KR20220122251A - Diagnostic methods for prognosis of non-small-cell lung cancer using BRD3 SNP - Google Patents

Abstract

The present invention relates to a method for diagnosing the prognosis of non-small cell lung cancer using a BRD3 polymorphism, and more specifically, to a method for diagnosing or predicting prognosis affecting the survival outcome of early-stage non-small cell lung cancer using the rs2427964C>T single nucleotide polymorphism of the BRD3 gene and uses thereof. The technology for predicting the survival prognosis of non-small cell lung cancer according to the present invention can easily and accurately predict the patient's prognosis for patients with non-small cell lung cancer, also be applied to methods for selection and evaluation of appropriate therapies, and further increase the survival rate of the patients with non-small cell lung cancer through novel targeted therapies for non-small cell lung cancer.

Description

Prognosis diagnosis method for non-small-cell lung cancer using single nucleotide polymorphism of BRD3 {Diagnostic methods for prognosis of non-small-cell lung cancer using BRD3 SNP}

The present invention relates to a method for diagnosing the prognosis of non-small cell lung cancer using a polymorphism of BRD3. Specifically, the present invention relates to a method for diagnosing or predicting the survival prognosis of non-small cell lung cancer using the rs2427964C>T single nucleotide polymorphism of BRD3, and uses thereof is about

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small-cell lung cancer (NSCLC) accounts for about 85% of primary lung cancers, and about 2/3 of NSCLCs have advanced disease.

Diagnosis of NSCLC is made by the pathologist's examination of the suspected tissue, such as a biopsy sample. After diagnosis of NSCLC, the patient's disease is assigned to the patient's overall health and age, the severity of symptoms such as cough and shortness of breath, the specific type of NSCLC and the prognosis (chance of recovery) using the staging of the cancer. The stage of progression takes into account the size of the tumor and whether the tumor exists only in the lungs or has spread to other parts of the body. Specific treatment options for patients with NSCLC are selected based on the above considerations, and the stage of cancer progression is an important factor for treatment selection. Patients with early NSCLC can potentially be cured by surgical resection to remove the tumor, but current diagnostic modalities cannot predict which patients will relapse after surgery.

The ideal treatment for lung cancer is to detect it early and completely remove the cancer with surgery. In addition, after lung cancer, especially non-small cell cancer, surgery is performed first if it has not progressed enough to be surgically performed, but radical resection is only possible in 30% of cases. The 5-year survival rate, which determines whether or not the patient is cured after surgery, differs depending on the degree of cancer progression, but in all patients who underwent radical resection, about 37% of squamous cell carcinomas, 27% of adenocarcinomas, and 27% of large cell carcinomas were cured. The majority of these patients die after surgical resection due to recurrence of a more aggressive disease.

Current clinical diagnostic methods cannot confirm early NSCLC prognosis with sufficient accuracy to instruct more aggressive therapy for patients with high relapse potential.

Therefore, it is necessary to identify high-risk early NSCLC patients who must receive chemotherapy or have a general re-evaluation of treatment findings, and if the prognosis of NSCLC can be determined so that the patient can easily decide whether to use additional treatment methods, the patient may increase the survival rate of

On the other hand, BET protein is known as an epigenetic leader that regulates gene expression by mobilizing chromatin regulatory enzymes of histone modification. The BET protein family consists of bromo domains including BRD2, BRD3, BRD4 and bromodomain testis specific protein (BRDT). However, it is known that the dysregulation of these BET family genes is related to the development of cancer.

However, it has not yet been reported that the specific rs2427964C>T polymorphism present in BRD3 among the BET protein family can be used for prognosis of non-small cell lung cancer.

Republic of Korea Patent Registration 10-2195591

Accordingly, the present inventors selected 77 potential functional single nucleotide polymorphisms in three genes, BRD2, BRD3, and BRD4, which are related to the development and progression of non-small cell lung cancer (NSCLC), and their genotypes were 349 of the discovery cohort that underwent radical resection. rs2427964C>T of BRD3, a single nucleotide polymorphism related to the prognosis of patients with non-small cell lung cancer, by analyzing the association with the clinical results of 424 patients in patients and validation cohorts and analyzing the genotype for the selected single nucleotide polymorphism. The present invention was completed by excavation.

Accordingly, it is an object of the present invention to provide a composition for a marker for prognosis and prediction of non-small cell lung cancer prognosis comprising the rs2427964 C>T polymorphism of the BRD3 gene.

Another object of the present invention is to provide a composition for predicting survival prognosis of non-small cell lung cancer patients, comprising an agent capable of detecting the rs2427964 C>T polymorphism of the BRD3 gene.

In order to achieve the above object of the present invention, the present invention provides a composition for a marker for diagnosis and prediction of prognosis of non-small cell lung cancer including a single nucleotide polymorphism (SNP) of a Bromodomain-containing protein 3 (BRD3) gene. The single nucleotide polymorphism provides a marker composition for non-small cell lung cancer prognosis diagnosis and prediction, characterized in that the rs2427964 C>T polymorphism located at the 201st base in the nucleotide sequence shown in SEQ ID NO: 1.

In addition, the present invention provides a composition for predicting survival prognosis of non-small cell lung cancer patients, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) of the 201st base in the nucleotide sequence represented by SEQ ID NO: 1.

In one embodiment of the present invention, the agent is a polynucleotide consisting of 10 to 100 consecutive bases including a single nucleotide polymorphism (SNP) of the 201st base in the nucleotide sequence consisting of SEQ ID NO: 1 or a polynucleotide complementary thereto It may be a primer or probe capable of amplifying

The present invention also provides a kit for predicting survival prognosis of non-small cell lung cancer patients comprising the composition of the present invention.

The present invention also provides a microarray for predicting survival prognosis of a non-small cell lung cancer patient comprising the composition of the present invention.

In addition, the present invention provides information for predicting survival prognosis of patients with non-small cell lung cancer, comprising the step of confirming the rs2427964 C>T polymorphism of the Bromodomain-containing protein 3 (BRD3) gene with respect to the nucleic acid extracted from the sample. provides

In one embodiment of the present invention, when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is TT, it may be determined as a group with a low survival prognosis.

In one embodiment of the present invention, when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is CC or CT, it may be determined as a group having a better survival prognosis than when the genotype is TT.

In one embodiment of the present invention, when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is CC or CT, SIN3A, a transcriptional repressor, binds to BRD3 to reduce or inhibit the expression of BRD3. can

In one embodiment of the present invention, when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is TT, the activity of the BRD3 promoter increases compared to the case where the genotype is CC or CT, thereby increasing the expression of BRD3. have.

