues of the three NDRG3 over-expressing transgenic mice TG-2, TG-8 and TG-13 prepared by the method described in Example 1 above After extraction, they were sectioned as described above. The sectioned liver tissue was then used to perform and visualize immunohistochemical staining as described above. PCNA and anti-CDNA for the detection of PCNA and Ki-67, hepatocellular carcinoma (HCC) markers, glutamine synthetase (GS) and heat shock protein 70 (HSP) -HSP70, anti-Ki-67 and anti-GS antibodies. In addition, the sectioned tissues were visualized by immunofluorescence staining using an anti-NDRG3 antibody and a confocal microscope by the method described in Example <4-1> (FIG. 21D).

In addition, in order to examine the effect of NDRG3 overexpression on liver tumors, hepatic tissues were isolated from NDRG3-overexpressing transgenic mice and control mice and frozen with liquid nitrogen. Then, RT-PCR was performed by the method described in Example <4-2> above. The above tissues were dissolved in the same manner as in Example 2, and Western blotting was performed using anti-NDRG3, anti-phosphorylated-ERK1 / 2 and anti-ERK1 / 2 antibodies (FIG. 21E).

As a result, as shown in Figs. 21A to 21C, tumors started to form in NDRG3 transgenic mice after 9 months (Fig. 21A), and tumors were found in various organs including lung, bowel and lower abdomen (Fig. 21B). In addition, lymphoma-expressing B-cells and T-cells were found in secondary lymphoid organs such as mesenteric lymph nodes and spleen as well as liver of NDRG3-overexpressing mice (Fig. 21c).

21d and 21e, the expression of the hepatocellular carcinoma marker and the cell proliferation marker was remarkably high in all three NDRG3 over-expressing transgenic mice (Fig. 20d), and the expression of IL- (Fig. 21E), confirming that the expression of IL-1α, IL-1β IL-6, COX-2 and PAI-1 mRNA was increased and ERK1 / 2 phosphorylation was increased Of the patients.

<10-2> In patients with cancer NDRG3  Expression and Kinase  Verifying Path Active

To confirm the clinical relevance of NDRG3 in cancer, a tissue microarray assay was performed using liver cancer tissue from human liver cancer patients.

Specifically, tissue samples were obtained from patients with pathologically defined HCC at Inje University Paik Hospital. All tissue samples were fixed with 10% buffered formalin and treated with paraffin. The paraffin-treated HCC tissue samples (donor blocks) were then subjected to a core tissue biopsy (diameter 2 mm) and analyzed using a trephine instrument (Superbiochips Laboratories, Seoul, Korea) And aligned to the receiver paraffin block (tissue array block). Results The tissue array block included 104 HCCs and 20 non-neoplastic tissues. Immunohistochemical staining was performed by the method described in the above Example <11-1> using the 4-section of the tissue array block. NDRG3, or phosphorylated-ERKl / 2, with anti-NDRG3 and anti-phosphorylated-ERK1 / 2 antibodies. In addition, normal saline was used as a negative control. Nuclear staining for> 10% medium grade cytoplasmic staining and / or NDRG3 for cell membrane staining and> 10% weakness for phosphorylated-ERK were scored positively. The statistical significance between the expression levels of NDRG3 protein and phosphorylated-ERK was evaluated in two tests (Figure 22).

As a result, as shown in Fig. 22, NDRG3 protein was hardly found in the normal liver, whereas NDRG3 protein expression was strongly observed in cytoplasm and plasma membrane of liver of HCC patients, and expression of phosphorylated ERK1 / 2 protein was remarkable . In particular, it was confirmed that ERK1 / 2 protein was phosphorylated in 19 (76%) HCC tissues among 25 HCC tissues expressing NDRG3 protein (FIG. 22). Thus, the above results confirm that abnormal expression of NDRG3 promotes tumorigenesis and activates the ERK1 / 2 pathway.

< Example  11> Hypoxia  In the reaction NDRG3 Confirm the control mechanism of

Based on the results of Examples 3 to 11, a schematic model of the role of NDRG3 in the hypoxic reaction was completed.

