KR20190079810A - Biomarker for lung cancer metastasis diagnosis comprising rip1 and use thereof - Google Patents

Abstract

The present invention relates to a biomarker for diagnosing lung cancer metastasis, which can determine lung cancer metastatic potential by the expression level of Receptor-interacting protein 1 (RIP1), a diagnostic kit for lung cancer metastasis using the same, and a method for providing information for diagnosing lung cancer metastasis. The present invention enables the rapid, accurate and easy prediction of lung cancer metastasis.

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker for lung cancer metastasis diagnosis including RIP1, and a biomarker for use in diagnosis of lung cancer metastasis,

The present invention relates to a biomarker for diagnosing lung cancer metastasis comprising RIP1 (Receptor-interacting protein 1) and a use thereof. More particularly, the present invention relates to a biomarker for lung cancer metastasis diagnosis, And a method for providing information for diagnosis of lung cancer metastasis.

According to 2008 statistics, one in every 70 people in the population was cancer survivors or survivors after cancer treatment. When Koreans survived to the average life expectancy (81 years), the likelihood of cancer was 36.9% It is estimated that cancer occurs in 1 out of every 10 people (30.5%) in one (37.2%) and women (83). The incidence of cancer in 2013 is 445.7 (449.9 male, 441.5 female) per 100,000 population. In addition, as cancer statistics show that age is increasing, the rapid aging of society is very likely to cause a surge in cancer patients.

Although the 5-year survival rate of cancer patients has increased remarkably over the last 20 years, the first cause of death is still cancer, especially the problem of improving the quality of life of cancer patients, As a reality, the reduction of social costs due to the improvement of cancer treatment rate and the quality of life of patients through continuous cancer research is becoming a national task.

Although the results of RIP1 studies on cancers are mainly due to the apoptosis mechanism, recent studies on the proliferation and survival mechanism of cancer cells by RIP1 have been reported. In addition, research on the mechanism of metastasis by RIP1 is in the stage of initiation, and inhibition of metastasis is considered to be important in cancer therapy, and many researchers are conducting research to prevent cancer metastasis.

Lung cancer is the most common cause of cancer-related deaths in both men and women. In the United States in 2005, lung cancer accounts for more deaths than both breast cancer, prostate cancer, and colon cancer combined. In these lung cancers, metastasis is considered to be important. Since genes involved in lung cancer metastasis are not known, it is necessary to develop a biomarker for lung metastasis diagnosis that enables target treatment by diagnosing metastasis of lung cancer.

Korean Patent Laid-Open Publication No. 2012-0077567 Korean Patent Laid-Open Publication No. 2013-0129347

The present inventors have found that RIP1 has a function of increasing the invasiveness and metastasis of lung cancer cells and completed the present invention.

Accordingly, an object of the present invention is to provide a biomarker for lung cancer metastasis diagnosis comprising RIP1.

It is also an object of the present invention to provide a lung cancer metastasis diagnostic kit comprising means capable of measuring the expression level of RIP1.

Another object of the present invention is to provide a method for providing information for diagnosis of lung cancer metastasis.

In order to achieve the above object, the biomarker for lung cancer metastasis diagnosis of the present invention may contain RIP1 (Receptor-interacting protein 1).

The lung cancer metastasis diagnostic kit of the present invention may comprise means for measuring the expression level of RIP1.

Said means may be to measure the expression level of RIP1 protein.

The means for measuring the expression level of the RIP1 protein may be selected from the group consisting of an antibody, an antibody fragment, a protein microarray, and an aptamer.

The means may be to measure the expression level of mRNA of RIP1.

The means for measuring the expression level of mRNA of RIP1 may be selected from the group consisting of primers and probes.

A method for providing information for lung cancer metastasis diagnosis according to the present invention may include the following steps.

(1) measuring the amount of RIP1 expression from a lung cancer patient sample;

(2) measuring the amount of RIP1 expression from a normal control sample; And

(3) comparing the expression amount of step (1) with the expression amount of step (2).

The method for providing information for diagnosis of lung cancer metastasis according to the present invention may further comprise the step of determining that the lung cancer metastatic potential is high when the expression level of step (2) is equal to or greater than the amount of metastasis of step (1) have.

The amount of RIP1 expression may be measured by measuring the amount of protein or mRNA.

The amount of the protein may be measured by an antigen-antibody reaction.

The antigen-antibody reaction may be measured by Western blotting, antigen-antibody reaction, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, immunohistochemistry or rocket immunization Electrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS or protein chip assays.

Measurement of the amount of mRNA may be performed by reverse transcriptase-polymerase chain reaction (RT-PCR), northern blotting, or nucleic acid microarray.

The present invention provides a biomarker for diagnosis of lung cancer metastasis, a kit for diagnosing lung cancer metastasis using the same, and a method for providing diagnosis information of lung cancer metastasis, thereby enabling rapid, accurate and easy prediction of the incidence of lung cancer metastasis, Can be used as a specific target in the development of antiretroviral drugs and can provide accurate baseline information on planning and performing effective protocols for future treatment by the clinician.

1 is a clinical data analyzing the expression of RIP1 and visentin in patients with lung cancer.
Figure 2 shows EMT expression analysis in MEF and lung cancer cells.
Figure 3 shows the analysis of the EMT activity and cell mobility increase of RIP1 overexpressing cells.
Fig. 4 shows the results of analysis of cell mobility by RIP1 kinase.
Figure 5 shows the results of analysis of cell mobility in RIP1 kinase inhibitory cells.
FIG. 6 shows the results of analysis of signal transduction involved in cell mobility by RIP1.
Figure 7 shows the results of analysis of EGFR signaling by RIP1.
Fig. 8 shows the results of analysis of the activation of the inflammatory reaction by RIP1.
Fig. 9 shows the results of analysis of the relationship between RIP1 and NF-κB signal transduction.
Figure 10 shows increased transit capacity and reduced survival rate by RIPl in animal models.
Fig. 11 shows the results of analysis of cell mobility activity by 5-aza.
Fig. 12 shows the results of analysis of RIP1 and vimentin expression by 5-aza.

