KR102514265B1 - Biomarkers in salvia for predicting prognosis of oral cancer - Google Patents

Abstract

The present invention relates to a method for predicting the prognosis of oral cancer by non-invasively measuring the amount of mRNA or protein in a sample, and more specifically, expression of one or more genes selected from the group consisting of BMP7, NAB2, MAOB, and NPIPB4 genes It relates to a method for predicting the prognosis of oral cancer by measuring the amount.

Description

Biomarkers in salvia for predicting prognosis of oral cancer {Biomarkers in salvia for predicting prognosis of oral cancer}

The present invention relates to a biomarker for predicting the prognosis of oral cancer by a non-invasive method.

Oral cancer is one of the 10 most frequent cancers worldwide, and oral cancer accounts for about 5% of all cancers in Korea. dying due to These oral cancers belong to a high degree of malignancy, and 80 to 90% can be cured if detected in the early stages, but since more than half of the patients are discovered after oral cancer has progressed to stage 3 or higher, the cure rate is low and the prognosis is poor. In particular, lymph node metastasis is an important prognostic factor for oral cancer, and patients with lymph node metastasis have a 5-year survival rate of 25 to 40%, whereas patients without lymph node metastasis have a survival rate of about 90%. Although lymph node metastasis is an important factor in determining the treatment method after oral cancer diagnosis, there is still a lack of methods for accurately diagnosing lymph node metastasis.

On the other hand, most cancer diagnosis methods are invasive methods such as biopsy, and are not easily accessible in that they require severe pain for the examiner, side effects due to infection, hospitalization, and recovery period after examination. Because of this, they are usually not tested until symptoms appear. Therefore, in most cases, most patients are diagnosed with cancer when the cancer has already progressed significantly. Therefore, recently, methods for analyzing proteins, DNA, etc. from blood, urine, feces, etc. are being actively developed for diagnosis of cancer through a non-invasive method. However, development of a biomarker that can be practically used to predict the prognosis of oral cancer is still incomplete.

Therefore, if a biomarker capable of accurately predicting the prognosis of oral cancer in a non-invasive manner is developed, it is expected that the treatment efficiency of oral cancer can be significantly increased.

Domestic Patent Publication 10-2015-0125030

The present invention has been made to solve the above problems in the prior art, and predicts the prognosis of oral cancer with high accuracy by checking the expression levels of BMP7, NAB2, MAOB, and/or NPIPB4 genes using saliva samples Its purpose is to provide a way to do so.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention (a) a group consisting of BMP7 (bone morphogenetic protein 7), NAB2 (NGFI-A-binding protein 2), MAOB (Monoamine oxidase B), and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4) from biological samples Measuring the expression level of any one or more genes selected from; and (b) classifying oral cancer as having a poor prognosis when the expression level of at least one of the four genes significantly changes compared to that of a control group.

In addition, the present invention is any one selected from the group consisting of BMP7 (bone morphogenetic protein 7), NAB2 (NGFI-A-binding protein 2), MAOB (Monoamine oxidase B), and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4) Provided is a composition for predicting the prognosis of oral cancer containing a substance for measuring the expression level of the above genes.

In addition, the present invention is any one selected from the group consisting of BMP7 (bone morphogenetic protein 7), NAB2 (NGFI-A-binding protein 2), MAOB (Monoamine oxidase B), and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4) Provided is a kit for predicting the prognosis of oral cancer containing a substance for measuring the expression level of the above genes.

In one embodiment of the present invention, the biological sample may preferably be saliva, blood, urine, feces, etc., more preferably saliva or a sample that can be obtained by a non-invasive method, but is not limited thereto. The composition and kit can diagnose oral cancer using the biological sample and also predict the prognosis of oral cancer.

In another embodiment of the present invention, the step of measuring the expression level (expression level) can be preferably measured by a method of measuring the expression level of mRNA or a method of measuring the expression level of protein, the mRNA Methods for measuring expression levels include reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), RNase protection assay ( RPA; RNase protection assay), Northern blotting, DNA microarray chip, etc., and methods for measuring the expression level of the protein include western blotting, enzyme linked immunosorbent assay (ELISA), radiation Radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, Rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complete fixation assay ), flow cytometry (FACS), protein chip, etc., but is not limited thereto as long as it can measure the expression level of the gene of the present invention.

In another embodiment of the present invention, the change may be that the expression level of the BMP7 or NAB2 gene is increased compared to the control group, and the expression level of the MAOB or NPIPB4 gene is decreased compared to the control group. The control group is preferably a patient with non-metastatic oral cancer.

