Application of p62 and / or DNA-PKcs in diagnosis, prognosis and treatment of cervical cancer

Abstract

The invention discloses an application of p62 and / or DNA-PKcs in diagnosis of cervical cancer, p62 or a combination of p62 and a DNA damage repair factor DNA-PKcs can be used as a prognosis biomarker of cervical cancer, and also discloses an application of p62 alone or a combination of p62 and DNA-PKcs as a target in screening of drugs for treating cervical cancer. The invention provides a novel cervical cancer diagnosis and prognosis marker which can clearly characterize the occurrence and development of cervical cancer; the marker shows a specific high expression phenomenon in human clinical cervical cancer tissues compared with normal tissues; high expression is also shown in a human cervical cancer cell line. Knock-down of p62 can inhibit proliferation and migration ability of cervical cancer cells, and significantly inhibit growth of subcutaneous transplantation tumors in nude mice. The invention provides a new marker and target for diagnosis, prognosis and treatment of cervical cancer, and can provide a new strategy and direction for treatment of cervical cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to the application of p62 and / or DNA-PKcs in the diagnosis, prognosis and treatment of cervical cancer. Background Art

[0002] Cervical cancer is the fourth most common cancer among women globally. 85% of cases occur in developing countries. Cervical cancer (CC) is one of the main causes of death among women worldwide, with approximately 530,000 new cases and 275,000 deaths each year. Persistent infection with high-risk human papillomavirus (HPV) is the main cause of cervical cancer. High-risk HPV infection causes normal epithelial tissues to develop into low-grade and high-grade cervical intraepithelial neoplasia (CIN), and further develop into cancer. Although HPV vaccination can prevent the occurrence of cervical cancer, it cannot completely cover all types of HPV infections. In addition, the vaccine itself has no therapeutic effect. For those patients who have been diagnosed with cancer, they can only be effectively treated through means such as surgery, radiotherapy and chemotherapy. Moreover, patients with advanced and high-grade tumors often have the risk of recurrence and metastasis, and the prognosis is poor. Therefore, to reduce the mortality rate of cervical cancer patients, develop new anti-cancer drugs, and find molecular targets in the process of cervical cancer formation and development is of great significance.

[0003] To study the targeted therapy of tumors and develop new drugs, in-depth understanding of the molecular mechanism of tumorigenesis is the basis. Research shows that autophagy plays different roles in the processes of tumor cell growth, development, maturation and death. It is reported that autophagy plays a dynamic tumor-suppressive or tumor-promoting role in different contexts and stages of tumor development. In early tumorigenesis, autophagy, as a survival pathway and quality control mechanism, can prevent tumorigenesis and inhibit cancer progression. Once the tumor progresses to the advanced stage and is under environmental stress, autophagy, as a dynamic degradation and recycling system, contributes to the survival and growth of established tumors and promotes the invasiveness of cancer by promoting metastasis. Autophagy is becoming an important biological mechanism for human cancers including cervical cancer. Some studies show that high-risk HPV promotes the development of cervical cancer by inhibiting autophagy. In human cervical squamous cell carcinoma, the expression of some proteins that play important roles in autophagy, such as Beclin-1 and LC3B, is downregulated. These research progress indicate that autophagy plays an important role in the development and treatment of cervical cancer.

[0004] p62 (sequestosome-1, SQSTM1) is a multifunctional ubiquitin-binding protein that participates in two protein degradation processes, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome system. It is an adaptor protein between autophagosomes and substrates and plays a role as a molecular regulator in the process of autophagy. Its abnormal accumulation can cause malignant transformation of cells and lead to the occurrence of tumors. p62 has multiple domains that can interact with a variety of proteins and regulate various cellular functions. Among them, the LIR region contributes to the direct interaction with LC3, enabling p62 to be specifically degraded by autophagy. During normal autophagy, p62 is continuously degraded. In addition, studies have shown that the expression rate of p62 in cervical cancer tissues and metastatic lymph node tissues is higher than that in normal tissues, indicating that p62 may be involved in the occurrence and development of cervical cancer. The follow-up results also show that the 3-year survival rate of patients with high p62 expression is significantly lower than that of patients with low expression. Therefore, studying the role and mechanism of p62 in cervical cancer has very important clinical significance. Summary of the Invention

[0005] The object of the present invention is to provide an application of p62 and / or DNA-PKcs in the diagnosis, prognosis and treatment of cervical cancer in view of the above problems.