BRIEF DESCRIPTION OF THE DRAWINGS The advantages and features of the present invention, and how to achieve them, will become apparent with reference to the embodiments described in detail below with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art. It is to be understood by one of ordinary skill in the art that the scope of the invention is to be fully understood and the invention is only defined by the scope of the claims.

Unless defined otherwise, all terms (including technical and scientific terms) used herein may be used in a sense commonly understood by one of ordinary skill in the art to which this invention belongs. Also, commonly used predefined terms are not ideally or excessively interpreted unless explicitly defined otherwise.

Hereinafter, a biomarker for diagnosing lung cancer metastasis including RIP1 according to the present invention, a kit for diagnosing lung cancer metastasis using the same, and a method for providing information for lung cancer metastasis diagnosis will be described in detail.

RIP1 is thought to be a kinase that plays an important role in determining cell death. RIP1 has three domains, a serine / threonine kinase domain necessary for necroticis, a medial domain containing a motif for the interaction of kinetic interaction, and a death domain for apoptotic activity. The ubiquitination of RIP1 promotes cell survival and diubiquitination reverses cell death in cells. Since there are several domains in RIP1, activation of RIP1 can result in a variety of consequences, including NF-κB, mitogen-activated protein kinase (MAPK), apoptosis or necroticis. Necrotostin-1 (nec-1) blocks RIP1 kinase activity and prevents necroticism induced by death. RIP3 regulates programmed neocorticity and increases RIP1 recruitment in the necromosome. In addition to RIP1 and RIP3 kinases, several other kinases are involved in the phosphorylation of RIP1 and RIP3.

As described above, studies on RIP1 in cancer have been mainly focused on apoptosis, but recently, studies on the proliferation and survival mechanism of RIP1-induced cancer cells have been reported, and the study on the transition mechanism by RIP1 have. In cancer treatment, metastatic suppression is considered to be important, and many researchers are conducting research to prevent cancer metastasis. In lung cancer, metastasis is also important, and there are many genes that are not known to be involved in metastasis. Therefore, it is necessary to find a gene that inhibits metastasis of lung cancer and to find out its function. Furthermore, Method development is needed.

Therefore, in the present invention, RIP1 is presumed to be a candidate for providing an important clue for the induction of lung cancer metastasis, that is, RIP1 has a function of increasing invasiveness and metastasis of lung cancer cells. The following are identified:

We have shown that RIP1 can induce EMT by confirming the relationship between RIP1 and EMT (epithelial-mesenchymal transition) in cells, and EMT induction by RIP1 increases the migration and invasion of cancer cells The signal transduction mechanism of RIP1 was investigated in terms of cell mobility. As a result, it was confirmed that the kinase effect of RIP1 induces cell mobility. The kinase effect of RIP1 confirmed that signal transduction was induced through EGFR-Src phosphorylation, and activation of STAT3 transcription factor through Src sub- Respectively. In addition, the present inventor confirmed that IL-1β, which is a representative marker of inflammatory response, is increased by RIP1, and that RIP1 is also involved in the inflammatory reaction related to proliferation and metastasis of cancer cells. As a result of observing the expression of EMT marker by 1L-1β in lung cancer cells overexpressing RIP1, expression of cell mobility and inflammatory response-related genes was increased by RIP1, and cell mobility was increased by IL-1β, , The use of RIP1 as a biomarker for diagnosing lung cancer metastasis could be confirmed.

Accordingly, the present invention provides a biomarker for lung metastasis diagnosis comprising RIP1 (Receptor-interacting protein 1).

The gene and protein information of RIP1 are registered in National Center for Biotechnology Information (NCBI). [Reference sequence No. NM_001354930.1 of RIP1 gene, reference sequence No. Q13546.3 of RIP1 protein]

In the present invention, the term " biomarker " refers to a molecular marker for the purpose of predicting the diagnosis of lung cancer metastasis. It is a marker of normal or abnormal progression in an individual or a marker of disease or other condition in an individual, Quot; is used interchangeably to refer to < / RTI > More specifically, the biomarker is an anatomical, physiological, biochemical or molecular parameter associated with the presence of chronic, acute, specific physiological condition or progression, if abnormal, whether normal or abnormal. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging.

In addition, the present invention provides a lung cancer metastasis diagnostic kit comprising means capable of measuring the expression level of RIP1.

In the present invention, "amount of expression of RIP1" means the expression amount of RIP1 protein or the amount of mRNA expression of RIP1.

The means may be a known molecular biological means or immunochemical means. In a preferred embodiment, the means may be a measuring means selected from the group consisting of primers, probes, antibodies, antibody fragments, protein microarrays and aptamers. Here, the antibody, the antibody fragment, the protein microarray or the aptamer corresponds to a means for measuring the expression level of the protein, and the primer, the probe or the nucleic acid microarray corresponds to a means for measuring the expression level of the mRNA.

The term "primer" refers to a single-stranded oligonucleotide capable of acting as a starting point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerization enzymes) within a suitable buffer and a suitable temperature it means. The appropriate length of the primer may vary depending on various factors, such as temperature and use of the primer. In addition, the sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer in the present invention does not need to have a perfectly complementary sequence to the nucleotide sequence of the RIP1 gene as a template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the gene sequence and acting as a primer.