In another embodiment of the present invention, the prediction of prognosis means predicting metastasis of oral cancer to the lymph nodes, that is, predicting or diagnosing a patient with metastatic oral cancer to the lymph nodes. In addition, when a patient is predicted to be a lymph node metastatic oral cancer patient, a poor prognosis may be predicted.

In another embodiment of the present invention, the material for measuring the expression level may be a primer or probe that specifically binds to the mRNA of the gene, or an antibody or probe that specifically binds to a protein expressed from the gene. It may be an aptamer or the like, but is not limited thereto as long as it can measure the expression level of mRNA or the expression level of protein.

In another embodiment of the present invention, the composition or kit may further include a material for measuring the expression level of genes that are constantly expressed, such as GAPDH, 18s rDNA, and β-actin, for normalization.

In another embodiment of the present invention, the one or more genes may be BMP7 and MAOB, BMP7 and NPIPB4, MAOB and NPIPB4, or a combination of BMP7, MAOB and NPIPB4, but is not limited thereto.

The biomarker composed of BMP7, NAB2, MAOB, and/or NPIPB4 according to the present invention can predict the prognosis of oral cancer with high accuracy using a non-invasive method such as saliva, and is an easily accessible diagnostic method. Through this, it is expected that the treatment efficiency and survival rate of oral cancer can be significantly increased by determining the treatment method for oral cancer.

1 is a view showing the results of identifying five genes whose expression levels were significantly changed in patients with metastatic oral squamous cell carcinoma of the lymph node according to an embodiment of the present invention.

The most effective way to reduce cancer mortality is to determine a treatment method by predicting the prognosis of cancer, and through this, it is expected that the recurrence rate and mortality due to cancer can be substantially reduced.

In the present invention, oral cancer can be diagnosed with high accuracy by checking the expression level of a gene using saliva, which is a non-invasive method, and by using a complex biomarker, the accuracy can be further improved, so early diagnosis of oral cancer It can be effectively used for, and through this, it is expected to significantly contribute to the improvement of survival rate through early diagnosis of oral cancer.

As used herein, "biological sample" is a sample that can be obtained non-invasively, preferably blood, plasma, serum, bone marrow, tissue, cell, saliva, sputum, hair, urine, feces, etc. And, more preferably saliva, etc., or samples capable of measuring the mRNA expression level or protein expression level of one or more biomarkers selected from the group consisting of BMP7, NAB2, MAOB, and NPIPB4 genes are not limited thereto. .

In the present specification, "method for providing information" is a method for providing information on the prognosis of oral cancer, and is a method for providing information on BMP7, NAB2, MAOB, and/or NPIPB4 genes in non-invasive samples such as saliva. When the expression level is changed compared to the control group, the patient is predicted to be a lymph node metastatic oral cancer patient, and a method of obtaining information that can predict that the patient's prognosis is poor.

In the present specification, "kit" is a prognosis of oral cancer by measuring the amount of mRNA or protein of one or more genes selected from the group consisting of BMP7, NAB2, MAOB, and NPIPB4 contained in a non-invasive sample It refers to a predictable diagnostic device, and is not limited as long as it can measure the expression level of the gene from a biological sample isolated from a patient. Preferably, it may include a probe or primer set having a sequence complementary to the mRNA, and the "probe or primer" refers to an oligonucleotide having a sequence complementary to the mRNA. However, any material capable of measuring the amount of mRNA of the gene is not limited thereto. Alternatively, it may include an antibody, an aptamer, etc. that specifically binds to the protein, but is not limited thereto as long as it can measure the amount of the protein of the gene.

Hereinafter, the following examples are presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example

Example 1: Patient saliva sample preparation

In order to select complex biomarkers for prognosis of oral cancer, saliva samples were prepared. All participants were recruited from those who visited the Department of Oral and Maxillofacial Surgery, Kyungpook National University Hospital from 2015 to 2020, and informed consent was obtained from all patients before saliva collection. Participants with a history of previous malignancy, osteomyelitis, human immunodeficiency virus (HIV) infection, or immunodeficiency were excluded from the study. To obtain a saliva sample, the mouth was rinsed with saline for 30 seconds and approximately 3 mL of saliva was collected in a 50 mL sterile tube. Then, after treatment with 3 μL of RNase inhibitor, centrifugation was performed at 7,600 rpm and 4° C. for 15 minutes. In addition, 500 μL of the supernatant was transferred into two Eppendorf tubes, respectively, and stored at -20 ° C until use. As a control group, saliva samples (n=18) from patients with non-metastatic oral squamous cell carcinoma (OSCC) were used, and as an experimental group, saliva samples from patients with metastatic oral squamous cell carcinoma (n=18) were used. was used.