[0006] In order to achieve its object, the technical solution adopted by the present invention is:

[0007] The first aspect of this aspect provides an application of a reagent for detecting the expression of the biomarker p62 in the preparation of a product for auxiliary diagnosis or diagnosis of cervical cancer subjects.

[0008] The method of the diagnosis includes: obtaining a test sample from a subject suspected of having cervical cancer, determining the expression level of p62 in the test sample, and if the expression of p62 is higher than that of the normal control, it indicates that the subject is a high-risk population or a patient with cervical cancer.

[0009] The second aspect of this aspect provides an application of a reagent for detecting the expression of a biomarker in the preparation of a product for prognosis of cervical cancer subjects, where the biomarker is p62, or p62 and the DNA damage repair factor DNA-PKcs are combined as a prognostic biomarker.

[0010] The method of the prognosis includes: obtaining a test sample from a subject with cervical cancer, determining the expression level of p62 or p62 and DNA-PKcs in the test sample, and high expression of p62 or p62 and DNA-PKcs indicates poor prognosis of the patient.

[0011] The third aspect of this aspect provides an application of p62 alone or p62 combined with DNA-PKcs as a target in screening drugs for the treatment of cervical cancer.

[0012] The drug inhibits the expression of p62 or inhibits the expression of p62 and DNA-PKcs, thereby inhibiting the viability, proliferation, invasion and migration of cervical cancer cells;

[0013] p62 affects the biological functions of cervical cancer cells by regulating DNA-PKcs in the NHEJ pathway and regulates DNA double-strand breaks.

[0014] The fourth aspect of this aspect provides the use of a p62 expression inhibitor in the preparation of a drug for treating cervical cancer.

[0015] The expression inhibitor includes nucleic acid molecules, proteins, and compounds.

[0016] Preferably, the nucleic acid molecule includes shRNA.

[0017] Preferably, the shRNA is shp62-1 or shp62-3 or shp62-4.

[0018] The sense strand sequence of shp62-1 is: 5'-CGAGGAATTGACAATGGCCAT-3';

[0019] The antisense strand sequence of shp62-1 is: 5'-ATGGCCATTGTCAATTCCTCG-3';

[0020] The sense strand sequence of shp62-3 is: 5'-GCAGATGAGAAAGATCGCCTT-3

[0021] The antisense strand sequence of shp62-3 is: 5'-AAGGCGATCTTTCTCATCTGC-3';

[0022] The sense strand sequence of shp62-4 is: 5'-CCGAATCTACATTAAAGAGAA-3'

[0023] The antisense strand sequence of shp62-4 is: 5'-TTCTCTTTAATGTAGATTCGG-3'.

[0024] The beneficial effects of the present invention are:

[0025] New cervical cancer diagnosis and prognosis markers are provided, which can clearly characterize the occurrence and development of cervical cancer; this marker shows a phenomenon of high specific expression in human clinical cervical cancer tissues compared with normal tissues; it also shows high expression in human cervical cancer cell lines. Knocking down p62 can inhibit the proliferation and migration ability of cervical cancer cells and significantly inhibit the growth of subcutaneous xenografts in nude mice. The present invention provides new markers and targets for the diagnosis, prognosis and treatment of cervical cancer, and can provide new strategies and directions for the treatment of cervical cancer. Brief Description of the Drawings

[0026] Figure 1 Shows the expression of p62 in cervical cancer cells: A. Detection results by immunohistochemistry; B. Protein expression of p62 in normal cervical epithelial cells, cervical squamous cancer cells and cervical adenocarcinoma cells; C. mRNA expression levels of p62 in normal cervical epithelial cells, cervical squamous cancer cells and cervical adenocarcinoma cells.