The term "probe" refers to a linear or linear oligomer of natural or modified monomer or linkages, which can specifically hybridize to the base sequence of RIP1, which contains deoxyribonucleotides and ribonucleotides and is a target nucleotide, Or artificially synthesized. The probes according to the present invention may be single-stranded, preferably oligodeoxyribonucleotides. In the present invention, the probes may include natural dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), nucleotide analogues or derivatives. In addition, the probe may also include ribonucleotides. For example, the probe can be a backbone modified nucleotide, such as a peptide nucleic acid (PNA), a phosphorothioate DNA, a phosphorodithioate DNA, a phosphoamidate DNA, an amide-linked DNA, an MMI- O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro RNA, 2'- 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and nucleotides with base modifications such as C-5 substituted pyrimidines Is selected from the group consisting of fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethytyl-, propynyl-, alkynyl-, thiazolyl-, imidazolyl-, Pyrrolyl, etc.), 7-deazapurines with C-7 substituents (substituents being fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, Neal, Alkenyl -, quinolyl and quinoxalyl thiazol-, quinolyl and quinoxalyl imidazolidin -, pyridyl-contained, etc.), and it may contain inosine-diamino-purine.

The "antibody" may include a polyclonal antibody, a monoclonal antibody, a recombinant antibody, or the like that can specifically bind to the RIP1 protein.

Since the antibody is commercially available and can be used, the amino acid sequence of the RIP1 protein is known, and the antibody can be directly prepared using the amino acid sequence. The form of the antibody is not particularly limited, and any polyclonal antibody, monoclonal antibody or antigen-binding antibody thereof may be included in the antibody of the present invention, and all immunoglobulin antibodies are included. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies. In addition, the antibody comprises a functional fragment of an antibody molecule as well as a complete form having two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen-binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.

For example, in the case of a polyclonal antibody, RIP1 antigen is injected into an animal and blood is collected from the animal to contain the antibody. Such polyclonal antibodies can be prepared from any animal species host, such as goat, rabbit, sheep, monkey, horse, pig, cow, dog, and the like. Monoclonal antibodies can be prepared using the hybridoma method (Kohler et al., European Jounal of Immunology, 6, 511-519, 1976) well known in the art, or can be prepared using the phage antibody library (Clackson et al., Nature, 352, 624-628, 1991, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991).

The term "antibody fragment" means a functional fragment of an antibody molecule. The functional fragment of an antibody molecule is an antibody fragment having at least an antigen binding function, for example, Fab, F (ab '), F ') 2 and Fv.

The "microarray" may be, for example, a nucleic acid microarray for detecting the RIP1 gene or a protein microarray for detecting the RIP1 protein.

In the present invention, a primer, a probe or an antibody capable of measuring the expression level of the RIP1 protein or a gene encoding the RIP1 protein is used as a hybridizable array element and is immobilized on a substrate. Preferred substrates may include, for example, membranes, filters, chips, slides, wafers, fibers, magnetic beads or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries, as suitable rigid or semi-rigid supports have. The hybridization array elements are arranged and immobilized on the substrate, and such immobilization can be performed by chemical bonding methods or covalent bonding methods such as UV. For example, the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface. In addition, the hybridization array element can be coupled to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).

The "aptamer" means a nucleic acid oligomer having affinity for the RIP1 protein. The aptamers synthesized randomly synthesized DNA oligomers having about 60 bases. Among them, the DNA oligomers binding to the RIP1 protein were separated into affinity columns, amplified and the separation process was repeated to obtain affinity for RIP1 Can be produced.

The lung cancer metastasis diagnostic kit of the present invention may be used for detecting RIP1 gene or detecting RIP1 protein.

When the lung cancer metastasis diagnostic kit of the present invention is used for gene amplification to be applied to the PCR amplification process, the kit of the present invention may optionally comprise a reagent necessary for PCR amplification such as a buffer, a DNA polymerase (for example, Thermus aquaticus (Taq) thermostable DNA polymerase obtained from Thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs, and the lung cancer metastasis diagnostic kit of the invention is immunologically For detection of RIP1 protein applied to the assay, the kit of the present invention may optionally comprise a secondary antibody and a labeling substrate. Further, the kit according to the present invention may be manufactured from a number of separate packaging or compartments containing the reagent components described above.

A method for providing information for lung cancer metastasis diagnosis according to the present invention may include the following steps.

(1) measuring the amount of RIP1 expression from a lung cancer patient sample;

(2) measuring the amount of RIP1 expression from a normal control sample; And

(3) comparing the expression amount of step (1) with the expression amount of step (2).

The method for providing information for diagnosis of lung cancer metastasis according to the present invention may further comprise the step of determining that the lung cancer metastatic potential is high when the expression level of step (2) is equal to or greater than the amount of metastasis of step (1) have.

The sample of the patient refers to a sample capable of measuring overexpression of RIP1, which is a biomarker for lung cancer metastasis diagnosis, and includes samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine. It does not.

In the present invention, the expression level of RIP1 in the normal control group can be compared with the amount of RIP1 expressed in patients suspected to have lung cancer or those with early symptoms of mild lung cancer, thereby diagnosing the actual lung cancer metastasis in patients suspected of having cancer or patients having mild symptoms . That is, when the amount of expression of RIP1 is measured from a sample of a patient, the amount of expression of RIP1 is measured from a sample of a normal person, and the amount of expression of RIP1 is compared with that of a normal sample Lung cancer metastasis can be predicted.

The amount of RIP1 expression is preferably measured by measuring the amount of protein or mRNA.

The amount of the protein is preferably measured by an antigen-antibody reaction.

The antigen-antibody reaction may be measured by Western blotting, antigen-antibody reaction, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, immunohistochemistry or rocket immunization Electrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS or protein chip assay.