The technology for predicting the survival prognosis of non-small cell lung cancer according to the present invention can predict the prognosis of a patient with non-small cell lung cancer easily and accurately, and can be applied to a method for selecting and evaluating an appropriate treatment, furthermore, non-small cell lung cancer New targeted therapy for non-small cell lung cancer can increase the survival rate of patients.

1 shows the bioinformatics annotation of the BRD3 promoter region and five types of polymorphisms of LD (linkage disequilibrium) using the UCSC Genome Browser, where A shows the positions on the chromosomes of rs2506711 and rs2427964, B is SNPinfo using JPT BRD3 tagged SNP plots generated by the web server-LD TAG SNP selection web-based software are shown, and C shows the rs2506711 and rs2427964 LD plots in 773 lung cancer patient genotypes generated by the Haploview software.
Figure 2 shows the overall survival curve according to the genotype of the BRD3 rs2427964 SNP, where A and B are the analysis results of the discovery cohort, C and D are the analysis results of the validation cohort, and E and F show the binding analysis results.
3 shows the results of ChIP-qPCR analysis for the BRD3 promoter region in H1299 and A549 cell lines (A), and shows the results of double luciferase reporter analysis according to the genotype of the rs2427964 SNP (B).
Figure 4 shows the results of EMSA (Electrophoretic mobility shift assay) analysis for BRD3 rs2427964 SNP, where A is an unlabeled rs2427964-C or rs2427964-T oligonucleotide to perform competition analysis, and B is SIN3A and ETS1 The results of Supershift analysis using the antibody are shown.
5 shows the expression level of BRD3 in cancer cell lines and the results of survival analysis using the TCGA database, where A is a box plot showing the RNA-seq mRNA expression level of CCLE (Cancer Cell Line Encyclopedia), and The dotted line shows the average, B shows the result of RT-PCR analysis of the expression level of BRD3 in various lung cancer cell lines, and C shows the BRD3 mRNA expression level in tumor and non-malignant lung tissues using the TCGA database. and D is a Kaplan-Meier plot showing the change in overall survival according to BRD3 mRNA expression using the TCGA database.
6 is an analysis of the effect of BRD3 gene silencing on the growth, migration and invasion of H1299 and A549 cancer cells. B is a Western blot confirmation of the BRD3 protein level according to siRNA treatment, C is an analysis of the effect of siRNA treatment on the proliferation of H1299 and A549 cell lines by cell counting, and D is an analysis of cell viability through MTT assay E is an analysis of the effect on cell migration through a transwell invasion assay, F is a soft agar colony formation assay that confirms the colony forming ability, and G is a wound caused by cell migration depending on whether siRNA is treated or not. Shows the results of observation of healing or not.
Figure 7 is an analysis of the effect of BRD3 gene silencing on apoptosis and cell cycle, A shows the degree of cell death according to whether siRNA (siBRD3) treatment for BRD3 was confirmed through flow cytometry, B is cells by cell cycle It shows the results of analysis of the distribution diagram with flow cytometry.

The present invention is characterized by providing a novel use of a single nucleotide polymorphism (SNP) of a Bromodomain-containing protein 3 (BRD3) gene for prognostic diagnosis and prediction of non-small cell lung cancer.

Specifically, the present invention can provide a marker composition for diagnosing and predicting the prognosis of non-small cell lung cancer including a single nucleotide polymorphism (SNP) of the Bromodomain-containing protein 3 (BRD3) gene, and in the present invention, The single nucleotide polymorphism is characterized in that the rs2427964 C>T polymorphism is located at the 201st base in the nucleotide sequence shown in SEQ ID NO: 1.

SEQ ID NO: 1 is as follows.

서열번호 1 : AACTCTTTTC CTCTTCTGTT CCTTCTCACA GGGTAGGGCG GGGGAGGACG GGACACACGA AGCTGCCTCT TGCAGTTCCA GCCCAATTAC TCTGCCCACC AATGCACTAC AGTCTCCCCT CCTGGTTAAC TTTTGCAGAC AATGACCAAG TTTAATGTAA GGGGGAGAAA TCCCAGTATC TATTTCTGAG CAGAAAGCTT C CAACCAAAA TTAAAGATCG CAACCACTCA AGGATTGGGT CGAACGGAGG AACCCACACG ATGGACGCCG GGTCCCTGCC CCCGGTGGGT CTCGGCGGGC CGAGTTCTCG CCCTCCAGGG AAAGGAGGCC GCGCGAAGCC CCCATCCCGG CCCTGCCCCC GAACGAGCAA CCGGTGGAAT AAGACTTCCC GCACGCCCCA GCTTTATTCA CAGAGAGCCC ACCAGGGCCA TGTGGGTGTA C

While the present inventors were studying to discover biomarkers that can rapidly and accurately diagnose and predict the prognosis of non-small cell lung cancer, 77 in three genes, BRD2, BRD3, and BRD4, which are related to the development and progression of non-small cell lung cancer (NSCLC). Potential functional single nucleotide polymorphisms were selected, and as a result of analyzing whether the selected 77 single nucleotide polymorphisms were correlated with the cancer outcome of 349 patients in the discovery cohort and 424 patients in the validation cohort who underwent non-small cell lung cancer resection, It was confirmed that the rs2427964C>T polymorphism of the BRD3 gene is significantly related to the prognosis of non-small cell lung cancer.

According to an embodiment of the present invention, as a result of analyzing the relationship between the genotype for the rs2427964C>T polymorphism in the BRD3 gene and the survival rate of patients with non-small cell lung cancer, when the genotype of the rs2427964C>T polymorphism is TT, the survival prognosis is low. In the case of the polymorphic genotype CC or CT, the survival prognosis was better than that of the TT genotype.

Therefore, through these results, it was found that the genotyping analysis of the rs2427964C>T polymorphism of the BRD3 gene could predict the survival prognosis of non-small cell lung cancer patients.

Furthermore, the present inventors analyzed the expression level of BRD3 in various lung cancer cell lines. As a result, it was found that the expression level of BRD3 was increased in lung cancer cells compared to normal lung cells, and in particular, in non-small cell lung cancer cell lines H1299 and A549 cells. The highest expression level was confirmed.

Therefore, it was found that the present inventors can predict or diagnose the onset of lung cancer, preferably non-small cell lung cancer, through BRD3 expression level analysis.

