Application of apolipoprotein APOD deletion as molecular marker for diagnosing cervical cancer

Abstract

The invention provides application of apolipoprotein APOD deletion as a molecular marker for diagnosing cervical cancer. The biomarker APOD can be used for preparing a reagent for diagnosing the occurrence and / or severity of the cervical cancer, or preparing a reagent for predicting the occurrence and / or severity of the cervical cancer, or preparing a reagent for evaluating the prognosis of the cervical cancer. It is found that deletion of the cancer suppressor gene APOD possibly plays an important role in the occurrence and development process of cervical cancer, molecular markers for diagnosing precancerous lesions of cervical cancer are expected to be revealed, a brand new strategy is provided for prevention, diagnosis, treatment and prognosis, and the application and popularization are suitable.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technologies, and particularly to the application of apolipoprotein APOD deletion as a molecular marker for diagnosing cervical cancer. Background Art

[0002] Cervical cancer is one of the most common malignant tumors among women in China, with over 150,000 new cases and over 50,000 death cases every year, threatening the lives and health of Chinese women. According to the WHO GLOBOCAN 2020 report, there are over 600,000 new cases and over 340,000 death cases among women globally every year. Persistent HPV infection is the main factor in the development of cervical cancer, but the etiological factors of cervical cancer in women without HPV infection are still unclear. Therefore, the occurrence and development of cervical cancer may be related to host genetic factors, and it is crucial to further explore the key molecular regulatory mechanisms of cervical cancer in the host. The prognosis of early-stage cervical cancer is good. Therefore, it is crucial for healthy women to undergo regular cervical cancer screening. The current technical defects and deficiencies in cervical cancer screening mainly include the following points: ① Limitations of screening methods: Visual inspection with acetic acid / Lugol's iodine solution staining: Low cost and simple operation, but with low sensitivity and specificity, and difficult quality control. Currently, it is only used in some low-resource countries and regions; Cytological examination: Simple operation, convenient sampling, and high detection rate of abnormal cells, but the diagnosis lacks objective criteria, is easily affected by subjective factors, has low reproducibility, and low accuracy; HPV DNA testing: Although simple in operation, objective and accurate, it has low specificity and cannot distinguish transient virus infection from persistent virus infection, which may lead to excessive colposcopy referrals, bringing great pressure to patients and society. ② Challenges in primary medical institutions: Weak health infrastructure in primary medical institutions, lack of trained cytologists, and inconsistent test results from different cytologists limit the wide application of cytological examination. ③ Insufficient screening coverage: The screening coverage rate of cervical cancer in China is only 40%-60%, far lower than the prevention and control target of over 80%. ④ Lack of data support for screening technologies and strategies: Appropriate screening technologies and strategies for China lack data support, which is related to the large population base in China and the uneven development of economic and medical levels in different regions. ⑤ Insufficient physician training: There is a lack of standardized training for cytology, histopathology, and colposcopy physicians. ⑥ Challenges in the application of AI technology in cervical cancer screening: Although the application of AI systems in cervical cancer screening has potential, the current research sample size is small and mostly for internal validation, and more persuasive and well-designed prospective clinical trials are needed for verification; Limitations of AI algorithms: Most studies are based on deep learning algorithms, and the training process is easily affected by hidden confounding factors, requires a large amount of data, and mainly uses image recognition as the main calculation method, unable to fully utilize patient information. These defects and deficiencies limit the accuracy, efficiency, and popularity of cervical cancer screening, and need to be overcome through efforts in technological progress, policy support, education and training, etc. Therefore, it is necessary to screen out new molecular markers for cervical cancer and develop a precise diagnostic kit for the auxiliary screening of cervical cancer to provide new strategies for the prevention, diagnosis, and treatment of cervical cancer.

[0003] Apolipoprotein D (APOD) is a glycoprotein with a molecular weight of approximately 29 - 33 kDa, encoded by the human APOD gene. Different from other lipoproteins mainly produced in the liver, APOD is mainly produced in the brain and testis. It is a component of high-density lipoprotein (HDL) and has no significant similarity with other apolipoprotein sequences. APOD is closely related to lecithin:cholesterol acyltransferase (LCAT), which is involved in lipoprotein metabolism. APOD is also an important link in the transient interaction between HDL and LDL particles, as well as between HDL particles and cells. APOD is a biomarker for androgen insensitivity syndrome (AIS) and is related to nerve disorders and nerve damage, especially myelin. In a rat model, its APOD level is elevated. The APOD level is also elevated in bipolar disorder. APOD is an atypical type of apolipoprotein and is classified into the lipocalin superfamily. Members of this family have similar structures and can act as carriers for small hydrophobic molecules. APOD is also found to have the structural characteristics of the lipocalin superfamily and can bind to small hydrophobic molecules such as cholesterol, progesterone, pregnenolone, heme-related compounds, and arachidonic acid. As an apolipoprotein in human plasma HDL and VHDL, APOD has the function of binding and transporting lipids. APOD in human plasma forms a complex with ApoA-I and lecithin:cholesterol acyltransferase (LCAT), and they can act synergistically: ApoA-I regulates the activity of LCAT, LCAT catalyzes the conversion of cholesterol to cholesterol esters, and finally APOD transports the cholesterol esters to LDL and VLDL. Since this complex undertakes the synthesis and transport of cholesterol esters and maintains homeostasis, it is called the "cholesterol ester transfer complex". At the same time, APOD can also stabilize the activity of LCAT and is a potential agonist of LCAT. Therefore, APOD plays an important role in regulating cholesterol metabolism. APOD exists in the central and peripheral nervous systems of normal people. Both plasma APOD and APOD synthesized by the brain itself can bind to cholesterol and its derivatives in plasma, the interstitial fluid, and cerebrospinal fluid, facilitating the free passage of hydrophobic substances such as cholesterol through the blood-brain barrier and playing an important role in lipid transport in the brain and maintaining lipid metabolic balance. Studies have shown that the lacrimal gland can also synthesize APOD, and APOD interacts with other lipids in tears, which can play a cleaning role and protect the cornea from damage by lipophilic molecules. In addition, APOD is also part of the antioxidant defense system, protecting cells and tissues from oxidative damage. There is currently no report on the relationship between APOD and cervical cancer. Summary of the Invention