The amount of the mRNA may be measured by reverse transcriptase-polymerase chain reaction (RT-PCR), Northern blotting or nucleic acid microarray.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

<Examples>

1. TCGA clinical data of epithelial-mesenchymal transition (EMT) controlled by RIP1

Experiments were performed to determine the relationship between RIP1 intracellular signal transduction and EMT. First, we confirmed that RIP1 and EMT are clinically relevant, and confirmed the correlation between RIP1 and vimentin expression, an EMT marker, in cancer tissues of lung cancer patients.

[Experimental Method]

Based on the TCGA clinical data, the relationship between the expression of RIP1 and visumin in the tissues of lung cancer patients (total: whole tissue in lung cancer patients, primary cancer tissue in NO: lung cancer patients) is shown in the graph of FIG.

[result]

As shown in FIG. 1, the correlation between RIP1 and Vimentin in lung cancer patients was confirmed by using TCGA clinical data. As a result, visemantin expression was high in tissues of patients with high expression of RIP1. These results suggest that RIP1 and Vimentin are correlated and that RIP1 affects the metastasis.

2. Epithelial-mesenchymal transition, which is regulated by RIP1 in MEF and lung cancer cells,

Based on previous clinical data, we have confirmed the relationship between RIP1 and EMT in cells. RIP1 has been reported to be related to TNF-α signaling, and it has been shown in other studies that RIP1 and vimentin expression and EMT-related expression are observed in knockout MEF cell line and lung cancer cell line.

1) The knockout of RIP1, TRADD, TNFR1, and TRAF2 expression in MEF cells was followed by the expression of RIP1 and visentin and the expression of TRAF2 and TRADD.

[Experimental Method]

MEF (WT), MEF (RIP1, TRADD, TNFR1, and TRAF2 knockout) cell lines were seeded at 1 × 10 ⁶ in a 60π dish, the cells were harvested after 24 hours and proteins were extracted, To confirm the expression of RIP1, visentin, MMP2, MMP9 and actin.

[result]

As shown in FIG. 2A, knockout of RIP1 in MEF cells indicates that the expression of MMP2, MMP9, and Vimentin, which are EMT markers, is weakened.

2) expression of Vimentin was detected by RIP1 expression in lung cancer cell line.

[Experimental Method]

A549, H460, H1299, and LU99 lung cancer cell lines were seeded at 1 × 10 ⁶ in a 60π dish, and after 24 hours, the cells were harvested and the proteins were extracted and then subjected to Western blotting to obtain RIP1, Vimentin, MMP2, MMP9 Expression was confirmed.

[result]

As shown in FIG. 2B, expression of RIP1 and EMT markers was detected in lung cancer cell lines using lung cancer cell lines H1299, LU99 and RIP1, which express RIP1 overexpressively, and lung cancer cell lines A549 and H460, which weakly express RIP1.

The cell lines overexpressing RIP1 showed strong vimentin expression and the cell lines weakly expressing RIP1 showed relatively weak expression of vimentin by Western blotting.

From the above results, the expression of visentin was altered by the expression of RIP1, confirming that RIP1 and visentin are correlated. The correlation between RIP1 and Vimentin has shown that RIP1 can induce EMT.

3. Effects of EMT induction by RIP1 on the migration and invasion of cancer cells

In order to examine whether EMT induction by RIP1 can enhance the migration and invasion of cancer cells, RIP1 overexpresses RIP1 in lung cancer cell lines A549 and H460 cells. After cell lines were prepared, invasion and migration analyzes were performed to observe whether cell mobility was promoted by increased expression of RIP1.

[Experimental Method]

1) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Each cell line was seeded at 1 x 10 &lt; ⁶ &gt; in a 60 [mu] dish, and after 24 hours, the cells were harvested and proteins were extracted and then Western blot was performed to express RIP1, His, Vimentin, MMP2, MMP9 and Actin And the results are shown at the top of FIG. 3A.

2) A549, then the trait implanting RIP1 in H460 create a RIP1-overexpressing cell line (A549 RIP1, H460 RIP1), seeded with the vector control cell line (A549 PC, H460 PC) to 5 × 10 ⁵ to 60π plate, 24 Subsequently, the supernatant was harvested after 18 hours to load the gelatin into gel, and the gel was stained with Coomassie Brilliant Blue to confirm the activity of MMP2 and MMP9. The results are shown in the lower part of FIG. 3A Respectively.

3) A RIP1-overexpressing cell line (A549 RIP1, H460 RIP1) was produced by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were seeded onto matigel coating trnaswell ⁴ , and 10% FBS medium was added to the lower chamber. After 24 hours, the cells passed through the transwell were stained to confirm the invasion. The results are shown in FIG. 3B.

4) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were put into the upper chamber 2 × 10 ⁴ in collagen- And 10% FBS medium was added to the lower chamber. After 24 hours, the cells passed through the transwell were stained to confirm cell migration. The results are shown in FIG. 3C.

[result]

As shown in FIG. 3A, the expression of RIP1 and EMT markers was observed by Western blotting in RIP1-overexpressing cell line, and the activity of MMP2 and MMP9 was measured by gelatin zymography assay. RIP1 and EMT markers And MMP2 and MMP9, respectively.

As shown in FIG. 3B and FIG. 3C, invasion and migration analysis using cells overexpressing RIP1 were performed to confirm cell mobility. As a result, it was found that cell mobility was increased as the expression of RIP1 was stronger Respectively.

From the above results, it was confirmed that genes involved in EMT were activated by RIP1, resulting in increased cell mobility.

4. Signal transduction mechanism of RIP1 affecting cell mobility

In the signal transduction pathway of RIP1, the kinase effect is known to be a major signal transduction mechanism, and the role of this kinase in inducing EMT and cell mobility has been observed. In other words, activation of EMT was observed after inhibition of kinase function using RIP1 kinase inhibitor.

