Marker for diagnosis and prognosis of ovarian cancer related to endometriosis

Abstract

The invention relates to a marker for diagnosis and prognosis of ovarian cancer related to endometriosis, which belongs to the field of medicine and biotechnology, and is characterized in that the marker is LncRNA BANCR / miRNA-612 / CPNE3. The ovarian cancer marker is a marker for diagnosis and prognosis of ovarian cancer related to endometriosis. The invention also relates to application of the marker in preparation of a kit for diagnosis and / or prognosis evaluation of endometriosis-related ovarian cancer, in diagnosis and prognosis of endometriosis-related ovarian cancer, as well as in preparation of a product with a function of inhibiting endometriosis-related ovarian cancer as an endometriosis canceration risk evaluation and the marker as a recombinant plasmid vector. According to the LncRNA BANCR / miRNA-612 / CPNE3 provided by the invention, an experiment shows that the LncRNA BANCR / miRNA-612 / CPNE3 is abnormally expressed in an ovarian endometrial malignant cancer tissue and an endometrial tissue of the LncRNA BANCR / miRNA-612 / CPNE3, and the purpose of diagnosing and / or prognostically evaluating the endometrial-related ovarian cancer can be achieved by performing specific detection on the expression level of the LncRNA BANCR / miRNA-612 / CPNE3 in an endometrial by utilizing the LncRNA BANCR / miRNA-612 / CPNE3 detection kit.

Description

Technical Field

[0001] The present invention belongs to the fields of medicine and biotechnology, and specifically relates to a LncRNA BANCR / miRNA-612 / CPNE3 regulatory axis as a marker for the diagnosis or prognosis of endometriosis malignant transformation-related ovarian cancer and its application. The diagnosis of endometriosis malignant transformation-related ovarian cancer and the judgment of the disease progression can be performed based on the individual LncRNA BANCR / miRNA-612 / CPNE3 uterine eutopic endometrial molecule expression level. Background Art

[0002] Endometriosis is a difficult and common disease with biological behaviors similar to malignant tumors, such as invasion, implantation, recurrence and extensive growth, and has a tendency to malignant transformation. The malignant transformation of endometriosis is an important factor in grasping the timing of medical intervention for endometriosis. The study of its occurrence mechanism is important but is constrained by insufficient basic research conditions. After the ectopic endometrium becomes malignant, the vigorously growing tumor tissue destroys the ectopic endometrial tissue of origin. In addition, the limitations of pathological sampling lead to many missed pathological diagnoses of endometriosis malignancy, and it is difficult to obtain tissue specimens and cell models of endometriosis malignancy. Basic research is relatively lagging, especially for type I endometrial cancer, which lacks the necessary detection and diagnosis. Therefore, new markers are needed to help predict the risk of endometriosis malignancy and diagnostic methods. Summary of the invention

[0003] The purpose of the present invention is to provide a marker for the diagnosis and prognosis of endometriosis-related ovarian cancer in view of the above problems. LncRNA BANCR is one of the differentially expressed LncRNAs in type I endometrial cancer, and the expression levels of LncRNA BANCR and CPNE3 molecules decrease in endometriosis malignant cancer tissue, endometriosis malignant eutopic endometrial tissue, endometriosis eutopic endometrial tissue, and normal endometrial tissue. On the contrary, the expression level of miRNA-612 increases in the above tissues. At the same time, the expression level of CPNE3 in the tissue is positively correlated with the level of LncRNA BANCR and negatively correlated with the level of miRNA-612. Therefore, the LncRNA BANCR / miRNA-612 / CPNE3 regulatory axis is involved in the occurrence of endometriosis-related ovarian cancer. Elucidating the role and mechanism of the LncRNA BANCR / miRNA-612 / CPNE3 regulatory axis in endometriosis-related ovarian cancer has extremely important significance and application prospects for the development of diagnosis and treatment of endometriosis-related ovarian cancer based on the LncRNA BANCR / miRNA-612 / CPNE3 molecule strategy.

[0004] Another object of the present invention is to provide the application of LncRNA BANCR / miRNA-612 / CPNE3 regulatory axis as a diagnostic or prognostic marker for endometriosis malignant transformation-related ovarian cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer, characterized in that the marker is LncRNA BANCR / miRNA-612 / CPNE3.

[0007] A method for extracting markers for diagnosis and prognosis of endometriosis-related ovarian cancer as described above, characterized in that: the extraction method is:

[0008] (1) Before operation, all items must be sterilized at high temperature and high pressure to remove enzymes, the experimental space and desktop must be sprayed with RNAse-removing reagents, and the centrifuge must be pre-cooled in advance for standby use;

[0009] (2) After the tissue was taken out of the -80°C freezer, it was placed on ice to thaw. The tissue was weighed using an electronic balance and placed in an enzyme-free EP tube. Trizol was added and the tissue was cut into a paste using ophthalmic scissors. After being vortexed thoroughly, it was placed on ice for 5 min.

[0010] (3) After centrifuging the paste obtained in step (2) for 5 minutes, collect the upper liquid in a new EP tube, add chloroform, mix thoroughly, and let stand on ice for 10 minutes;

[0011] (4) After the upper layer liquid obtained in step (3) is centrifuged in an ultracentrifuge for 15 minutes, the uppermost layer liquid is transferred to a new EP tube with a pipette, and an equal volume of isopropanol is added to the liquid in the tube, and the tube is placed on ice for 10 minutes;

[0012] (5) Aspirate the supernatant obtained in step (4). The white precipitate at the bottom of the tube is the total RNA.

[0013] (6) Add the total RNA to the pre-prepared 75% ethanol at 4°C, mix well and place on ice for 30 min;

[0014] (7) Centrifuging the product obtained in step (6) for 5 min and collecting the precipitate;

[0015] (8) Wash with 75% ethanol and centrifuge for 5 min to retain the precipitate;

[0016] (9) Leave the EP tube open and dry for 10 minutes to fully evaporate the residual ethanol;

[0017] (10) Add DEPC water and mix thoroughly by pipetting;

[0018] (11) RNA concentration determination and quality assessment The RNA concentration was determined using Nano Drop 2000 ssRNA mode and the concentration was adjusted to 200-500 ng / ul using DEPC water. A 260 / 280 value of 2.0 was considered to be relatively pure.

[0019] A method for extracting markers for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above, characterized in that steps (1)-(11) are all performed at 4°C.

