JP2016198094A - Prognosis inspection and diagnostic method of ovarian cancer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a prognosis inspection method of ovarian cancer which is capable of predicting prognosis of ovarian cancer by using a blood sample.SOLUTION: A prognosis inspection method of ovarian cancer of the present invention consists of comparing an expression level of miR-135 of miRNA in a blood sample at the point of prediction inspection and diagnosis of ovarian cancer with a reference expression level of miR-135 in a comparison sample, inspecting that the expression level of miR-135 is reduced, and predicting prognosis of ovarian cancer. Further, the invention is characterized in that the reference expression level of the miR-135 is the expression level of miR-135 after an extraction surgery of corresponding ovarian cancer and/or an expression index of miR-135 on an ROC curve is the expression level of miR-135 corresponding to 0.40.SELECTED DRAWING: Figure 6

Description

  The present invention relates to a prognostic test / diagnosis method for ovarian cancer. More specifically, the present invention relates to a prognostic test / diagnosis method for ovarian cancer in which microRNA-135 (miR-135) is used as a tumor marker to test / diagnose the prognosis of ovarian cancer.

  Main gynecological cancers include cervical cancer, uterine body cancer, ovarian cancer, etc. In the United States, 98,280 people developed in 2014 and 30,440 people died. Of these, ovarian cancer has developed in 21,290 people and 14,180 have died (Non-patent Document 1).

  The mortality rate of ovarian cancer is high because ovarian cancer is difficult to detect by screening because subjective symptoms such as bleeding and pain are difficult to occur. One of the causes is that metastasis is often found in a state of progressing in stages 3 and 4 where it has spread widely.

  However, ovarian cancer has a very poor 5-year survival rate of about 20% to 30% in the advanced stage of disease progression, whereas the cure rate is about 70% in the early stage of the first and second stages. % -90% and very high.

  In this way, ovarian cancer has a close relationship between the stage of progression at the time of its visit and the survival rate, so if early detection of ovarian cancer can be achieved, reduction in mortality and long-term postoperative management can be achieved. I can expect to do it.

  In addition, ovarian cancer differs from other gynecological cancers such as cervical cancer and endometrial cancer that can be diagnosed by biopsy before surgery, unlike cytology and histology before surgery. At present, there is no reliable method for early detection of ovarian cancer because the stage cannot be determined until the results of histopathological diagnosis of the ovaries removed by laparotomy are obtained. .

  Ovarian cancer is also rarely cured by removal surgery alone in an advanced state, and is generally treated with chemotherapy with an anticancer agent such as a platinum preparation after the operation.

  On the other hand, ovarian cancer has a feature that anticancer drugs are relatively effective, and recently, new anticancer drugs have increased and treatment options have increased.

  However, although the types of anticancer drugs effective for ovarian cancer are increasing, a method for selecting a therapeutic agent according to the type of cancer of the patient has not yet been established.

  Furthermore, ovarian cancer is one of the malignant tumors that has a high possibility of recurrence, is difficult to treat, and has a very difficult prognosis. The prognosis for ovarian cancer is currently predicted based on the surgical resection rate, clinical progression stage, and pathological examination, but there is no way to predict the prognosis of ovarian cancer by blood tests. Patent Document 1).

  However, extensive experience to date has shown that the patient's postoperative results improve if the lesions are completely removed by surgical weight loss or if few cells are left behind. (Non-Patent Document 2).

  Even if cancer treatment advances in this way, there is no accurate tool for diagnosing early ovarian cancer that can be cured, so the prognosis of ovarian cancer cannot be predicted.

  Thus, since ovarian cancer is often an advanced stage that has already metastasized at the time of consultation, various screening methods have been developed for early detection of ovarian cancer.

  The main screening methods include cancer antigen 125 (CA-125) measurement, ultrasound imaging, biomarker search, and the like.

  CA-125 is a high molecular compound glycoprotein expressed in a high proportion of epithelial ovarian cancer (Non-patent Document 3), and it has been established as a tumor marker for epithelial ovarian cancer. Sex is inadequate.

  CA-125 is only expressed in about 50% of stage I epithelial ovarian cancer and about 75% to 90% of advanced stage ovarian cancer (Non-Patent Documents 4, 5, and 6).

  CA-125 is also expressed not only in epithelial ovarian cancer, but also in other pathologies or normal tissues (Non-Patent Document 4), and false-positive results are observed in malignant and benign disorders. (Non-Patent Documents 7 and 8). Therefore, CA-125 is not a specific cancer antigen for epithelial ovarian cancer.