[Experimental Method]

1) A549, by transfection implanting RIP1 to H460 in RIP1-overexpressing cell line (A549 RIP1, H460 create a RIP1), the vector control cell line (A549 PC, H460 PC) to Marti gel coating 5 × 10 ⁴ per upper chamber in the transwell , Treated with necro-statin, and 10% FBS medium was added to the lower chamber. After 24 hours, the cells passed through the transwell were stained to confirm infiltration. The results are shown in FIG. 4A.

2) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were put into the upper chamber at 2 × 10 ⁴ in collagen- Treated with necro-statin, and 10% FBS medium was added to the lower chamber. After 24 hours, cells passed through the transwell were stained to confirm cell migration. The results are shown in FIG. 4B.

3) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at ⁶⁰ x 10 &lt; ⁶ &gt; dish and treated with necrotostin. After 24 hours, the cells were harvested and then the protein was extracted and then Western blot was performed to obtain RIP1, Vimentin, MMP2 , And the expression of MMP9 was confirmed. The results are shown in the upper part of FIG. 4C.

4) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were seeded at 5 × 10 ⁵ in a 60π dish, The supernatant was harvested after 18 hours, and the gelatin was loaded on the gel. The gel was stained with Coomassie Brilliant Blue to confirm the activity of MMP2 and MMP9. The results were shown in the lower part of FIG. 4C Respectively.

[result]

As shown in FIGS. 4A and 4B, infiltration and migration analysis were performed after necrostatin treatment, which is a kinase inhibitor of RIP1, to observe cell infiltration and cell migration. As a result, it was confirmed that infiltration and migration were reduced by necrostatin .

As shown in FIG. 4C, the expression of EMT markers was observed by Western blotting upon inhibition of the kinase effect of RIP1, and the activities of MMP2 and MMP9 were measured by gelatin gimography test. In inhibition of the kinase effect, EMT Decreased expression of the marker, and decreased MMP2 and MMP9 activity.

From the above results, it was confirmed that the increase in cell mobility due to RIP1-mediated EMT activation was due to the activation of EMT by the kinase signal transduction activity of RIP1, thereby increasing cell mobility.

5. Increased cell mobility by RIP1 kinase through EGFR-Src signaling

In this experiment, the RIP1 kinase inhibitory cell line as well as RIP1 kinase inhibitor treatment, as well as the inhibition of kinase function of RIP1 at the cellular level, induced EMT induction and cell mobility, thereby confirming the kinase effect of RIP1 by EGFR-Src phosphorylation And to induce signal transduction.

[Experimental Method]

1) RIP1 over-expressing cell lines (A549 RIP1, H460 RIP1) and kinase inhibiting cell line (K45A) were prepared by transfecting A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) × 10 ⁴ into the upper chamber, and 10% FBS medium was placed in the lower chamber. After 24 hours, the cells passed through the transwell were stained to confirm cell infiltration. The results are shown in FIG. 5A.

(A549 PC, H460 PC) were transfected into collagen-coated transwells with 2 × (1 × 10 5 cells / well), and transfected with A549 and H460 to produce RIP1 over- expressing cell lines (A549 RIP1, H460 RIP1) and kinase- 10 ⁴ into the upper chamber, and 10% FBS medium was placed in the lower chamber. After 24 hours, the cells passing through the transwell were stained to confirm cell migration. The results are shown in FIG. 5B.

3) A549 and H460 were transfected with RIP1 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and kinase inhibitory cell line (K45A), and vector control cell lines (A549 PC, H460 PC) were also prepared. Then, each cell line was seeded at a density of 1 x 10 &lt; ⁶ &gt; in a 60 [pi] dish, and after 24 hours, the cells were harvested and the protein was extracted, and then Western blotting was performed to obtain RIP1, Vimentin, MMP2, MMP9, Src, p-EGFR, and EGFR. The results are shown in Fig. 5C (left).

4) A549, by transfection implanting RIP1 in H460 RIP1-overexpressing cell line (A549 RIP1, H460 RIP1) and the kinase create and suppress cell line (K45A), the vector control cell line (A549 PC, H460 PC) to 5 × 10 ⁵ to 60π plate 24 hours after seeding, the medium was changed to a serum-free culture medium, and the supernatant was harvested after 18 hours. Then, the gelatin was loaded on the gel, and the gel was stained with Coomassie Brilliant Blue to confirm the activity of MMP2 and MMP9. The results are shown in FIG. 5C (right).

[result]

As shown in FIGS. 5A and 5B, infiltration and migration analysis was performed in the kinase inhibitory cell line of RIP1 to observe cell infiltration and cell migration. As a result, it was confirmed that cell infiltration and cell migration were reduced by RIP1 kinase inhibition.

As shown in FIG. 5C, when kinase inhibition of RIP1 was observed, EMT marker and EGFR signal transduction were observed by Western blotting, and the activity of MMP2 and MMP9 was measured by gelatin gimography test. As a result, it was confirmed that RIP1 kinase inhibition decreased the expression of EMT marker and FR-Src, decreased the activity of MMP2 and MMP9, and the increase of cell mobility by RIP1 induced EMT through RIP1 kinase activity , And EGFR-Src signaling.

6. Activation of STAT3 transcription factor by EGFR-Src

In this experiment, we examined whether STAT3 transcription factor is activated through down-signaling of EGFR-Src. In other words, it was reported that STAT3, which is a transcription factor, is activated in EMT signal transduction, and activation of STAT3 by EGFR-Src was observed.