[0027] Figure 2 Shows the results of Western Blot detection of the protein level of p62 in the cell line with knocked-down p62 (A) and the results of fluorescence quantitative PCR detection (B).

[0028] Figure 3 Shows: A. EDU experiment to detect the effect of knocking down p62 on the proliferation ability of SiHa and HeLa cells; B. Transwell experiment to detect the changes in the migration and invasion ability of SiHa and HeLa cells after knocking down p62.

[0029] Figure 4 Shows the experiment to verify the effect of p62 on DNA damage in cervical cancer cells: A. IF staining results; B. Comet assay results.

[0030] Figure 5 Shows that Western blot detected the changes in related genes in the HR (A) and NHEJ (B) pathways and the change in the protein level of DNAPKcs in SiHa cells after knocking down p62 (C).

[0031] Figure 6 Is the survival analysis result of cervical cancer patients.

[0032] Figure 7 Shows the CCK-8 experiment results of DNAPKcs inhibiting the proliferation of cervical cancer cells.

[0033] Figure 8 Shows the effect that knocking down p62 significantly inhibits the growth of subcutaneous xenografts of SiHa cells in nude mice.

[0034] Figure 9Shows the effect of knocking down p62 on the formation of subcutaneous xenografts in nude mice: A. HE staining results; B. IHC results. Detailed implementation methods

[0035] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereby.

[0036] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0037] Embodiment 1

[0038] 1 Materials

[0039] 1.1 Tissue samples

[0040] Pathological sections of human cervical cancer tissues, cervical intraepithelial neoplasia (CIN) tissues, and normal cervical tissues were provided by the Second Affiliated Hospital of Chongqing Medical University, and the tissue samples have been clearly diagnosed by the Pathology Department of the Second Affiliated Hospital of Chongqing Medical University. The Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University has approved the use authorization of the tissues in this study. All cervical cancer patients have been informed and consented before this study.

[0041] 1.2 Cells

[0042] The immortalized human normal cervical epithelial cells H8 were purchased from Shanghai Qingqi Biotechnology Co., Ltd.; the human cervical cancer cells SiHa and HeLa were purchased from Wuhan Punosai Life Science Co., Ltd.

[0043] 1.3 Molecular biology related sequences

[0044] 1) The shRNA plasmid targeting p62 was purchased from Sigma. The sequence of shRNA is as follows:

[0045]

[0046] 2) The sequences of primers for RT-qPCR were designed using the NCBI website and synthesized by Tsingke Biotechnology. The primer sequences are as follows:

[0047]

[0048] 2 Methods

[0049] 2.1 Immunohistochemistry (IHC)

[0050] (1) Baking the slices: Place the paraffin sections in an oven at 60 °C for 1 h.

[0051] (2) Dewaxing and Hydration: Immediately after taking out the sections, perform dewaxing and hydration in the following order: xylene (I) for 15 min, xylene (II) for 15 min, 100% ethanol for 5 min, 95% ethanol for 5 min, 80% ethanol for 3 min, 75% ethanol for 3 min, and finally wash with tap water for 3 min.

[0052] (3) Antigen Retrieval: In accordance with the instruction manual of the antibody to be detected (p62, 66184-1-Ig), place the tissue sections rinsed with tap water in the heated and boiling EDTA retrieval solution (pH 9.0, Solarbio, C1034) and perform retrieval in a boiling water bath for 20 min. Then turn off the induction cooker and wait for the retrieval solution to cool naturally to room temperature. Wash three times with PBS (3 min each time).

