CN108315423A - Purposes of the serum excretion body aHIF as ovarian cancer diagnosis and the marker of outcome - Google Patents

Abstract

The present invention provides the use of serum exosomal aHIF as a marker for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer. The present invention also provides the use of serum exosomal aHIF as a marker molecule in the preparation of a kit for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer. The present invention also provides a kit for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer, which contains a reagent for detecting serum exosomal aHIF. It is proved by experiments that exosomal aHIF can become a new non-invasive marker for tumor diagnosis of epithelial ovarian cancer and prognosis prediction of epithelial ovarian cancer.

Description

Use of serum exosomal aHIF as a marker for the diagnosis and prognosis of ovarian cancer

technical field

The invention belongs to the field of medical detection, and relates to a serum exosome aHIF, specifically the use of the serum exosome aHIF as a marker for the diagnosis and prognosis of ovarian cancer.

Background technique

Malignant ovarian tumors are the female reproductive system tumors with the highest mortality rate. At present, the pathogenesis of ovarian cancer is still unclear. Although surgical treatment and chemotherapy are improving, the prognosis of ovarian cancer patients is still poor. The survival rate is still only 46%, and 60% of the patients are already in the advanced stage when they are diagnosed (FIGO III-IV stages of the International Federation of Gynecology and Obstetrics Ovarian Cancer Surgery-Pathological Staging System), and the 5-year survival rate of patients with advanced ovarian cancer is only 28%. .

Predicting the prognosis of patients with malignant tumors can help clinicians and patients choose the most appropriate treatment, which is beneficial to the management of the disease, thereby improving the quality of life of patients and even prolonging the survival time of patients to a certain extent. Traditional ovarian cancer prognostic factors are mainly tumor stage, pathological type, grade and so on. However, due to the heterogeneity of the disease, there may be large differences in the survival time of different patients even if the above clinicopathological features are similar. With the advancement of medical technology and the popularization of concepts such as targeted chemotherapy and personalized treatment, the theory of molecular typing has gradually matured, and its clinical significance has become more and more prominent. In recent years, liquid biopsy-related research, such as circulating tumor cells/DNA, exosome-related biomarkers, etc., has opened up new ideas for the diagnosis and prognosis prediction of ovarian cancer, and is expected to further improve the diagnosis and prognosis of ovarian cancer. Prognosis accuracy, perfect diagnosis and treatment strategy.

Exosomes, one of the important components of the extracellular environment, were first discovered during the study of the maturation of reticulocytes. They are small vesicles with a diameter of 30-150 nm and a double-layered lipid membrane. At present, it is generally believed that exosomes are early endosomes formed by cell membrane invagination, and some cytokines and membrane surface receptors also enter early endosomes; Vesicles (multivesicular bodies, MVBs), MVB anchored with the cell membrane under the action of Ras superfamily GTPase Rab, Ca ^2+, etc., fuse to form exosomes and release into the extracellular matrix. Normal cells such as mesenchymal stem cells, T cells, and various tumor cells can secrete exosomes, which exist in most body fluids such as blood, urine, and ascites. Exosomes can carry a variety of biologically active substances, including proteins, lipids, and genetic materials such as messenger RNA (messenger RNA, mRNA), microRNA (microRNA, miRNA) and long non-coding RNA (longnon-coding RNA, lncRNA )Wait.

Exosomes are extremely stable and can be stored for several years under various freezing and thawing conditions. The content is also extremely rich, with 10^ ^8-13 exosomes per milliliter of plasma. These advantages endow exosomes with the possibility of becoming new tumor markers, and some studies in recent years have also proved this well. For example, in pancreatic cancer research, it can be detected that cell surface glycoprotein (glypican-1, GPC1) is specifically expressed in exosomes derived from pancreatic cancer cells. By detecting GPC1+ exosomes in the serum of pancreatic cancer patients, pancreatic cancer patients of different stages can be distinguished from pancreatitis patients and healthy people, and the specificity and sensitivity are close to 100%. Further inventions also found that the content of GPC1+ exosomes is significantly correlated with the tumor burden and survival rate of pancreatic cancer patients. The zoological invention proves that GPC1+ exosomes can detect pancreatic intraepithelial neoplasia earlier than MRI, which has the value of early diagnosis of pancreatic cancer. It can be seen that exosomes have the potential to become new biomarkers of tumors.

