KR102492149B1 - MicroRNA-1246 for diagnosing of ovarian cancer and use thereof - Google Patents

Abstract

The present invention relates to microRNA-1246 (microRNA-1246, miR-1246) and its use for the diagnosis or differential diagnosis of ovarian cancer. Since its expression significantly increases in ovarian cancer patients, miR-1246 is expected to be effectively used in clinical practice as a useful biomarker for diagnosis or differential diagnosis of ovarian cancer.

Description

MicroRNA-1246 for diagnosing or differential diagnosis of ovarian cancer and its use {MicroRNA-1246 for diagnosing of ovarian cancer and use thereof}

The present invention relates to microRNA-1246 (microRNA-1246, miR-1246) and its use for diagnosing or differentially diagnosing ovarian cancer, and more particularly, microRNA-1246 useful for diagnosing ovarian cancer or differentially diagnosing benign ovarian tumors. A biomarker composition containing RNA-1246, a composition and kit for diagnosing or differential diagnosis of ovarian cancer including an agent for measuring the expression level of the microRNA-1246, and a method for diagnosing or differentially diagnosing ovarian cancer using the biomarker It is about.

Ovarian cancer is one of the three major female cancers along with breast cancer and cervical cancer. The 5-year survival rate for breast cancer and cervical cancer is 90-91%, whereas ovarian cancer is 62%, which is relatively low compared to other gynecological cancers. , this is maintained at almost the same level as the 5-year survival rate for ovarian cancer in the 1990s.

The reason for the low 5-year survival rate of ovarian cancer is that, unlike other gynecological cancers, early diagnosis is difficult because there are few specific symptoms. It is characterized by high

Currently, CA-125, a tumor marker, is commonly performed as an early screening test for ovarian cancer, but it is elevated only in about 50% in the early stage, so the early diagnosis rate is low, and not only ovarian cancer, but also depending on the menstrual cycle or pregnancy and pregnancy It is elevated even in the same normal state, and it is also elevated in benign diseases such as endometriosis, myoma, or pelvic inflammatory disease, so it is difficult to distinguish it from malignant.

To compensate for this, the ROMA score test, which analyzes the risk according to pre- and post-menopause by calculating the protein substance secreted from the epithelium of malignant ovarian tumor called HE4 together with CA125, has been in the limelight recently, but its sensitivity and specificity are 50% and 63.4, respectively. Its effectiveness and efficiency are low compared to the cost, and the early diagnosis rate of ovarian cancer is still low.

Therefore, it is necessary to establish a more effective early screening test for ovarian cancer. In addition, for effective treatment of ovarian cancer, differential diagnosis from benign ovarian tumor before surgery is very important. This is because the gynecological oncologist's operation has a direct causal relationship with the patient's prognosis.

Republic of Korea Patent Registration No. 10-1784714 (2017.10.12. Notice)

In order to solve the above conventional problems, the present invention analyzes the exosome-derived microRNA (microRNA) released from ovarian cancer to understand the pathophysiology of ovarian cancer and analyzes the cancer cell exchange network. It aims to develop and commercialize early diagnosis or differential diagnosis technology.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosis or differential diagnosis of ovarian cancer, including microRNA-1246.

In addition, the present invention provides a composition for diagnosing or differential diagnosis of ovarian cancer, including an agent for measuring the expression level of microRNA-1246, and a kit using the same.

In addition, the present invention provides an information providing method for diagnosing or differentially diagnosing ovarian cancer, comprising measuring the expression level of microRNA-1246 in a biological sample isolated from a subject.

The present invention analyzed microRNAs contained in exosomes isolated from the blood of patients with benign ovarian tumors and ovarian cancer before surgery, and as a result, the expression was significantly increased in ovarian cancer patients compared to benign ovarian tumors, As a result of analyzing the expression in the patient's tissues, microRNA-1246 (miR-1246), whose expression is significantly increased in ovarian cancer, was discovered. As a useful biomarker for differential diagnosis of cancer, it is expected to be effectively used in clinical practice.