[0004] The present invention provides the application of apolipoprotein APOD deletion as a molecular biomarker for the diagnosis of cervical cancer. The present invention clarifies that the expression of APOD is related to the degree of cervical lesions, and is expected to improve the accuracy of cervical cancer diagnosis. The present invention proves that APOD can be used as a diagnostic indicator for cervical lesions and cervical cancer, providing a strong theoretical basis for the method of diagnosing cervical cancer. The present invention confirms the role of APOD in the process of cervical lesions. APOD affects the occurrence and development of cervical cancer, and is expected to become a new means of cervical cancer screening and a new target for precision treatment of cervical cancer in clinical practice.

[0005] On the one hand, the present invention provides the application of a biomarker APOD in the preparation of a reagent for diagnosing cervical cancer or evaluating the prognosis of cervical cancer.

[0006] In one embodiment of the present invention, the reagent is used for diagnosing the occurrence and / or severity of cervical cancer, or predicting the occurrence and / or severity of cervical cancer, or evaluating the prognosis of cervical cancer.

[0007] In one embodiment of the present invention, the reagent is used for the detection, diagnosis, classification, prediction, treatment monitoring, prognosis or other evaluation of the stage or invasive level or risk of cervical cancer, or related diseases.

[0008] In one embodiment of the present invention, the reagent is used for detecting the expression level of the biomarker APOD in a sample, specifically including the presence, absence or excess of the biomarker APOD, and / or comparing the amount of the biomarker APOD in the test sample with the normal or control expression level or standard.

[0009] In one embodiment of the present invention, the reagent includes detecting the biomarker APOD at the protein level; or detecting the biomarker APOD at the nucleic acid level.

[0010] In one embodiment of the present invention, the reagent includes at least one of a reagent for detecting the copy number of the APOD gene level, a reagent for quantitatively detecting the RNA transcription level of APOD, and a reagent for quantitatively detecting the protein expression level of APOD.

[0011] In one embodiment of the present invention, the reagent is a reagent capable of detecting the mRNA level of the biomarker APOD. Such reagents are well known in the art and include, but are not limited to, nucleic acid probes that specifically bind to the target sequence, primers for amplifying the target sequence, non-specific fluorescent dyes (e.g., SYBR Green I), or combinations thereof. In certain embodiments, the nucleic acid probe can be a single-labeled nucleic acid probe, such as a probe labeled with a radionuclide (such as 32P, 3H, 35S, etc.), a biotin-labeled probe, a horseradish peroxidase-labeled probe, a digoxin-labeled probe, or a fluorescent group (such as FITC, FAM, TET, HEX, TAMRA, Cy3, Cy5, etc.); the nucleic acid probe can also be a double-labeled nucleic acid probe, such as a Taqman probe, a molecular beacon, a displacement probe, a scorpion primer probe, a QUAL probe, a FRET probe, etc. In certain embodiments, the reagent includes a Taqman probe. In certain embodiments, the primers for amplifying the target sequence include F: 5’-ACAAGCATTTCATCTTGGGAAGT-3’; R: 5’-CATCAGCTCTCAACTCCTGGT-3’.

[0012] In one embodiment of the present invention, the reagent is an antibody or an antigen-binding fragment thereof capable of detecting the biomarker APOD protein. In a specific embodiment of the present invention, the antibody or its antigen-binding fragment is specific for the epitope of the biomarker APOD or its functional fragment. The detection methods are well known to those skilled in the art and include, but are not limited to, mass spectrometry, one-dimensional or two-dimensional gel analysis systems, chromatography, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), Western blot, immunoprecipitation, and immunohistochemistry. These methods use antibodies or antibody equivalents to detect proteins, or biophysical techniques. Antibody arrays or protein chips can also be used.

[0013] In one embodiment of the present invention, the sample includes one of blood, cells, and tissues. In a specific embodiment of the present invention, the tissue includes cervical cancer tissue, tissue adjacent to cervical cancer, etc.

[0014] In one embodiment of the present invention, the diagnosis or evaluation includes the following steps:

[0015] Step a) Detect the expression level of APOD in the sample;

[0016] Step b) Based on the detection result of a), diagnose cervical cancer or evaluate the prognosis of cervical cancer.

[0017] In a specific embodiment of the present invention, in step a), the sample includes a control tissue (such as normal cervical tissue) and a tumor tissue (such as cervical tumor region tissue).

[0018] In a specific embodiment of the present invention, step b) includes comparing the expression levels of APOD in tumor tissues and control tissues.

[0019] i. If the expression level of APOD in tumor tissues is significantly lower than that in control tissues, it is diagnosed as cervical cancer.