In another embodiment of the present invention, as it was confirmed that BRD3 was overexpressed in the non-small cell lung cancer cell line, an experiment was performed to confirm whether there was anticancer activity according to the inhibition of the expression of BRD3. Expression of the BRD3 gene by siRNA treatment for BRD3 was induced to suppress or decrease silencing, and changes in cancer cell proliferation, cell migration, apoptosis, and cell cycle were analyzed in non-small cell lung cancer cell lines treated with siRNA.

As a result, the group treated with BRD3 siRNA suppressed cell proliferation, cell migration, and apoptosis induction and cell cycle arrest (G2/M cell cycle arrest) compared to the group not treated with siRNA. ) has been shown to have anticancer activity.

Through this, the present inventors found that an inhibitor capable of inhibiting the expression or activity of BRD3 can be used as a therapeutic agent for non-small cell lung cancer.

In addition, in order to confirm the effect of the BRD3 rs2427964C>T polymorphism in non-small cell lung cancer, the present inventors confirmed the effect of the rs2427964C>T polymorphism on BRD3 promoter activity through a double luciferase reporter assay.

As a result, it was found that the rs2427964 T allele had significantly higher promoter activity than the rs2427964 C allele in the non-small cell lung cancer cell line.

This result means that the expression of BRD3 may be different depending on the genotype of the allele in the BRD3 rs2427964C>T polymorphism. It was found that the expression of

In addition, binding of transcription factors according to the genotype of the allele in the BRD3 rs2427964C>T polymorphism was analyzed. As a result, transcription regulator family member A (SIN3A), known as a transcriptional repressor, was found to selectively bind only to the C allele.

Through these results, the present inventors found that the C allele of the BRD3 rs2427964 polymorphism is reduced or suppressed by the binding between the transcriptional repressor SIN3A, and the expression of BRD3 is reduced or suppressed. , it was found that the expression of BRD3 was reduced or suppressed compared to the case where the genotype was TT, so that the survival prognosis of non-small cell lung cancer patients was good. On the other hand, when the genotype is TT, compared to the case where the genotype is CC or CT, the expression of BRD3 is increased due to the enhancement of the promoter activity of BRD3, so that the survival prognosis of non-small cell lung cancer patients is low.

Therefore, the present invention can provide a composition for a marker for diagnosing and predicting the prognosis of non-small cell lung cancer including the rs2427964C>T single nucleotide polymorphism of the BRD3 gene. It is possible to provide a composition for predicting survival prognosis of patients with non-small cell lung cancer, including an agent that can be used.

In addition, the agent capable of detecting the rs2427964C>T single nucleotide polymorphism of the BRD3 gene includes a single nucleotide polymorphism (SNP) of the 201st base in the nucleotide sequence consisting of SEQ ID NO: 1 polymorph consisting of 10 to 100 consecutive bases It may be a primer or probe capable of amplifying a nucleotide or its complementary polynucleotide.

In the present invention, the "prognosis" refers to the progress and cure of diseases such as the onset, recurrence, metastatic spread, and the possibility of lung cancer-induced death or progression, including drug resistance, of a disease such as lung cancer. For the purposes of the present invention, the prognosis means the risk of developing lung cancer, preferably non-small cell lung cancer, and the prognosis for survival after the onset.

"Prediction" means determining whether a patient is likely to develop lung cancer, preferably non-small cell lung cancer, and responding favorably or unfavorably to therapy, such as chemotherapy or radiation therapy, to treat the patient, e.g., a specific with respect to whether and/or the likelihood of survival after treatment with a therapeutic agent, and/or surgical removal of the primary tumor, and/or chemotherapy for a specified period of time without cancer recurrence.

The prediction method of the present invention is a patient with a high risk of developing non-small cell lung cancer for any specific patient, and through special and appropriate management, the onset time is delayed or prevented, or the most appropriate treatment method for the non-small cell lung cancer patient is selected. can be used clinically to make treatment decisions. The predictive method of the present invention determines whether a patient responds favorably to a treatment regimen, such as a prescribed treatment regimen, including, for example, administration of a given therapeutic agent or combination, surgical intervention, chemotherapy, etc. It is possible to predict whether or not survival is possible.

"Genetic polymorphism" refers to the occurrence of a genetic mutation with a frequency of at least 1% or more in a population. The insertion, deletion, or substitution of one nucleotide in DNA is called single nucleotide polymorphism (SNP).

The term "polymorphism" refers to the placement in the sequence of a gene that varies within a population. Polymorphisms consist of different “alleles”. The placement of such polymorphisms can be identified by their position in the gene and the different amino acids or bases found therein. These amino acid variations are the result of two different alleles, two possible variant bases, C and T. Because the genotype consists of two different distinct alleles, any of several possible variants can be observed in any one individual (eg, CC, CT or TT). Individual polymorphisms are also known to those skilled in the art and are used, for example, in the Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation available on the NCBI website. An identifier ("reference SNP", "refSNP" or "rs#").

"Single nucleotide polymorphism (SNP)" is the diversity of DNA sequences that occurs when a single nucleotide (A, T, C, or G) in the genome differs between members of a species or between pairs of chromosomes in an individual. means For example, when three DNA fragments from different individuals (eg, AAGT[A/A]AG, AAGT[A/G]AG, AAGT[G/G]AG) contain differences in a single base, Called two alleles (A or G), in general almost all SNPs have two alleles. Within a population, SNPs can be assigned a minor allele frequency (MAF; the lowest allele frequency at a locus found in a particular population). A single base can be changed (replaced), removed (deleted), or added (inserted) into the polynucleotide sequence. SNPs can cause changes in the translation frame.

The term “genotype” refers to a specific allele of a particular gene in a cell or tissue sample.

"Allele" or "allele" means one of two or more alternative forms of a gene that occupy the same chromosomal locus.

"Diagnosing" means identifying the presence or character of a pathological condition. Among them, the present invention particularly includes predicting recurrence, progression, metastasis, and the like of non-small cell lung cancer. Therefore, an indicator that can accurately predict the recurrence and progression of non-small cell lung cancer is very important, and a factor that can predict the response to treatment while supplementing clinical indicators such as the degree of tissue differentiation and stage is needed. Since T SNP functions as such an indicator, it can be used as a diagnostic factor for non-small cell lung cancer. That is, measurement of polymorphism of these genes can be used as a useful indicator (diagnostic marker) for predicting the degree of differentiation, stage, and progression of non-small cell lung cancer.

"Diagnostic marker or diagnostic marker" is a substance that can distinguish and diagnose non-small cell lung cancer cells from normal cells, and a polypeptide or nucleic acid that shows an increase in non-small cell lung cancer cells compared to normal cells organic biomolecules such as (eg, mRNA, etc.), lipids, glycolipids, glycoproteins, sugars (monosaccharides, disaccharides, polysaccharides, etc.), and the like.