[0053] (4) Blocking: Gently blot the excess washing solution PBS on the tissue sections with lens paper, circle the tissue using an immunohistochemistry pen, and add a sufficient amount of endogenous peroxidase blocking solution (Beyotime, P0100B) to cover the tissue and incubate at room temperature for 10 min. After the blocking process is completed, immerse the tissue sections in PBS and wash three times (3 min each time).

[0054] (5) Sealing: Remove the excess PBS outside the tissue, add the immunostaining sealing solution (Beyotime, P0260) to completely cover the tissue, place the sections flat on a wet box, and incubate in an incubator at 37°C for 30 min.

[0055] (6) Incubating with Primary Antibody: Carefully blot the excess sealing solution on the sections with lens paper, add the diluted primary antibody (p62, 1:200) to completely cover the tissue, and incubate in a 4°C refrigerator overnight.

[0056] (7) Take out the wet box the next day. After rewarming the tissue sections at 37°C for 30 min, wash three times with PBS (3 min each time).

[0057] (8) Incubating with Secondary Antibody: Discard the excess washing solution outside the tissue, add the HRP-labeled goat anti-mouse / anti-rabbit IgG secondary antibody (Zsbio, PV-6000) to cover the tissue, place it flat in a wet box, and incubate in an incubator at 37°C for 30 min. After the incubation of the secondary antibody is completed, wash three times with PBS (3 min each time).

[0058] (9) DAB Chromogenic Reaction: Prepare the DAB substrate solution (Zsbio, PV-8000) according to the instruction manual, add it to the tissue, observe and control the chromogenic time under a microscope. When the antibody staining shows a brownish yellow color at about 30 s - 40 s, immediately terminate the chromogenic reaction with tap water.

[0059] (10) Nuclear counterstaining: The nuclei of tissue sections were stained with hematoxylin (Solarbio, G1140). After observing that the nuclei showed blue-violet color for about 45 s - 60 s, they were immediately immersed in tap water for blueing.

[0060] (11) Dehydration, clearing, mounting and imaging: The sections were dehydrated using gradient ethanol and xylene (in the reverse order of dewaxing). After the tissue sections were air-dried naturally in a fume hood, they were mounted with neutral resin (Biosharp, BL704A). After the sections were dried, the images were observed and photographed under an OLYMPUS BX53 microscope.

[0061] 2.2 Immunoblotting (Western blot)

[0062] 2.2.1 Preparation of cell protein samples

[0063] The culture medium was discarded, and the cells were washed 1 - 2 times with pre-cooled PBS. Then the cells were digested with 0.25% trypsin, and the cell pellet was collected after centrifugation. RIPA lysis buffer (Solarbio, R0010) containing protease inhibitor and phosphatase inhibitor PIC (MCE, HY-K0013) was added to lyse the cells on ice for 30 min. Vortex on ice every 10 min, centrifuge at 12,000 rpm and 4 °C for 10 min, collect the supernatant lysate, quantify the protein concentration using a BCA protein assay kit (Beyotime, P0010S), add 5× protein loading buffer, denature at 95 °C for 10 min, and the protein sample after brief centrifugation can be directly used for Western Blot or stored at -80 °C.

[0064] 2.2.2 Preparation of SDS-PAGE gel

[0065] 2.3 RT-PCR

[0066] Total RNA in cells was extracted by the Trizol method. Referring to the instruction manual of the kit (ABclonal, RK20429), the residual genomic DNA in the RNA template was removed, and the RNA was reverse transcribed into cDNA. Then it was detected by real-time fluorescence quantitative PCR (probe method). The designed specific primer sequences were synthesized by Tsingke Biological Company (China). Subsequently, the cDNA template, specific primers, and SYBR Green Fast qPCR Mix reagent (RK21203, ABclonal, China) were mixed to amplify the target gene, and RT-qPCR reaction was carried out on a Roche LightCycler 480 II system. The expression of p62 was normalized to GAPDH or β-actin. The relative expression was calculated using the 2-ΔΔCT method.