1 schematically shows a microRNA (miRNA) selection process according to an embodiment of the present invention.
Figure 2 confirms the difference in expression levels in tissues using real-time polymerase chain reaction of three miRNAs selected according to Example 2-1 of the present invention.
Figure 3 confirms the difference in the expression level of miR-1246 according to Example 2-2 of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors collected preoperative serum from benign ovarian tumor and ovarian cancer patients, isolated exosome RNA from it, and compared and analyzed the expression level of exosome microRNA through NGS analysis. microRNAs whose expression was significantly increased in ovarian cancer patients and whose expression was significantly increased in ovarian cancer tissues compared to benign ovarian tumor tissues, and based on this, the present invention was completed.

Accordingly, the present invention provides a biomarker composition for diagnosis or differential diagnosis of ovarian cancer, including microRNA-1246 (miR-1246).

As used herein, the term "ovarian cancer" includes primary and recurrent cancers occurring in the ovary, including epithelial cell carcinoma, germ cell tumor, and sex cord stromal tumor depending on the tissue in which the cancer occurs. The epithelial ovarian cancer is further classified into serous ovarian cancer, mucinous ovarian cancer, endometrioid carcinoma, clear cell carcinoma, and Brenner tumor. Brenner tumor) and undifferentiated cell cancer (undifferentiated carcinoma). In addition, ovarian cancer may be classified into stages 1, 2, 3, 3A, 3B, 3C, and 4 depending on the stage, and such ovarian cancer may also be included in the scope of the present invention.

As used in the present invention, the term "diagnosis" in a broad sense means to judge the actual condition of a patient's disease in all aspects, and the content of the judgment includes the name of the disease, etiology, disease type, severity, detailed aspects of the disease, and The presence or absence of complications may be included. Diagnosis in the present invention may include determining whether or not ovarian cancer has developed or progressing, or differentially diagnosing benign ovarian tumors or ovarian cancer.

As used in the present invention, the term “biomarker” is an indicator that can detect changes in the body, and indicates the normal or pathological state of an organism, that is, the diagnosis of ovarian cancer, the degree of response to drugs, etc. It means a substance that can be diagnosed separately from the control group, and is a polypeptide or nucleic acid (eg, mRNA, etc.), lipid, glycolipid, glycoprotein, sugar (monosaccharide, disaccharide, oligosaccharides, etc.) and the like.

In the biomarker composition according to the present invention, the miR-1246 may consist of the nucleotide sequence of SEQ ID NO: 1.

* miR-1246: 5'-AAUGGAUUUUUGGAGCAGG-3' (SEQ ID NO: 1)

The biomarker composition may further include at least one of CA125 and HE4.

Since the biomarker according to the present invention shows the same results even in repeated experiments, and the change in its expression level shows significant results, it can be regarded as a highly reliable marker, and thus the predicted results can be reasonably trusted.

The present invention provides a composition for diagnosis or differential diagnosis of ovarian cancer, including an agent for measuring the expression level of miR-1246.

The agent may be sense and antisense primers or probes that complementarily bind to miR-1246, but are not limited thereto.

As used herein, the term "primer" is a short gene sequence that is the starting point of DNA synthesis, and refers to oligonucleotides synthesized for the purpose of diagnosis, DNA sequencing, and the like. The primers may be synthesized and used in a length of 15 to 30 base pairs, but may vary depending on the purpose of use, and may be modified by methylation, capping, or the like by a known method.

As used herein, the term “probe” refers to a nucleic acid capable of specifically binding to an mRNA having a length of several to several hundred bases prepared through enzymatic chemical separation and purification or synthesis. The presence or absence of mRNA can be confirmed by labeling a radioactive isotope or an enzyme, and it can be designed and modified using a known method.

The composition may further measure the expression level of one or more of CA125 or HE4. By including the expression level of at least one of CA125 and HE4, the diagnostic efficiency of CA125 or HE4, which is an existing ovarian cancer blood biomarker, can be improved.