"Marker for predicting survival prognosis of patients with non-small cell lung cancer" means a marker having polymorphism that can predict the risk of non-small cell lung cancer, whether or not the onset of non-small cell lung cancer is cured, or the course, preferably the above-mentioned nucleotides means In addition, the patient refers to a patient for determining the risk of developing non-small cell lung cancer or a patient who has undergone chemotherapy for non-small cell lung cancer.

"Subject" or "patient" means any single individual in need of treatment, including humans, cattle, dogs, guinea pigs, rabbits, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial that do not show any clinical manifestations of any disease, or subjects who participated in epidemiological studies or subjects used as controls.

"Tissue or cell sample" means a collection of similar cells obtained from the tissue of a subject or patient. Sources of tissue or cell samples may include solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; blood or any blood component; The cells may be at any time of conception or development in a subject. A tissue sample may also be a primary or cultured cell or cell line.

"Nucleic acid" is meant to include any DNA or RNA, eg, chromosomal, mitochondrial, viral and/or bacterial nucleic acids present in a tissue sample. It includes one or both strands of a double-stranded nucleic acid molecule, and includes any fragment or portion of an intact nucleic acid molecule.

By “gene” is meant any nucleic acid sequence or portion thereof that has a functional role in protein coding or transcription or in the regulation of other gene expression. A gene may consist of any nucleic acid encoding a functional protein or only a portion of a nucleic acid encoding or expressing a protein. Nucleic acid sequences may include gene abnormalities within exons, introns, initiation or termination regions, promoter sequences, other regulatory sequences, or unique sequences adjacent to the gene.

A "primer" is an oligonucleotide sequence that hybridizes to a complementary RNA or DNA target polynucleotide and serves as a starting point for the stepwise synthesis of polynucleotides from mononucleotides, for example, by the action of nucleotidyl transferases occurring in the polymerase chain reaction. means

"Protein" also includes fragments, analogs and derivatives of proteins that retain essentially the same biological activity or function as a reference protein.

"Treatment" means an approach for obtaining beneficial or desirable clinical results. For the purposes of the present invention, beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, reduction of the extent of disease, stabilization (ie, not worsening) of disease state, delay or rate of disease progression, disease state improvement or temporary relief and alleviation (partial or total), detectable or non-detectable. "Treatment" may also mean increasing survival as compared to the expected survival rate if not receiving treatment. Treatment refers to both therapeutic treatment and prophylactic or prophylactic methods. Such treatments include the treatment required for the disorder being prevented as well as the disorder that has already occurred. "Palliating" a disease means that the extent and/or undesirable clinical signs of the disease state are reduced and/or the time course of progression is delayed or prolonged, compared to no treatment. means to lose

In addition, the present invention can provide a kit for predicting the survival prognosis of a non-small cell lung cancer patient comprising the composition for predicting the survival prognosis of a non-small cell lung cancer patient according to the present invention, and furthermore predicting the survival prognosis of a non-small cell lung cancer patient according to the present invention It is possible to provide a microarray for predicting survival prognosis of a patient with non-small cell lung cancer comprising a composition for the present invention.

The kit of the present invention can be used to predict the survival prognosis of non-small cell lung cancer by identifying the rs2427964 C>T polymorphic region (SNP) of the BRD3 gene, which is a marker for predicting the survival prognosis of non-small cell lung cancer. The kit for predicting the survival prognosis of non-small cell lung cancer of the present invention includes a polynucleotide, primer, or probe for confirming the rs2427964 C>T SNP of the BRD3 gene, as well as one or more other component compositions, solutions or devices suitable for the analysis method may be included.

In addition, the kit of the present invention may be a kit including essential elements necessary for performing PCR. The PCR kit contains, in addition to polynucleotides, primers or probes specific for the SNP, a test tube or other suitable container, reaction buffer (with varying pH and magnesium concentrations), deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase. enzymes such as DNase, RNAse inhibitors, DEPC-water and sterile water, and the like.

In addition, the microarray of the present invention may be composed of a conventional microarray except for containing the polynucleotide, primer or probe of the present invention.

Hybridization of nucleic acids on microarrays and detection of hybridization results are well known in the art. The detection is, for example, labeling the nucleic acid sample with a labeling material capable of generating a detectable signal including a fluorescent material, for example, a material such as Cy3 and Cy5, and then hybridizing on a microarray and the labeling material. A hybridization result can be detected by detecting a signal generated from

The microarray method can simultaneously study the RNA expression of thousands or even tens of thousands of genes within a tumor, allowing more effective insight into the molecular basis of human disease.

It can also be used to evaluate gene expression patterns in tumor classification, clinical outcomes, and response to chemotherapy.

Furthermore, the present invention can provide a method of providing information for predicting survival prognosis of a patient with non-small cell lung cancer, wherein the method preferably includes rs2427964 C of the Bromodomain-containing protein 3 (BRD3) gene with respect to the nucleic acid extracted from the sample. identifying the >T polymorphism.

At this time, when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is TT, it can be determined as a group with a low survival prognosis, and when the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is CC or CT, the genotype is Compared to the case of TT, it can be determined as a group with a better survival prognosis.

In addition, in the method of providing information for predicting the survival prognosis of the non-small cell lung cancer patient according to the present invention, the method of confirming the rs2427964 C>T polymorphism of the BRD3 gene is a method of confirming the genotype of the rs2427964 C>T polymorphism. The genotype can be confirmed by sequencing analysis, sequencing analysis using an automatic sequencing analyzer, pyrosequencing, hybridization by microarray, PCR-RELP method (restriction fragment length polymorphism), PCR-SSCP method (single strand conformation polymorphism), PCR-SSO method (specific sequence oligonucleotide), ASO (allele specific oligonucleotide) hybridization method combining PCR-SSO method and dot hybridization method, TaqMan-PCR method, MALDI-TOF/MS method, RCA method (rolling circle amplification), HRM (high resolution melting) method, primer extension method, Southern blot hybridization method, it can be carried out by a known method such as a dot hybridization method.

In addition, the results of the SNP polymorphism can be statistically processed using statistical analysis methods commonly used in the art, for example, Student's t-test, Chi-square test (Chi-). square test), linear regression line analysis, and multiple logistic regression analysis, such as continuous variables, categorical variables, odds ratios, and 95 It can be analyzed using variables such as % confidence interval.