[0020] ii. If the expression level of APOD in tumor tissues is comparable to that in control tissues, cervical cancer is excluded.

[0021] In an embodiment of the present invention, compared with control tissues, the more obvious the decrease in the expression level of the said APOD, the greater the severity of cervical cancer and the greater the risk of metastasis.

[0022] In an embodiment of the present invention, the expression of the said APOD gene is significantly down-regulated in the cervical cancer tissues of cervical cancer patients compared with the paired normal cervical tissues distal to the cancer; the expression of the said APOD protein is significantly down-regulated in the cervical cancer tissues of cervical cancer patients compared with the paired normal cervical tissue samples distal to the cancer.

[0023] In a specific embodiment of the present invention, the sample in step a) is cervical cancer tissue.

[0024] In a specific embodiment of the present invention, step b) includes the auxiliary staging of cervical cancer based on the expression level of APOD in cervical cancer tissues:

[0025] i. If APOD is positively expressed in cervical cancer tissues, the patient tends to have an early T stage (T1-T2) and no lymph node metastasis (N0);

[0026] ii. If APOD is negatively expressed in cervical cancer tissues, the patient tends to have a late T stage (T3-T4) and lymph node metastasis (N1).

[0027] In an embodiment of the present invention, the said reagent can be used to determine the stage of cervical cancer and thus can be used to (for example, in the early stage of the disease or using histologically negative samples) predict disease progression, malignant phenotype and metastasis, provide accurate staging, facilitate rational treatment and develop drugs or compositions for the specific treatment of cervical cancer. In an embodiment of the present invention, the stage of cervical cancer includes T stage and N stage. In an embodiment of the present invention, the T stage and N stage of cervical cancer are significantly correlated with the positive expression of APOD. The positive expression rate of APOD is higher in patients with an early T stage (T1-T2) and no lymph node metastasis (N0); the positive rate of APOD is lower in patients with a late T stage (T3-T4) and lymph node metastasis (N1).

[0028] In one embodiment of the present invention, the overall survival rate of patients is significantly correlated with the positive expression of APOD. Patients with negative APOD expression have a worse overall survival rate (poor prognosis), while patients with positive APOD expression have a better prognosis. The APOD expression level may be related to disease progression or deterioration.

[0029] In a second aspect of the present invention, there is provided an application of a biomarker APOD regulator in the preparation of a drug for preventing and / or treating cervical cancer.

[0030] In one embodiment of the present invention, the regulator includes, but is not limited to, a promoter, agonist or activator of the biomarker APOD.

[0031] In one embodiment of the present invention, the drug includes a promoter, agonist or activator of the APOD gene and / or its expression product; the promoter, agonist or activator is a reagent for promoting the expression of the APOD gene, or a reagent for promoting the stability of the APOD gene expression product, or a reagent for increasing the activity of the APOD gene expression product, or a reagent for enhancing the function of the APOD gene expression product; the reagent for promoting the expression of the APOD gene is a reagent for promoting the transcription of the APOD gene, or a reagent for promoting the translation of the APOD gene, or a reagent for promoting the content of the APOD protein, or a reagent containing the APOD gene, or a reagent formed by a vector carrying the APOD gene, or a reagent formed by a host cell carrying the APOD gene, or a reagent containing the APOD protein.

[0032] In one embodiment of the present invention, overexpression of APOD in cervical cancer cells by constructing an APOD overexpression vector system can significantly inhibit the proliferation of cervical cancer cells, promote apoptosis of cervical cancer cells, inhibit the migration ability of cervical cancer cells, inhibit the invasion ability of cervical cancer cells, interfere with the formation or stability of F-actin (filamentous actin) in the cytoskeleton, inhibit the occurrence of epithelial-mesenchymal transition, and inhibit the self-renewal of cervical cancer cells.

[0033] In a third aspect of the present invention, there is provided a method for screening a candidate drug for preventing and / or treating cervical cancer and / or improving the prognosis of cervical cancer, the method comprising detecting the effect of the candidate drug on the level of the biomarker APOD in a sample of a subject, wherein, after using the candidate drug, if the biological function of the biomarker APOD is improved or enhanced, it indicates that the candidate drug has the effect of preventing and / or treating cervical cancer and / or improving the prognosis of cervical cancer; the improvement or enhancement of the biological function of the biomarker APOD includes an increase or rise in the expression level of the biomarker APOD.

[0034] In the fourth aspect of the present invention, a method for predicting or prognosticating whether a patient or subject is suitable for APOD-targeted therapy is provided, and the method includes: predicting or prognosticating whether the patient or subject is suitable for APOD-targeted therapy based on the APOD level in the blood sample of the patient or subject.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The present invention first uses RNA-sequence to screen out the molecule APOD with differential expression between cervical cancer and adjacent tissues. Through cell experiments, it is found that compared with human primary normal cervical squamous epithelial cells, APOD is not expressed in cervical cancer cells. Further, a cervical cancer tissue microarray is used to detect the expression of APOD in cervical cancer tissues and adjacent tissues, indicating that APOD is highly expressed in cervical squamous epithelium, but lowly expressed in cervical cancer tissues, and the low expression of APOD is related to poor prognosis. In order to explore the effect of APOD on the malignant phenotype of cervical cancer, APOD is overexpressed in cervical cancer cells, indicating that APOD inhibits the proliferation, migration, invasion, and self-renewal ability of cervical cancer cells and promotes apoptosis. It is suggested that APOD inhibits the malignant phenotype of cervical cancer cells. The present invention proposes a new application of APOD, which can be used as a molecular marker for detecting cervical cancer diagnosis and is suitable for popularization and application.