[0067] 2.4 Lentivirus packaging

[0068] Feeder cells: Digest HEK 293T cells in the logarithmic growth phase with trypsin digestion solution, resuspend them in the culture medium, take 1 / 5 of the total volume of the suspension and seed it in a 6-cm culture dish, and supplement with complete culture medium.

[0069] After culturing for about 24 h, perform lentivirus packaging according to the following system.

[0070] Reagent 1 Dosage pMD2.G 0.5 μg psPAX2 2.25 μg Empty plasmid / Target plasmid 2.25 μg FBS-free DMEM 250 μL Reagent 2 Dosage PEI 15 μL FBS-free DMEM 250 μL

[0071] After preparing the above two reagents separately, let them stand at room temperature for 5 minutes. Mix the prepared reagent 1 and reagent 2 together, pipette more than fifteen times to make them fully mixed evenly. Let it stand at room temperature for 15 minutes to allow the PEI transfection reagent to fully bind to the added plasmid. Add a total of 500 μl of the above mixed solution to the culture dish containing HEK 293T cells, mix crosswise in the biosafety cabinet, and place it in an incubator at 37 °C for culture. Replace the medium with fresh medium after 6 - 8 h. After 48 h and 72 h of transfection, transfer all the cell supernatant into a 15-mL centrifuge tube, centrifuge at 4 °C and 1000 rpm for 5 minutes, retain the supernatant, and filter the supernatant with a 0.22 uM filter. Then aliquot the virus supernatant and place it in a sterile 1.5-mL EP tube, and transfer it to a -80 °C refrigerator for storage.

[0072] 2.5 Lentivirus infection and construction of stable cell lines

[0073] (1) Feeder cells: Digest HeLa and SiHa cells in the logarithmic growth phase, add the culture medium to make a cell suspension, and take about 1.25×10 6 Seed it in a 6-cm culture dish.

[0074] (2) Infect with lentivirus: After 24 h, when the cell density is about 40 - 50%, thaw the virus supernatant on ice, aspirate the original medium completely, then add 2.75 mL of fresh complete culture medium and 1.25 mL of virus supernatant to the cell culture dish, and at the same time add 5 μl of 1 mg / mL polybrene. Mix crosswise and continue to culture in a 37 °C cell culture incubator. Replace the medium with fresh medium after 6 - 8 h.

[0075] (3) Resistance screening: After 48 h of infecting the cells with lentivirus, change the medium to complete culture medium containing puromycin (HeLa: 1 μg / μL and SiHa: 1 μg / μL), and perform resistance screening on the cells with puromycin. Screen for about one week. When no more cells die, obtain stable cell lines for subsequent experiments.

[0076] 2.6 CCK8 assay

[0077] (1) Select SiHa and HeLa sh-NC, shp62-1, and shp62-3 cells in the logarithmic growth phase, digest them into cell suspensions with 0.25% trypsin, count the cells using a cell counting plate, and then inoculate 3000 cells / well into a 96-well plate. Generally, set 5 replicates, with each well containing 100 μL of complete medium. Place it in a 5% CO2, 37 °C incubator.

[0078] (2) After the cells are completely adherent 6 h after inoculation, add 10 μL of CCK-8 solution to each well, incubate at 37 °C for 1 h, place the 96-well plate on a multifunctional microplate reader, and measure the absorbance at 450 nm.

[0079] (3) According to the experimental requirements, set up blank wells (medium without cells and the substance to be tested), control wells (medium containing cells, CCK-8, without the substance to be tested), and experimental wells (medium containing cells, CCK-8, and the substance to be tested). Continuously monitor the absorbance of the cells at 0 h, 24 h, 48 h, 72 h, and 96 h.

[0080] (4) Import the OD450 values obtained from different groups into GraphPad Prism 8, plot the cell proliferation curve, and perform statistical analysis.