[Experimental Method]

1) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at 1x10 &lt; ⁶ &gt; in a 60 [pi] dish, and after 24 hours, the cells were harvested and then proteins were extracted and then Western blot was performed to obtain p-Src, Src, p- And the results are shown in Fig. 6A.

2) A549 and H460 were transfected with RIP1 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Each cell line was then seeded at 1 × 10 ⁶ in a 60π dish and then treated with necrotostin. After 24 hours, the cells were harvested and then the protein was extracted and then Western blotted to obtain p-Src, Src, p-STAT3, and STAT3. The results are shown in Fig. 6B.

3) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were injected into the upper chambers 5 × 10 ^{4 in} Matrigel- And treated with STAT3 inhibitor. 10% FBS medium was added to the lower chamber, and cells passed through the transwells after 24 hours were stained to confirm cell infiltration. The results are shown in FIG. 6C.

4) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were put into the upper chamber 2 × 10 ⁴ in collagen- , Treated with STAT3 inhibitor. 10% FBS medium was added to the lower chamber, and the cells passed through the transwells after 24 hours were stained to confirm cell migration. The results are shown in FIG. 6D.

5) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Each cell line is then seeded at 1 x 10 ⁶ in a 60 pi dish and then treated with a STAT3 inhibitor (Fig. 6E), a Src inhibitor (Fig. 6F), harvesting the cells after 24 hours and then extracting the protein (STAT3 inhibitor) and FIG. 6F (Src inhibitor), respectively, by Western blotting. The expression of RIP1, Vimentin, MMP2, MMP9, p-Src, Src, p- Respectively.

6) A549, by transfection implanting RIP1 in H460 RIP1-overexpressing cell line (A549 RIP1, H460 RIP1) to create, and vector control cell line in (A549 PC, H460 PC) after the 5 × 10 ⁵ seeded by a 60π plate necromancer statin treatment After 24 hours, the medium was replaced with serum-free culture medium, and the supernatant was harvested after 18 hours. Then, the gelatin was loaded on the gel, and the gel was stained with Coomassie Brilliant Blue to confirm the activity of MMP2 and MMP9, and the result is shown at the bottom of FIG. 6E.

[result]

As shown in FIG. 6A, a signal transduction mechanism to increase cell invasion and cell migration by RIP1 was observed. Activation of the Src-STAT3 signaling mechanism by RIP1 was confirmed by western blotting.

As shown in FIG. 6B, the signal transduction mechanism by the inhibition of the kinase effect of RIP1 was observed, and the decrease of Src-STAT3 signaling was confirmed by Western blotting.

As shown in FIGS. 6C and 6D, infiltration and migration analysis during STAT3 inhibitor treatment was performed to observe cell infiltration and cell migration, and it was confirmed that infiltration and migration were inhibited during the treatment with STAT3 inhibitor.

As shown in FIGS. 6E and 6F, Western blotting was performed on STAT3 inhibitor and Src inhibitor treatment for EMT marker expression and signal transduction. As a result, it was confirmed that expression of EMT marker and expression of Src and STAT3 were decreased.

From the above results, it was confirmed that EMT activation by RIP1 is induced by EGFR-Src-STAT3 signaling and ultimately increased cell mobility.

7. Activation of EGFR signaling by RIP1

EGFR signaling mechanism is an important signal transduction mechanism in cell mobility signaling mechanism. In the above example, EGFR signaling mechanism was confirmed and it was confirmed that EGFR signaling was activated by RIP1.

[Experimental Method]

1) A549, by transfection implanting RIP1 to H460 in RIP1-overexpressing cell line (A549 RIP1, H460 create a RIP1), the vector control cell line (A549 PC, H460 PC) to Marti gel coating 5 × 10 ⁴ per upper chamber in the transwell And treated with EGFR inhibitor. Then, 10% FBS medium was added to the lower chamber, and 24 hours later, cells passed through the transwell were stained to confirm cell infiltration. The results are shown in FIG. 7A.

2) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were put into the upper chamber at 2 × 10 ⁴ in collagen- , Treated with EGFR inhibitor. Then, 10% FBS medium was added to the lower chamber, and after 24 hours, cells passed through the transwell were stained to confirm cell migration. The results are also shown in FIG. 7A.

3) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Each cell line was then seeded at a density of 1x10 &lt; ⁶ &gt; cells in a 60 [mu] dish, and after 24 hours, the cells were harvested and proteins were extracted and then Western blotted to confirm expression of p-EGFR, EGFR The results are shown in FIG. 7B.

4) A549 and H460 were transfected with RIP1 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at ⁶⁰ x 10 &lt; ⁶ &gt; dish and treated with necrotostin. After 24 hours, the cells were harvested and then the protein was extracted and then Western blot was performed to obtain RIP1, Vimentin, MMP2 , MMP9, p-EGFR, EGFR, p-Src, Src, p-STAT3 and STAT3. The results are shown in Figs. 7C, 7D and 7E, respectively.

5) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at 1 x 10 &lt; ⁶ &gt; in a 60 [mu] dish, treated with necro-statin, STAT3 inhibitor, and the cells were harvested after 24 hours and then proteins were extracted and then Western blot was performed to obtain p-EGFR , Expression of EGFR was confirmed, and the results are shown in Figs. 7F and 7G, respectively.

[result]

As shown in FIG. 7A, infiltration and migration analysis during EGFR inhibition were performed to observe cell infiltration and cell migration, and it was confirmed that cell infiltration and cell migration were suppressed upon treatment with EGFR inhibitor.

As shown in FIG. 7B, a signal transduction mechanism for increasing cell infiltration and cell migration by RIP1 was observed by western blotting, and EGFR signaling mechanism was activated by increasing EGFR phosphorylation by RIP1.