[0020] A use of the marker for diagnosis and prognosis of endometriosis-related ovarian cancer as described above in the preparation of a kit for diagnosis and / or prognosis evaluation of endometriosis-related ovarian cancer.

[0021] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above, as a biomarker for assessing the risk of malignant transformation of endometriosis.

[0022] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above is used as a recombinant plasmid vector to prepare a product with the function of inhibiting endometriosis-related ovarian cancer.

[0023] A marker for diagnosis and prognosis of endometriosis-related ovarian cancer as described above is used as a recombinant plasmid vector to prepare a drug for preventing and / or treating endometriosis-related ovarian cancer.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0025] The LncRNA BANCR / miRNA-612 / CPNE3 provided by the present invention is found through experiments to be abnormally expressed in ovarian endometriosis malignant cancer tissue and its eutopic endometrial tissue. The specific detection of the expression level of LncRNA BANCR / miRNA-612 / CPNE3 in the endometrium using the LncRNA BANCR / miRNA-612 / CPNE3 detection kit can achieve the purpose of diagnosing and / or prognostic evaluation of endometriosis-related ovarian cancer. In addition, the risk of malignant transformation of endometriosis can be evaluated; LncRNA BANCR and CPNE3 can significantly enhance the proliferation, migration and invasion capabilities of endometrial cells, and targeted knockdown of lncRNA BANCR or CPNE3 can inhibit the proliferation, invasion and migration of endometriosis-related ovarian cancer cells by inhibiting the expression of LncRNA BANCR or CPNE3; miRNA-612 has the opposite effect to the above-mentioned effect, and upregulating the expression of miRNA-612 can inhibit the proliferation, migration and invasion of cells. The recombinant lentiviral vector of LncRNA BANCR / miRNA-612 / CPNE3 is introduced into the body as a drug, which will have a preventive and therapeutic effect on endometriosis-related ovarian cancer. Therefore, LncRNA BANCR / miRNA-612 / CPNE3 can be used as a new biomarker for the diagnosis and / or prognosis evaluation of endometriosis-related ovarian cancer; and the corresponding recombinant lentiviral vector of LncRNA BANCR / miRNA-612 / CPNE3 can be used as a new drug with potential preventive and therapeutic value for endometriosis-related ovarian cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :The expression levels of LncRNA BANCR in endometriosis malignant carcinoma tissue (MTE), endometriosis malignant eutopic endometrium tissue (MEU), endometriosis eutopic endometrium tissue (euEM), and normal endometrium tissue (NE) decreased in turn, and the differences were statistically significant (P<0.05);

[0027] Figure 2 : The expression levels of miRNA-612 in endometriosis malignant carcinoma tissue (MTE), endometriosis malignant eutopic endometrium tissue (MEU), endometriosis eutopic endometrium tissue (euEM), and normal endometrium tissue (NE) increased in turn, which was statistically significant (P<0.05);

[0028] Figure 3 : The expression levels of CPNE3 mRNA and protein in endometriosis malignant carcinoma tissue (MTE), endometriosis malignant eutopic endometrium tissue (MEU), endometriosis eutopic endometrium tissue (euEM), and normal endometrium tissue (NE) decreased, which was statistically significant (P<0.05);

[0029] Figure 4 : The relative expression correlation index between CPNE3mRNA and LncRNA BANCR was R=0.811, which was a positive correlation, p<0.01; the relative expression correlation index between CPNE3mRNA and miRNA-612 was R=-0.867, which was a negative correlation, p<0.01;

[0030] Figure 5 : Dual luciferase reporter gene experiments confirmed that LncRNA BANCR and CPNE3 are direct target genes of miRNA-612;

[0031] Figure 6 : Compared with the Vector group, the proliferation ability of normal endometrial stromal cells overexpressing BANCR or CPNE3 was enhanced, p < 0.05. After co-transfection with BANCR + miRNA-612mimics or CPNE3 + miRNA-612mimics, it was found that compared with the single overexpression of BANCR or CPNE3, the cell proliferation ability was inhibited, p < 0.05, and there was no significant difference compared with the Vetor group, p > 0.05; compared with SH-NC, the cells transfected with SH- The proliferation of EESCs cells transfected with BANCR or SH-CPNE3 decreased, p < 0.05. The proliferation ability of cells co-transfected with SH-BANCR + miRNA-612inhibitor or SH-CPNE3 + miRNA-612inhibitor was restored compared with EESCs cells transfected with single knockdown plasmid SH-BANCR or SH-CPNE3, p < 0.05, while there was no significant difference in the proliferation ability between the co-transfection group and the SH-NC group, p > 0.05;

[0032] Figure 7: Compared with the Vector group, the scratch healing ability of cells overexpressing BANCR or CPNE3 genes was enhanced, p < 0.05, and the scratch healing ability of cells co-transfected with BANCR + miRNA-612mimics or CPNE3 + miRNA-612mimics was restored compared with single overexpression of BANCR or CPNE3, p < 0.05, and there was no significant difference compared with the Vetor group, p > 0.05; compared with SH-NC, cells transfected with SH-BANCR The scratch healing and migration abilities of cells co-transfected with SH-BANCR+miRNA-612inhibitor or SH-CPNE3+miRNA-612inhibitor were decreased, p<0.05. The scratch healing abilities of cells co-transfected with SH-BANCR+miRNA-612inhibitor or SH-CPNE3+miRNA-612inhibitor were enhanced compared with cells transfected with single knockdown plasmid SH-BANCR or SH-CPNE3, p<0.05. There was no significant difference in the scratch healing and migration abilities of cells between the co-transfection group and the SH-NC group, p>0.05.