  If ultrasound is used, detailed image analysis of the ovary and morphological changes indicating the degree of malignant progression can be detected. There is a report that a transvaginal ultrasound (TVUS) screening method is a feasible method for early detection of ovarian cancer (Non-patent Document 9).

  However, there have been many studies on the results of ultrasound image analysis of the ovary, but there are considerable differences among observers in image analysis in ultrasound image diagnosis. It is not enough to predict the prognosis of ovarian cancer because it cannot be diagnosed automatically.

  Therefore, because ovarian cancer has already metastasized at the first visit, ovarian cancer can be detected by a combination of blood test CA-125 measurement and transvaginal ultrasonography for people with no symptoms. Many studies aimed at early detection were conducted, especially in the West.

  Unfortunately, however, there was no difference in mortality between ovarian cancer patients who were screened and those who did not. As a result, a guideline has been issued in the West that does not recommend that asymptomatic women undergo ovarian cancer screening. In addition, the American Preventive Medicine Working Group has taken the opposite position even to regularly screen for ovarian cancer (Non-patent Document 10).

  Because of this difficulty in early detection of epithelial ovarian cancer, research on early detection of ovarian cancer continues, and biomarkers that improve susceptibility to ovarian cancer use various materials and methods. Have been explored.

  The most prominent approach in biomarker search is to use proteomics. As a result, many biomarkers were detected, but their function was unfortunately similar to CA-125 at best. Therefore, it is unlikely that a single biomarker alone can be used clinically for diagnosis of epithelial ovarian cancer (Non-patent Document 11).

  So far, screening methods for ovarian cancer have been eagerly studied, but unfortunately, a general ovarian cancer screening method that can be applied clinically has not yet been completed (Non-Patent Documents). 12).

  That is, at this point, although there is rudimentary evidence that screening methods can improve survival, it is still unclear whether ovarian cancer mortality can be improved. Moreover, although the initial knowledge which hope can have is obtained by the proteomic method, these knowledge has not yet reached the stage which can be utilized clinically (Non-patent Document 12).

  In recent years, due to dramatic progress in gene research, endogenous microRNA (miRNA), one of short RNAs, is one of the most important regulators of post-transcriptional expression regulation in eukaryotes. It has become clear.

  This miRNA is a 18 to 25 base (nt) long single-stranded RNA molecule that does not encode a protein, and regulates the expression of about 30% or more of the gene encoding the protein (Non-patent Document 13). ) In addition, the human genome is thought to encode more than 1000 miRNAs. Subsequent studies revealed that miRNA regulates target mRNA expression by suppressing protein production resulting from translational suppression of target mRNA, mRNA cleavage, and mRNA degradation.

  In addition, recent studies have shown that certain miRNAs are involved in the regulation of cell proliferation and apoptosis, which are important for cancer development. Moreover, since miRNA shows a specific expression pattern in a specific cancer type and stage, it is suggested that miRNA functions as a biomarker for cancer diagnosis by profiling miRNA (Non-patent Document 14). ).

  Several techniques, including Northern blot analysis, real-time PCR, and miRNA microarrays, may have several types of miRNAs directly involved in human cancers such as lung cancer, breast cancer, brain tumor, liver cancer, colon cancer, and leukemia It became clear (for example, nonpatent literature 15).

  In addition to these cancers, there are reports of miRNAs related to gynecological cancers that use cancer cell lines (see Non-Patent Documents 15 and 16) and patient clinical specimens (Non-Patent Documents). 17).

  Non-Patent Document 12 describes that 13 types of miRNAs were identified by comparing the expression of mRNA and miRNA between endometrial cancer tissues of various stages and grades and normal endometrial tissues. Yes. However, Non-Patent Document 12 describes that among 13 types of miRNAs, 8 types of miRNAs are increased in endometrial cancer and 5 types of miRNAs are decreased in endometrial cancer. There is no mention that these 13 miRNAs can be used to diagnose endometrial cancer. Moreover, this non-patent document does not describe or even suggest any ovarian cancer.

  Patent Document 1 (Japanese Patent Application Laid-Open No. 2010-154843) describes numerous miRNA expression profiles and their uses in gynecological cancers that do not contain the 13 types of miRNA identified in Non-Patent Document 13. Thus, a method for using a specific miRNA as a biomarker for gynecological cancer is described.

