KR20130022204A - Method of using ant2 mrna in exosome for breast cancer diagnosis - Google Patents
Method of using ant2 mrna in exosome for breast cancer diagnosis Download PDFInfo
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Abstract
Description
본 발명은 혈액으로부터 분리된 엑소좀(exosome)을 이용하여 유방암의 발병 여부를 측정하는 방법에 관한 것으로, 보다 구체적으로는 adenine nucleotide translocater 2(ANT2) mRNA를 포함하고 있는 엑소좀의 이용 방법에 관한 것이다.The present invention relates to a method for measuring the incidence of breast cancer using exosomes isolated from blood, and more particularly, to a method for using exosomes containing adenine nucleotide translocater 2 (ANT2) mRNA. will be.
엑소좀은 대부분의 세포에서 분비되는 작은 형태의 소포체(membrane vesicle)이다. 이 엑소좀 안에는 그 세포에서 유래된 다양한 종류의 단백질, 유전물질(DNA, mRNA, miRNA), 지질 등이 포함되어 있는 것이 보고된 바 있다. 특히 혈청 내에는 과량의 RNA 분해 효소(RNase)가 존재하므로 세포로부터 분비되는 유전물질들이 쉽게 분해되어 측정하기 쉽지 않은 반면, 엑소좀 안에 존재하는 RNA의 경우에는 RNA 분해효소로부터 보호되어 안정적으로 존재하는 것이 알려져 있다. 이와 같이, 조직 유래 엑소좀은 엑소좀을 분비한 조직의 상태를 반영하기 때문에, 질병의 진단에 이용될 수 있음이 보고된 바 있다.Exosomes are small forms of membrane vesicles that are secreted by most cells. These exosomes have been reported to contain various kinds of proteins, genetic material (DNA, mRNA, miRNA), lipids, etc. derived from the cell. In particular, since there is an excessive amount of RNA degrading enzyme (RNase) in the serum, the genetic material secreted from the cell is not easily decomposed and easy to measure, whereas in the exosomes, RNA is protected from the RNA degrading enzyme and stably exists. It is known. As such, since tissue-derived exosomes reflect the state of tissues secreting exosomes, it has been reported that they can be used for the diagnosis of diseases.
종양(Tumor)은 비정상적인 세포의 과잉으로 인하여 발생하는 비정상적이고, 비제어적이며, 무질서한 세포증식의 산물로써, 이러한 종양이 파괴적인 증식성, 침윤 및 전이성을 가지게 되면 악성종양(malignant tumor)으로 분류하게 된다. 특히, 분자생물학적인 관점에서 볼 때 유전자의 변이에 의하여 발생하는 유전적 질환이라고 할 수 있다. 각각의 종양은 정상 세포에는 존재하지 않는 돌연변이 유전자를 평균적으로 50 내지 80개를 보유하고 있는 것으로 알려진바 있다. 이러한 돌연변이 유전자를 검출하는 현 기법으로는 생검 시료의 분석이 가장 일반적이다. 그러나 생검 시료의 분석은 침습성이기 때문에 대상체에게 유해할 수 있다. 또한, 제한 부위로부터 조직 시료를 채취하기 때문에, 종양이 정상 조직 내에 분산되어 있는 경우에는 결과가 정확하지 않을 수 있다. 그래서 최근에는 혈액과 같은 체액을 순환하고 있는 돌연변이 유전자를 검출하는 비침습성 방법도 제기되고 있으나, 유전자의 복제수가 극도로 낮기 때문에 검출이 어렵다는 단점이 있다.Tumors are the product of abnormal, uncontrolled, and disordered cell proliferation caused by the excess of abnormal cells, which can be classified as malignant tumors if they have destructive proliferative, invasive, and metastatic properties. do. In particular, it may be regarded as a genetic disease caused by mutation of a gene from a molecular biological point of view. Each tumor is known to have on average 50 to 80 mutant genes that are not present in normal cells. Analysis of biopsy samples is the most common technique for detecting such mutant genes. However, the analysis of biopsy samples can be harmful to the subject as it is invasive. In addition, since a tissue sample is taken from the restriction site, the result may not be accurate if the tumor is dispersed in normal tissue. Therefore, in recent years, non-invasive methods for detecting mutant genes circulating in body fluids such as blood have been proposed. However, since the number of copies of genes is extremely low, detection is difficult.