[0033] Figure 8 :Compared with the Vector group, the Transwell migration ability of cells overexpressing BANCR or CPNE3 genes was enhanced, p<0.05. Compared with cells overexpressing BANCR or CPNE3 alone, cells co-transfected with BANCR+miRNA-612mimics or CPNE3+miRNA-612mimics showed a restorative inhibition of Transwell migration ability, p<0.05, and there was no significant difference compared with the Vetor group, p>0.05. Compared with SH-NC, cells transfected with SH-BAN The Transwell migration ability of cells transfected with CR or SH-CPNE3 decreased, p < 0.05. The Transwell migration ability of cells co-transfected with SH-BANCR + miRNA-612inhibitor or SH-CPNE3 + miRNA-612inhibitor was restored compared with cells transfected with single knockdown plasmid SH-BANCR or SH-CPNE3, p < 0.05, while there was no significant difference in the scratch healing migration ability of cells between the co-transfection group and the SH-NC group, p > 0.05;

[0034] Fig. 9:Compared with the Vector group, the CPNE3 mRNA level of normal endometrial stromal cells overexpressing BANCR or CPNE3 was significantly upregulated, p<0.05, while co-transfection of BANCR+miRNA-612mimics or CPNE3+miRNA-612mimics cells could inhibit the expression of CPNE3 mRNA compared with the single overexpression group, p<0.05, and there was no statistical difference with the Vector group, p>0.05; compared with SH-NC, cells transfected with SH-BANCR or SH-CPNE 3, the CPNE3 mRNA expression level in the cells co-transfected with SH-BANCR+miRNA-612inhibitor or SH-CPNE3+miRNA-612inhibitor was decreased, p<0.05, and the CPNE3 mRNA expression level in the cells co-transfected with SH-BANCR+miRNA-612inhibitor or SH-CPNE3+miRNA-612inhibitor was significantly increased compared with the cells transfected with single knockdown plasmid SH-BANCR or SH-CPNE3, p<0.05, while the CPNE3 mRNA expression level in the co-transfection group and SH-NC group was not significantly different, p>0.05;

[0035] Fig.10 : Compared with the Vector group, the expression level of CPNE3 protein in normal endometrial stromal cells in the BANCR overexpression or CPNE3 overexpression groups was increased, p < 0.05, and the expression level of CPNE3 protein in cells co-transfected with BANCR + miRNA-612mimics or CPNE3 + miRNA-612mimics was decreased compared with the single overexpression group, p < 0.05, and there was no statistical difference between the co-transfection group and the Vector group, p > 0.05; compared with the SH-NC group, cells transfected with SH-BANCR or The expression level of CPNE3 protein in SH-CPNE3 cells decreased (p<0.05). The expression level of CPNE3 protein in co-transfected SH-BANCR+miRNA-612inhibitor or SH-CPNE3+miRNA-612inhibitor cells increased compared with cells transfected with single knockdown plasmid SH-BANCR or SH-CPNE3 (p<0.05). However, there was no significant difference in the expression level of CPNE3 protein between the co-transfection group and the SH-NC group (p>0.05).

[0036] Fig.11: Compared with the Vector group, the survival of normal subcutaneous stromal cells in the BANCR overexpression group and the CPNE3 overexpression group increased, and the H-score of CPNE3 protein in the tissue increased significantly, p < 0.05; while the number of subcutaneous cells in the co-transfection BANCR + miRNA-612mimics group and the CPNE3 + miRNA-612inhibitor group was significantly reduced compared with the single BANCR overexpression group and the CPNE3 overexpression group, and the H-score of CPNE3 protein was significantly reduced, p < 0.05;

[0037] Fig.12 : Compared with the Vector group, the tumor growth rate of the Ishikawa cell overexpression BANCR group and the CPNE3 overexpression group was faster (p < 0.05), and the final tumor volume and mass were larger (p < 0.05); while the growth rate, final tumor volume and mass of the co-transfection BANCR + miRNA-612mimics group and the CPNE3 + miRNA-612inhibitor group showed a reversion inhibition compared with the respective single overexpression groups, and there was no statistical difference with the Vector group (p > 0.05);

[0038] Fig.13 :In the overexpression of BANCR group and the overexpression of CPNE3 group, the expression of CPNE3 protein in the tumor was increased compared with the vector group, p<0.05; the protein expression in the co-transfected BANCR+miRNA-612mimics group and the CPNE3+miRNA-612inhibitor group was reduced compared with the single overexpression group, and the difference was not significant with the vector group, p>0.05. In the immunohistochemical experiment of tumor tissue, the CPNE3 protein staining in the overexpression of BANCR group and the overexpression of CPNE3 group was deeper than that in the vector group, and the staining in the co-transfected BANCR+miRNA-612mimics group and the CPNE3+miRNA-612inhibitor group was lighter than that in the single overexpression group, and the staining depth was close to that of the vector group. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with specific embodiments. The following embodiments will contribute to the understanding of the present invention, but these embodiments are only for the purpose of illustrating the present invention, and the present invention is not limited to these contents. The operating methods not specifically described in the embodiments are all conventional operating methods in the art.

[0040] BANCR is a BRAF-activated non-protein coding RNA (BANCR), a 693bp long lncRNA. It was first named in a study in 2012 to evaluate gene expression in malignant melanoma cells associated with BRAF mutations. Since its discovery, more and more studies have confirmed its important role in tumorigenesis. In retinoblastoma, hepatocellular carcinoma, and esophageal squamous cell carcinoma, increased expression levels of lncRNA BANCR are closely associated with high tumor grade, large tumor volume, severe venous infiltration, and advanced TNM stage. LncRNA BANCR can be used as an independent prognostic marker for overall survival in patients with primary hepatocellular carcinoma. Compared with the control group, downregulation of lncRNA BANCR can inhibit the proliferation of liver cancer tissues and liver cancer cells and promote cell apoptosis. The results of this study showed that the expression level of LncRNA BANCR was significantly increased in endometriosis malignant tissue and its paired eutopic endometrium, suggesting that LncRNA BANCR also plays an important role in the malignant transformation of ectopic endometrium, and the quantitative accumulation of LncRNA BANCR in eutopic endometrium may be an important marker for endometriosis and even malignant transformation of normal endometrium.

[0041] As a member of the miRNA family, miRNA-612 mainly functions by regulating the expression of downstream target genes. Studies have shown that miRNA-612 can negatively regulate the formation of invasive pseudopodia, extracellular matrix degradation, EMT and distant metastasis of liver cancer through HADHA-mediated lipid reprogramming. The reduction of miRNA-612 in liver cancer cells leads to upregulation of HADHA and initiation of β-oxidation activity of fatty acids, providing sufficient acetyl-CoA and ATP for cholesterol biosynthesis through the SREBP2 / HMGCR cascade reaction, thereby affecting the metastasis of hepatocellular carcinoma through lipid rafts and pseudopodia. At the same time, miRNA-612 can inhibit the proliferation, migration, invasion and distant metastasis of liver cancer, colorectal cancer and head and neck squamous cell carcinoma by downregulating the expression of AKT2, and the expression of miRNA-612 in tissues is negatively correlated with tumor size, pathological grade, local invasion depth and distant metastasis. It can be seen that miRNA-612 plays a role in tumorigenesis and development mainly by participating in the regulation of the expression or function of downstream target genes, but there are few studies on endometrial cancer or endometriosis malignancy, which deserves attention. In our study, the expression level of miRNA-612 in endometrial malignant tissue was lower than that in the paired malignant group eutopic endometrium, and was also lower than that in endometrium eutopic endometrium and normal endometrium, indicating that miRNA-612 may play an inhibitory role in the process of endometriosis malignancy, which is similar to other reports.