  In this patent document, one or more miRNAs selected from a large number of miRNA groups including miR-135 are used as biomarkers for gynecological cancer, a method for determining gynecological cancer, and miR-135 Numerous miRNAs are composed of RNA in which one or more nucleotides are deleted, substituted, or added in each nucleotide sequence, and are controlled in the blood of gynecological cancer tissues or gynecological cancer subjects The method of using RNA whose expression is increased or decreased as compared with the above as a biomarker for gynecological cancer and a method for determining gynecological cancer are described.

  However, this patent document only describes that many miRNAs, including miR-135, have increased or decreased expression compared to controls in the blood of gynecological cancer tissues or gynecological cancer subjects, There is no description on how to specifically determine the increase / decrease in the expression of such miRNA and use it as a biomarker.

  It is generally well-known that miRNAs increase or decrease in cancer tissues and blood compared to controls, but which specific miRNAs have increased expression for which specific cancers Or it will not be known at all, and it cannot be predicted.

  Patent Document 1 does not describe at all what specific gynecological cancers miR-135 has increased or decreased, and it is unpredictable to increase or decrease ovarian cancer expression. Is possible. Moreover, of course, no predictions have been made about the prognosis of ovarian cancer.

  Therefore, in order to investigate the recurrence of ovarian cancer after surgery or the presence or absence of metastasis to other organs, there is a demand for means and methods that can predict the prognosis of ovarian cancer.

JP 2010-154843 A

CA CANCER J CLIN 2015; 65: 5-29 Cannistra, S.A., N. Engl. J. Med. 2004; 351: 2519-2529 Bast, R.C., et al. J Clin Invest. 1981; 68: 1331-1337 Jacobs, I., et al. Hum Reprod. 1989 Jan; 4 (1): 1-12 Woolas, R.P., et al. J Natl Cancer Inst. 1993; 85: 1748-1751 Fritsche, H.A., et al. Clin Chem 1998; 44: 1379-1380 Ozguroglu, M., et al. Am J Clin Oncol. 1999; 22: 615-618 Meden, H., et al. Int J Biol Markers. 1998; 13: 231-237 Sato, S., et al. Cancer. 2000; 89: 582-588 http://www.cancer.gov/cancertopics. NCI Ovarian Cancer Screening (PDQ) Sasaroli, D., et al. Biomark Med. 2009 Jun 1; 3 (3); 275-288 Rauh-Hain, J.A., Rev Obstet Gynecol. 2011; 4 (1): 15-21 Lynam-Lennon, N., et al. Biol Rev Camb Philos Soc. 2009 Feb; 84 (1): 55-71) Zhang, B., et al. J Cell Mol Med. 2008 Jan-Feb; 12 (1): 3-21 Jiang J, et al. Nucleic Acids Res. 2005; 28: 5394-5403 Tsuda N, et al. Int J Oncol. 2005; 27: 1299-1306 Boren T, et al. Gynecol Oncol. 110 (2008) 206-215

  Therefore, in order to examine and diagnose the prognosis of ovarian cancer, which was impossible in the past, the present inventor, based on the sensitivity of miR-135 to ovarian cancer to anticancer drugs, If the sensitivity to anticancer drugs is low, that is, if miR-135 is overexpressed, ovarian cancer has high resistance to anticancer drugs, so in this case miR-135 As a prognostic marker, the present inventors have found that the prognosis of ovarian cancer can be predicted.

  In the present specification, the term “prognosis” is used to mean the progress prediction after the initial treatment. For ovarian cancer, initial treatment is generally a combination of weight loss surgery and platinum chemotherapy (Ann Oncol (January 2005) 16 (1): 4-6). In this specification, unless otherwise specified, it means the progress prediction after initial treatment with ovarian cancer weight loss surgery and platinum preparation.

  In addition, in this specification, the term “reference expression level of miR-135” refers to the expression level of miR-135 after ovarian cancer resection surgery to be compared in predicting the prognosis of postoperative ovarian cancer, or in FIG. As described, the expression level of miR-135 corresponding to the expression index of miR-135 of 0.40 on the ROC curve is meant. In other words, if the expression level of miR-135 at the time of predicting the prognosis of postoperative ovarian cancer is lower than the comparative expression level, it may be judged or diagnosed that the prognosis is poor. it can. In other words, in this case, it can be determined or diagnosed that ovarian cancer has a high risk of recurrence and / or metastasis and / or anticancer agents are not effectively acting. Conversely, if the miR-135 expression level at the time of prognosis prediction is equal to or higher than the reference expression level, the prognosis for ovarian cancer is good, and ovarian cancer has relapsed and / or metastasized and / or Or it can be judged or diagnosed that the anticancer agent is acting effectively.