23, the accumulation of HIF-1 alpha protein is induced by the inactivation of PHD2 in the early stage of hypoxic state, and as a result, genes (LDHA, PDK1, etc.) related to metabolic adaptation of cells due to hypoxia are raised And the corresponding process is activated. Thereafter, expression of NDRG3 protein was increased by lactate produced / accumulated by the increased process, along with 294th proline hydroxylation inhibition of hypoxic target site of NDRG3 by hypoxic PHD2 inactivation.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Methods of quantitative analysis for lactic acid content using          NDRG3 protein <130> 2015P-03-022 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 1128 <212> DNA <213> Homo sapiens <400> 1 atggatgaac ttcaggatgt tcagctcaca gagatcaaac cacttctaaa tgataagaat 60 ggtacaagaa acttccagga ctttgactgt caggaacatg atatagaaac aactcatggt 120 gtggtccacg tcactataag aggcttaccc aaaggaaaca gaccagttat actaacatat 180 catgacattg gcctcaacca taaatcctgt ttcaatgcat tctttaactt tgaggatatg 240 caagagatca cccagcactt tgctgtctgt catgtggatg ccccaggcca gcaggaaggt 300 gcaccctctt tcccaacagg gtatcagtac cccacaatgg atgagctggc tgaaatgctg 360 cctcctgttc ttacccacct aagcctgaaa agcatcattg gaattggagt tggagctgga 420 gcttacatcc tcagcagatt tgcactcaac catccagagc ttgtggaagg ccttgtgctc 480 attaatgttg acccttgcgc taaaggctgg attgactggg cagcttccaa actctctggc 540 ctgacaacca atgttgtgga cattattttg gctcatcact ttgggcagga agagttacag 600 gccaacctgg acctgatcca aacctacaga atgcatattg cccaagacat caaccaagac 660 aacctgcagc tcttcttgaa ttcctacaat ggacgcagag acctggagat cgaaagaccc 720 atactgggcc aaaatgataa caaatcaaaa acattaaagt gttctacttt actggtggta 780 ggggacaatt cgcctgcagt tgaggctgtg gtcgaatgca attcccgcct gaaccctata 840 aatacaactt tgctaaagat ggcggactgt gggggactgc cccaggtagt tcagcctggg 900 aagctcaccg aggccttcaa gtactttttg cagggaatgg gctacatacc atctgccagc 960 atgactcggc tcgcccgatc acgaacccac tcaacctcga gtagcctcgg ctctggagaa 1020 agtcccttca gccggtctgt caccagcaat cagtcagatg gaactcaaga atcctgtgag 1080 tcccctgatg tcctggacag acaccagacc atggaggtgt cctgctaa 1128 <210> 2 <211> 284 <212> PRT <213> Homo sapiens <400> 2 Glu His Asp Ile Glu Thr Thr His Gly Val Val His His Val Thr Ile Arg   1 5 10 15 Gly Leu Pro Lys Gly Asn Arg Pro Val Ile Leu Thr Tyr His Asp Ile              20 25 30 Gly Leu Asn His Lys Ser Cys Phe Asn Ala Phe Phe Asn Phe Glu Asp          35 40 45 Met Gln Glu Ile Thr Gln His Phe Ala Val Cys His Val Asp Ala Pro      50 55 60 Gly Gln Gln Glu Gly Ala Pro Ser Phe Pro Thr Gly Tyr Gln Tyr Pro  65 70 75 80 Thr Met Asp Glu Leu Ala Glu Met Leu Pro Pro Val Leu Thr His Leu                  85 90 95 Ser Leu Lys Ser Ile Gly Ile Gly Val Gly Ala Gly Ala Tyr Ile             100 105 110 Leu Ser Arg Phe Ala Leu Asn His Pro Glu Leu Val Glu Gly Leu Val         115 120 125 Leu Ile Asn Val Asp Pro Cys Ala Lys Gly Trp Ile Asp Trp Ala Ala     130 135 140 Ser Lys Leu Ser Gly Leu Thr Thr Asn Val Val Asp Ile Ile Leu Ala 145 150 155 160 His His Phe Gly Gln Glu Glu Leu Gln Ala Asn Leu Asp Leu Ile Gln                 165 170 175 Thr Tyr Arg Met His Ile Ala Gln Asp Ile Asn Gln Asp Asn Leu Gln             180 185 190 Leu Phe Leu Asn Ser Tyr Asn Gly Arg Arg Asp Leu Glu Ile Glu Arg         195 200 205 Pro Ile Leu Gly Gln Asn Asp Asn Lys Ser Lys Thr Leu Lys Cys Ser     210 215 220 Thr Leu Le Val Val Gly Asp Asn Ser Pro Ala Val Glu Ala Val Val 225 230 235 240 Glu Cys Asn Ser Arg Leu Asn Pro Ile Asn Thr Thr Leu Leu Lys Met                 245 250 255 Ala Asp Cys Gly Gly Leu Pro Gln Val Val Gln Pro Gly Lys Leu Thr             260 265 270 Glu Ala Phe Lys Tyr Phe Leu Gln Gly Met Gly Tyr         275 280 <210> 3 <211> 12 <212> PRT <213> Homo sapiens <400> 3 Gln Asn Asp Asn Lys Ser Lys Thr Leu Lys Cys Ser   1 5 10 <210> 4 <211> 58 <212> RNA <213> Artificial Sequence <220> <223> shNDRG3 <400> 4 ccggacattg gcctcaacca taaatctcga gatttatggt tgaggccaat gttttttg 58 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siNDRG3 <400> 5 cgccugaacc cuauaaaua 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siNDRG3 <400> 6 gcuaaaggcu ggauugacu 19 <210> 7 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siNDRG3 <400> 7 aaacagacca guuauacua 19 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siNDRG3 <400> 8 gaucaaacca cuucuaaau 19 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 forward primer <400> 9 aaccataaat cctgtttcaa tg 22 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 reverse primer <400> 10 tccacaacat tggttgtcag g 21 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 (N66D) sense primer <400> 11 gaccataaat cctgtttcaa tgcattcttt 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 (N66D) antisense primer <400> 12 gaggccaatg tcatgatatg ttagtataac 30 <210> 13 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siGFP sense <400> 13 guucagcgug uccggcgagt t 21 <210> 14 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siGFP antisense <400> 14 cucgccggac acgcugaact t 21 <210> 15 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD1 sense <400> 15 caucgagcca cucuuugact t 21 <210> 16 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD1 antisense <400> 16 gucaaagagu ggcucgaugt t 21 <210> 17 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD2 sense <400> 17 aacggguuau guacgucaut t 21 <210> 18 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD2 antisense <400> 18 augacguaca uaacccguut t 21 <210> 19 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD3 sense <400> 19 ccagauaugc uaugacugut t 21 <210> 20 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siPHD3 antisense <400> 20 acagucauag cauaucuggt t 21 <210> 21 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siP4HTM sense <400> 21 gagugucggc ucaucaucct t 21 <210> 22 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siP4HTM antisense <400> 22 ggaugaugag ccgacacuct t 21 <210> 23 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siP4HA1 sense <400> 23 gaucugguga cuucucugat t 21 <210> 24 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> siP4HA1 antisense <400> 24 ucagagaagu caccagauct t 21 <210> 25 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 (G138W) sense primer <400> 25 gtttgggctg gagcttacat cctcagc 27 <210> 26 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NDRG3 (G138W) antisense primer <400> 26 tccaattcca atgatgcttt tcaggct 27