종양 중 유방암은 조기 진단에 성공하면, 다른 악성 종양에 비하여 치료할 수 있는 확률이 높다. 현재, 가장 일반적으로 사용되는 유방암 진단 방법은 기계적인 검사를 통하여 유방에 존재하는 이질적인 조직을 검출하는 방법인데, 이러한 방법은 검사과정이 비교적 간단하다는 장점에도 불구하고, 진단 성공률이 높지 않아서 생검 시료의 분석과 같은 방법과 병행되어 수행되고 있다.Breast cancer in tumors, if diagnosed early, is more likely to be treated than other malignancies. Currently, the most commonly used method for diagnosing breast cancer is to detect heterogeneous tissues present in the breast through mechanical examination. Despite the advantage that the examination process is relatively simple, the success rate of diagnosis is not high. It is performed in parallel with the same method as the analysis.
따라서 비침습성이며, 고감도 및 고특이성으로 유방암을 조기에 진단할 수 있는 방법의 개발이 요구되고 있는 실정이다.Therefore, the development of a method for early diagnosis of breast cancer with non-invasive, high sensitivity and high specificity is required.
본 발명은 상기와 같은 종래 기술상의 문제점을 해결하기 위해 안출된 것으로, 유방암의 조기 진단에 응용될 수 있는 방법으로, 혈액으로부터 분리된 엑소좀을 이용하여 유방암의 발병여부를 측정하는 방법에 관한 것으로, 보다 구체적으로는 엑소좀에 포함된 adenine nucleotide translocater 2(ANT2) mRNA의 발현수준을 측정하여 유방암의 진행 여부를 확인하는 것을 그 목적으로 한다.The present invention has been made to solve the above-mentioned problems in the prior art, a method that can be applied to the early diagnosis of breast cancer, relates to a method for measuring the development of breast cancer using exosomes separated from blood More specifically, the aim of the present invention is to determine the progression of breast cancer by measuring the expression level of adenine nucleotide translocater 2 (ANT2) mRNA contained in exosomes.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은 생물학적 시료로부터 엑소좀을 분리하는 단계, 및 상기 엑소좀 내의 ANT2(adenine nucleotide translocater 2) mRNA 발현 수준을 확인하는 단계를 포함하는 유방암의 발병 여부 측정을 위한 엑소좀의 이용 방법을 제공한다.The present invention provides a method of using an exosome for measuring the onset of breast cancer comprising the step of separating the exosomes from the biological sample, and confirming the expression level of adenine nucleotide translocater 2 (ANT2) mRNA in the exosomes. .
본 발명의 일 구현예로, 상기 생물학적 시료는 혈액 또는 혈청인 것을 특징으로 한다.In one embodiment of the invention, the biological sample is characterized in that the blood or serum.
본 발명의 다른 구현예로, 상기 ANT2 mRNA 발현 수준을 확인하는 단계는 엑소좀으로부터 ANT2 mRNA 분리 및 증폭에 의해 수행되는 것을 특징으로 한다.In another embodiment of the present invention, the step of identifying the ANT2 mRNA expression level is characterized in that it is performed by ANT2 mRNA separation and amplification from the exosomes.
또한 본 발명은 혈청으로부터 엑소좀을 분리한 후, 상기 엑소좀 내의 ANT2(adenine nucleotide translocater 2) mRNA의 농도를 측정하여 유방암 발병 여부를 측정하는 것을 특징으로 하는 유방암 진단 키트를 제공한다.In another aspect, the present invention provides a breast cancer diagnostic kit, characterized in that after separating the exosomes from the serum, by measuring the concentration of ANT2 (adenine nucleotide translocater 2) mRNA in the exosomes to determine the onset of breast cancer.