  Furthermore, the miR-135 expression level corresponding to an expression index of miR-135 of 0.40 in the ROC curve was used as the reference expression level for miR-135 because the sensitivity and specificity for ovarian cancer was 60%. It is because it can be judged that it is high risk and has high resistance to anticancer agents. In the present invention, the expression level of miR-135 is based on the ROC curve expression index of 0.40, and when the expression index is lower than 0.40, the prognosis is poor, and the expression index is 0.40. If it is higher, it is judged that the prognosis is good. That is, when ovarian cancer has a poor prognosis, it can be judged that there is a suspicion that ovarian cancer has recurred or metastasized, and the anticancer drug administered after surgery is not acting effectively. Conversely, when ovarian cancer has a good prognosis, it can be judged that the risk of recurrence or metastasis of ovarian cancer is low, and that the anticancer drug administered after surgery is likely to be effective.

  Therefore, an object of the present invention is to provide a prognostic test / diagnosis method for ovarian cancer, which comprises testing / diagnosis prognosis of ovarian cancer using miR-135 as a prognostic marker for ovarian cancer. .

  In order to achieve the above object, the present invention provides a semi-quantitative measurement of the expression level of serum miR-135 in a blood sample at the time of prognosis test for ovarian cancer by the PCR method. The object is to provide a prognostic method for ovarian cancer, which comprises testing for a decrease in miR-135 expression level in a comparative sample after cancer removal surgery.

  The present invention also reduces the prognosis of ovarian cancer patients by reducing the expression level of serum miR-135 in the blood sample compared to the reference expression level of miR-135 in the comparative sample. A prognostic method for ovarian cancer comprising predicting prognosis.

  Furthermore, the present invention provides a test or diagnosis that the prognosis of ovarian cancer is poor when the expression level of miR-135 at the time of predicting the prognosis of ovarian cancer is lower than the reference expression level of miR-135. Prognostic and diagnostic methods for ovarian cancer. More specifically, the present invention relates to a case where the expression level of miR-135 at the time of predicting the prognosis of ovarian cancer is lower than the reference expression level of miR-135, that is, after the corresponding ovarian cancer is removed. If the expression level of miR-135 and / or the expression index of miR-135 on the ROC curve is lower than the expression level of miR-135 corresponding to 0.40, ovarian cancer has relapsed and / or metastasized and Provided is a prognostic test / diagnosis method for ovarian cancer, which comprises testing or diagnosing high risk that an anticancer drug administered after surgery is not effective.

  Furthermore, according to the present invention, the reference expression level of miR-135 is such that the expression level of miR-135 and / or miR-135 expression index on the ROC curve after the corresponding ovarian cancer excision surgery is 0.40. Prognostic and diagnostic methods for ovarian cancer comprising miR-135 expression levels corresponding to

  Since the prognostic test / diagnosis method for ovarian cancer according to the present invention can predict the prognosis of ovarian cancer, which has been impossible until now, ovarian cancer with a high risk of recurrence due to postoperative metastasis, etc. It can be detected early with only a blood test. Therefore, since the present invention makes it possible to predict the prognosis of ovarian cancer, in some cases, there is a great advantage that an existing treatment policy can be changed to another treatment policy at an early stage. As a result, even in advanced stage ovarian cancer, the prognosis can be improved, and the QOL of ovarian cancer patients can be remarkably improved.

FIG. 1 is an explanatory diagram describing a nucleic acid drug using miR-135. FIG. 2 is a diagram showing case information of serum samples used for miRNA screening. FIG. 3 is a diagram showing the results of comprehensive analysis of miRNA expression levels by microarray for specific miRNAs. FIG. 4 is a diagram showing the results of examination of serum miR-135, miR-630 and miR-1207 by the real-time PCR method (Taqman method). The vertical axis represents the expression index (Expression Index, standardized miR-135 expression level). (Example 2) FIG. 5 shows a study of serum miR-135 taking benign disease into consideration. FIG. 6 is a diagram showing the results of comparative studies on serum miR-135 depending on the presence or absence of recurrence. FIG. 7 shows miR-135 Receiver Operating Characteristic curves (ROC curves) in benign disease patients and ovarian cancer patients (clinical progression stage III / IV). FIG. 8 shows the results of a cell survival test examining the platinum sensitivity of miR-135 in the ovarian cancer cell line SK-OV-3. FIG. 9 is a diagram showing the results of investigating tumorigenicity by transplanting an ovarian cancer cell line to the back of a mouse.

