Molecular marker for screening and / or diagnosing cervical cancer and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a molecular marker for screening and / or diagnosing cervical cancer and application of the molecular marker. The molecular marker for screening and / or diagnosing cervical cancer is 2 '-5'-oligonadenylate synthase 1, and tests show that the content of 2 '-5'-oligonadenylate synthase 1 in tissues and serum of a patient with cervical cancer in the early stage is remarkably increased compared with that of healthy people and is maintained until the later stage of disease progression. Therefore, the expression quantity of the 2 '-5'-oligonadenylate synthase 1 in the cervical tissue and / or the content of the 2 '-5'-oligonadenylate synthase 1 in the serum can be detected to identify whether the examined person suffers from the cervical cancer or not, so as to diagnose or assist the clinical pathological diagnosis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to a molecular marker for screening and / or diagnosing cervical cancer and an application thereof. Technical Background

[0002] Tumor cells can generate and release a variety of metabolites, proteins, enzymes, exosomes and other products of life activities. Using the unique biochemical or immune characteristics of these products produced specifically by tumor cells to identify them can assist in the diagnosis of tumors. As a common malignant tumor of the female reproductive system, cervical cancer faces many challenges in the diagnosis process, especially in the detection of markers, where there are many problems to be solved.

[0003] The commonly used cervical cancer screening marker, HPV-PCR test, has high sensitivity but insufficient specificity, which can easily lead to excessive colposcopy referrals and psychological panic in patients. Cytology examination requires experienced cytology experts to make judgments, which is highly subjective, has poor consistency, and has low detection sensitivity, which can easily lead to missed detection. Providing new analytical markers is a breakthrough in improving cervical cancer diagnosis technology and an effective means to improve the accuracy and sensitivity of cervical cancer diagnosis.

[0004] 2'-5'-Oligodenylate synthase 1 (OAS1) is an antiviral enzyme induced by interferon and activated by double-stranded RNA (dsRNA), and occupies a central position in the innate immune defense mechanism of cells. In addition, OAS1 may also be involved in regulating the delicate balance of cell apoptosis, cell proliferation, differentiation process and gene expression. It synthesizes high-level 2'-5'-linked oligoadenylate chains by catalyzing ATP, which in turn bind to the inactive monomers of ribonuclease L (RNase L), inducing its dimerization and activation. After RNase L is activated, it can degrade cellular and viral RNA, effectively blocking the protein synthesis pathway, thereby curbing the replication process of the virus. It is worth noting that OAS1 has shown the ability to mediate antiviral effects through the classic RNase L-dependent pathway and the alternative antiviral pathway that is independent of RNase L. Its secreted form can fight against a variety of viruses such as vesicular stomatitis virus, herpes simplex virus type 2 and encephalomyocarditis virus, and may stimulate the initiation of other alternative antiviral mechanisms.

[0005] However, although OAS1 has been studied in the antiviral field, its mechanism of action and application in the context of cancer, especially its relationship with tumorigenesis closely related to viral infection, are still in the early stages of exploration, and more in-depth scientific research is urgently needed to reveal its mysteries. Summary of the invention

[0006] One of the purposes of the present invention is to provide the use of 2'-5'-oligoadenylate synthase 1 as a molecular marker for cervical cancer screening and / or diagnosis.

[0007] The second object of the present invention is to provide the use of 2'-5'-oligoadenylate synthase 1 in the preparation of products for screening and / or diagnosing cervical cancer.

[0008] Preferably, the product comprises a reagent and a kit.

[0009] The third object of the present invention is to provide a product for screening and / or diagnosing cervical cancer, wherein the product is a reagent for detecting 2'-5'-oligoadenylate synthase 1, or the product contains a reagent for detecting 2'-5'-oligoadenylate synthase 1.

[0010] Preferably, cervical cancer is screened and / or diagnosed or assisted in diagnosis by detecting the expression of 2'-5'-oligoadenylate synthase 1 in cervical tissue and / or detecting the content of 2'-5'-oligoadenylate synthase 1 in serum.

[0011] Preferably, 2'-5'-oligoadenylate synthase 1 is expressed in cervical tissue, and the corresponding detection subject suffers from cervical cancer.

[0012] Preferably, the cervical tissue includes any one or a combination of squamous epithelial cells, glandular epithelial cells, and epidermal basal cells.