[0081] 2.7 Transwell Assay

[0082] (1) Coat the basement membrane with Matrigel: In the invasion assay, add 30 - 50 μL of diluted Matrigel to the upper layer of the Transwell chamber to completely cover the basement membrane, incubate in a 37 °C incubator for 1 - 2 h to polymerize Matrigel into a gel, and aspirate the residual liquid on the upper layer of the basement membrane (this step can be skipped in the migration assay). Hydrate the basement membrane: Add 50 μL of serum-free medium.

[0083] (2) Seed cells: To exclude the influence of serum, after trypsinizing SiHa and HeLa cells in the logarithmic growth phase, prepare cell suspensions with serum-free medium. After counting the cells, seed 150 μL of 5×10 4 cells on the upper chamber, and 600 μL of medium containing 20% FBS on the lower chamber. Then place it back in a 37 °C cell incubator and continue culturing for 48 h.

[0084] (3) Fix the cells: Fix with 4% paraformaldehyde at room temperature for 20 min, and wash 3 times with PBS.

[0085] (4) Crystal violet staining: Discard the fixing solution, add crystal violet to the 24-well plate, stain the migrated cells for 15 min, use forceps to pick up the chamber and rinse it moderately with ddH 2 2O to remove the floating color, and air-dry the chamber naturally.

[0086] (5) Photographing and result analysis: Place the stained chamber on a clean glass slide and collect images under an upright microscope. When taking pictures, select 3-5 fields of view under the microscope to photograph the bottom surface of the upper chamber. Use Image J software to analyze the number of migrated / invasive cells, perform statistical analysis on the data of 3 independent replicates, and import the data into GraphPad Prism 8 to draw a bar chart.

[0087] 2.8 Comet assay experiment

[0088] (1) Select a comet assay kit (Trevigen, 4250-050-K). Before the experiment, the following need to be prepared: PBS (without Ca 2+ and Mg 2+ ), lysis buffer (pre-cooled at 4 °C for at least 30 min), low melting point agarose LMA (heated in a water bath until melted and kept at 37 °C), Gold staining solution (diluted with DMSO and stored in the dark at 4 °C).

[0089] (2) Cell sample preparation: Mix PBS containing 1 x 10 5 / ml cells and pre-melted low melting point agarose LMA at 37 °C in a volume ratio of 1:10. Then pipette 50 μL of the cell-containing LMA onto the frosted surface of a pre-warmed glass slide and let it solidify at 4 °C for 30 min - 45 min.

[0090] (3) Immerse the glass slide in pre-cooled cell lysis buffer at 4 °C and incubate overnight.

[0091] (4) DNA unwinding: After cell lysis, take out the glass slide, rinse it twice with PBS to remove the high-concentration salt on the surface of the glass slide, and then place the glass slide in neutral electrophoresis buffer for 0.5 h to unwind the DNA to form single-stranded DNA, which is easy to migrate in the electrophoresis field.

[0092] (5) Single cell electrophoresis: Prepare a Comet ES device, add 850 mL of freshly prepared pre-cooled electrophoresis buffer at 4 °C to the electrophoresis tank, and ensure that the liquid level covers the glass slide by 0.5 cm. Set the electrophoresis conditions at 21 V and 4 °C for 45 min.

[0093] (6) DNA precipitation: After electrophoresis, blot the residual electrophoresis buffer outside the glass slide with filter paper, and immerse the glass slide in DNA precipitation solution for 30 min.

[0094] (7) Gold staining: Immerse the glass slide in 70% ethanol and incubate at room temperature for 30 min. Place the glass slide in a 37 °C oven to dry for 10 - 15 min. Then add 100 μL of the diluted Gold staining solution, stain at room temperature in the dark for 30 min, wash away the SYBR staining solution at the edge of the sample with distilled water, and place the glass slide in a 37 °C incubator to dry.