Example 2: Total RNA extraction and real-time qRT-PCR

In order to extract total RNA from the saliva sample prepared by the method of Example 1, after mixing the same volume of TRIzol solution with 500 μL of the saliva sample, the mixture was reacted at room temperature for 5 minutes. Then, 200 μL of 1-bromo-2 chloropropane was added and mixed, followed by reaction at room temperature for 3 minutes. After the reaction was completed, centrifugation was performed at 12,000 rpm and 4° C. for 15 minutes, and 800 μL of the supernatant was transferred to two new Eppendorf tubes in 400 μL each. Then, after adding 800 μL of isopropanol, the mixture was stirred and reacted at -20°C. After reacting for 16 hours, left at room temperature for 10 minutes, centrifuged at 12,000 rpm and 4 ° C for 10 minutes, the supernatant was removed, and the pellet was washed with 75% ethanol and at room temperature. dried for 10 minutes. The dried RNA pellet was dissolved using 20 μL of DEPC-treated distilled water. The concentration of extracted RNA was measured using NanoDrop (Thermo Fisher Scientific).

After the extracted salivary RNA was transcribed into cDNA using a cDNA synthesis kit (Applied Biosystems), real-time qRT-PCR was performed. qRT-PCR was performed three times using the ABI 7500 real-time PCR system (Applied Biosystems), and after normalizing the average Ct value to the expression level of the GAPDH gene, the Δ cycle threshold determined by calculating the 2- ^ΔΔCt value ( The expression level was quantified based on the value of Δ Primer sequences used for qRT-PCR are listed in Table 1.

[Table 1]

Example 3: Selection of biomarkers for prognosis of oral cancer

In order to select biomarkers for prognosis of oral cancer, qRT-PCR was performed in the same manner as in Example 2, and mRNA in saliva and metastasis of oral squamous cell carcinoma were confirmed. More specifically, a receiver operating characteristic (ROC) curve was confirmed for each result value, and AUC for each biomarker was calculated. Hereinafter, all analyzes were calculated using the R program (R.3.6.1, https://www.r-project.org/), and it was considered statistically significant when the P value was 0.05 or less. For fold change, the fold of the gene expression level of patients with metastatic oral squamous cell carcinoma to the lymph nodes was calculated (meta/non-meta) based on the patients with non-metastatic oral squamous cell carcinoma.

As a result of qRT-PCR, among 30 candidate genes, BMP7 (bone morphogenetic protein 7), NAB2 (NGFI-A-binding protein 2), MAOB (monoamine oxidase B), and NPIPB4 (nuclear pore complex interacting protein family member B4), 4 It was confirmed that the expression level of the dog's gene was significantly changed in patients with oral squamous cell carcinoma. The results are shown in Figure 1 and Table 2.

Gene Fold change P-value Sensiti-vity Specifi-city AUC Gene Accession Cytoband BMP7 2.18 0.00216 0.67 0.83 0.78 NM_001719 20q13 NAB2 1.85 0.00874 0.72 0.78 0.74 NM_005967 12q13.3 MAOB 0.37 0.03071 0.78 0.39 0.60 NM_000898 Xp11.23 NPIPB4 0.40 0.01723 0.72 0.61 0.65 OTTHUMT00000402426 16p12.2

As shown in Table 2 and FIG. 1, as a result of analyzing the amount of mRNA in the saliva of lymph node metastatic oral squamous cell carcinoma patients, the expression levels of BMP7 and NAB2 genes were significantly increased compared to non-metastatic oral squamous cell carcinoma patients. , it was confirmed that the expression levels of the MAOB and NPIPB4 genes were significantly decreased.

Example 4: Selection of composite biomarkers for prognosis of oral cancer

In order to select composite biomarkers for prognosis of oral cancer, the accuracy of diagnosing lymph node metastasis of oral squamous cell carcinoma was confirmed using various combinations of biomarkers in saliva mRNA in the same manner as in Example 3. The results are shown in Table 3.

Gene AUC 95% CI BMP7+MAOB 0.88 0.58~0.89 BMP7+NPIPB4 0.82 0.56~0.89 BMP7+MAOB+NPIPB4 0.80 0.53~0.94

As shown in Table 3, it was confirmed that all of the composite markers using the three combinations of BMP7, MAOB, and NPIPB4 exhibited an AUC of 0.8 or higher. In particular, it was confirmed that an AUC of 0.88 was shown when a composite marker of BMP7 and MAOB was used. That is, it was confirmed that the prognosis prediction of oral cancer, that is, the accuracy of prediction of lymph node metastasis can be remarkably improved by using the combination of the biomarkers.