Among the delivery components of exosomes, studies have shown that exosomes can be used as transport carriers for functional lncRNAs, induce phenotypic changes in recipient cells, and participate in the occurrence and development of tumors; and can become potential candidates for tumor diagnosis and prognosis prediction. landmark. Antisense hypoxia inducible factor (aHIF) is an lncRNA first discovered by Thrash-Bingham et al. ) natural antisense RNA. (The gene sequence is shown in SEQ ID NO.1)

Contents of the invention

The purpose of the present invention is to provide the use of serum exosomal aHIF as a marker for the diagnosis and prognosis of ovarian cancer, and the use of the serum exosomal aHIF as a marker for the diagnosis and prognosis of ovarian cancer

To solve the technical problem in the prior art that the means for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer are effective but not effective.

The present invention provides the use of serum exosomal aHIF as a marker for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer.

The present invention also provides the use of serum exosomal aHIF as a marker molecule in the preparation of a kit for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer.

The present invention also provides a kit for diagnosing epithelial ovarian cancer or the prognosis of epithelial ovarian cancer, which contains a reagent for detecting serum exosomal aHIF.

The present invention also provides a use of an inhibitor of serum exosome aHIF in the preparation of a drug for treating or preventing epithelial ovarian cancer.

Compared with the prior art, the present invention has positive and obvious technical effects. It is proved by experiments that exosomal aHIF can become a new non-invasive marker for tumor diagnosis of epithelial ovarian cancer and prognosis prediction of epithelial ovarian cancer.

Description of drawings

Figure 1 shows that the isolated serum exosomes seen under the transmission electron microscope are in the shape of round particles, with a size between 30-100nm (Figure A, scale bar: 0.2um); the extracted serum exosomes were analyzed by NanoSight nanoparticle tracking technology The particle size distribution of exosomes shows that the peak value is around 100nm (Figure B); Western blot can detect the expression of CD63, TSG101, HSP70, and HSP90 on the surface of exosomes (Figure C).

Figure 2 shows the expression of serum exosomal aHIF in the ovarian cancer group and the control group.

Detailed ways

Example 1 Identification of Serum Exosomes

Serum exosome extraction process:

1) Take the serum sample (about 200ul/portion) out of the -80°C refrigerator and centrifuge at 3000g for 15 minutes;

2) Transfer the supernatant to another clean sterilized tube, add 50ul of ExoQuick exosome precipitation solution, and mix by inverting up and down.

3) Incubate for 30 minutes, centrifuge at 1500g at room temperature for 30 minutes, and a precipitate can be seen at the bottom of the tube.

4) Remove the supernatant, continue to centrifuge at 1500g for 5 minutes, carefully remove the liquid components in the upper layer, and add 20ul sterile water to resuspend the precipitate.

The isolated serum exosomes were identified with a transmission microscope. It can be seen that the exosomes isolated under the electron microscope are round and uniform in size, with a diameter of 30-150nm. Through the NanoSight nanoparticle tracking technology Analysis of the particle size distribution of exosomes shows that the peak value is about 100nm. Subsequently, Western blot was performed to detect the expression of CD63, TSG101, HSP70, and HSP90 on the surface of exosomes. The above results show that the present invention has successfully extracted exosomes in serum.

Example 2 Expression of serum exosomal aHIF in ovarian cancer patients and control groups

Detection process:

Real-time fluorescence quantitative PCR method:

The primer sequences are as follows: aHIF:

Upstream primer 5'-CTACCACGTACTGCTGGCAA-3';

Downstream primer: 5'-CATCATGATCATAGGCAGTTG-3';

GAPDH:

Upstream primer: 5'-TGACTTCAACAGCGACACCCA-3';

Downstream primer: 5'-CACCCTGTTGCTGTAGCCAAA-3';

The reaction system is as follows: SYBR○R Premix Ex Taq 10ul; upstream primer 0.8ul; downstream primer 0.8ul; cDNA template 2ul; ROX Reference Dye II 0.4ul; RNase free water 6ul.