본 발명에 따른 엑소좀을 이용하여 유방암을 진단하는 것은, 비침습성 방법이다. 또한 상기 엑소좀은 세포에서 생성되어 분비된 RNA를 포함하고 있기 때문에, 고밀도로 응집이 가능하며 혈액내의 RNA 분해효소로부터 RNA를 보호할 수 있다. 따라서 본 발명에 따른 혈액으로부터 분리된 엑소좀의 이용 방법은 비침습성이며, 고감도 및 고특이성 유방암 조기 진단법에 응용할 수 있는 기술로 기대된다.Diagnosis of breast cancer using the exosomes according to the present invention is a non-invasive method. In addition, since the exosomes contain the RNA produced and secreted in the cell, it is possible to aggregate at high density and protect the RNA from RNA degrading enzymes in the blood. Therefore, the method of using the exosomes isolated from the blood according to the present invention is non-invasive and is expected to be a technology that can be applied to high sensitivity and high specific breast cancer early diagnosis.
도 1 은 혈액에서 분리한 엑소좀 내에 포함되어 있는 ANT2 mRNA의 양을 RT-qPCR로 측정한 결과를 보여주는 그래프이다.1 is a graph showing the results of measuring the amount of ANT2 mRNA contained in the exosomes isolated from blood by RT-qPCR.
본 발명은 생물학적 시료로부터 엑소좀을 분리하는 단계, 및 상기 엑소좀 내의 ANT2 mRNA 발현 수준을 확인하는 단계를 포함하는 유방암의 발병 여부 측정을 위한 엑소좀의 이용 방법을 제공한다.The present invention provides a method of using an exosome for measuring the onset of breast cancer comprising the step of separating the exosome from the biological sample, and confirming the ANT2 mRNA expression level in the exosome.
본 발명자들은 유방암 환자의 혈액 또는 혈청에서 분리한 엑소좀에 대하여 연구하던 중, 유방암 세포에서 과발현되는 것으로 알려진 ANT2 유전자가 유방암 세포에서 분비되는 엑소좀 내에 포함되어 있을 경우, ANT2 mRNA가 혈액 내의 RNA 분해효소에 의하여 분해되지 않고 고밀도로 보존되어 있을 것으로 기대하였다. 따라서 본 발명자들은 정상인과 유방암 판정을 받은 환자들의 혈액 또는 혈청을 채취하여, 엑소좀을 분리하였다. 그리고 분리된 엑소좀으로부터 mRNA를 추출하여 ANT2 mRNA의 양을 RT-qPCR로 정량하였다. 그리고 RT-qPCR 결과를 통계 처리하여 정상인과 유방암 환자간의 ANT2 유전자의 발현 수준 차이의 유의성을 확인하였다.While the present inventors have studied exosomes isolated from blood or serum of breast cancer patients, when ANT2 genes known to be overexpressed in breast cancer cells are contained in exosomes secreted from breast cancer cells, ANT2 mRNA is degraded in the blood. It was expected to be stored at high density without being degraded by enzymes. Therefore, the present inventors collected blood or serum of normal people and patients diagnosed with breast cancer, and isolated exosomes. And mRNA was extracted from the isolated exosomes and the amount of ANT2 mRNA was quantified by RT-qPCR. RT-qPCR results were statistically analyzed to confirm the significance of ANT2 gene expression level differences between normal and breast cancer patients.
또한 본 발명은 혈청으로부터 엑소좀을 분리한 후, 상기 엑소좀 내의 ANT2 mRNA의 농도를 측정하여 유방암 발병 여부를 측정하는 것을 특징으로 하는 유방암 진단 키트를 제공한다.In another aspect, the present invention provides a breast cancer diagnostic kit, characterized in that after separating the exosomes from the serum, by measuring the concentration of ANT2 mRNA in the exosomes to determine whether the breast cancer.