In addition, the diagnostic method relates to examining the expression characteristics of specific markers according to non-small cell lung cancer, and the method disclosed herein is a method for obtaining useful data and information when evaluating appropriate or effective therapies for treating patients with non-small cell lung cancer. It would be possible to provide a convenient, efficient and cost effective means.

Nucleic acids from non-small cell lung cancer patients can be obtained from samples such as tissues, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine obtained from these patients, and the nucleic acid sample is synthesized from DNA, mRNA, or mRNA. contains cDNA.

The nucleic acid of the non-small cell lung cancer patient can be obtained by conventional methods and separation methods such as phenol/chloroform extraction and protease K treatment, and also by amplifying the target nucleic acid through PCR and purifying it.

In addition, the present invention can also provide information for determining the amount and administration method of an anticancer agent necessary to prevent recurrence or metastasis after chemotherapy treatment for non-small cell lung cancer.

That is, the present invention confirms the rs2427964 C>T polymorphism of the BRD3 gene from a biological sample isolated from a non-small cell lung cancer patient. may be applied differently to suitably according to the type of the corresponding lung cancer.

As such, the present invention includes all uses of the BRD3 rs2427964 C>T polymorphic expression profile as a prognostic diagnostic and predictive marker for non-small cell lung cancer in patients with non-small cell lung cancer.

Hereinafter, the components and technical features of the present invention will be described in more detail through the following examples. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited by the examples.

<Test materials and methods>

1. Selection of research subjects

The Discovery cohort enrolled 349 non-small cell lung cancer (NSCLC) patients who underwent resection at Kyungpook National University Hospital from September 1998 to December 2007. The validation cohort included 424 patients who underwent NSCLC treatment surgery at Seoul National University Bundang Hospital from September 2005 to March 2012. All patients in this study were Korean, and blood samples were collected for genotyping with written consent prior to surgery. In addition, this study was conducted with the approval of the institutional review committees of both hospitals.

2. Polymorphism Selection and Genotyping

For the collection of polymorphisms, the rSNPBase database (http://rsnp.psych.ac.cn/result) was used, which provides a curated human SNP resource analyzing regulatory functions for all SNPs in the human genome. Polymorphisms targeting the BRD2, BRD3 and BRD4 genes were searched for through the "List Search" module of rSNPBase, but BRDT, another member of the BET family, was excluded from this study because it is a testis-specific chromatin protein. A total of 5669, 2025 and 2624 BRD2, BRD3 and BRD4 polymorphisms, respectively, were selected. Next, we used the "TagSNP" utility for LD tag SNP selection on the SNPinfo web server (https://snpinfo.niehs.nih.gov/) with a small allele frequency of less than 0.1 in HapMap JPT and a strong linkage disequilibrium ( LD) (r2>0.8) was excluded. Finally, 77 SNPs were selected (59 from BRD2, 7 from BRD3, 11 from BRD4). The selected polymorphisms were genotyped by MassARRAY iPLEX analysis (Sequenom) according to the manufacturer's instructions.

3. The Cancer Genome Atlas (TCGA) data analysis

Data on mRNA sequencing gene expression from The Cancer Genome Atlas (TCGA) were obtained through the Broad Institute's data portal for TCGA squamous cell carcinoma and adenocarcinoma cohorts. In total, normal tissue samples from 51 lung squamous cell carcinoma and 58 lung adenocarcinoma tumor pairs were included. Statistical analysis was performed using statistical software (SAS, version 9.4, SAS Laboratories).

4. Protein Extraction and Western Blot

The H1299 and A549 cell lines were washed twice with pre-chilled phosphate buffered saline (PBS), and then the cells were lysed by the addition of mammalian protein extraction reagent (Thermo Scientific). Protein concentration was determined using a Pierce BCA protein assay kit (Thermo Scientific). The total protein lysate was separated by 10% SDS-PAGE electrophoresis, then transferred to a polyvinylidene difluoride membrane (Millipore) and blocked with blocking buffer. Thereafter, the reaction was performed using BRD3 (abcam) as a primary antibody, and β-actin antibody (primary antibody; Santa Cruz Biotech) was used as an internal control. Thereafter, immunoreactive proteins were detected by Immobilon Western Chemiluminescent HRP Substrate (Millipore).

5. Cell Culture

Human lung cancer cell lines H1299 and A549 were purchased from the American Type Culture Collection and used. Each cell line was cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and penicillin G (100 U/mL) in a 37 °C, 5% CO ₂ incubator.

6. BRD3 siRNA and transfection

Knockdown of BRD3 in human lung cancer cells was performed by transfection using BRD3 siRNA, siBRD3 (Bioneer). The siRNA sequences targeting BRD3 are as described below. In this case, siCtrl, a non-specific siRNA oligonucleotide provided by Bioneer, was used as a negative control, and cells not treated with siRNA or cells treated with only the transfection reagent were used as a control (Mock). Cells were transfected with 100 nM siRNA using Effectene (Quiagen) reagent according to the manufacturer's protocol.

BRD3 siRNA sense: 5'-CUGAUGUCCGGCUGAUGUU-3'

BRD3 siRNA antisense: 5'-AACAUCAGCCGGACAUCAG-3'

7. Cell proliferation assay

Cell viability was analyzed using MTT (3-(4,5-demerthylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) reagent. Specifically, cells were aliquoted in a 96-well plate at the number of 4 x 10 ³ cells per well, the medium was removed after 24 hours, and transfected with siBRD (Bioneer) using Effectene transfection reagent (Qiagen). Thereafter, cell viability was analyzed using a Multiskan™ FC Microplate Photometer (Thermo Scientific). Cell viability of each well was confirmed by measuring absorbance at a wavelength of 570 nm, and this experiment was repeated 8 times.

8. Transwell invasion assay

Cell invasion assay was performed using a BioCoat Matrigel invasion chamber (24-well, 8-μm pore size; Corning, #354480). Prior to the experiment, Matrigel-coated transwell inserts were rehydrated with 500 µL of serum-free RPMI-1640 medium and incubated at 37 °C for 2 h. H1299 and A549 cells were trypsinized and washed with PBS. A total of 2.5 x 10 ⁴ cells were suspended in 500 μL of RPMI-1640 medium without FBS and the suspension was added to the upper chamber, and 750 μL of RPMI-1640 medium containing 10% FBS was added to the lower chamber. After maintaining for 12 hours in a state where the cells were able to migrate, they were fixed with 3.7% paraformaldehyde for 20 minutes, washed with PBS, and then stained with a 20% methanol solution containing 0.05% crystal violet for 30 minutes. Thereafter, the transwell was washed with PBS and the non-invasive cells in the upper chamber were removed with a cotton swab. Then, images of cell invasion (cell migration) were observed using a microscope (Nikon).