[0042] CPNE3 has been reported to be a functional protein for many diseases, especially in tumor research. For example, CPNE3 molecules and their encoded proteins are highly expressed in non-small cell lung cancer, breast cancer, and prostate cancer, mainly by promoting cell invasion and migration, leading to the occurrence of malignant tumors and poor prognosis. In 2018, Sun et al. found that high expression of CPNE3 mRNA and protein was a biomarker for poor prognosis of colorectal cancer through qRT-PCR and Western blot detection of colorectal cancer plasma exosomes. Another study that collected data from 272 patients with acute myeloid leukemia found that the prognosis of patients with high CPNE3 expression was significantly worse than that of the low expression group, and CPNE3 was an independent prognostic factor for AML. In this study, it was found that the expression of CPNE3 mRNA and protein in endometriosis and endometriosis malignant tissues was increased, and it was speculated that CPNE3 may promote the occurrence and malignant transformation of endometriosis. Co-expression bioinformatics analysis of endometrial cancer showed that the expression levels of LncRNA BANCR and CPNE3 were increased and significantly correlated (p < 0.05). The detection of endometriosis malignant tissue also proved that CPNE3 was significantly positively correlated with the expression level of LncRNA BANCR, and negatively correlated with the relative expression level of miRNA-612.

[0043] Endometriosis-related ovarian cancer is a malignant tumor that is closer to the endometrium. Genetic changes in the eutopic endometrium are correlated with the malignant transformation of the ectopic endometrium. Under the same high-risk factors, the eutopic endometrium and the ectopic endometrium have consistent pathological proliferation trends, and the ectopic endometrium is even more sensitive to high-risk factors than the eutopic endometrium. Therefore, risk assessment of molecular markers related to the malignant transformation of the eutopic endometrium in patients with endometriosis, early intervention, and timely treatment of endometriosis for the purpose of preventing malignant transformation will play a preventive role in the malignant transformation of endometriosis.

[0044] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer, characterized in that the marker is LncRNA BANCR / miRNA-612 / CPNE3.

[0045] A method for extracting markers for diagnosis and prognosis of endometriosis-related ovarian cancer as described above, characterized in that: the extraction method is:

[0046] (1) Before operation, all items must be sterilized at high temperature and high pressure to remove enzymes, the experimental space and desktop must be sprayed with RNAse-removing reagents, and the centrifuge must be pre-cooled in advance for standby use;

[0047] (2) After the tissue was taken out of the -80°C freezer, it was placed on ice to thaw. The tissue was weighed using an electronic balance and placed in an enzyme-free EP tube. Trizol was added and the tissue was cut into a paste using ophthalmic scissors. After being vortexed thoroughly, it was placed on ice for 5 min.

[0048] (3) After centrifuging the paste obtained in step (2) for 5 minutes, collect the upper liquid in a new EP tube, add chloroform, mix thoroughly, and let stand on ice for 10 minutes;

[0049] (4) After the upper layer liquid obtained in step (3) is centrifuged in an ultracentrifuge for 15 minutes, the uppermost layer liquid is transferred to a new EP tube with a pipette, and an equal volume of isopropanol is added to the liquid in the tube, and the tube is placed on ice for 10 minutes;

[0050] (5) Aspirate the supernatant obtained in step (4). The white precipitate at the bottom of the tube is the total RNA.

[0051] (6) Add the total RNA to the pre-prepared 75% ethanol at 4°C, mix well and place on ice for 30 min;

[0052] (7) Centrifuging the product obtained in step (6) for 5 min and collecting the precipitate;

[0053] (8) Wash with 75% ethanol and centrifuge for 5 min to retain the precipitate;

[0054] (9) Leave the EP tube open and dry for 10 minutes to fully evaporate the residual ethanol;

[0055] (10) Add DEPC water and mix thoroughly by pipetting;

[0056] (11) RNA concentration determination and quality assessment The RNA concentration was determined using Nano Drop 2000 ssRNA mode and the concentration was adjusted to 200-500 ng / ul using DEPC water. A 260 / 280 value of 2.0 was considered to be relatively pure.

[0057] A method for extracting markers for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above, characterized in that steps (1)-(11) are all performed at 4°C.

[0058] A use of the marker for diagnosis and prognosis of endometriosis-related ovarian cancer as described above in the preparation of a kit for diagnosis and / or prognosis evaluation of endometriosis-related ovarian cancer.

[0059] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above, as a biomarker for assessing the risk of malignant transformation of endometriosis.

[0060] A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer as described above is used as a recombinant plasmid vector to prepare a product with the function of inhibiting endometriosis-related ovarian cancer.

[0061] A marker for diagnosis and prognosis of endometriosis-related ovarian cancer as described above is used as a recombinant plasmid vector to prepare a drug for preventing and / or treating endometriosis-related ovarian cancer.

[0062] Example

[0063] Specimen selection.

[0064] Tissue experiment: Patients who underwent surgery from September 2017 to September 2021 were selected. The experimental group: 12 patients with ovarian cancer who were confirmed to have ovarian cancer by intraoperative freezing and accompanied by endometriotic cysts. At the same time, malignant cancer tissue and paired endometrial tissue were obtained. Postoperative pathology showed that there were 8 cases of clear cell carcinoma and 4 cases of endometrioid carcinoma. Control group 1: 20 patients with ovarian endometriosis surgery, in situ endometrial tissue was obtained, and postoperative pathology confirmed endometriosis; control group 2: 20 patients who underwent hysterectomy or conization and curettage due to cervical HSIL during the same period were selected, and normal endometrial tissue was obtained. All tissues were stored in a -80℃ refrigerator after acquisition. The average age of the three groups of patients was (48.37±5.38), (38.52±5.35) and (40.32±6.12) years old, and there was no statistically significant difference (p>0.05). All patients did not take hormone drugs within 6 months of surgery and signed informed consent.