본 발명의 일 실시예에서는 유방암 환자의 혈액에서 분리된 엑소좀은 정상인의 혈액에서 분리된 엑소좀에 비하여 ANT2 mRNA를 고농도로 포함하고 있음을 확인하였다(실시예 1 참조).In one embodiment of the present invention, it was confirmed that the exosomes isolated from the blood of a breast cancer patient contained a high concentration of ANT2 mRNA compared to the exosomes isolated from the blood of a normal person (see Example 1).
상기 결과로부터, 혈액으로부터 분리된 엑소좀 내의 ANT2 유전자를 정량하는 방법은 유방암을 진단하는데 있어 이용될 수 있을 것으로 기대되며, 특히 ANT2 shRNA를 이용한 유전자 치료법을 시행하기 전 환자의 ANT2 발현 수준을 확인함으로써 치료법의 적합성을 판단하는데 응용될 수 있을 것으로 기대된다.From the above results, the method of quantifying ANT2 gene in exosomes isolated from blood is expected to be used to diagnose breast cancer. In particular, by confirming the level of ANT2 expression in patients before performing gene therapy with ANT2 shRNA It is expected that it may be applied to determine the suitability of therapy.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.
Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.
[실시예][Example]
실시예Example 1. 혈액에서 분리한 1. Separated from blood 엑소좀Exosomes 내의 undergarment ANT2ANT2 mRNAmRNA 정량 dose
혈액으로부터 엑소좀을 분리하기 위하여, 임상시험심사위원회(Institutional Review Board, IRB)를 통과한 유방암 환자의 혈액을 준비하였다. 준비된 3cc의 혈액을 상온에서 30분간 방치한 후, 3,000rpm으로 10분간 원심분리하여 250uL의 혈청을 획득하였다. 획득한 혈청 250uL에 엑소좀 분리시약(Expquick, Exosome precipitation solution)을 첨가하고, 4℃에서 12시간 이상 보관 후, 1,500×g로 30분간 원심분리하여 상층액을 제거하였다. 획득한 엑소좀에 Trizol(Invitrogen) 용액 1mL을 첨가하여, 엑소좀으로부터 RNA를 분리하였다. 분리된 RNA는 Nanodrop을 이용하여 농도, 순도 및 무결성을 확인하였다. 엑소좀으로부터 분리된 RNA를 이용하여 RT-qPCR(SYBR Takara one step RT-qPCR kit)을 수행하여 ANT2 mRNA를 정량하였다. 이때 RNA 농도를 보정하기 위하여, GAPDH(house keeping gene) 유전자를 비교군으로 사용하였다. 그리고 정상인과 유방암 환자로부터 분리한 엑소좀 내의 ANT2 유전자의 상대적인 양을 비교하기 위하여 RT-qPCR 결과를 하기 공식을 이용하여 분석하였다.To separate the exosomes from the blood, blood from breast cancer patients who passed the Institutional Review Board (IRB) was prepared. The prepared 3cc of blood was left at room temperature for 30 minutes, and then centrifuged at 3,000 rpm for 10 minutes to obtain 250 uL of serum. An exosome separation reagent (Expquick, Exosome precipitation solution) was added to 250uL of the obtained serum, and stored at 4 ° C. for at least 12 hours, followed by centrifugation at 1,500 × g for 30 minutes to remove the supernatant. RNA was isolated from exosomes by adding 1 mL of Trizol (Invitrogen) solution to the obtained exosomes. Isolated RNA was checked for concentration, purity and integrity using Nanodrop. ANT2 mRNA was quantified by performing the SYBR Takara one step RT-qPCR kit (RT-qPCR) using RNA isolated from exosomes. At this time, to correct RNA concentration, GAPDH (house keeping gene) gene was used as a comparison group. And RT-qPCR results were analyzed using the following formula to compare the relative amount of ANT2 gene in exosomes isolated from normal and breast cancer patients.