9. Soft Agar Colony Formation Assay

Transfected A549 cells (1 × 10 ⁴ ) were mixed with 0.3% soft agar, and the cells were plated on 0.6% bottom agar dispensed on a 6-well plate. Then, the plate was incubated in 1 mL of medium containing 10% FBS at 37°C, 5% CO ₂ in an incubator. Cells were stained with 0.05% crystal violet to count colonies formed while culturing for 3 weeks, and images of colonies formed were obtained.

10. Wound Healing Assays

H1299 and A549 cells were each seeded in a 6-well plate and cultured until the cells were confluent. The monolayer of cells was then scratched using a 20 μL pipette tip and washed with PBS to remove cell debris. Then, the cells were treated with siRNA. 6-well plates were incubated at a temperature of 37 °C in 5% CO ₂ for 0, 24 and 48 h, then the same wound site was observed at each time point and images were obtained under a microscope. In addition, the distance between the scratches was also measured by time.

11. Cell Apoptosis Analysis Using Annexin V

H1299 and A549 cells transfected with siBRD3 or scrambled siRNA were treated with trypsin/EDTA and annexin V and PI staining was performed using Annexin V-FITC Apoptosis Detection Dit (abcam, # ab14085) according to the manufacturer's instructions. Stained cells were immediately analyzed by flow cytometry (FACS Calibur, BD Biosciences).

12. Cell Cycle Arrest Analysis

For cell cycle analysis, cells were collected by trypsinization and centrifugation. After washing with PBS, pre-cooled 70% ethanol was added to the cells to fix them. The fixed cells were washed with PBS and then resuspended in PI/RNase staining buffer (BD Pharmingen™, # 550825) at room temperature under dark conditions for 30 minutes. Stained cells were analyzed by flow cytometry (FACS Calibur, BD Biosciences), and the results were expressed as a percentage of total cells in cells in G1, S or G2 phase of the cell cycle.

13. Chromatin Immunoprecipitation by Quantitative PCR

Chromatin immunoprecipitation (ChIP) analysis was performed using the Pierce Magnetic ChIP Kit (Thermo science) according to the manufacturer's instructions. Specifically, when the NSCLC cell lines (H1299 and A549) reached the number of 4 × 10 ⁶ cells, the cells were cross-linked with a medium supplemented with 1% formaldehyde at room temperature for 10 minutes, and then quenched with a glycine solution. . Then, the cells were washed twice with cold PBS. 1 mL of cold PBS containing 10 μL of halt cocktail was added to the cells and the cells were detached by scraping. Cell DNA was digested from cell lysates by sonication using MNase and Vibra-Cell Ultrasonic Liquid Processors (Sonics & Materials). DNA was digested to an average length of 300 bp and centrifuged at 10,000 xg for 10 min to remove cell debris. Then, the cell lysate was reacted with target-specific IPs anti-SIN3A (10 ug, abcam) and anti-ETS1 (10 ug, abcam), in which case IP normal rabbit IgG was used as a negative control, and a positive control As an IP anti-RNA polymerase II antibody, these antibodies were reacted at 4°C for 24 hours. Magnetic beads were added to each IP and incubated for 2 h at 4 °C with mixing. After the reaction, the beads were collected on a magnetic stand and the supernatant was carefully removed and discarded. Collected beads were eluted with 1X IP elution buffer. Finally, DNA was purified by spin column from each eluted IP, and genomic DNA was obtained through a similar procedure. All DNA samples were quantified using a NanoDrop 2000 spectrophotometer, and ChIP DNA was analyzed by quantitative PCR (qPCR) using a QuantiFast SYBR Green PCR Master Mix (QIAGEN) on a LightCycler 480 (Roche Applied Science).

The primers used for the PCR are as follows.

BRD3 Forward Primer: 5'-CCCAGTATCTATTTCTGAGCAG-3'

BRD3 reverse primer: 5'-TTCCTCCGTTCGACCCAATCCT-3'

14. Promoter-Luciferase Constructs and Luciferase Assays

Luciferase analysis was performed to confirm whether rs2427964C>T of the BRD3 transcription start site (TSS) region regulates the promoter activity of the gene. A 791 bp fragment containing rs2427964C>T (a fragment corresponding to -390 to +400 in rs2427964) was synthesized by PCR using the genomic DNA of a donor carrying the heterozygote. A forward primer containing a KpnI restriction enzyme site (5'-GGGGTACCGGTTCAGACCTTCCTTAGACC -3') and a reverse primer containing an XhoI restriction enzyme site (5'-CCGCTCGAGAGTTCCCGTTTTGAAATCAGCC-3') were used. The PCR product was cloned into the KpnI / XhoI site of the pGL3-basic vector (Promega) to prepare a pGL3-Basic-BRD3 plasmid construct containing the rs2427964 C or T allele. The exact sequence of all clones was confirmed by genomic sequencing. H1299 and A549 cell lines were transfected with pRL-SV40 vector (Promega) and pGL3-basic vector using Effectene (Qiagen). 48 hours after transfection, cells were harvested and cell lysates were prepared using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was assayed using a Synergy HTX Multi-Mode Microplate Reader (BioTek Instruments). The results were measured by normalizing to pRL-SV40 Renilla luciferase activity.

15. Electrophoretic mobility shift assay and supershift assay

Electrophoretic mobility shift assay (EMSA) analysis was performed using the LightShift Chemiluminescent EMSA Kit (Pierce Biotechnology Inc.). To this end, nuclear extracts were first obtained from H1299 and A549 cells using Pierce NE-PER nuclear extraction reagent and cytoplasmic extraction reagent. A complementary 31 bp oligonucleotide (based on the sequence -15 to +15 of rs2427964) was synthesized as follows: 5'-CTGAGCAGAAAGCTT C CAACCAAAATTAAAG-3' and 5'-CTGAGCAGAAAGCTT T CAACCAAAATTAAAG-3' ( Polymorphic alleles are underlined in bold letters). After the oligonucleotide probe was labeled with biotin, 5 μg of the nuclear extract was reacted with the probe at room temperature for 20 minutes. DNA-protein binding specificity analysis was analyzed using a binding reaction in which 2- and 5-fold unlabeled specific competitive oligonucleotides or non-specific competitive oligonucleotides were added to the labeled probe and reacted. The reaction mixture was separated on a non-denaturing 6% acrylamide gel, and then the gel was transferred to a positively charged nylon membrane. Membranes were UV cross-linked and biotin-labeled probes were detected using a stabilized streptavidin-horseradish peroxidase conjugate. Bands of DNA-protein complexes were quantified by densitometry analysis using ScionImage (Scion Corporation). 10 μg of nuclear extract reacted with SIN3A and ETS1 antibodies before the probe labeling step was used for super shift analysis.