[0065] Primary culture cells: normal endometrial stromal cells NESCs, endometriosis eutopic endometrial stromal cells EESCs cells were extracted from fresh tissues after surgery, and the cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% penicillin / streptomycin. Cell culture incubator parameters: 37°C, 5% CO2, 95% humidity.

[0066] Cell line: Ishikawa cell line was purchased from Shanghai Cell Bank, Chinese Academy of Sciences, and cells were cultured in RPMI-1640 medium with 10% FBS and 1% double antibody; 293T cells were from Shanghai Cell Bank, Chinese Academy of Sciences, and cultured in DMEM high glucose medium with 10% FBS and 1% double antibody; cell culture incubator conditions were the same as above.

[0067] Experimental animals: BALB / c female nude mice were purchased from Liaoning Changsheng Biotechnology Co., Ltd., 6 weeks old, weighing 14±1.5g, for a total of 40 mice. All nude mice were raised in an SPF barrier environment with constant temperature and humidity, 12 hours of light and 12 hours of darkness, and feed and water were sterilized by high pressure. Before the experiment, the mice were randomly distributed into 5 groups, each of which was implanted with BANCR / miRNA-612 / CPNE3 overexpression / co-transfection cells, 8 mice in each group, see Table 2.1. All experimental operations have been approved by the Ethics Committee of the Experimental Animal Department of China Medical University.

[0068] Total RNA extraction from tissues:

[0069] (1) Before operation, all items must be sterilized at high temperature and high pressure to remove enzymes, the experimental space and desktop must be sprayed with RNase-removing reagents, and the centrifuge must be pre-cooled to 4°C for standby use;

[0070] (2) After the tissue was taken out of the -80°C freezer, it was placed on ice to thaw. About 100 mg of tissue was weighed using an electronic balance and placed in an enzyme-free EP tube. 1 ml of Trizol was added and the tissue was cut into a paste using ophthalmic scissors. After being thoroughly vortexed, it was placed on ice for 5 min.

[0071] (3) After centrifugation at 12000g for 5 min at 4°C, collect the upper layer into a new EP tube, add 200 μl of chloroform, mix thoroughly, and place on ice for 10 min;

[0072] (4) After centrifugation at 12000g for 15 min at 4°C, carefully transfer the top layer of liquid to a new EP tube using a pipette. Add 500 μl of isopropanol (equal to the volume of the liquid in the tube) to each tube and place on ice for 10 min.

[0073] (5) Centrifuge at 12000g for 10 min at 4°C and aspirate the supernatant. The white precipitate at the bottom of the tube is the total RNA.

[0074] (6) Add 1 ml of 75% ethanol prepared at 4°C to each tube, mix well and place on ice for 30 min;

[0075] (7) Centrifuge at 7500 g for 5 min at 4°C and collect the precipitate;

[0076] (8) Wash with 1 ml of 75% ethanol, centrifuge at 7500 g for 5 min at 4 °C, and retain the precipitate;

[0077] (9) Leave the EP tube open and dry for 10 minutes to fully evaporate the residual ethanol;

[0078] (10) Add about 30 μl of DEPC water to each tube and mix thoroughly by pipetting;

[0079] (11) RNA concentration determination and quality assessment The RNA concentration was determined using Nano Drop 2000 ssRNA mode and DEPC water was used to adjust the concentration to 200-500 ng / ul. A 260 / 280 value of around 2.0 was considered to be relatively pure.

[0080] RNA Reverse Transcription:

[0081] The experiment was conducted using TAKARA 047A as an example

[0082] (1) Remove gDNA and operate on ice as follows

[0083]

[0084] Reaction conditions: 42℃2min→4℃Forever volume 10ul

[0085] (2) The RNA reverse transcription reaction system was prepared as follows

[0086]

[0087]

[0088] After preparing the liquid, mix it thoroughly, add it to the reaction solution in Step 1, mix it thoroughly, and then start the reaction on the machine. Reaction conditions: 37℃15min→85℃5s→4℃Forever Volume 20ul

[0089] (3) DNA concentration determination and quality assessment The DNA concentration was determined using the Nano Drop 2000 dsDNA mode. The DNA concentration should not be higher than 1000 ng / ul, and the 260 / 280 value should be around 1.8.

[0090] qRT-PCR experiments:

[0091] (1) The primers required for PCR were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences are shown in the table below. All primers were diluted to 10 uM for later use;

[0092] Table 1.1 Primer list

[0093]

[0094] The reverse transcription primers and amplification primers of miRNA-612 and internal reference U6 were designed and synthesized by Guangzhou Ruibo Company;

[0095] (2) RT-PCR reaction solution was prepared on ice using TAKARA 820A as shown in the table below.

[0096]

[0097] The liquid is placed in the PCR eight-row array and reacted as follows:

[0098] Pre-denaturation: 95℃30s

[0099] PCR amplification: 95℃5s→60℃30s→72℃30s×39cycles

[0100] Melting curve: 95℃10s→65℃5s+0.5℃ / s plate read→95℃

[0101] (3) After the reaction is completed, confirm the amplification curve. If the melting curve is a single peak, the product specificity is good;

[0102] (4) The internal reference gene Actin was used as the target gene for relative quantification, and the calculation method was 2 ﹣ΔΔCt Law.

[0103] Immunohistochemistry:

[0104] (1) Cut 4 μm thick slices from paraffin specimens, dry the slices with a slide spreader, and bake the slices in an oven at 60°C for 30 min;

[0105] (2) Xylene dewaxing: xylene I for 5 min, xylene II for 5 min, xylene III for 5 min;

[0106] (3) hydration: 100% ethanol I for 20 min, 100% ethanol II for 20 min, 90% ethanol for 20 min, and 75% ethanol for 20 min;

[0107] (4) After dewaxing, soak the glass slide in tap water for 3 min × 2 times, and wipe the liquid on the glass slide with paper towels;

[0108] (5) Wash the slides in PBS for 3 min × 3 times;

[0109] (6) Antigen repair: Dissolve 1.5 g of trisodium citrate dihydrate in 500 ml of double distilled water. Soak the rack in the repair solution, seal the beaker with plastic wrap, microwave on high for 6 min until the water boils for 7 min, open the door and let it dry for 5 min, then low heat for 5 min. Repeat this three times and let it cool at room temperature.