[0037] The present invention discovers that the deletion of the tumor suppressor gene APOD may play an important role in the occurrence and development of cervical cancer. The present invention originates from clinical samples. By sequencing clinical samples, the APOD gene is screened out. Further in vitro studies show that APOD inhibits the occurrence and development of cervical cancer, and comprehensively and deeply clarifies the molecular mechanism of APOD regulating the malignant phenotype of cervical cancer in combination with clinical data and prognostic indicators, etc. It is expected to reveal molecular markers for diagnosing precancerous lesions of cervical cancer and provide new strategies for prevention, diagnosis, treatment, and prognosis, and is suitable for popularization and application. Description of the Drawings

[0038] Figure 1 It is a diagram for the expression analysis of APOD in cervical cancer tissues. A is for verifying the expression of the APOD gene in cervical cancer tissues by RT-qPCR; B is for detecting the expression of APOD in adjacent cervical tissues and paired cervical cancer tissues by Western Blot; C is for detecting the expression of APOD in primary cervical squamous cells PNCSCs and cervical cancer cells by RT-qPCR; D is for detecting the expression of APOD in primary cervical squamous cells PNCSCs and cervical cancer cells by Western Blot.

[0039] Figure 2This is the analysis diagram of APOD and cervical cancer prognosis. A shows the expression of APOD detected by IHC in adjacent tissues and cervical cancer tissues; B shows the progression-free survival curve (PFS); C shows the overall survival curve (OS).

[0040] Figure 3 This is the result diagram of stable overexpression of APOD in cervical cancer cells. A shows the expression of APOD protein detected by Western Blot in cervical cancer cells; B shows the expression of APOD detected by RT-qPCR in primary cervical squamous cells PNCSCs and cervical cancer cells.

[0041] Figure 4 This is the result diagram of the effects of APOD on the proliferation and apoptosis of cervical cancer cells. A shows the detection of cell proliferation phenotype by plate clone colony formation assay; B shows the detection of cell apoptosis by AnnexinV-PE / 7-AAD staining.

[0042] Figure 5 This is the result diagram of APOD inhibiting the migration and invasion phenotypes of cervical cancer cells. A shows the detection of cell migration phenotype by scratch assay; B shows the detection of cell migration phenotype by Transwell chamber migration assay; C shows the detection of cell invasion phenotype by Transwell chamber invasion assay; D shows the detection of changes in the cell skeleton by F-actin staining of the cytoskeleton protein; E shows the detection of epithelial-mesenchymal transition-related markers by Western Blot.

[0043] Figure 6 This is the result diagram of the detection of the ability of APOD to inhibit the self-renewal of cervical cancer cells by the spheroid formation assay.

[0044] Figure 7 This is the ROC curve diagram for predicting cervical cancer by the expression level of APOD. Detailed implementation methods

[0045] The technical solutions of the present invention will be further described in detail below in combination with specific embodiments. It should be understood that the following embodiments are only used to illustrate and explain the present invention exemplarily, and should not be construed as limiting the protection scope of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection of the present invention.

[0046] Unless otherwise specified, the raw materials and reagents used in the following embodiments are all commercially available products, or can be prepared by known methods.

[0047] The term "APOD" (Apolipoprotein D) in the present invention is a small molecule glycoprotein belonging to the apolipoprotein family. Apolipoproteins are a class of proteins that bind to lipids (such as cholesterol and phospholipids) and participate in lipid metabolism. APOD is a lipid-binding protein that is widely expressed in different tissues and cells, especially in the central nervous system and plasma. Its structural characteristics enable it to bind to various lipid molecules, such as cholesterol and cholesterol esters.

[0048] "APOD gene", whose gene sequence includes but is not limited to: human APOD gene, NCBI Gene ID: 347, Ensembl ID: ENSG00000189058; mouse APOD gene, NCBI Gene ID: 11810, Ensembl ID: ENSMUSG00000020113. The amino acid sequence of the biomarker APOD includes but is not limited to that shown in SEQ ID NO.1. The nucleotide sequence of the biomarker APOD includes but is not limited to that shown in SEQ ID NO.2.

[0049] A "promoter", "agonist" or "activator", such as a compound or an antibody, is an agent that induces (e.g., increases) one or more activities or functions of the target of the agent (e.g., APOD) after the agent binds to the target.

[0050] The term "APOD promoter, agonist or activator" in the present invention refers to a molecule, drug or conditional factor that can enhance or activate the function or expression of apolipoprotein D (APOD). These promoters or agonists may regulate the expression of the APOD gene, protein activity or its binding ability to ligands directly or indirectly, thereby affecting its biological function. An APOD promoter or agonist refers to any compound or molecule that can enhance the function or expression of APOD. These compounds include, but are not limited to, the following categories: (1) Agonistic anti-APOD antibodies or fragments thereof: By directly binding to the APOD protein, enhancing its lipid-binding ability or antioxidant function. APOD-Ig fusion proteins: For example, an APOD polypeptide or fragment thereof is linked to the Fc region of an antibody (such as the Fc fragment of IgG1, IgG2, IgG3 or IgG4), enhancing its stability and function. (2) Multimeric forms of APOD: Comprising 2, 3, 4, 5 or more APOD protein molecules or fragments, a complex formed by linking through oligomeric or polymeric domains to enhance its lipid transport ability. (3) Nucleic acids that promote APOD expression: Including mRNA encoding APOD or vectors, such as plasmids or viral vectors expressing APOD, for gene therapy. Antisense nucleic acids and ribozymes: Indirectly increase the level of APOD by targeting and inhibiting factors that negatively regulate APOD expression. (4) Small molecule compounds that activate APOD function: These molecules may act by regulating the ligand binding or protein activity of APOD. Short peptides that mimic APOD function: Short peptides designed based on the key active sites of APOD can directly replace or activate APOD function. (5) Glycoalkaloids and retinoids: Certain natural compounds may promote the expression or activity of APOD by regulating upstream signaling pathways. (6) Transfected cells expressing APOD: For example, cell lines into which the APOD gene has been introduced can be used for research and applications. Engineered proteins that modify APOD domains: Improve its binding ability or functional activity by modifying specific regions.

