A Protease 3 molecular marker for ovarian cancer immunotherapy and molecular diagnosis

Abstract

The present invention provides a Protease 3 molecular marker for ovarian cancer immunotherapy and molecular diagnosis. The present invention discovers that the expression level of PR3 in ovarian cancer patients is significantly higher than that in the healthy population, and identifies that PR3 can be a potential molecular marker for ovarian cancer diagnosis and / or prognosis. Moreover, the present invention further confirms that PR3 can also be used as a therapeutic target, and the expression of PRTN3 in ovarian cancer cell lines can be inhibited by sivelestat, and the migration ability of ovarian cancer cell lines can also be weakened. In summary, PR3 is a potential molecular marker related to the diagnosis and treatment of ovarian cancer, and sivelestat also has the prospect of being used as an anti-ovarian cancer drug, providing a reference for the early diagnosis, prognosis analysis and early treatment of ovarian cancer patients.

Description

Technical Field

[0001] The present invention relates to the technical field of ovarian cancer diagnosis aids, and in particular to a Protease 3 molecular marker used in immunotherapy and molecular diagnosis of ovarian cancer. Background Art

[0002] Ovarian cancer (OC) is a very deadly gynecological malignancy. The latest statistics show that in 2022 alone, there were nearly 320,000 new cases of OC worldwide, and more than 200,000 women died from OC each year, ranking fifth among female cancers in terms of mortality. In China, there were nearly 60,000 new cases of OC in 2022, and OC is becoming more and more malignant and younger.

[0003] The current first-line treatment for OC is based on tumor reduction surgery combined with platinum drugs and paclitaxel. However, more than half of patients will relapse within 2 years, and the patient's survival rate has hardly improved. However, it is worth noting that the five-year survival rate of early OC is as high as 92%, while that of patients with late OC is only 29%. Regrettably, more than 70% of OC cases are detected in the late stage, which may be due to the fact that the early screening of OC is still mainly based on imaging diagnosis, and the existing molecular marker (CA125) as a single value lacks sufficient specificity and sensitivity.

[0004] Therefore, there is an urgent need to develop new, reliable, non-invasive early diagnostic markers to promote early intervention and treatment of ovarian cancer and prolong the survival of ovarian cancer patients. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a molecular marker of Protease 3 (also referred to as PRTN3 or PR3) for use in immunotherapy and molecular diagnosis of ovarian cancer. The present invention uses PR3 as an auxiliary diagnostic marker for ovarian cancer, and can quickly achieve auxiliary detection of ovarian cancer by analyzing its expression level, providing a reference for early diagnosis and early intervention treatment of ovarian cancer patients.

[0006] One of the purposes of the present invention is to provide a marker for early diagnosis or prognosis analysis of ovarian cancer, wherein the marker is Protease 3.

[0007] Another object of the present invention is to provide the use of Protease 3 as a diagnostic and / or prognostic marker in the preparation of reagents for early diagnosis or prognostic analysis of ovarian cancer.

[0008] Preferably, the expression level of PR3 in ovarian cancer patients is detected to assist in early diagnosis or prognosis analysis of ovarian cancer.

[0009] Another object of the present invention is a kit for early diagnosis or prognostic analysis of ovarian cancer, and the kit contains reagents for quantitatively analyzing the PR3 gene or its expression level.

[0010] Preferably, the reagent is achieved by means such as PCR, immunohistochemistry or immunoblot analysis.

[0011] Another object of the present invention is the use of PR3 in the preparation of a kit for early diagnosis or prognostic analysis of ovarian cancer.

[0012] Another object of the present invention is the application of PR3 as a therapeutic target in the preparation of a drug for treating ovarian cancer.

[0013] Preferably, the application of an inhibitor of PR3 in the preparation of a drug for treating ovarian cancer, the drug taking PR3 as a therapeutic target and being used for knocking out or reducing the expression level of the gene.

[0014] Preferably, the inhibitor includes but is not limited to sivelestat.

[0015] Another object of the present invention is to provide a method for screening drugs for treating ovarian cancer, which is characterized in that the method screens drugs capable of inhibiting its expression or activity by the interaction between a candidate drug and PR3.

[0016] One object of the present invention is a therapeutic drug for ovarian cancer, the drug taking PR3 as a therapeutic target and being used for knocking out or reducing the expression level of the gene.

[0017] Preferably, the drug includes sivelestat.

[0018] Preferably, the drug includes siRNA for targeting and inhibiting the expression of the PR3 gene.

[0019] Preferably, the siRNA sequences are as shown in SEQ ID NO.1-2.

[0020] Preferably, the drug includes a pharmaceutically acceptable carrier or excipient.

[0021] Another object of the present invention is to provide the use of sivelestat and siRNA in combination for the preparation of a therapeutic drug for ovarian cancer.