16. Statistical processing

Overall survival (OS) and disease-free survival (DFS) were analyzed by the Kaplan-Meier method. OS was calculated from the date of surgery to the date of death or the last follow-up. DFS was counted as recurrence or death from any cause from the day of surgery. HRs and CIs were calculated for a multivariate statistical model (Cox proportional hazards model) with adjustments for age, sex, smoking status, pathological stage and adjuvant therapy. P values less than 0.05 were considered statistically significant, and all analyzes were performed using Statistical Analysis System version 9.4 for Windows (SAS Institute).

<Example 1> Patient characteristics and clinical predictors

The clinical characteristics of patients and their association with overall survival (OS) and disease-free survival (DFS) were investigated, and the results are shown in Table 1 below. Pathological stage had a statistically significant effect on overall survival and disease-free survival in both cohort groups. Younger patients were associated with better overall survival, and women, naive smokers, and adenocarcinomas were found to be associated with better overall survival in the validation cohort (Table 1).

<Example 2> Relation between SNPs and survival outcome

Among the 77 genotyped SNPs, 59 were analyzed in the present invention except for 18 that showed Sequenom failure or deviation from the Hardy-Weinberg equilibrium. Eleven SNPs were found to be associated with OS or DFS with a P value less than 0.05 (see Table 2). Among these 11 SNPs, the BRD3 rs2506711C>T polymorphism was replicated in the validation cohort and in the same direction as the discovery cohort. In addition, the BRD3 rs2506711C>T polymorphism was found to be significantly correlated with OS in the discovery cohort, validation cohort, and combinatorial analysis (under a recessive model, HR = 1.79, 95% CI = 1.17 - 2.74, P = 0.01; HR = 1.97, 95% CI = 1.18 - 3.28, P = 0.01; and HR = 1.91, 95% CI = 1.38 - 2.65, P = 9 × 10 ^-5 , respectively; see Table 3).

According to bioinformatics predictions, rs2506711C>T is present in heterochromatin and inhibitory regions (Fig. 1A), and thus the effect of rs2506711C>T in OS may be due to other polymorphisms in LD. Although rs2506711C>T is a labeled polymorphism, four other polymorphisms are in strong LD relationship (r2 ≥ 0.9) with rs2506711 (Fig. 1B). The functional content of the five polymorphisms of LD is given in Table 4 below. Among them, BRD3 rs2427964 had a strong LD together with rs2506711 (D' and r2 = 0.98), was located in the TSS region, and was expected to serve as a promoter (see Table 4 and Fig. 1). BRD3 rs2427964C>T was found to be associated with poor OS in the discovery cohort, validation cohort and binding analysis (under a recessive model, HR = 1.70, 95% CI = 1.11 - 2.61, P = 0.02; HR = 1.97, 95% CI = 1.18 - 3.28, P = 0.01; and HR = 1.92, 95% CI = 1.39 - 2.65, P = 8 × 10 ^-5 , respectively; Table 5 and Figure 2).

In addition, stratified analysis showed that the clinical effect of BRD3 rs2427964 differed only by histological cell type (P value <0.05 for homogeneity test, Table 4). A significant association between BRD3 rs2506711 or rs2427964 and OS was found only in adenocarcinoma and not in squamous cell carcinoma (SCC).

<Example 3> ChiP-qPCR and promoter analysis for BRD3 rs2427964C>T

ROADMAP epigenomics project and ENCODE data showed that rs2427964 is located in the transcription factor binding region in A549 cell line (Fig. 3A). Therefore, the present inventors analyzed whether rs2427964 is located in the transcription factor binding region in A549 cells and H1299 cells through ChiP-qPCR.

As a result, as shown in Figure 3A, it was possible to confirm a higher input (input) value in rs2427964 compared to the control (IgG) in both H1299 and A549 cell lines.

In addition, the effect of rs2427964C>T on BRD3 promoter activity was evaluated in vitro through a double luciferase reporter assay. and promoter activity was significantly higher in the rs2427964 T allele than in the rs2427964 C allele in both H1299 and A549 cell lines (P = 0.046 and 2×10 ⁻⁶ , respectively; FIG. 3B ).

<Example 4> Effect of rs2427964 on transcriptional activity

Since the rs2427964C>T polymorphism is located in the promoter region of the BRD3 gene, we expected that this variant could affect BRD3 transcription by regulating the binding of sequence-specific transcription factors. Accordingly, the effect on the binding to transcription factors according to the rs2427964C>T allele was analyzed through EMSA analysis.

As a result, as shown in Fig. 4A, the rs2427964 C allele was shown to bind more transcription factors than the rs2427564 T allele. It was found that the migration intensity of the biotin-labeled T allele probe was decreased in the group to which the competitive C allele was added (lane 10) compared to the group to which the competitive T allele was added (lane 8). On the other hand, it was shown that the migration intensity of the biotin-labeled C allele probe was increased in the group to which the competitive T allele (lane 5) was added compared to the competitive C allele (lane 3).

On the other hand, the transcription factor ChIP-seq cluster showed that the transcription factors ETS1 and SIN3A bind to the rs2427964 region in the A549 cell line (Fig. 1A). Accordingly, the present inventors performed Supershift analysis to determine whether the degree of binding to ETS1 and SIN3A is different depending on the rs2427964 C or T allele.

As a result, as shown in Fig. 4B, the EST1 antibody was shown to bind both the C and T alleles, whereas the SIN3A antibody selectively bound only the C allele. Since SIN3A is known as a transcriptional repressor, binding between the BRD3 rs2427964 C allele and SIN3A could be expected to suppress the expression of BRD3.

<Example 5> Analysis of BRD3 expression level in various cancer cell lines

Cancer cell encyclopedia data showed that the mRNA expression level of BRD3 was increased in various human cancer cell lines, including lung cancer cells (FIG. 5A). To confirm this, the present inventors measured the mRNA expression of BRD3 in various lung cancer cell lines using RT-PCR.

As a result, BDR3 mRNA expression was found to be increased in various lung cancer cell lines compared to MRC5, a normal lung cell ( FIG. 5B ), and in particular, the BDR3 mRNA expression level was highest in non-small cell lung cancer cell lines H1299 and A549 cells. . Therefore, in the following experiments, experiments were performed using H1299 and A549 cell lines.