Through the above results, by measuring the expression levels of BMP7, NAB2, MAOB, and/or NPIPB4 genes in saliva using the amount of mRNA or protein, not only can the diagnostic accuracy of lymph node metastatic oral cancer be increased, but also BMPB7 and MAOB It was confirmed that the prognosis prediction accuracy could be further increased by using a combination of BMP7 and NPIPB4, a combination of MAOB and NPIPB4, a combination of BMP7, MAOB and NPIPB4, and the like. In addition, since the method of the present invention uses a saliva sample, it is easy to access as a non-invasive diagnosis method, so it is expected that not only the prognosis prediction efficiency of oral cancer can be improved, but also the treatment efficiency can be significantly improved through this. .

The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Biomarkers in salvia for predicting prognosis of oral cancer <130> MP19-193P1 <150> KR 10-2019-0135250 <151> 2019-10-29 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> artificial sequence <220> <223> NAB2 forward primer <400> 1 cacatccctg ctaaagctga a 21 <210> 2 <211> 21 <212> DNA <213> artificial sequence <220> <223> NAB2 reverse primer <400> 2 gtcgaaacgg ccatagatga t 21 <210> 3 <211> 22 <212> DNA <213> artificial sequence <220> <223> BMP7 forward primer <400> 3 tcgtggaaca tgacaaggaa tt 22 <210> 4 <211> 22 <212> DNA <213> artificial sequence <220> <223> BMP7 reverse primer <400> 4 tggaaagatc aaaccggaac tc 22 <210> 5 <211> 20 <212> DNA <213> artificial sequence <220> <223> NPIPB4 forward primer <400> 5 acctttgggt gtctctcctg 20 <210> 6 <211> 20 <212> DNA <213> artificial sequence <220> <223> NPIPB4 reverse primer <400> 6 cctcagccct tcctgtctac 20 <210> 7 <211> 20 <212> DNA <213> artificial sequence <220> <223> MAOB forward primer <400> 7 tctgctctct ggttcctgtg 20 <210> 8 <211> 20 <212> DNA <213> artificial sequence <220> <223> MAOB reverse primer <400> 8 gggaggtccat tatccgctca 20 <210> 9 <211> 22 <212> DNA <213> artificial sequence <220> <223> GAPDH forward primer <400> 9 agatcatcag caatgcctcc tg 22 <210> 10 <211> 21 <212> DNA <213> artificial sequence <220> <223> GAPDH reverse primer <400> 10 atggcatgga ctgtggtcat g 21

Claims (12)

(a) bone morphogenetic protein 7 (BMP7) and monoamine oxidase B (MAOB) from biological samples; or measuring gene expression levels of BMP7 and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4); and
(b) the BMP7 and MAOB; or predicting lymph node metastasis of oral cancer when the gene expression levels of BMP7 and NPIPB4 significantly change compared to the control group. According to claim 1,
The biological sample is characterized in that saliva, blood, urine or feces, information providing method. According to claim 1,
The step of measuring the expression level is RT-PCR, competitive RT-PCR (Real-time RT-PCR), RNase protection assay (RPA; RNase protection assay), Northern blotting (northern blotting), DNA microarray chip, western blotting, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion method, rocket (Rocket) At least one selected from the group consisting of immunoelectrophoresis, immunohistochemical staining, immunoprecipitation assay, complete fixation assay, flow cytometry (FACS), and protein chip Characterized in that, the information providing method. According to claim 1,
Characterized in that the control group is a non-metastatic oral cancer patient, information providing method. According to claim 1,
The change is characterized in that the expression level of the BMP7 gene is increased compared to the control group, and the expression level of the MAOB or NPIPB4 gene is decreased compared to the control group. delete bone morphogenetic protein 7 (BMP7) and monoamine oxidase B (MAOB); Or BMP7 and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4) containing a substance measuring the expression level of the gene, a composition for predicting lymph node metastasis of oral cancer. According to claim 7,
Characterized in that the material for measuring the expression level is a primer or probe specifically binding to mRNA of the gene, or an antibody or aptamer binding specifically to a protein expressed from the gene, composition. According to claim 7,
The composition is characterized in that using a biological sample separated from saliva, blood, urine or feces, composition. bone morphogenetic protein 7 (BMP7) and monoamine oxidase B (MAOB); Or BMP7 and NPIPB4 (Nuclear Pore Complex Interacting Protein Family Member B4) genes for measuring the expression level, the kit for predicting lymph node metastasis of oral cancer. According to claim 10,
Characterized in that the material for measuring the expression level is a primer or probe that specifically binds to mRNA of the gene, or an antibody or aptamer that specifically binds to a protein expressed from the gene, the kit. According to claim 10,
Characterized in that the kit further comprises a material for measuring the expression level of GAPDH, 18s rDNA or β-actin.

Images