[0110] (7) Wash with PBS for 3 min × 3 times and wipe the glass slide dry;

[0111] (8) Add H2O2 and incubate the glass slide in a humidified chamber at 37°C for 30 min;

[0112] (9) Wash with PBS for 3 min × 3 times and wipe the glass slide dry;

[0113] (10) Add blocking solution to the tissue block on the glass slide and incubate in a humidified chamber at 37°C for 30 min.

[0114] (11) Primary antibody CPNE3: PBS = 1:200;

[0115] (12) Wipe off the blocking solution, add the diluted antibody, and place in a humidified box at 4°C overnight;

[0116] (13) The wet box was placed at room temperature for 30 min, washed with PBS for 3 min × 3 times, and then dried;

[0117] (14) Add goat anti-rabbit and anti-mouse secondary antibody and incubate the glass slide in a humidified chamber at 37°C for 30 min;

[0118] (15) Wash with PBS for 5 min × 3 times and wipe dry;

[0119] (16) Add HRP labeling solution and incubate in a humidified chamber at 37°C for 30 min;

[0120] (17) Wash with PBS for 5 min × 3 times and wipe dry;

[0121] (18) DAB color development: Add DAB color development solution and rinse the slide after 90 seconds to terminate the color development;

[0122] (19) Add hematoxylin solution to the counterstained slide for 1 minute, and then rinse the slide with running water to stop staining;

[0123] (20) Dehydration and sealing: 75% ethanol for 30 min, 90% ethanol for 30 min, 100% ethanol for 30 min, xylene for 30 min, coverslip with neutral gum, and place in a fume hood to dry;

[0124] (21) After drying, place the slide under a microscope and take a picture.

[0125] (22) Histochemical semi-quantitative scoring:

[0126] ①Positive cell percentage: no positive cells were assigned 0 points, positive cells <10% were assigned 1 point, 10% < positive cells >50% were assigned 2 points, 50% < positive cells >80% were assigned 3 points, and positive cells >80% were assigned 4 points;

[0127] ② Color intensity: no staining is assigned 0 points, weak staining is assigned 1 point, moderate staining is assigned 2 points, and strong staining is assigned 3 points;

[0128] ③The product of the staining intensity and percentage of positive cells: 0-3 points are negative, 4-12 points are positive.

[0129] Total protein extraction from tissues:

[0130] (1) Weigh about 100 mg of frozen tissue and place it in an EP tube on ice to thaw. Wash it three times with PBS to remove blood stains.

[0131] (2) Add 500 μl of prepared cell lysis buffer (RIPA:PMSF=100:1), cut the tissue into a paste with ophthalmic scissors, and lyse on ice for 30 min;

[0132] (3) Ultrasonic wall breaking machine on ice for 20 times;

[0133] (4) Centrifuge at 12000g for 15 min at 4°C and transfer the supernatant to a new EP tube;

[0134] (5) Determination of protein concentration by BCA method;

[0135] (6) Calculate the amount of protein sample, Loading Buffer, and ddH2O according to the 20ug / 20ul system, add them into new EP tubes and mix well;

[0136] (7) Denature the sample at 100°C for 5 min on a metal heater. Cool the sample on ice and store it at -80°C until use.

[0137] Western Blot Experiment:

[0138] (1) Take a clean and dry glass plate and clamp it on the glue rack;

[0139] (2) Prepare the lower layer of 10% separation gel (two-layer gel) according to the table below:

[0140]

[0141] After thorough mixing, quickly inject between the glass plates, cover the upper layer with isopropanol, and let stand at room temperature for 30 min;

[0142] (3) Carefully suck out the isopropyl alcohol with a 1 ml syringe, rinse with pure water 3 to 5 times, wipe off the water droplets with a paper towel, and let dry;

[0143] (4) Prepare the upper layer of 5% concentrated gel (two-plate gel) according to the table below:

[0144]

[0145]

[0146] After fully mixing, quickly inject into the glass plate, insert the tooth comb into the glass plate, and leave at room temperature for 30 minutes;

[0147] (5) 1L of electrophoresis solution and transfer solution were prepared according to the following table.

[0148]

[0149] After the transfer solution is mixed, place it in a refrigerator at 4°C for precooling;

[0150] (6) Clamp the glass plate and fix it in the electrophoresis tank. Use a glass rod to remove the bubbles between the plates. Carefully remove the tooth comb to avoid causing the gel column to fall over.

[0151] (7) Add 20ul of sample to each lane in the order of loading, add 5ul of colored protein marker to the lane, connect the electrophoresis cover, and perform electrophoresis at 80V for 30min;

[0152] (8) After all proteins have entered the separation gel, adjust the voltage to 120 V and continue electrophoresis for about 1 hour;

[0153] (9) Cut off several pieces of PVDF membrane of about 8 cm × 5 cm and filter paper of 10 cm × 10 cm for later use;

[0154] (10) Activate the PVDF membrane in methanol for 1 min and set aside. Place the transfer chucks in order, cut the concentrated gel with a gel cutting board, and smoothly transfer the entire gel to the filter paper that has been laid.

[0155] (11) Clamp the transfer clip onto the transfer rack and place it in the transfer tank. Then place the entire clip into an ice-water mixture and fill it with ice-cold transfer solution. Transfer the film at a constant voltage of 80 V for 2 h.

[0156] (12) Preparation of TBST: 10 ml Tris-HCl pH 7.5, 8 g NaCl, 1 ml Tween-20, add water to 1 L, and mix by magnetic stirring;

[0157] (13) Wash the PVDF membrane after transfer with TBST for 5 min × 3 times;

[0158] (14) 5% skim milk, shake slowly for 1 hour to seal;

[0159] (15) After blocking, wash with TBST for 5 min × 3 times;

[0160] (16) Primary antibody dilution solution: CPNE3 (1:1000) and Actin (1:1000) primary antibody incubation solution;

[0161] (17) Place the PVDF membrane in the primary antibody incubation solution and shake slowly at 4°C overnight;

[0162] (18) The next day, the primary antibody was recovered and washed with TBST for 15 min × 3 times;

[0163] (19) Prepare the secondary antibody incubation solution corresponding to the primary antibody species, and incubate the PVDF membrane at room temperature for 1 h;

[0164] (20) Wash with TBST for 15 min × 3 times;

[0165] (21) Prepare the developing liquid in equal proportions using the ECL luminescence kit, drop it onto the PVDF membrane, and incubate at room temperature for about 30 s to begin developing;

[0166] (22) Developed with Bio-rad instrument and Image-lab software, grayscale analysis and image processing. Primary endometrial stromal cell extraction:

[0167] (1) Take freshly excised postoperative endometrial tissue, about 200 mg in size, soak it in DMEM / F12 complete medium and transfer it to the laboratory for operation within 2 hours;

[0168] (2) Wash the tissue three times with sterile PBS in a clean bench to remove blood stains;

[0169] (3) Use ophthalmic scissors to cut the tissue into a paste, add 10 ml of prepared 0.1% type I collagenase, stir well, and place in a shaker at 37°C for full digestion for 1 hour. To reduce the adhesion of damaged cell DNA, 0.025% DNase can be added for digestion;

[0170] (4) After digestion, add an equal volume of 10 ml of complete medium to terminate digestion, filter with an 80-mesh sieve and discard the residue. At this time, the cells are suspended in the filtrate;

[0171] (5) Centrifuge at 800 g for 5 min at 25°C and resuspend the cells;

[0172] (6) Filter through a 400-mesh sieve, centrifuge the filtrate at 800 g for 5 min at 25°C, and discard the supernatant;

[0173] (7) resuspending the cells again, inoculating them into a culture flask, and placing them in an incubator for further culture;

[0174] (8) After 12 h, observe the cell adhesion, discard the culture medium in the bottle, and add fresh complete culture medium.

[0175] (9) Cell subculture:

[0176] ① If the cells are growing well, discard the original culture medium and rinse with PBS;

[0177] ②Add trypsin for digestion;

[0178] ③ Stop digestion when the cells become brighter and rounder, and add 1 ml of complete medium;

[0179] ④Puff and collect the liquid into a 15ml centrifuge tube;

[0180] ⑤ Centrifuge at 800g for 5 min at 25℃ and retain the precipitate;

[0181] ⑥ Resuspend the cells and inoculate them into new culture flasks according to the proportion.

[0182] (10) Cell cryopreservation:

[0183] ① When the cell density is about 80%, discard the original culture medium and digest the cells with trypsin;

[0184] ② Prepare cell freezing solution with serum and DMSO components at a ratio of 9:1;

[0185] ③ After centrifugation, the cell pellet is resuspended in cell freezing solution, divided into cell freezing tubes, and marked with cell name and time;

[0186] ④Use a gradient freezing box and place it in a -80℃ refrigerator overnight.

[0187] Renilla-firefly dual luciferase assay:

[0188] (1) 293T cells in good condition were counted at 2×10 4 Cells / well were plated in 24-well plates;

[0189] (2) After 24 hours, prepare the transfection solution with 500 ng of plasmid and 1 μl of Lipo3000, incubate at room temperature for 15 min, add dropwise into the 24-well plate, and replace the solution after 6 hours;

[0190] (3) Dual luciferase assay was started after 48 h of continuous culture;

[0191] (4) Add 500 μl of 1×PLB to a 24-well plate and shake slowly for 15 min at room temperature. Transfer the cell lysate to a 1.5 ml EP tube and centrifuge at 12,000 g for 10 min at 4°C to collect the supernatant.

[0192] (5) Add 100 μl of Luciferase Assay Reagent II working solution into the 96-well plate;

[0193] (6) Add 20 μl of cell lysis buffer and mix by pipetting 2 to 3 times. Measure the data after 2 seconds and record the FireflyLuciferase value.

[0194] (7) Add 100 μl Stop&Glo Reagent and mix by pipetting 2 to 3 times. Measure the data after 2 seconds and record the Renilla Luciferase value.

[0195] (8) After subtracting the background value from the fluorescence value of each group, the firefly fluorescence value / Renilla fluorescence value was calculated, and NC was used as the reference.

[0196] Plasmid transfection:

[0197] (1) Cells in good growth condition were counted as 18×10 4 Cells / well were plated in 6-well plates so that the cell density reached about 70% after 24 hours;

[0198] (2) Change the medium 2 h before transfection and return the cells to the incubator for continued culture;

[0199] (3) After preliminary experiments, the optimal ratio for primary cultured cells was 2500ng and 5ul Lipo3000, and the co-transfection group was a premix of 1250ng of plasmid and 5ul Lipo3000;

[0200] (4) Prepare transfection solution according to the above ratio, mix well and incubate at room temperature for 15 min;

[0201] (5) After incubation, add the solution dropwise to the well plate and shake gently. Return the 6-well plate to the incubator and continue incubating for 6 h. Do not move the cells during this period.

[0202] (6) After 6 hours, discard the original culture medium and replace with new complete culture medium. Continue culturing for 72 hours before conducting subsequent experiments.

[0203] Cell proliferation assay:

[0204] (1) After cell digestion, the cells were counted and plated in a 96-well plate at 5000 cells / well for 4 time points. Each group of cells had 3 replicate wells surrounded by PBS to prevent the evaporation of the liquid in the side wells from affecting the experimental results. The blank control group was filled with complete culture medium without cells.

[0205] (2) Prepare 10% CCK8 reaction solution on-site, add 100 μl of the reaction solution to each well at 24, 48, 72, and 96 h, and incubate in a cell culture incubator for 2-4 h in the dark;

[0206] (3) Detect the absorbance at 450 nm using an ELISA reader under light-protected conditions;

[0207] (4) Statistical analysis of data and drawing of cell proliferation curves.

[0208] Cell scratch assay:

[0209] (1) Cells were seeded in six-well plates and cultured in a cell culture incubator for 24 h;

[0210] (2) When the cells have grown to about 90%, use a 200 μl pipette tip to draw a straight line on the well plate;

[0211] (3) Wash the plate three times with PBS to remove detached and floating cells;

[0212] (4) Add serum-free medium and return to the incubator for further culture;

[0213] (5) Take photos under a microscope at 0, 24, and 48 hours after streaking;

[0214] (6) The size of the area covered by the line during cell migration can be used to evaluate the migration ability of the cells.

[0215] Transwell experiment:

[0216] (1) Digest cells that are growing well, centrifuge and resuspend, and dilute the cells to 4 × 10 5 cells / ml;

[0217] (2) Add 200ul of cell suspension to the upper chamber and 600ul of complete culture medium containing 20% ​​FBS to the lower chamber, and place the upper chamber steadily on the liquid, taking care to remove bubbles from the bottom of the upper chamber;

[0218] (3) Place the cells in a cell culture incubator and continue culturing. After 24 hours, observe the cells every 4 hours until intact cells attached to the wall are seen in the lower chamber.