As shown in FIGS. 7C, 7D and 7E, signaling mechanisms by EGFR inhibition were observed by western blotting. EGFR inhibition decreased signaling of EMT markers and Src, STAT3, and decreased EGFR signaling Respectively.

As shown in FIGS. 7F and 7G, EGFR signaling mechanism was observed by Western blotting when treating RIP1 kinase effect and STAT3 inhibitor, but it was confirmed that phosphorylation of EGFR did not decrease.

From the above results, it was confirmed that EGFR phosphorylation is induced by RIP1, activation of Src-STAT3 by EGFR phosphorylation occurs, and cell mobility is increased by EMT induction.

8. Activation of inflammatory response by RIP1

RIP1 is known to be involved not only in the function of kinase but also in the inflammatory response. According to recent studies, it has been reported that the inflammatory response is related to the proliferation and metastasis of cancer cells. In the present invention, activation of RIP1 inflammatory response was confirmed and experiments were conducted to determine the effect on cell mobility.

[Experimental Method]

1) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1 and H460 RIP1), and chip analysis was carried out to perform gene analysis. The results are shown in FIG. 8A.

2) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and kinase inhibitory cell line (K45A) and vector control cell lines (A549 PC, H460 PC). Each cell line was then seeded at a density of 1x10 &lt; ⁶ &gt; in a 60 [mu] dish. After 24 hours, the cells were harvested and the protein was extracted. Western blotting was then performed to confirm the expression of IL- Is shown in Fig. 8B.

3) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at a density of 1 x 10 &lt; ⁶ &gt; in a 60 [pi] dish, and then the cells were harvested after 24 hours of IL-1 [beta] treatment. Then, the protein was extracted, and then Western blotting was performed to obtain RIP1, MMP2, Vimentin, and beta -actin, and the results are shown in Fig. 8C.

4) A549, H460, and H1299 cell lines were seeded at 1 × 10 ⁶ in a 60π dish, then cells were harvested 24 hours after IL-1β treatment, and then proteins were extracted and then subjected to Western blot Menthyl, RIP1, IL-1 beta, MMP2, MMP9, and beta-actin, and the results are shown in FIG.

5) Cell lines A549, H460, and H1299 were placed in an upper chamber at 5 × 10 ^{4 in} a Matrigel-coated transwell and 10% FBS medium was placed in the lower chamber. After 24 hours, the cells passed through the transwells were stained for cell infiltration And the results are shown in Fig. 8E.

(A549 PC, H460 PC, H1299 PC) were transfected into collagen-coated transwells with 2 &lt; (R) &gt; × 10 ⁴ into the upper chamber, and 10% FBS medium was placed in the lower chamber. After 24 hours, cells passing through the transwell were stained to confirm cell migration. The results are also shown in FIG. 8E.

[result]

As shown in FIG. 8A, gene analysis was performed by performing chip arrays with A549, H460 cells and A549 and H460 cells overexpressing RIP1. As a result, it was confirmed that expression of cell mobility gene and inflammatory response gene was increased in RIP1 overexpressed cells.

As shown in FIG. 8B, activation of inflammatory response by RIP1 was confirmed by Western blotting, and it was confirmed that IL-1β (Interleukin 1β), which is a typical marker of inflammatory response, is increased by RIP1.

As shown in FIG. 8C, the expression of EMT marker by IL-1β in A549 and H460 cells overexpressing RIP1 was observed by Western blotting, and as a result, the expression of EMT marker was increased in IL-1β treatment .

As shown in FIG. 8D, EMT markers by IL-1? In A549 and H460 cells were observed by western blotting, and infiltration and migration analysis were performed to observe cell infiltration and cell migration. As a result, it was confirmed that expression of EMT marker and invasion and migration were increased by IL-1β.

From the above results, it was confirmed that the expression of the cell mobility and inflammatory response-related genes was increased by RIP1, and the cell mobility was increased by IL-1β, which is one of inflammatory reaction genes. In other words, it was confirmed that the increase of IL-1β activity by RIP1 increased the inflammatory response by RIP1.

9. Effect of RIP1 on NF-κB activation mechanism and cell mobility

IL-1β is known to be activated by NF-κB, and RIP1 is known to activate NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation mechanism and cell mobility.

[Experimental Method]

1) RIP1 was transfected into A549 and H460 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at a density of 1 x 10 &lt; ⁶ &gt; in a 60 [pi] dish, and after 24 hours, the cells were harvested and then the protein was extracted and then subjected to Western blotting to obtain p-IKBa, p-P65, p50, p105 And the results are shown in Fig. 9A.

2) RIP1 over-expressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were injected into the upper chambers 5 × 10 ^{4 in} Matrigel- And treated with NF-κB inhibitor. 10% FBS medium was added to the lower chamber, and cells passed through the transwells after 24 hours were stained to confirm cell infiltration. The results are shown in FIG. 9B.

3) RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) were prepared by transfection of A549 and H460 with RIP1 and vector control cell lines (A549 PC, H460 PC) were put into the upper chamber 2 × 10 ⁴ in collagen- , And treated with NF-κB inhibitor. 10% FBS medium was added to the lower chamber, and the cells passed through the transwell after 24 hours were stained to confirm cell migration. The results are shown in FIG. 9C.

4) A549 and H460 were transfected with RIP1 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was seeded at a density of 1 x 10 &lt; ⁶ &gt; in a 60 [mu] dish and treated with an NF- [kappa] B inhibitor. After 24 hours, the cells were harvested and then proteins were extracted. Western blotting was then performed to obtain RIP1, , MMP2, MMP9, and actin. The results are shown in FIG. 9D.