[0013] Preferably, the ELISA method is used to detect the content of 2'-5'-oligoadenylate synthase 1 in the sample serum; if the content of 2'-5'-oligoadenylate synthase 1 in the serum is greater than 3.97 ng / ml, the corresponding test subject suffers from cervical cancer.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 2'-5'-Oligodenylate synthase 1 (OAS1) has long been used in antiviral research. This application first discovered that it can be used as a molecular marker for diagnosing cervical cancer.

[0016] Through in-depth exploration of large-scale sample databases in the field of clinical biomedicine, this application experiment revealed the significant high expression of the OAS1 gene in tissue samples of cervical cancer patients, and this high expression state remained stable in different pathological stages of the disease. Furthermore, the results of immunohistochemical analysis clearly pointed out that OAS1 showed negative expression in various cervical tissue cells of healthy individuals, while it frequently showed positive expression characteristics in tissue cells of cervical cancer patients. The ELISA method showed that the OAS1 content in the serum of cervical cancer patients was significantly increased compared with that of healthy people.

[0017] Through in-depth research at the gene and protein expression levels, this application experiment has strongly demonstrated the potential value of OAS1 as a diagnostic marker for cervical cancer. OAS1 has good application value in the field of cervical cancer screening and diagnosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Comparison results of the gene expression of OAS1 in normal and cervical cancer tumor tissues of all pathological stages (two-sample T-test statistical test).

[0019] Figure 2 Figure 2 shows the gene expression of OAS1 in cervical cancer tumor tissues at each pathological stage (one-way Annova test).

[0020] Figure 3 The figure shows the protein immunohistochemical staining of OAS1 in healthy people (A) and cervical tissues of cervical cancer patients (B). Number 1 in the figure represents subcutaneous soft tissue, 2 represents epidermal basal cells, 3 represents squamous epithelial cells, 4 represents cervical squamous cell carcinoma cells, and 5 represents cervical adenocarcinoma cells.

[0021] Figure 4 The results of serum test for OAS1, where A is the comparison of serum OAS1 protein levels between cervical cancer and healthy subjects, and B is the receiver operating characteristic curve (ROC curve) for distinguishing cervical cancer from healthy subjects based on OAS1 protein levels. DETAILED DESCRIPTION

[0022] The technical solution of the present application is described in more detail below in conjunction with analytical tests and drawings.

[0023] Unless otherwise specified, the terms used herein have the meanings commonly understood by those skilled in the art.

[0024] 1. OAS1 gene is highly expressed in cervical tissue of patients with cervical cancer

[0025] Using the second-generation sequencing data resources of cervical cancer patients in the TCGA (The Cancer Genome Atlas) database and the gepia2 online analysis tool, a comprehensive and in-depth analysis of the gene expression pattern of OAS1 in 306 cervical cancer tissues and 13 normal cervical tissues was conducted. Figure 1 As shown, the analysis results clearly revealed that the gene expression of OAS1 in cervical cancer tissue was significantly increased compared with that in normal cervical tissue.

[0026] On this basis, further grouping research was conducted according to the pathological grade of cervical tumor tissue. The results are as follows Figure 2As shown in the figure, it can be seen that regardless of the pathological stage of the disease (i.e., stage I, stage II, stage III, or stage IV), the content of OAS1 is maintained at a relatively constant and high level. This finding strongly suggests that OAS1 plays a highly expressed role in all stages of cancer progression, providing important clues for understanding its potential role in the occurrence and development of cervical cancer.

[0027] 2. Expression of OAS1 protein in cervical cancer tissues

[0028] HPA (Human Protein Atlas) database contains immunohistochemical data of various normal human tissues and various cancer samples. We extracted the immunohistochemical data of OAS1 in normal human cervical tissue and tumor tissue of cervical cancer patients from HPA database, and found that OAS1 protein was negative in normal cervical tissue of healthy people (such as Figure 3 In the figure, the tumor tissues of patients with cervical cancer showed generally positive staining (Atlas Antibodies antibody) (as shown in Figure 2A). Figure 3 (shown in B).

[0029] Analysis shows that OAS1 is not expressed in various types of cells (squamous epithelial cells, glandular epithelial cells, epidermal basal cells) in normal human cervical tissue, but is expressed at low to moderate levels in cancer cells of cervical tumor tissue of cervical cancer patients. Detecting the expression of OAS1 in cervical tissue is expected to be a method to distinguish healthy people from cervical cancer patients.

[0030] 3. OAS1 protein levels in the serum of patients with cervical cancer

[0031] 46 healthy individuals and 62 cervical cancer patients were selected as research subjects. Fasting serum samples were collected from all subjects through strict operating procedures. The collected serum samples were properly stored in an ultra-low temperature environment of -80°C to ensure their biological activity and stability. In the detection process, the highly sensitive ELISA (enzyme-linked immunosorbent assay) technology was used to accurately determine the content of OAS1 protein in serum samples. The ELISA kit was purchased from Boster.