  In the present invention, the prognosis of ovarian cancer is defined by sensitivity to an anticancer agent called a platinum preparation used for maintenance therapy after surgery. In basic experiments, it has been proved that the presence of a large amount of miR-135 extracellularly contributes to the sensitivity to the anticancer drug. From this, it can be predicted that the miR-135 expression level is platinum resistance when the expression level is low, and the prognosis is poor with standard treatment.

  In other words, the prognostic test method for ovarian cancer according to the present embodiment measures miR-135 contained in a serum sample collected from a living body, and the miR-135 concentration is lower than the reference expression level. It can be said that this is a method for examining a poor prognosis of ovarian cancer by associating it with resistance to an anticancer drug after surgery for ovarian cancer.

  The present specification also provides a nucleic acid drug using miR-135 for suppressing cell growth of ovarian cancer and improving sensitivity to an anticancer drug such as cisplatin, and a method for activating the nucleic acid drug. .

  Nucleic acid drugs using miR-135 are based on the results of diligent research by the present inventors. Specifically, miR-135 is administered in vivo to suppress cell growth of ovarian cancer. It was completed based on the knowledge that it will be done.

  In realizing a nucleic acid drug using miR-135, for example, in order to enable intravenous administration, miR-135 is subjected to a predetermined modification or formed into a complex with another physical configuration. It is also possible. The nucleotide sequence of miR-135 is shown as SEQ ID NO: 1 in the attached sequence listing. When used as a nucleic acid drug, it is not necessarily the same as the base sequence described in SEQ ID NO: 1, and deletion, substitution or addition has been made as long as the same effect as miR-135 can be exhibited. It goes without saying.

  Examples of the predetermined modification applied to miR-135 include phosphorothioation and 2′-position o-Methylation. By applying these modifications, miR-135 is cleaved in vivo. Or the degradation can be suppressed to improve the in vivo stability or to reduce the immunostimulatory property.

  In addition, examples of forming a complex with another physical configuration include, for example, a complex of so-called microbubbles and nanobubbles, and miR-135.

  As shown in FIG. 1, problems with general nucleic acid drugs include in vivo instability, transferability to target organs, and uptake efficiency into cells. According to a nucleic acid drug comprising a complex with 135, miR-135 itself is protected by microbubbles, so that stability in the living body can be improved. Reaching is encouraged.

  It should also be noted that when microbubbles break in the vicinity of an organ or cell, a small hole is formed in the cell by the impact, and miR-135 encapsulated in the microbubble through the hole is efficiently introduced into the cell. It becomes possible to introduce well.

  More specifically, miR-135 is concentrated at the target site in the vicinity of the target ovarian cancer cell by causing a trigger means to break up the microbubbles of the complex containing miR-135. Can be transferred into cancer cells.

  Such trigger means is not particularly limited as long as fine bubbles can be broken. For example, ultrasonic waves can be employed. That is, a complex of microbubbles and miR-135 is administered intravenously, for example, and by irradiating the target site with ultrasonic waves, the microbubbles are broken in the vicinity of the target site and efficiently in the cell. miR-135 can be introduced to effectively suppress the growth of ovarian cancer cells and to effectively improve the sensitivity of anticancer agents.

  In addition, it is possible to provide a nucleic acid drug that forms the core of a highly efficient drug delivery system for the treatment of ovarian cancer.

  Next, experiments conducted to estimate the prognosis of ovarian cancer patients by semiquantitatively measuring mRNA miR-135 extracted from patient serum by real-time PCR will be described below.

  FIG. 2 shows case information of a serum sample collected from the ovarian cancer patient shown in FIG. 2 used for miRNA screening. In the figure, case numbers 1 to 6 were classified as a poor prognosis group in cases relapsed within the latter half of the initial treatment, and case numbers 7 to 12 were classified as a good prognosis group in cases having no relapse within half a year.

  Serum samples were prepared from blood collected from each patient, and miRNA expression levels were comprehensively analyzed using a microarray according to a conventional method.