Claims (13)

1. A method for quantifying intracellular lactic acid comprising the step of measuring the expression level of NDRG3 (N-myc downstream-regulated gene 3) protein consisting of the amino acid sequence of SEQ ID NO: 1 in a cell,
Wherein the NDRG3 protein binds to and accumulates in the intracellular lactic acid.
The method according to claim 1, wherein the cells are collected from any one selected from the group consisting of tissue, blood, plasma, serum, saliva, semen and urine.
delete The method according to claim 1, wherein the lactic acid is bound to the 62nd aspartic acid, the 138th glycine, the 139th alanine or the 229th tyrosine of the amino acid sequence of the NDRG3 protein.
1) treating the cultured cell with the NDRG3 protein consisting of the amino acid sequence of SEQ ID NO: 1; And
2) measuring the expression level of NDRG3 protein in the cells of step 1), comprising the steps of:
Wherein the NDRG3 protein binds to and accumulates in the intracellular lactic acid.
6. The method for quantifying lactic acid according to claim 5, wherein the cultured cells are cultured in a biological cell or an animal cell collected from any one selected from the group consisting of tissue, blood, plasma, serum, saliva, semen and urine.
6. The method according to claim 5, wherein the lactic acid is bound to the 62nd aspartic acid, the 138th glycine, the 139th alanine or the 229th tyrosine in the amino acid sequence of the NDRG3 protein.
6. The method according to claim 5, wherein the method for quantifying lactic acid comprises comparing an expression level of a normal NDRG3 protein with an expression level of a mutated NDRG3 protein at a lactic acid binding site.
delete delete [Claim 6] The method according to claim 5, wherein the NDRG3 protein is an NDRG3 recombinant protein fused with a fluorescent protein.
The method of claim 11, wherein the fluorescent protein is selected from the group consisting of Red Fluorescent Protein (RFP), Enhanced Red Fluorescent Protein (ERFP), Cyan Fluorescent Protein (CFP), Enhanced Blue Fluorescent Protein (YFP), Enhanced Yellow Fluorescent Protein (EYFP), mtFP (monomeric teal Fluorescent Protein), Green Fluorescent Protein (ECF) GFP), and a modified green fluorescent protein (EGFP).
6. The method according to claim 1 or 5, wherein the expression level is determined by an in-vitro binding assay, an enzyme immunoassay (ELISA), an immunohistochemical staining, Western blotting, a protein chip, And a real-time fluorescence imaging method. 2. A method for quantifying lactic acid, comprising the steps of:

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