Fold change = 2-ΔΔCt(threshold cycle number)Fold change = 2 -ΔΔCt (threshold cycle number)
ΔΔCt=(Ct of gene of ANT2 breast cancer patient serum - Ct of GAPDH breast cancer patient serum)-(Ct of ANT2 normal serum - Ct of GAPDH normal serum)ΔΔCt = (Ct of gene of ANT2 breast cancer patient serum-Ct of GAPDH breast cancer patient serum)-(Ct of ANT2 normal serum-Ct of GAPDH normal serum)
분석한 결과를 가지고 Student's t test를 시행하였고, p<0.05인 경우 의미있는 차이라고 판단하였다. 그 결과는 도 1에 나타내었다.Student's t test was performed with the analysis, and p <0.05 was determined to be a significant difference. The results are shown in FIG.
유방암 환자의 경우 정상인에 비하여, ANT2 유전자의 발현이 7.3배 이상 증가되어 있으며, 특히 유방암 초기인 1기에 해당하는 환자의 경우에도 4.8배 이상 증가되어 있음을 확인할 수 있었으며, 이는 통계적으로 유의한 수준이다. 상기 결과는 유방암 환자의 혈액에서 분리한 엑소좀은 ANT2 유전자를 고농도로 포함하고 있다는 것을 시사한다.In the case of breast cancer patients, the expression of ANT2 gene was increased by 7.3 times or more, compared with the normal person, especially in patients with
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예 및 실험예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.
The above description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. It is therefore to be understood that the embodiments and experiments described above are illustrative in all aspects and not restrictive.
Claims (4)
(b) 상기 엑소좀 내의 ANT2(adenine nucleotide translocater 2) mRNA 발현 수준을 확인하는 단계를 포함하는 유방암의 발병 여부 측정을 위한 엑소좀의 이용 방법.
(a) separating the exosomes from the biological sample; And
(b) a method for using exosomes for determining the incidence of breast cancer comprising the step of confirming the expression level of adenine nucleotide translocater 2 (ANT2) mRNA in the exosomes.
상기 생물학적 시료는 혈액 또는 혈청인 것을 특징으로 하는, 방법.
The method of claim 1,
The biological sample is blood or serum.
상기 (b) 단계의 ANT2 mRNA 발현 수준의 확인은 엑소좀으로부터 ANT2 mRNA 분리 및 증폭에 의해 수행되는 것을 특징으로 하는, 방법.
The method of claim 1,
Identification of the ANT2 mRNA expression level of step (b) is characterized in that it is performed by ANT2 mRNA separation and amplification from exosomes.
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| WO2014193159A1 (en) * | 2013-05-28 | 2014-12-04 | 주식회사 바이오인프라 | Method for predicting therapeutic responsiveness of lung cancer with respect to egfr-targeting therapeutic agent |
| US9213029B2 (en) | 2013-06-25 | 2015-12-15 | Samsung Electronics Co., Ltd. | Method for diagnosing breast cancer by detection of polymeric immunoglobulin receptor in vesicles isolated from patients |
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| KR101968046B1 (en) * | 2018-07-19 | 2019-04-11 | (주) 바이오인프라생명과학 | Complex biomarkers for early diagnosis of cancer |
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| KR20070101610A (en) * | 2006-04-11 | 2007-10-17 | 주식회사 바이오인프라 | Gene therapy for cancer using small interfering rna specific to ant2 and a method to overcome tolerance to antitumor agent |
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| WO2014193159A1 (en) * | 2013-05-28 | 2014-12-04 | 주식회사 바이오인프라 | Method for predicting therapeutic responsiveness of lung cancer with respect to egfr-targeting therapeutic agent |
| US9213029B2 (en) | 2013-06-25 | 2015-12-15 | Samsung Electronics Co., Ltd. | Method for diagnosing breast cancer by detection of polymeric immunoglobulin receptor in vesicles isolated from patients |
| EP3544605A4 (en) * | 2016-11-22 | 2020-11-25 | Prime Genomics, Inc. | Methods for cancer detection |
| KR20200015101A (en) * | 2018-08-02 | 2020-02-12 | 엑소젠 피티이. 엘티디 | Method for early diagnosis of breast cancer and monitoring after treatment using liquid biopsy multi-cancer gene biomarkers |
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