[0051] The term "statistically significant", or "statistical significance", is well known in the art and can be determined using methods known in the art, such as those described herein. In some embodiments, statistical significance means, for example, p < 0.1, p < 0.05, p < 0.04, p < 0.03, p < 0.02 or p < 0.01 relative to a baseline.

[0052] "Expression level" refers to the measurable amount of a gene product produced by a target gene in a sample from a subject, where the gene product can be a transcriptional product or a translational product. Thus, the statement "determining the expression level of a target gene" can refer to determining the level of the mRNA of the gene or a fragment of the mRNA, or the level of the cDNA of the gene or a fragment of the cDNA, or the level of the protein encoded by the gene or a polypeptide fragment thereof.

[0053] "Control" refers to a sample or data that is compared with an experimental group (i.e., the group of samples in which the expression level of a cancer biomarker is detected). The purpose of the control is to provide a benchmark to help evaluate whether the expression level of the biomarker in the experimental group is significantly higher or lower than the normal level or other comparison conditions. In one embodiment of the present invention, specimens of normal cervical tissue or healthy individuals are used as the control group to compare the biomarker expression with that of the cervical cancer tissue group (experimental group). Preferably, the control is a normal cervical tissue sample.

[0054] Example 1 Expression analysis and verification of APOD in cervical cancer tissues and cells

[0055] 1. Fifteen cases each of cervical cancer tissues and paired normal cervical tissues distal to the cancer were collected from cervical cancer patients, and human primary normal cervical squamous epithelial cells (PNCSC) and cervical cancer cell lines (HeLa, SiHa, and C33A) were obtained. Total RNA in the above tissues and cells was extracted using TRIZOL, and the total RNA was reverse transcribed into cDNA using reverse transcriptase. The target fragment was amplified using the APOD primer sequences for qPCR (F: 5'-ACAAGCATTTCATCTTGGGAAGT-3' (SEQ ID NO: 3); R: 5'-CATCAGCTCTCAACTCCTGGT-3' (SEQ ID NO: 4)). The fluorescence signal was monitored in real time using a real-time PCR instrument, and data analysis was performed. The amount of target DNA present in the original sample was calculated based on the detected Ct value for quantitative gene expression analysis. The results showed that the APOD gene was significantly downregulated in cervical cancer tissues and cells ( Figure 1 A and C).

[0056] 2. The cervical cancer tissues and paired normal cervical tissues distal to the cancer of cervical cancer patients were lysed with lysis buffer to lyse tissues and cells and release proteins. The protein concentration in the samples was determined using a BCA protein quantification kit. The samples were mixed with SDS-PAGE loading buffer and heated to 100 °C to denature the proteins. The samples were loaded onto a polyacrylamide gel and electrophoresed in an electrophoresis apparatus to separate the proteins according to their molecular weights. The proteins in the gel were transferred to a PVDF membrane using a wet transfer membrane apparatus. After transfer, the membrane was washed 3 times with TBST (phosphate buffer containing Tween) buffer for 5 minutes each time. After washing the membrane, the membrane was blocked with 5% skim milk in TBST to prevent non-specific binding. The membrane was incubated with a diluted specific APOD primary antibody to allow the primary antibody to bind to the target protein. The membrane was washed multiple times with TBST to remove the unbound primary antibody. The membrane was incubated with a secondary antibody (usually conjugated with an enzyme or a fluorescent label) that matches the primary antibody, and the secondary antibody would bind to the primary antibody. The membrane was washed again with TBST to remove the unbound secondary antibody. Chemiluminescence was used, and an ECL developer was added to the membrane to react with the enzyme to produce a light signal, and then an imaging device was used to capture the image. The obtained image was analyzed, and the expression level of APOD was evaluated by comparing the band intensity of the APOD protein with that of the control group. The results showed that the APOD protein was significantly downregulated in cervical cancer tissues and cells ( Figure 1 B and D).

[0057] 3. Validation of the molecular marker APOD

[0058] Cervical cancer tissues and normal cervical tissues distal to the cancer of cervical cancer patients meeting the standards were collected according to the standard operating procedure (SOP), and complete clinical case information was systematically collected. A total of 15 cervical cancer tissue samples and 15 normal cervical tissue samples adjacent to the cancer were collected. Gene expression quantitative analysis was performed on the clinical samples according to the detection method in step 1. And statistical analysis was performed on the data to explore the predictive diagnostic value of the APOD expression level for cervical cancer.