The present invention provides a kit for diagnosing or differentially diagnosing ovarian cancer, including the composition for diagnosing or differentially diagnosing ovarian cancer.

The diagnostic or differential diagnosis kit may be composed of one or more other component compositions, solutions or devices suitable for the analytical method.

For example, in order to perform PCR, the kit of the present invention includes genomic DNA derived from a sample to be analyzed, a primer set specific for the biomarker gene of the present invention, an appropriate amount of DNA polymerase, a dNTP mixture, a PCR buffer, and water. It may be a kit that includes. The PCR buffer may contain KCl, Tris-HCl and MgCl2. In addition, the kit of the present invention may additionally include components necessary for performing electrophoresis to confirm whether the PCR product is amplified or not.

In addition, the kit of the present invention may be a kit containing essential elements necessary for performing RT-PCR. The RT-PCR kit contains, in addition to each primer pair specific for a biomarker gene, a test tube or other suitable container, reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase and RNase inhibitors, DEPC-water, sterile water, and the like. It may also include a primer pair specific to a gene used as a quantitative control.

In addition, the present invention provides an information providing method for diagnosing or differentially diagnosing ovarian cancer, comprising measuring the expression level of miR-1246 in a biological sample isolated from a subject.

The term “information provision method for diagnosis or differential diagnosis of ovarian cancer” used in the present invention is a preliminary step for diagnosis or differential diagnosis, which provides objective basic information necessary for the diagnosis or differential diagnosis of ovarian cancer, Clinical judgment or opinion is excluded.

In the present invention, when the expression level of miR-1246 in exosomes isolated from a biological sample derived from a patient suspected of having ovarian cancer according to the above method is higher than the expression level of a normal control group without ovarian cancer or a benign ovarian tumor patient group It can be diagnosed as ovarian cancer, and the patient's prognosis can be improved by applying to a gynecological oncologist for appropriate surgery before surgery.

The biological sample may include tissue, cell, whole blood, blood, saliva, sputum, cerebrospinal fluid or urine, and more preferably blood or serum-derived exosomes, but is not limited thereto.

The expression level of miR-1246 is measured by next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase by conventional methods known in the art. It can be measured through one or more methods selected from the group consisting of Real-time PCR, RNase protection assay (RPA), microarray, and northern blotting, but , but is not limited thereto.

Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the contents of the present invention, but the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

<Example 1> Experiment preparation and experiment method

1-1. Recruitment of analysis subjects

In order to discover useful biomarkers for early diagnosis and differential diagnosis of ovarian cancer, the present inventors collected serum samples and tissue samples from patients with benign and ovarian cancer and found biomarkers that show differences between them. wanted to find

To this end, serum and tumor tissues were provided from 35 patients diagnosed with ovarian cancer and 16 patients with benign ovarian tumors at Soonchunhyang University Cheonan Hospital and Gangnam Severance Hospital. This study was conducted after receiving approval from the Clinical Research Ethics Board (IRB) of Soonchunhyang University.

1-2. RNA isolation and sequencing performed from serum

Figure 1 schematically shows the microRNA (miRNA) selection process according to an embodiment of the present invention. First, samples collected from ovarian cancer patients and benign ovarian tumor patients according to Example 1-1 Exosomes were isolated from serum samples using Exoquick (SBI, USA). Total RNA was isolated from the isolated exosomes using the Qiagen miRNeasy kit, and the quality of the isolated total RNA was measured using an Agilent RNA Bioanalyzer according to the manufacturer's instructions.

Then, using the isolated RNA, a library was prepared using the Takara SMARTer smRNA for illumina kit from Macrogen, followed by next-generation sequencing analysis (NGS) with Hiseq 2500, and seven miRNAs showing significant expression were secured. .

<Example 2> Discovery of miRNA for diagnosis or differential diagnosis of ovarian cancer

2-1. Patients with benign ovarian tumors vs. Detection of miRNAs with differences in expression levels among ovarian cancer patients before surgery

In order to develop a biomarker for diagnosis of ovarian cancer as well as differential diagnosis from benign ovarian tumors, the present inventors compared and analyzed the expression of miRNAs in tissues and exosomal miRNAs in the serum of patients with benign ovarian tumors.