5) A549, 1 × 10 ⁶ to the by transfection implanting RIP1 in H460 RIP1-overexpressing cell line (A549 RIP1, H460 RIP1) and kinase create and suppress cell line (K45A), the vector control cell line (A549 PC, H460 PC) to 60π plate After 24 hours, the cells were lysed, and the cytoplasm and nuclei were separated. The radioactive isotope-labeled NF-κB probe was inserted into the nucleus and reacted with NF-κB present in the nucleus. , And the position of the band after gel loading was confirmed to confirm NF-κB present in the nucleus. The result is shown in FIG. 9E.

[result]

As shown in FIG. 9A, NF-κB signal transduction in cells overexpressing RIP1 was observed by Western blotting, and it was confirmed that the expression of molecules involved in NF-κB signal transduction was increased by RIP1.

9B, 9C and 9D, changes in cell infiltration and cell migration by NF-κB inhibitor treatment were confirmed by infiltration and migration analysis, and EMT marker expression was observed by western blotting , Decreased cell invasion and cell migration upon treatment with NF-κB inhibitor, and decreased EMT marker expression.

As shown in Fig. 9E, the activation of NF-κB was observed by performing EMSA (Electrophoric Mobility Shift Assay). As a result, it was confirmed that NF-κB was activated by RIP1 and NF-κB activity was decreased by kinase inhibition Respectively.

From the above results, it was confirmed that RIP1 activates NF-κB using the kinase effect and NF-κB activity enhances cell mobility.

10. Activation of EMT by RIP1 in animal models

In previous cell experiments, it was confirmed that EMT was activated by RIP1 and cell mobility was increased. Animal experiments were conducted using animal models to determine whether these cell experiments were also occurring in animal models. In addition, experiments were conducted to determine the effect of the A549 RIP1 over-expressing cell line on the animal survival rate due to an increase in metastatic ability and an increase in the metastatic potential of cancer cells.

[Experimental Method]

A549 and H460 were transfected with RIP1 to generate RIP1-overexpressing cell lines (A549 RIP1, H460 RIP1) and vector control cell lines (A549 PC, H460 PC). Then, each cell line was transfected with a luciferase into a cell line, and the cell line thus prepared was intravenously injected at 1 × 10 ⁶ into the tail of the nude mouse at 5 weeks of age. Then, weekly luciferase (100 ug) was infused by intraperitoneal injection, and the activity of luciferase was measured by a fluorescence machine and the survival rate of the mice was measured. The results are shown in Figs. 10A and 10B.

[result]

As shown in FIGS. 10A and 10B, the transfer ability of RIP1 and the survival rate of the animal were observed in an animal model using a mouse animal model. RIP1 increased the cancer cell metastasis capacity by 20% in vivo, Was reduced by 25%.

From the above results, it was found that the increase of cell mobility of RIP1 using cell experiments was observed in an animal model experiment, so that RIP1 increased the cancer cell metastasis ability in vivo and ultimately decreased the survival rate.

11. Mechanisms affecting the expression of RIP1

In the previous experiments, the mechanism of EMT activation by RIP1 and the mechanism of cell mobility by inflammatory response were identified. However, the expression of RIP1 was not strong in A549 and H460 cells. Therefore, an experiment was conducted to determine which mechanism controls the expression of RIP1. Based on the recent research that RIP1 regulation is regulated by methylation in RIP families, the expression of RIP1 is regulated by methylation and its effect on cell mobility is observed. The results are shown in Figs. 11 and 12 Respectively.

11A, RIP1 and EMT markers were observed in 5-aza-treated A549 and H460 cells. In 5-aza treatment, the expression of RIP1 was increased and EMT was activated.

As shown in Fig. 11B, cell death by 5-aza was observed, but it was confirmed that 5-aza did not induce apoptosis by treatment with 5-aza.

As shown in FIG. 11C, cell invasion and cell migration were observed during 5-aza treatment, and cell invasion and cell migration were increased by 5-aza.

FIG. 12 shows the results of observation of 5-aza-induced expression of RIP1 and visentin in a cell using a fluorescence microscope, showing that RIP1 and vimentin expression increased and complexed in the cells .

From the above results, it was confirmed that expression of RIP1 is regulated by methylation in A549 and H460 cells, and EMT is activated and cell mobility is increased when RIP1 expression is suppressed.

Claims (12)

A biomarker for lung cancer metastasis diagnosis including RIP1 (Receptor-interacting protein 1). A kit for diagnosing lung cancer metastasis, comprising means capable of measuring the expression level of RIP1. 3. The method of claim 2,
Wherein said means measures the expression level of RIP1 protein. The method of claim 3,
Wherein the means is selected from the group consisting of an antibody, an antibody fragment, a protein microarray, and an aptamer. 3. The method of claim 2,
Wherein said means measures the expression level of mRNA of RIP1. 6. The method of claim 5,
Wherein the means is selected from the group consisting of a primer and a probe. (1) measuring the amount of RIP1 expression from a lung cancer patient sample;
(2) measuring the amount of RIP1 expression from a normal control sample; And
(3) comparing the expression level of the step (1) with the expression level of the step (2). 8. The method of claim 7,
And determining that the lung cancer metastatic potential is high when the amount of expression in step (2) is equal to or greater than the amount of methionine in step (1). 8. The method of claim 7,
Wherein said RIP1 expression level measurement measures the amount of protein or mRNA. 10. The method of claim 9,
Wherein the amount of the protein is measured by an antigen-antibody reaction. 11. The method of claim 10,
The antigen-antibody reaction may be measured by Western blotting, antigen-antibody reaction, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunoprecipitation, Ouchteroni immunodiffusion, immunohistochemistry or rocket immunization Electrophoresis, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS or protein chip assays. 10. The method of claim 9,
Wherein the measurement of the amount of mRNA is performed by RT-PCR (reverse transcriptase-polymerase chain reaction), Northern blotting or nucleic acid microarray.

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