The following reactions were carried out in the ABI 7900 real-time fluorescent quantitative PCR instrument:

Step 1: Pre-denaturation at 95°C for 30 seconds;

The second step: PCR reaction, a total of 40 cycles, each cycle is denatured at 95°C for 5 seconds, and extended at 60°C for 34 seconds.

As shown in Figure 2, the serum exosomal aHIF of 62 ovarian cancer patients and 20 control groups were detected by qRT-PCR, and the results showed that the serum exosomal aHIF content of ovarian cancer patients was significantly higher than that of the control group (P<0.05).

Example 3 Relationship between serum exosomal aHIF expression level and clinicopathological factors in patients with ovarian cancer

The present invention included 62 patients with epithelial ovarian cancer who underwent surgery in the Obstetrics and Gynecology Hospital of Fudan University from 2012 to 2014. Their age, stage, pathological type, histological grade, residual lesion size, ascites, and preoperative CA125 The 62 patients with ovarian cancer were divided into the low exosomal aHIF expression group (31 cases) and the high exosomal aHIF expression group based on the median of serum exosomal aHIF expression level. In the expression group (31 cases), the relationship between the expression level of serum exosomal aHIF and various clinicopathological factors was analyzed by χ2 test and Fisher test. The results are shown in Table 1. We found that the expression level of serum exosomal aHIF was related to the FIGO stage and histological grade of the patients, all P<0.01; but it had no significant relationship with the patient's age, pathological type, residual lesion size, ascites, and preoperative CA125 level (P >0.05).

Table 1 Relationship between exosomal aHIF expression level and clinicopathological factors in patients with ovarian cancer

Example 4 The relationship between serum exosomal aHIF and the prognosis of patients with ovarian cancer

We followed up the survival of these 62 patients with ovarian cancer. The follow-up time was 45.37±21.88 months, the shortest was 9 months and the longest was 79 months. As of December 2017, a total of 37 people died. The shortest survival time of the dead patients after surgery was 9 months, and the longest survival time was 59 months.

The Kaplan-Meier survival analysis method was used to conduct a univariate analysis of the factors that may affect the postoperative OS of patients (Table 2). The results showed that the OS of ovarian cancer patients was related to tumor FIGO stage, histological grade, size of residual lesions and serum exosomes. The aHIF level was significantly correlated (P<0.01), but had nothing to do with patient age, tumor pathological type, ascites, and preoperative CA125 level (P>0.05).

Table 2 Kaplan-Meier single factor analysis of the influence of each factor on the OS of patients with ovarian cancer

In summary, exosomal aHIF can be detected in the serum of patients with epithelial ovarian cancer, and its expression level is significantly higher than that of normal healthy women, which has the potential to diagnose epithelial ovarian cancer; serum exosomal aHIF in patients with epithelial ovarian cancer The expression level of aHIF is closely related to its FIGO stage and histological grade; the high expression of serum exosomal aHIF is an independent factor affecting the poor prognosis of patients with epithelial ovarian cancer. Exosomal aHIF could become a novel non-invasive biomarker for tumor diagnosis and prognosis prediction of epithelial ovarian cancer.

sequence listing

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Claims (4)

1. use of the serum excretion body aHIF as diagnosis ovarian epithelial carcinoma or the marker of ovarian epithelial carcinoma outcome On the way.

2. serum excretion body aHIF is preparing diagnosis ovarian epithelial carcinoma or the pre- aftereffect of ovarian epithelial carcinoma as mark molecule Purposes in the kit of fruit.

3. a kind of kit for diagnosing ovarian epithelial carcinoma or ovarian epithelial carcinoma outcome, it is characterised in that：Contain There is the reagent of detection serum excretion body aHIF.

4. a kind of purposes of inhibitor of serum excretion body aHIF in preparing treatment or preventing the drug of ovarian epithelial carcinoma.