[0059] Results: Using the normal cervical tissues adjacent to the cancer as the control, the predictive value of the APOD expression level for positive cervical cancer was analyzed by ROC curve. As Figure 7 , shown in Table 1, the results showed that the overall performance of the model was good, the AUC value was 0.876, the sensitivity was as high as 92.9%, and the accuracy was 83.7%. These all indicated that the model could effectively classify, especially being very powerful in identifying positive samples.

[0060] Table 1 ROC comparison of the detection results of 15 samples

[0061]

[0062] Correlation between Example 2 APOD and cervical cancer staging and overall survival of cervical cancer patients

[0063] Cervical cancer tissue microarrays with a 5-year survival period purchased from different companies (95 cases of adjacent cancer tissues and 185 cases of cancer tissues, with 5 cases of cancer tissue spots excluded, a total of 180 cases of cancer tissues) were subjected to APOD immunohistochemical staining. The specific steps are as follows: Dewax the purchased microarray, and use antigen retrieval solution (pH 9.0) to perform antigen retrieval for 20 minutes by microwave heating to improve the binding efficiency of antibodies. Block with 5% BSA blocking solution at room temperature for 15 minutes to reduce background staining. Use the primary antibody of APOD, and after diluting it with a 1:2000 dilution solution, evenly coat it on the tissue section and incubate it overnight at 4°C to allow the primary antibody to fully bind to the antigen. After washing multiple times the next day, incubate with a biotin-labeled secondary antibody at room temperature for 10 minutes. After washing thoroughly again, a chemical reaction occurs under the action of the enzyme on the substrate to form a colored precipitate, showing brown. Then use hematoxylin staining solution for counterstaining, perform dehydration and clearing treatments, and use a mounting medium to mount the slides, and observe and analyze them under a microscope. The results showed that APOD was expressed in both squamous epithelial cytoplasm and cell membrane ( Figure 2 A). Positive expression of APOD obtained a longer progression-free survival (PFS), but there was no statistical significance ( Figure 2 B); positive expression of APOD obtained a longer overall survival (OS), with statistical significance ( Figure 2 C); indicating that patients with positive expression of APOD had a better overall survival rate (better prognosis), while patients with negative expression of APOD had a worse prognosis. The expression level of APOD may be related to disease progression or deterioration. Analyze the relationship between these indicators and APOD expression for the clinical indicators (age and TN staging) of 180 cervical cancer patients. The results showed that there was a statistical significance between APOD expression and TN staging (Table 2). The positive expression rate of APOD was higher in patients with early-stage tumors (T1-T2 stage) and no lymph node metastasis (N0). While the expression of APOD was significantly decreased in patients with advanced tumors (T3-T4 stage) and lymph node metastasis (N1). Thus, it can be seen that the APOD molecule can be used as a potential marker for early diagnosis or tumor staging.

[0064] Table 2 Relationship between APOD expression and clinicopathological parameters in cervical cancer patients

[0065]

[0066]

[0067] T stage: Primary tumor stage; N stage: Regional lymph nodes stage.

[0068] Example 3: Construction of cervical cancer cells with stable overexpression of APOD

[0069] By lentiviral transfection, a cell line C33A-APOD and its negative control C33A-EV (cells expressing the empty vector in the C33A cell line) were established using an overexpression vector system. The above cells were cultured in DMEM medium containing 10% fetal bovine serum at 37 °C and 5% CO 2 in an incubator. When the cell confluence reached 80 - 90%, the cells were digested with 0.25% trypsin (containing 0.02% EDTA) for subculture. Logarithmically growing C33A cells were seeded in a 24-well plate. When the cell growth reached 50% confluence, a mixture of virus solution (the lentivirus for overexpressing APOD was provided by Shanghai GeneChem Co., Ltd.) and 5 μg / ml polybrene was dropped into the cell culture medium. After 72 hours, green fluorescence was observed under a fluorescence microscope. Puromycin was added for further screening to obtain C33A cells overexpressing APOD, which were verified by Western blot and RT-qPCR. The results showed that the C33A cell line overexpressing APOD was successfully constructed ( Figure 3 A and B).

[0070] Example 4: APOD inhibits the proliferation of cervical cancer cells and promotes apoptosis of cervical cancer cells

[0071] To observe the effect of APOD on the proliferation and apoptosis of cervical cancer cells, the cervical cancer cells with stable overexpression of APOD obtained in Example 3 were subjected to in vitro colony formation assay and AnnexinV-PE / 7-AAD staining assay.

[0072] 1. Colony formation assay: Cells in the logarithmic growth phase of each group were digested with trypsin, centrifuged, and counted. 200 cells were added to each well (6-well plate or 30-mm culture dish) and incubated in an incubator for 2 weeks. After observation, when visible clones appeared, the culture was terminated, the supernatant was discarded, and the cells were carefully washed twice with PBS. 2 ml of pure methanol or acetic acid / methanol prepared in a ratio of 1:3 was added for fixation for 15 minutes. The fixing solution was removed, an appropriate amount of Giemsa staining solution was added for 30 minutes, and then the staining solution was slowly washed away with running water and dried upside down on filter paper. The culture plate / dish was inverted, and the clones were directly counted with the naked eye and photographed. Colony formation rate = (number of clones / number of inoculated cells) × 100%. The results showed that APOD inhibited the proliferation of cervical cancer cells ( Figure 4 A).