[0032] The ELISA test steps are as follows:

[0033] Step S1: First, set up blank control wells and test sample wells on the ELISA coated plate. Then, accurately add 100 μL of serum sample diluted 10 times to each test sample well, and add an equal amount of PBS solution to the control well as a reference. To ensure that the sample is evenly distributed, gently shake the ELISA plate to fully mix the sample.

[0034] Step S2: Seal the ELISA plate tightly with a sealing film and incubate it at a constant temperature of 37° C. for 2 hours to promote full binding of the sample and the coated antigen.

[0035] Step S3: After the incubation, carefully remove the sealing film and completely discard the liquid in the wells, then remove the residue by spinning. Next, fill each well with PBS washing solution, let it stand for 30 seconds and then discard the washing solution again. This washing process needs to be repeated 3 to 5 times to ensure that unbound impurities and antibodies are completely removed.

[0036] Step S4: Add 100 μL of biotin-crosslinked antibody to each well and continue incubating at 37° C. for 1 hour to allow the biotin antibody to specifically bind to the target antigen in the sample.

[0037] Step S5: After the incubation is completed, the solution in the wells is discarded and the washing process of step S3 is repeated. Subsequently, 100 μL of horseradish peroxidase-labeled avidin (HRP-avidin) is added to each well and incubated again at 37° C. for 1 hour to form an enzyme-labeled complex.

[0038] Step S6: Finally, add substrate solution (tetramethylbenzidine) to each well and perform color development reaction under appropriate conditions. After the reaction is sufficient, add H2SO4 to terminate the reaction. At this time, use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450nm, and judge the content of OAS1 protein in the sample based on the size of the OD value.

[0039] ELISA test results Figure 3 As shown, the details are as follows:

[0040] Depend on Figure 4 As can be seen from Part A, the OAS1 protein levels in the serum of healthy individuals and cervical cancer patients were compared, and the results showed that the OAS1 level in the serum of cervical cancer patients was significantly higher than that of healthy people (P<0.0001). Figure 4 As can be seen from Part B, the area under the receiver operating characteristic (ROC) curve (AUC) is as high as 0.8983, showing its excellent sensitivity and specificity. When 3.97ng / ml is used as the critical value of serum OAS1 concentration, the detection of cervical cancer shows 100% specificity while maintaining a sensitivity of 61.29% or more. It is speculated that this is because the occurrence and development of cervical cancer is closely related to the persistent infection of human papillomavirus HPV, and this process causes increased OAS1 reactivity. According to the above analysis, it can be seen that detecting the content of OAS1 in serum can be used as an effective method to distinguish healthy people from cervical cancer patients.

[0041] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of 2'-5'-oligoadenylate synthase 1 as a molecular marker for cervical cancer screening and / or diagnosis.

2. Use of 2'-5'-oligoadenylate synthase 1 in the preparation of products for screening and / or diagnosing cervical cancer.

3. The use according to claim 2, characterized in that The products include reagents and kits.

4. A product for screening and / or diagnosing cervical cancer, characterized in that: The product is a reagent for detecting 2'-5'-oligoadenylate synthase 1, or the product contains a reagent for detecting 2'-5'-oligoadenylate synthase 1.

5. A product for screening and / or diagnosing cervical cancer according to claim 4, characterized in that: Screening and / or diagnosing cervical cancer is achieved by detecting the expression of 2'-5'-oligoadenylate synthase 1 in cervical tissue and / or detecting the content of 2'-5'-oligoadenylate synthase 1 in serum.

6. A product for screening and / or diagnosing cervical cancer according to claim 5, characterized in that: 2'-5'-oligoadenylate synthase 1 is expressed in cervical tissue, and the corresponding test subject suffers from cervical cancer.

7. A product for screening and / or diagnosing cervical cancer according to claim 5 or 6, characterized in that: The cervical tissue includes any one or a combination of squamous epithelial cells, glandular epithelial cells, and epidermal basal cells.

8. A product for screening and / or diagnosing cervical cancer according to claim 5, characterized in that: The content of 2'-5'-oligoadenylate synthase 1 in the serum of the sample is detected by enzyme-linked immunosorbent assay; if the content of 2'-5'-oligoadenylate synthase 1 in the serum is greater than 3.97 ng / ml, the corresponding test subject suffers from cervical cancer.

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