As a result, as shown in Table 1 below, seven miRNAs whose expression was increased or decreased more than twice (p < 0.05) in serum or tumor tissue of ovarian cancer patients compared to benign ovarian tumors were detected.

Among them, three miRNAs, miR-1246, miR-1290, and miR-29c-3p, which show common expression patterns in serum and tissues, were selected as final candidate miRNAs that can be used for early diagnosis of ovarian cancer.

In particular, the expression of miR-1246 increased more than 6 times compared to benign ovarian tumors in tissues, and the expression increased more than 27 times compared to benign ovarian tumors in serum.

* miR-1246: 5'-AAUGGAUUUUUGGAGCAGG-3' (SEQ ID NO: 1)

2-2. Analysis of miRNA expression using real-time polymerase chain reaction

As a result of comparing the relative expression changes by performing qRT-PCR in tissues for the three miRNAs (miR-1246, miR-1290, and miR-29c-3p) selected as candidates according to Example 2-1, As shown in Figure 2, a significant difference in expression was confirmed in miR-1246 and miR-1290.

In addition, as a result of comparative analysis of the expression of miR-1246 in serum and tissues of a number of benign ovarian tumor patients and ovarian cancer patients, as shown in FIG. A significant increase was confirmed.

The above results show that miR-1246 can be usefully used as a biomarker for early diagnosis and differential diagnosis of ovarian cancer.

Having described specific parts of the present invention in detail above, it is clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> Soonchunhyang University Industry Academy Cooperation Foundation <120> MicroRNA-1246 for diagnosing of ovarian cancer and its use <130> ADP-2020-0515 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> artificial sequence <220> <223> miR-1246 <400> 1 aauggauuuu uggagcagg 19

Claims (13)

A biomarker composition for differential diagnosis of ovarian cancer from benign ovarian tumors, comprising microRNA-1246 (miR-1246). According to claim 1,
The miR-1246,
A biomarker composition for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that it consists of the nucleotide sequence of SEQ ID NO: 1. According to claim 1,
The biomarker composition,
A biomarker composition for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that it further comprises at least one of CA125 and HE4. A composition for differential diagnosis of ovarian cancer from benign ovarian tumors, comprising an agent for measuring the expression level of microRNA-1246 (miR-1246). According to claim 4,
The miR-1246,
A composition for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that it consists of the nucleotide sequence of SEQ ID NO: 1. According to claim 4,
The formulation,
A composition for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that the sense and antisense primers or probes bind complementary to miR-1246. According to claim 4,
The composition,
A composition for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that it further comprises an agent for additionally measuring the expression level of at least one of CA125 and HE4. A kit for differentially diagnosing ovarian cancer from benign ovarian tumors, comprising the composition according to claim 4. A method for providing information for differential diagnosis of ovarian cancer from benign ovarian tumors, comprising measuring the expression level of microRNA-1246 (miR-1246) in a biological sample isolated from a subject. According to claim 9,
If the expression level of miR-1246 measured in the biological sample isolated from the subject is higher than that of the normal control group or the benign ovarian tumor group, the differential diagnosis of ovarian cancer from benign ovarian tumors (benign) is characterized in that, benign ovarian Information provision method for differential diagnosis of ovarian cancer from benign. According to claim 9,
The miR-1246,
An information providing method for differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that it consists of the nucleotide sequence of SEQ ID NO: 1. According to claim 9,
The biological sample,
A method for providing information for the differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that they are blood or serum-derived exosomes. According to claim 9,
The expression level of the miR-1246,
Next generation sequencing (NGS), polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time polymerase chain reaction (Real-time PCR), RNase protection assay For differential diagnosis of ovarian cancer from benign ovarian tumors, characterized in that measured by one or more methods selected from the group consisting of RPA), microarray, and northern blotting How to provide information.

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