  Specifically, 300 μL of serum centrifuged at 1,000 × g, 4 ° C., 10 minutes was centrifuged at 16,000 × g, 4 ° C., 10 minutes, and 200 μL of the supernatant was used as a serum sample. Next, RNA was extracted from 200 μL of serum sample using miRNeasy Mini Kit (Qiagen, model number: # 217004), and miRNA Complete Labeling Regent and Hyb Kit (Agilent Technologies, model number) : # 5190-0456), labeling and hybridization were performed, and analysis was performed with a SurePrint G3 Human miRNA microarray 8 × 60K (Agilent Technologies, model number: # 031181). The analysis results were digitized with Feature Extraction software (Agilent Technologies) and annotated with GeneSpring software (Agilent Technologies).

  As a result, the miRNAs that differed between the poor prognosis group and the good prognosis group were miR-135, miR-630, and miR-1207. miR-135 and miR-630 were low in the poor prognosis group. miR-1207 was high in the poor prognosis group. FIG. 3 shows these expression levels.

  As a result of exhaustive analysis of miRNA expression level by microarray for miRNA according to Example 1, serum miR-135, which was low in serum samples in the poor prognosis group, was subjected to real-time PCR (Taqman (registered trademark) method). And examined. In this example, a real-time PCR method using sera from patients with benign ovarian tumors, patients with uterine fibroids, uterine prolapse, etc. and clinical stage I / II (58 cases) and III / IV (40 cases) cases ( Taqman (registered trademark) method) was carried out according to a conventional method.

  Specifically, 300 μL of serum centrifuged at 1,000 × g, 4 ° C., 10 minutes was centrifuged at 16,000 × g, 4 ° C., 10 minutes, and 200 μL of the supernatant was used as a serum sample. Next, a solution in which 200 μL of serum sample and cel-miR-39 were mixed was extracted using miRNeasySerum / Plasma Kit (Qiagen, model number: # 217184) and purified to a 50 μL solution. Using 5 μL of this, TaqMan (registered trademark) probe of each miRNA (model number: hsa-miR-135a *, # 002232; hsa-miR-630, # 001563; hsa-miR-1207-5p, # 241060_mat; cel -MiR-39, # 000200) and TaqMan (registered trademark) MicroRNA Reverse Transcription Kit (model number: # 4366596) After reverse transcription, TaqMan (registered trademark) Universal Master Mix II, no UNG (model number: # 4440048) Was used for real-time PCR analysis using 7500 Fast real-time PCR system (Applied Biosystems). Correction was made by dividing the signal value of each miRNA by the signal value of cel-miR-39.

  As a result, compared with benign disease patients and clinically advanced stage I / II ovarian cancer patients, miR-135 alone had a lower serum level of clinically advanced stage III / IV patients as well as screening results (Mann-Whitney U Test). The result is shown in FIG.

  FIG. 5 shows the results of studies on serum miR-135 taking into account benign diseases. In this example, patients with benign diseases (benign ovarian tumors, uterine fibroids, uterine prolapse, 50 cases) and clinically advanced stage I / II ovarian cancer patients (58 cases) and stage III / IV ovarian cancer patients (40 cases) ) Was used for the real-time PCR method (Taqman (registered trademark) method) in the same manner as described above. Serum miR-135 expression was lower in sera of patients with clinically advanced stage III / IV compared to patients with benign disease and patients with clinically advanced stage I / II (Mann-Whitney U test).

  FIG. 6 shows a comparison result of the expression level of miR-135 in serum depending on the presence or absence of recurrence. In this example, ovarian cancer patients with a follow-up period of 1 year or more after the end of chemotherapy using cisplatin as a platinum preparation were relapsed after treatment (relapsed group) and non-relapsed group (non-relapsed group). In the same manner as above, the expression of serum miR-135 was examined by real-time PCR (Taqman (registered trademark) method). As a result, the expression of miR-135 in serum in the relapse group was lower than that in the non-relapse group (Mann-Whitney U test). In addition, as a matter of course, the anticancer agent includes an anti-ovarian cancer agent in addition to other platinum-based preparations that can be used for the treatment of ovarian cancer. Furthermore, stabilized miR135a can be formulated with 2′-O-methyl oligonucleotide to make an antisense drug that suppresses the growth of ovarian cancer cells.