[0073] 2. AnnexinV-PE / 7-AAD Staining Experiment: Take the cells in the logarithmic growth phase of each group. After digestion with trypsin without EDTA, centrifuge at 300g for 5 minutes to collect the cells. Dilute 4×Binding Buffer (4 mL binding buffer + 12 mL deionized water) 4-fold with deionized water. Wash the cells 2 times with PBS pre-cooled at 4°C, each time centrifuging at 300g, 4°C for 5 minutes. Resuspend the cells with 250 μL of 1×Binding Buffer and adjust its concentration to 1×106 cells / mL. Take 100 μL of the cell suspension into a 5 mL flow tube, add 5 μL of Annexin V / PE and 10 μL of 7-AAD, mix gently, react in the dark at room temperature for 15 minutes, and then add 400 μL of 1×Binding Buffer and mix well. The sample is detected within 1 hour. The results show that APOD promotes apoptosis of cervical cancer cells ( Figure 4 B).

[0074] Example 5 APOD Inhibits Migration and Invasion Phenotypes of Cervical Cancer Cells

[0075] To investigate the effect of APOD on the migration and invasion of cervical cancer cells, in vitro scratch assay, Transwell chamber migration and invasion assay, F-actin staining of cytoskeletal proteins, and Western Blot were performed to detect the expression of epithelial-mesenchymal transition-related proteins.

[0076] 1. Scratch Assay: Seed the cells in a 6-well plate until the cells reach about 80%-90% confluence. Treat with 1 μg / mL mitomycin for 1 hour. Use a 200 μL pipette tip to draw a straight line or multiple straight lines on the cell monolayer to form a "scratch" or "wound". Gently rinse the cells with phosphate buffer to remove cell debris and loose cells in the scratch, and then replace the phosphate buffer with serum-free medium to reduce the impact of cell proliferation on the experimental results. Take pictures of the scratch area at 0 hours, 24 hours, 48 hours, and 72 hours of culture using a microscope to monitor the dynamic process of cell migration. Use image analysis software to measure the width of the scratch area and calculate the distance and speed of cell migration. The results show that the healing rate of cervical cancer cells overexpressing APOD is slower than that of the control group ( Figure 5 A), indicating that APOD inhibits the migration ability of cervical cancer cells.

[0077] 2. Transwell chamber migration assay: Add 50 μL of serum-free culture medium to each well and hydrate the basement membrane at 37°C for 30 minutes. Culture cervical cancer cells until the logarithmic growth phase, digest the cells, wash them once with PBS and then once with serum-free medium, resuspend the cells with serum-free medium, and count. Add 200 μL of cell suspension to the upper chamber of each well, seed 200,000 cells per well, add 500 μL of 2×FBS complete medium to the lower chamber, place the Transwell chamber in the incubator, and continue culturing for 72 hours. Take out the chamber, wash it twice with PBS, fix it with 4% paraformaldehyde for 15 minutes, then stain it with crystal violet for 20 minutes, wash it twice with PBS, take pictures at 100× and then perform cell counting to evaluate the cell migration ability. The results showed that the number of cells migrating in cervical cancer cells overexpressing APOD was less than that in the control group ( Figure 5 B), indicating that APOD inhibits the migration ability of cervical cancer cells.

[0078] 3. Transwell chamber invasion assay: Put Matrigel matrix gel in the refrigerator at 4°C overnight to melt it one night in advance. Dilute Matrigel matrix gel with serum-free medium (operate on ice and pre-cool the pipette tips), add 100 μL of matrix gel to each upper chamber, and place it in the incubator at 37°C for 1 hour to allow it to solidify. Culture cervical cancer cells until the logarithmic growth phase, digest the cells, wash them once with PBS and then once with serum-free medium, resuspend the cells with serum-free medium, and count. Add 200 μL of cell suspension to the upper chamber of each well, seed 200,000 cells per well, add 500 μL of 2×FBS complete medium to the lower chamber, place the Transwell chamber in the incubator, and continue culturing for 72 hours. Take out the chamber, wash it twice with PBS, carefully wipe off the matrix gel in the chamber with a cotton swab, fix it with 4% paraformaldehyde for 15 minutes, then stain it with crystal violet for 20 minutes, wash it twice with PBS, take pictures at 100× and then perform cell counting to evaluate the cell invasion ability. The results showed that the number of cells invading in cervical cancer cells overexpressing APOD was less than that in the control group ( Figure 5 C), indicating that APOD inhibits the invasion ability of cervical cancer cells.

[0079] 4. F-actin staining experiment of cytoskeletal protein: Culture cells until 70-80% confluence, fix them with 4% paraformaldehyde for 20 minutes, wash them 3 times with PBS, add PBS containing 0.1% Triton X-100 to the fixed cells, let it stand for 5 minutes to increase permeability, and then wash the cells 3 times with PBS. Add the working solution of phalloidin conjugate, incubate at room temperature for 60 minutes, and then wash 3 times with PBS. Add the mounting medium and take pictures for observation. The results showed that the F-actin staining in cells overexpressing APOD was weakened ( Figure 5 D).

[0080] 5. Detection of the expression of epithelial-mesenchymal transition-related proteins by Western Blot: For the specific steps, please refer to Step 2 of Example 1. The primary antibodies used were APOD, E-cadherin, Claudin-1, N-cadherin, Vimentin, β-catenin, Snail, and β-actin, and they were incubated with the secondary antibodies matching the primary antibodies. The results showed that APOD inhibited the occurrence of epithelial-mesenchymal transition ( Figure 5 E).