In addition, TCGA data analysis showed that the relative mRNA expression of BRD3 was significantly higher in lung cancer than in adjacent normal tissues (P = 0.019, FIG. 5C ). Therefore, according to these results, it can be seen that overall survival may be worse for patients with high BRD3 expression compared to patients with low BRD3 expression, and BRD3 functions as a potential oncogene.

<Example 6> Effect of BRD3 gene silencing on proliferation and migration of cancer cell lines

In order to evaluate the knockdown efficacy of BRD3, three siRNAs were prepared and used. After transfection with siBRD3, as a result of analyzing the BRD3 mRNA expression level, it was found that the expression of BRD3 was significantly reduced compared to the control group (FIG. 6A). Western blot results also showed that BRD3 expression was significantly reduced in H1299 and A549 cells after siBRD3 treatment ( FIG. 6B ).

Next, since BRD3 is expressed at a high level in lung cancer, the number of cells per hour was counted for the siRNA-treated group and the untreated group to determine whether the knockdown of BRD3 affects the growth of lung cancer cells.

As a result, as shown in FIG. 6C , in the group transfected with siBRD3 for 72 hours, cancer cell growth was significantly reduced over time in both H1299 and A549 cell lines. Cell viability results by MTT assay also showed decreased cell viability in siBRD3-transfected H1299 and A549 cells ( FIG. 6D ). These results suggest that BRD3 has a significant effect on H1299 and A549 cancer cell growth.

Furthermore, transwell invasion analysis was performed to confirm that suppression of BRD3 expression also affects cell migration ability. As a result, H1299 and A549 cells transfected with siBRD3 showed that cell migration was significantly inhibited compared to Mock and siCtrl. appeared (FIG. 6E). Inhibition of the expression of BRD3 also affected the cell colony-forming ability, and colony formation was reduced in siBRD3-treated A549 cells compared to the control group ( FIG. 6F ). In the wound healing assay, the group transfected with siBRD3 for 48 hours showed reduced cell migration compared to the control group (Fig. 6G).

<Example 7> Effect of BRD3 gene silencing on cell apoptosis and cell cycle

Flow cytometry was performed to determine if BRD3 gene silencing affects apoptosis.

As a result, it was found that the percentage of cell death in H1299 and A549 cancer cells treated with BRD3 siRNA was increased compared to the control group not treated with siRNA ( FIG. 7A ).

Accordingly, the present inventors analyzed the cell cycle arrest period by additionally measuring the DNA content by flow cytometry to confirm the mechanism by which the induction of apoptosis by BRD3 silencing is caused.

As a result, as shown in 7B, in the siBRD3-treated group, G2/M cell cycle arrest was observed in both H1299 and A549 cells along with a decrease in G0/G1 phase. These results suggest that inhibition of gene expression of BRD3 induces apoptosis of cancer cells by arresting the G2/M cell cycle of cancer cells.

So far, with respect to the present invention, the preferred embodiments have been looked at. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Diagnostic methods for prognosis of non-small-cell lung cancer using BRD3 SNPs <130> PN2102-629 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 441 <212> DNA <213> Artificial Sequence <220> <223> BRD3 <400> 1 aactcttttc ctcttctgtt ccttctcaca gggtagggcg ggggaggacg ggacacacga 60 agctgcctct tgcagttcca gcccaattac tctgcccacc aatgcactac agtctcccct 120 cctggttaac ttttgcagac aatgaccaag tttaatgtaa gggggagaaa tcccagtatc 180 tatttctgag cagaaagctt ccaaccaaaa ttaaagatcg caaccactca aggattgggt 240 cgaacggagg aacccacacg atggacgccg ggtccctgcc cccggtgggt ctcggcgggc 300 cgagttctcg ccctccaggg aaaggaggcc gcgcgaagcc cccatcccgg ccctgccccc 360 gaacgagcaa ccggtggaat aagacttccc gcacgcccca gctttattca cagagagccc 420 accagggcca tgtgggtgta c 441

Claims (10)

A composition for markers for diagnosis and prediction of prognosis of non-small cell lung cancer including single nucleotide polymorphism (SNP) of BRD3 (Bromodomain-containing protein 3) gene,
The single nucleotide polymorphism of the BRD3 gene is an rs2427964 C>T polymorphism located at the 201st base in the nucleotide sequence shown in SEQ ID NO: 1, A composition for a marker for diagnosing and predicting prognosis of non-small cell lung cancer. A composition for predicting survival prognosis of non-small cell lung cancer patients, comprising an agent capable of detecting a single nucleotide polymorphism (SNP) of the 201st base in the nucleotide sequence consisting of SEQ ID NO: 1. 3. The method of claim 2,
The agent is a primer or probe capable of amplifying a polynucleotide consisting of 10 to 100 consecutive bases including a single nucleotide polymorphism (SNP) of the 201st base in the nucleotide sequence consisting of SEQ ID NO: 1 or a complementary polynucleotide thereof A composition for predicting survival prognosis of non-small cell lung cancer patients, characterized in that. A kit for predicting survival prognosis of a patient with non-small cell lung cancer, comprising the composition according to claim 2 or 3. A microarray for predicting survival prognosis of a non-small cell lung cancer patient comprising the composition according to claim 2 or 3. A method of providing information for predicting survival prognosis of non-small cell lung cancer patients, comprising the step of confirming the rs2427964 C>T polymorphism of a Bromodomain-containing protein 3 (BRD3) gene with respect to a nucleic acid extracted from a sample. 7. The method of claim 6,
When the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is TT, a method of providing information for predicting survival prognosis of non-small cell lung cancer patients, characterized in that the group is determined as a group with a low survival prognosis. 7. The method of claim 6,
When the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is CC or CT, information for predicting survival prognosis of non-small cell lung cancer patients, characterized in that it is determined as a group with a better survival prognosis than when the genotype is TT How to provide. 7. The method of claim 6,
When the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is CC or CT, SIN3A, a transcriptional repressor, binds to BRD3 and reduces or suppresses the expression of BRD3. A method of providing information for predicting survival prognosis. 7. The method of claim 6,
When the genotype of the rs2427964 C>T polymorphism of the BRD3 gene is TT, the activity of the BRD3 promoter increases compared to the case where the genotype is CC or CT, and the expression of BRD3 is increased, characterized in that the survival of patients with non-small cell lung cancer A method of providing information for predicting prognosis.

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