  FIG. 7 shows a Receiver Operating Characteristic curve (ROC curve) of miR-135 in benign disease patients and ovarian cancer patients (clinical progression stage III / IV). In this example, ROC curves were calculated based on the expression of serum miR-135 in patients with benign diseases (benign ovarian tumors, uterine fibroids, uterine prolapse, 50 cases) and clinically advanced stage III / IV ovarian cancer patients (40 cases). Was drawn. A cut-off value of about 60% was set for both sensitivity and specificity with an expression index of 0.40.

  In this example, a cell survival test was performed to examine the platinum sensitivity of miR-135 in the ovarian cancer cell line SK-OV-3 (FIG. 8). In the cell survival test, miR-135 was forcibly expressed in ovarian cancer cell line SK-OV-3 and exposed to cisplatin, a platinum anticancer agent, and then the real-time PCR method (Taqman (registered) in Example 2). (Trademark) method). As a result, forced miR-135 enhanced cisplatin sensitivity in ovarian cancer cell lines compared to controls.

  In this example, an ovarian cancer cell line was transplanted on the back of a mouse and examined for tumorigenicity. As a control, miR-blank-introduced cells and miR-135a-introduced cells were used. As a result of observation for 10 weeks, tumors hardly grew in mice transplanted with cells overexpressing miR-135a (FIG. 9).

  The prognostic test / diagnosis method for ovarian cancer according to the present invention enables postoperative ovarian cancer recurrence or metastasis to other organs, thereby enabling prognostic prediction, so that administration after ovarian cancer removal surgery It will be possible to test the effectiveness of the platinum product. The present invention can be expected to develop to the development of a technology that leads to early detection of ovarian cancer.

Claims (11)

  Test that miR-135 expression level in blood samples at the time of ovarian cancer prognosis test is lower than the reference expression level of miR-135 in comparative samples after ovarian cancer removal surgery A prognostic method for ovarian cancer characterized by comprising:   The prognostic test method for ovarian cancer according to claim 1, wherein the reference expression level of miR-135 is the expression level and / or ROC curve of miR-135 after the corresponding ovarian cancer removal operation. A method for prognosing ovarian cancer, wherein the expression level of miR-135 corresponds to an expression index of miR-135 of 0.40.   The prognosis test for ovarian cancer according to claim 1 or 2, wherein the expression level of serum miR-135 in the blood sample is measured by PCR method and microarray method. Method.   The ovarian cancer prognosis test method according to any one of claims 1 to 3, wherein the PCR method is performed using a real-time PCR method or a TaqMan (registered trademark) method. Prognostic inspection method.   The expression level of miR-135 in the blood sample at the time of prognosis of ovarian cancer is lower than the reference expression level of miR-135 in the comparative sample after ovarian cancer removal surgery, A prognosis method for ovarian cancer, comprising prognosing that the prognosis of ovarian cancer has decreased.   The prognosis method for ovarian cancer according to claim 5, wherein the reference expression level of miR-135 is the expression level and / or ROC curve of miR-135 after the corresponding ovarian cancer removal operation. A prognosis method for ovarian cancer, wherein the expression index of miR-135 corresponds to an expression index of miR-135 of 0.40.   The prognosis method for ovarian cancer according to claim 5 or 6, wherein the expression level of serum miR-135 in a blood sample is measured by a PCR method and a microarray method. Method.   The ovarian cancer prognosis method according to any one of claims 5 to 7, wherein the PCR method is performed using a real-time PCR method or a TaqMan (registered trademark) method. Prognosis diagnosis method.   The miR-135 expression was selected by comprehensive analysis of miRNA expression levels by microarray using blood samples, and the miR-135 expression level decreased compared to the reference expression level of miR-135 in the comparative sample A prognostic test method for ovarian cancer, characterized by checking that the patient is doing.   The prognosis method for ovarian cancer according to any one of claims 5 to 8, wherein the expression level of miR-135 at the time of prognosis diagnosis of ovarian cancer is the reference expression level of miR-135. Lower than that, the recurrence and / or metastasis of ovarian cancer after surgery and / or the poor prognosis of ovarian cancer due to low efficacy of postoperatively administered anticancer drugs A prognosis method for ovarian cancer characterized by the above.   Measure miR-135 contained in serum samples collected from living organisms, and determine if miR-135 concentration is lower than the reference expression level and resistance to anticancer drugs after surgery for ovarian cancer. A method for examining poor prognosis of ovarian cancer by associating with it.