[0081] Example 6 APOD inhibits the self-renewal ability of cervical cancer cells

[0082] To investigate the effect of APOD on the self-renewal ability of cervical cancer cells, the inventors conducted a spheroid formation assay. The specific steps were as follows: Cervical cancer cells in the logarithmic growth phase were digested and centrifuged, and then resuspended with serum-free conditioned medium (DMEM / F12 + B27 + EGF + bFGF). 100 cells were added to each well, and the cells were cultured in suspension in a low-adhesion round-bottom 96-well cell culture plate. After 7 days, tumor cell spheres were formed, and the area of the cell spheres was measured by photographing under a microscope. The results showed that the two-dimensional area of the tumor spheres overexpressing APOD was smaller than that of the control group ( Figure 6 ). It is indicated that APOD inhibits the self-renewal of cervical cancer cells.

[0083] Based on the above examples, it is shown that APOD can be used as a molecular marker for regulating the malignant phenotype of cervical cancer and can be used for the diagnosis, treatment, and prognosis evaluation of cervical cancer, providing a new diagnosis and treatment strategy for cervical cancer patients.

[0084] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, 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 spirit and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

[0085] Sequence Listing

[0086] SEQ ID NO:1

[0087] MVMLLLLLSALAGLFGAAEGQAFHLGKCPNPPVQENFDVNKYLGRWYEIEKIPTTFENGRCIQANYSLMENGKIKVLNQELRADGTVNQIEGEATPVNLTEPAKLEVKFSWFMPSAPYWILATDYENYALVYSCTCIIQLFHVDFAWILARNPNLPPETVDSLKNILTSNNIDVKKMTVTDQVNCPKLS

[0088] SEQ ID NO:2

[0089]

Claims

1. Use of a biomarker APOD in preparing a reagent for diagnosing the occurrence and / or severity of cervical cancer, or preparing a reagent for predicting the occurrence and / or severity of cervical cancer, or preparing a reagent for evaluating the prognosis of cervical cancer.

2. The use according to claim 1, characterized in that: The reagent is used to detect the expression level of the biomarker APOD in a sample, preferably including the presence, absence or amount of the biomarker APOD, and / or comparing the amount of the biomarker APOD in the detected sample with a normal or reference expression level or standard.

3. The use according to claim 2, characterized in that: The detection of the biomarker APOD includes detection at the protein level; or detection at the nucleic acid level; Preferably, the reagent comprises at least one of a reagent for detecting the horizontal copy number of the APOD gene, a reagent for quantitatively detecting the RNA transcription level of APOD, and a reagent for quantitatively detecting the expression level of the APOD protein; Preferably, the reagents include primers, and the sequences of the primers are shown in SEQ ID NO:3 and SEQ ID NO:

4.

4. The use according to claim 2, characterized in that: The sample includes one of blood, cells and tissues, and preferably the tissue includes cervical cancer tissue and cervical cancer para-tissue.

5. The use according to claim 1, characterized in that: The diagnosis or assessment includes the following steps: Step a) detecting the expression level of APOD in the sample; Step b) diagnosing cervical cancer or evaluating the prognosis of cervical cancer based on the test results of step a); It is preferably used to determine the cervical cancer staging, which includes T staging and N staging.

6. The use according to claim 5, characterized in that: The sample in step a) includes control tissue (such as normal cervical tissue) and tumor tissue (such as cervical tumor area tissue); Step b) comprises comparing the expression level of APOD in the tumor tissue with that in the control tissue, i. If the expression level of APOD in tumor tissue is significantly lower than that in control tissue, cervical cancer is diagnosed; ii. If the expression level of APOD in tumor tissue is comparable to that in control tissue, cervical cancer is excluded; Preferably, compared with the control tissue, the more obvious the reduction in the expression level of APOD is, the greater the severity of cervical cancer is, and the greater the risk of metastasis is; Preferably, the APOD gene is significantly down-regulated in cervical cancer tissues of cervical cancer patients compared with paired normal cervical tissues distal to the cancer; the APOD protein is significantly down-regulated in cervical cancer tissues of cervical cancer patients compared with paired normal cervical tissue samples distal to the cancer.

7. The use according to claim 5, characterized in that: The sample in step a) is cervical cancer tissue; Step b) comprises performing auxiliary staging of cervical cancer based on the expression level of APOD in cervical cancer tissue: i. If APOD is positively expressed in cervical cancer tissue, the patient tends to have early T stage (T1-T2) and no lymph node metastasis (N0); ii. If APOD expression is negative in cervical cancer tissue, the patient tends to have advanced T stage (T3-T4) and lymph node metastasis (N1).

8. Use of a biomarker APOD regulator in the preparation of a drug for preventing or treating cervical cancer, in, The regulator includes a promoter, agonist or activator of the APOD gene or its expression product.

9. A method for screening candidate drugs for preventing and / or treating cervical cancer, and / or improving the prognosis of cervical cancer, characterized in that: The method includes detecting the effect of the candidate drug on the level of the biomarker APOD in the sample of the user, wherein, after using the candidate drug, the biological function of the biomarker APOD is improved or enhanced, indicating that the candidate drug has the effect of preventing and / or treating cervical cancer, and / or improving the prognosis of cervical cancer; the improvement or enhancement of the biological function of the biomarker APOD includes an increase in the expression level of the biomarker APOD.

10. A method for predicting or evaluating the prognosis of whether a patient or subject is suitable for APOD targeted therapy, the method comprising: The APOD level in the blood sample of the patient or subject is used to predict or evaluate the prognosis of whether the patient or subject is suitable for APOD targeted therapy.

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