Protease 3 molecular marker applied to ovarian cancer immunotherapy and molecular diagnosis

Abstract

The invention provides a Protease 3 molecular marker applied to immunotherapy and molecular diagnosis of ovarian cancer. The invention finds that the expression level of PR3 in ovarian cancer patients is obviously higher than that in healthy people, and identifies that PR3 can be a potential ovarian cancer diagnosis and / or prognosis molecular marker. The invention further proves that the PR3 can also be used as a therapeutic target, and the sivelestat can be used for inhibiting the expression of the PRTN3 in the ovarian cancer cell line and weakening the migration ability of the ovarian cancer cell line. In conclusion, PR3 is a potential molecular marker related to diagnosis and treatment of ovarian cancer, and sivelestat also has a prospect of serving as an anti-ovarian cancer drug, and provides reference for 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 prognosis analysis of ovarian cancer, which contains a reagent for quantitative analysis of the PR3 gene or its expression level.

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

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

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

[0013] Preferably, the inhibitor of PR3 is used in the preparation of a drug for treating ovarian cancer, wherein the drug uses PR3 as a therapeutic target and is used to knock out or reduce the expression 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, characterized in that the method is to screen drugs that can inhibit the expression or activity of PR3 by interacting with candidate drugs.

[0016] One of the purposes of the present invention is to provide a therapeutic drug for ovarian cancer, wherein the drug takes PR3 as a therapeutic target and is used to knock out or reduce the expression level of the gene.

[0017] Preferably, the drug comprises sivelestat.

[0018] Preferably, the drug comprises siRNA that targets and inhibits the expression of PR3 gene.

[0019] Preferably, the siRNA sequence is shown as SEQ ID NO.1-2.

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

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

[0022] The advantages of the present invention are as follows: The present invention found that the expression level of PR3 in ovarian cancer patients was significantly higher than that in healthy people, and identified PR3 as a potential molecular marker for diagnosis and / or prognosis of ovarian cancer. The present invention also further confirmed that PR3 can also be used as a therapeutic target. The use of sivelestat can inhibit the expression of PRTN3 in ovarian cancer cell lines, and can also weaken the migration ability of ovarian cancer cell lines. 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 an anti-ovarian cancer drug, providing a reference for early diagnosis, prognosis analysis and early treatment of ovarian cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the prognostic analysis of PR3 in patients with ovarian cancer; Figure 2 is the expression analysis of PR3 in ovarian cancer; Figure 3 It is an analysis of the proliferation and migration ability of PR3 on ovarian cancer cells; Figure 4 This is an analysis of the combined use of sivelestat and siRNA. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0025] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0026] In the examples 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 examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0027] Example 1: Screening of differentially expressed genes The expression profile data and clinical information data of ovarian cancer patients were obtained from the GEO and TCGA databases, and the differentially expressed genes in the two groups of data were screened using the R language limma package. The screening criteria were |log2fold-change|>1, and P<0.05 for significantly differentially expressed genes. Using univariate Cox analysis, genes significantly associated with ovarian cancer prognosis were screened from the differentially expressed genes. The results showed ( Figure 1), Protease 3 (PRTN3, PR3) ranked first is closely related to the prognosis of ovarian cancer. The high expression of PR3 will significantly reduce the survival of ovarian cancer patients, proving that it can be used as a potential diagnostic molecular marker.

[0028] Example 2: Analysis of PR3 protein expression levels All the subjects were diagnosed with ovarian cancer by clinicians from March to September 2023, and their cancer tissues and adjacent tissues were collected for future use. At the same time, ovarian cancer cell lines SKOV3 and OVCAR8 and normal ovarian cell line IOSE80 were selected for future use. Immunohistochemistry and immunoblotting were used to analyze PR3 expression and distribution at the histological and cytological levels, respectively.

[0029] The results showed that compared with normal ovarian tissue, PR3 was highly expressed in ovarian cancer tissue ( Figure 2 In vitro, Western blotting (WB) was used to verify 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 ( Figure 2 (C, D) (*p>0.05, **p>0.01). The above results further confirmed that PR3 can be used as a molecular marker for the diagnosis of ovarian cancer.

[0030] Example 3: Analysis of the effect of PR3 on the proliferation and migration of ovarian cancer cells Sivelecstat is a neutrophil elastase (NE) inhibitor that can be used to inhibit the expression of PR3. In the present invention, Sivelecstat was used to inhibit the expression of PR3 in ovarian cancer cell lines SKOV3 and OVCAR8, and the inhibitory effect of Sivelecstat on PR3 was verified by performing protein immunoblotting experiments on SKOV3 and OVCAR8. The effect of Sivelecstat on the migration ability of SOC cell lines SKOV3 and OVCAR8 was verified by cell scratch experiments.

[0031] The results showed that compared with the control group, the expression of PR3 was significantly reduced in SKOV3 cells after being cultured in the presence of 0.64 μM and 1.28 μM sivelestat for 48 hours ( Figure 3 A, C) (**p<0.01, ***p<0.001). In the experiment on OVCAR8 cell line, 1.28μM sivelestat could significantly inhibit the expression of PR3 after 48 hours ( Figure 3 B, D) (*p<0.05). Figure 3As shown in Figures E and G, sivelestat can inhibit the migration ability of SKOV3 cells at concentrations of 0.64μM and 1.28μM, and the best effect is achieved at 48 hours and 1.28μM (***p<0.001). In the OVCAR8 cell line, sivelestat at a concentration of 1.28μM can also significantly inhibit the migration ability of cells after 24 hours or 48 hours (*p<0.05, **p<0.01).

[0032] To further study the credibility of PR3 as a therapeutic target for ovarian cancer, siRNAs that specifically target and inhibit PR3 gene expression were designed. 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). Figure 4 As shown in the figure, compared with single administration, the combination of siRNA and sivelestat can significantly inhibit the proliferation activity of ovarian cancer cells, and the two have a synergistic effect. The above results further confirm 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.

[0033] The above embodiments are intended to illustrate the essential content of the present invention, but are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and protection scope of the technical solution of the present invention.

Claims

1. A marker for early diagnosis or prognosis analysis of ovarian cancer, characterized in that: The marker is Protease 3.

2. Application of Protease 3 as a diagnostic marker in the preparation of reagents for early diagnosis or prognosis analysis of ovarian cancer.

3. The use according to claim 2, characterized in that The method is to detect the expression level of Protease 3 in ovarian cancer patients to assist in the diagnosis and / or evaluation of ovarian cancer.

4. A kit for assisting in the diagnosis and / or assessment of ovarian cancer, characterized in that: The kit contains reagents for quantitatively analyzing the PR3 gene or its expression level.

5. The kit according to claim 4, characterized in that This is achieved by PCR, immunohistochemistry or immunoblot analysis.

6. Use of Protease 3 in the preparation of a kit for assisting in the diagnosis and / or assessment of ovarian cancer.

7. Application of Protease 3 as a therapeutic target in screening drugs for treating ovarian cancer or preparing drugs for treating ovarian cancer.

8. Use of a Protease 3 inhibitor in the preparation of a drug for treating ovarian cancer, characterized in that: The drug takes Protease 3 as a therapeutic target and is used to knock out or reduce the expression level of the gene.

9. A drug for treating ovarian cancer, characterized in that: The drug takes Protease 3 as a therapeutic target and is used to knock out or reduce the expression level of the gene.

10. The therapeutic drug according to claim 9, characterized in that The therapeutic drug includes sivelestat and siRNA, and the siRNA sequence is shown in SEQ ID NO.1-2.

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