[0022] The advantages of the present invention are as follows: The present invention discovers that the expression level of PR3 in ovarian cancer patients is significantly higher than that in the healthy population, and identifies that PR3 can be a potential molecular marker for ovarian cancer diagnosis and / or prognosis. The present invention also further confirms that PR3 can also be used as a therapeutic target. Using sivelestat can inhibit the expression of PRTN3 in ovarian cancer cell lines, and can also weaken the migration ability of ovarian cancer cell lines. To sum up, PR3 is a potential molecular marker related to the diagnosis and treatment of ovarian cancer, and sivelestat also has the prospect of being used as an anti-ovarian cancer drug, providing a reference for the early diagnosis, prognosis analysis and early treatment of ovarian cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the prognosis analysis of PR3 in ovarian cancer patients;

[0024] Figure 2 is the expression analysis of PR3 in ovarian cancer;

[0025] Figure 3 is the analysis of the proliferation and migration ability of PR3 on ovarian cancer cells;

[0026] Figure 4 is the analysis of the combined use of sivelestat and siRNA. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] The following further describes the present invention in detail with reference to specific embodiments, so that those skilled in the art can understand the present invention more clearly.

[0028] The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the protection scope of the present invention.

[0029] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically specified, the technical means used are all conventional means well known to those skilled in the art.

[0030] Example 1: Screening of Differentially Expressed Genes

[0031] Obtain the expression profile data and clinical information data of ovarian cancer patients from the GEO and TCGA databases, and use the limma package in R language to screen the differentially expressed genes in the two groups of data. The screening criteria are |log2fold-change| > 1 and P < 0.05 for significantly differentially expressed genes. Use univariate Cox analysis to screen genes significantly related to the prognosis of ovarian cancer from the differentially expressed genes. The results show ( Figure 1), the top-ranked Protease 3 (PRTN3, PR3) is closely associated with the prognosis of ovarian cancer. High expression of PR3 significantly reduces the survival of ovarian cancer patients, demonstrating its potential as a diagnostic molecular marker.

[0032] Example 2: Analysis of PR3 protein expression level

[0033] All experimental subjects were clinically diagnosed with ovarian cancer from March 2023 to September 2023. Their cancer tissues and adjacent tissues were collected for later use. Meanwhile, ovarian cancer cell lines SKOV3 and OVCAR8 and normal ovarian cell line IOSE80 were selected for later use. Immunohistochemistry and immunoblotting were used to analyze the expression and distribution of PR3 at the histological and cytological levels respectively.

[0034] The results showed that: compared with normal ovarian tissues, PR3 was highly expressed in the tissues of ovarian cancer patients ( Figure 2 A, B in Figure 2 )(*p>0.05). In vitro, immunoblotting (WB) verified the expression of PR3 in ovarian cancer cell lines. Compared with the normal ovarian cell line IOSE80, PR3 was highly expressed in the ovarian cancer cell lines SKOV3 and OVCAR8 (

[0035] C, D in

[0036] )(*p>0.05, **p>0.01). The above results further confirmed that PR3 can be used as a diagnostic molecular marker for ovarian cancer.

[0037] The results showed that: compared with the control group, after culturing the SKOV3 cell line in the environment of sivelestat at concentrations of 0.64 μM and 1.28 μM for 48 hours, the expression of PR3 was significantly reduced ( Figure 3 A, C in Figure 3 )(**p<0.01, ***p<0.001). In the experiment on the OVCAR8 cell line, sivelestat at a concentration of 1.28 μM could significantly inhibit the expression of PR3 after 48 hours ( Figure 3As shown in E and G, sivelestat was able to inhibit the migration ability of SKOV3 cells at concentrations of 0.64 μM and 1.28 μM, and the effect was the best at 48 hours and 1.28 μM (***p < 0.001). In the OVCAR8 cell line, sivelestat at a concentration of 1.28 μM could also significantly inhibit the migration ability of cells after 24 hours or 48 hours (*p < 0.05, **p < 0.01).

[0038] To further investigate the credibility of PR3 as a therapeutic target for ovarian cancer, siRNAs specifically targeting and inhibiting its gene expression were designed based on the PR3 gene sequence, and the sequences are shown in SEQ ID NO.1 - 2. The MTT assay was used to evaluate the targeted therapeutic effect of siRNA (0.2 μM) combined with sivelestat (0.64 μM). The results are as Figure 4 shown. Compared with single drug administration, the combination of siRNA and sivelestat could significantly inhibit the proliferation activity of ovarian cancer cells, and there was a synergistic effect between the two. The above results further confirmed that PR3 is a key factor involved in regulating the proliferation and migration ability of ovarian cancer cells, and it can be used as a potential therapeutic target to achieve the treatment of ovarian cancer by targeting PR3.

[0039] The role of the above embodiments is to illustrate the substantial content of the present invention, but does not limit the protection scope of the present invention. Those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and protection scope of the technical solutions of the present invention.

Claims

1. Use of a reagent for detecting the expression level of the Protease3 gene in the preparation of a kit for ovarian cancer prognosis analysis, characterized in that, The kit is used for PCR, immunohistochemistry or immunoblot analysis.

2. Use of sivelestat in combination with siRNA in the preparation of a medicament for treating ovarian cancer, characterized in that, The siRNA sequences are as shown in SEQ ID NO.1-2.

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