[0219] (4) Wash the chamber with PBS for 3 min × 2 times, and wipe with a cotton swab to remove cells that have not penetrated the membrane in the upper chamber;

[0220] (5) Fix with 4% paraformaldehyde for 20 min and air-dry at room temperature for 20 min;

[0221] (6) Stain with crystal violet staining solution for 30 min at room temperature;

[0222] (7) Wash the chamber with PBS for 3 min × 3 times;

[0223] (8) Take photos of the transmembrane cells at 200x magnification in 5 random fields of view using a microscope and perform statistical analysis.

[0224] In vivo experiments:

[0225] Endometrial cell subcutaneous survival experiment in nude mice

[0226] (1) Normal endometrial stromal cells with good growth status were grouped as follows: Vector, BANCR, BANCR+miRNA-612mimics, CPNE3, and CPNE3+miRNA-612mimics were transfected with plasmid DNA, respectively, and inoculated subcutaneously after 24 h of culture;

[0227] (2) Press 10 6 Cells were inoculated into the subcutaneous tissues of the bilateral axillae and bilateral groins of nude mice, with 2 nude mice in each group;

[0228] (3) Preliminary experiments revealed that cells gradually died 7 days after subcutaneous injection. Therefore, nude mice were killed by cervical dislocation 10 days after injection, and the implanted foci were isolated along the injection needle track for the next histological experiment.

[0229] (4) Histochemical staining was scored using the H-score: H-score = Σpi(i+1), where pi represents the percentage of positive cells in the total number of cells in the section; i represents the staining intensity. No staining was 0 points, weak staining was 1 point, moderate staining was 2 points, and strong staining was 3 points.

[0230] Ishikawa cell subcutaneous tumor bearing experiment

[0231] (1) Ishikawa cell stable transfection cell lines were established according to the grouping of Vector, BANCR, BANCR+miRNA-612mimics, CPNE3, and CPNE3+miRNA-612mimics and cultured to the logarithmic growth phase;

[0232] (2) Collect cells from each group and 6 Cells / mouse were inoculated subcutaneously in the right axilla of nude mice, and 6 nude mice were set up in each group;

[0233] (3) On the 6th day after implantation, when the subcutaneous tumor was observed with the naked eye, the size of the tumor was measured every 3 days. The volume was calculated using the formula v = (width 2 × length) / 2;

[0234] (4) To comply with animal ethics, nude mice were killed by cervical dislocation 21 days after injection, and all tumors were freed, weighed, and photographed. All tumors were smaller than 2 cm 3 , in line with the ethics of the experiment;

[0235] (5) The tumor was transferred to -80°C for storage, with one part used for tissue protein detection and the other part used for histological experiments.

[0236] Data Statistical Analysis:

[0237] Data results are expressed as mean ± standard error. SPSS23 data analysis software was used for analysis. Two independent sample t-tests were used to compare the differences between the two groups. Variance homogeneity and normal distribution tests were performed before analysis, and Pearson's correlation analysis was used for correlation analysis. p < 0.05 was considered statistically significant.

Claims

1. A marker for the diagnosis and prognosis of endometriosis-related ovarian cancer, characterized in that The marker is LncRNA BANCR / miRNA-612 / CPNE3.

2. A method for extracting markers for diagnosis and prognosis of endometriosis-related ovarian cancer according to claim 1, characterized in that: The extraction method is: (1) Before operation, all items must be sterilized at high temperature and high pressure to remove enzymes, the experimental space and desktop must be sprayed with RNAse-removing reagents, and the centrifuge must be pre-cooled in advance for standby use; (2) After the tissue was taken out of the -80°C freezer, it was placed on ice to thaw. The tissue was weighed using an electronic balance and placed in an enzyme-free EP tube. Trizol was added and the tissue was cut into a paste using ophthalmic scissors. After being vortexed thoroughly, it was placed on ice for 5 min. (3) After centrifuging the paste obtained in step (2) for 5 minutes, collect the upper liquid in a new EP tube, add chloroform, mix thoroughly, and let stand on ice for 10 minutes; (4) After the upper layer liquid obtained in step (3) is centrifuged in an ultracentrifuge for 15 minutes, the uppermost layer liquid is transferred to a new EP tube with a pipette, and an equal volume of isopropanol is added to the liquid in the tube, and the tube is placed on ice for 10 minutes; (5) Aspirate the supernatant obtained in step (4). The white precipitate at the bottom of the tube is the total RNA. (6) Add the total RNA to the pre-prepared 75% ethanol at 4°C, mix well and place on ice for 30 min; (7) Centrifuging the product obtained in step (6) for 5 min and collecting the precipitate; (8) Wash with 75% ethanol and centrifuge for 5 min to retain the precipitate; (9) Leave the EP tube open and dry for 10 minutes to fully evaporate the residual ethanol; (10) Add DEPC water and mix thoroughly by pipetting; (11) RNA concentration determination and quality assessment The RNA concentration was determined using Nano Drop 2000 ssRNA mode and the concentration was adjusted to 200-500 ng / ul using DEPC water. A 260 / 280 value of 2.0 was considered to be relatively pure.

3. The method for extracting markers for diagnosis and prognosis of endometriosis-related ovarian cancer according to claim 2, characterized in that: Steps (1)-(11) are all carried out at 4°C.

4. Use of the marker for diagnosis and prognosis of endometriosis-related ovarian cancer as claimed in claim 1 in the preparation of a kit for diagnosis and / or prognosis evaluation of endometriosis-related ovarian cancer.

5. A marker for diagnosis and prognosis of endometriosis-related ovarian cancer as claimed in claim 1, used as a biomarker for assessing the risk of malignant transformation of endometriosis.

6. Use of the marker for diagnosis and prognosis of endometriosis-related ovarian cancer as claimed in claim 1 as a recombinant plasmid vector in the preparation of a product having the function of inhibiting endometriosis-related ovarian cancer.

7. The marker for diagnosis and prognosis of endometriosis-related ovarian cancer according to claim 6 is used as a recombinant plasmid vector for preparing a drug for preventing and / or treating endometriosis-related ovarian cancer.

8. A marker for diagnosis and prognosis of endometriosis-related ovarian cancer according to claim 1, characterized in that Application of lncRNA BANCR / miRNA-612 / CPNE3 regulatory axis as a diagnostic or prognostic marker for endometriosis-related ovarian cancer.

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