[0095] (8) Observation of experimental results and data processing: Observe and take pictures of the glass slide under an upright fluorescence microscope, and use CASP comet analysis software to analyze the percentage of DNA in the comet tail, which is used to evaluate the comet formation rate of cells.

[0096] 2.9 EdU experiment

[0097] For experimental procedures, refer to the instruction manual of EdU Imaging Kits (Cy3) (APExBIO K1075).

[0098] 2.10 Establishment of tumor animal models

[0099] (1) After one week of adaptive growth of nude mice, weigh the mice and sort them. Combine with the use of a random number table method to randomly divide them into two groups, n = 6 mice / group, and label them as the Scramble group and the shp62 group respectively.

[0100] (2) Take 100 μL of SiHa cells with stable knockdown of p62 and SiHa cells with empty vector control, mix them with Matrigel matrix glue at a ratio of 1:1, and inject them subcutaneously into the left posterior back of nude mice. Inject 6×10 6 cells into each nude mouse.

[0101] (3) Every 4 days after injecting tumor cells, use a vernier caliper to measure the long diameter and short diameter of the tumor, calculate the volume of the tumor mass, and the calculation formula: tumor volume = long diameter × short diameter 2 / 2, and draw a tumor growth curve according to the calculation results. At an appropriate time point, euthanize the nude mice by cervical dislocation. After taking pictures of the gross morphology of the nude mice, take out the tumor mass, weigh it and take pictures.

[0102] 2.11 HE staining

[0103] 3 Results

[0104] 3.1 Expression of p62 in cervical cancer cells

[0105] First, we used immunohistochemistry to detect the protein levels in normal cervical tissues, precancerous tissues and cancer tissues. The results are as Figure 1 shown in A. The protein level of p62 in cancer tissues is higher than that in normal tissues and precancerous tissues. We also detected the expression levels of p62 in various cervical cancer cells, where H8 is normal cervical epithelial cells, and SiHa and HeLa are squamous cell carcinoma and adenocarcinoma cells positive for HPV respectively. The results showed ( Figure 1In B-C), compared with normal cervical epithelial cells, both the protein and mRNA levels of HPV-positive cells were elevated.

[0106] 3.2 Successfully constructed a cell line with stable knockdown of p62

[0107] To explore the relationship between p62 and the occurrence and development of cervical cancer, we constructed a cell line with stable knockdown of p62 and detected the protein level of p62 by Western Blot. The results showed that the protein level of p62 was significantly lower than that of the control group ( Figure 2 A). The experimental detection by fluorescence quantitative PCR yielded consistent results ( Figure 2 B). A series of results indicated that the cell line with stable knockdown of p62 had been successfully constructed and subsequent experiments could be carried out.

[0108] 3.3 Knockdown of p62 inhibits the proliferation, migration and invasion of cervical cancer cells

[0109] To study whether p62 regulates the occurrence and development of cervical cancer, the EDU experiment was used to detect the effect of p62 knockdown on the proliferation ability of SiHa and HeLa cells. The experimental results showed that p62 knockdown significantly inhibited the growth rate of SiHa and HeLa cells ( Figure 3 A). Meanwhile, we also used the Transwell experiment to detect the changes in the migration and invasion abilities of SiHa and HeLa cells after p62 knockdown ( Figure 3 B). The Transwell experiment showed that the number of cells migrating from the upper chamber to the lower chamber in the p62 knockdown group was less than that of the control group, and the difference was statistically significant. The above results indicated that p62 knockdown significantly inhibited the proliferation, migration and invasion of cervical cancer cells.

[0110] 3.4 p62 can regulate DNA double-strand breaks

[0111] To verify the effect of p62 on DNA damage in cervical cancer cells, we first used the western blot experiment to detect the effect of p62 knockdown on DNA double-strand breaks in cervical cancer cells. As Figure 4 A shows, compared with the control group, p62 knockdown increased DNA double-strand breaks in SiHa cells. In addition, the comet assay experiment showed that p62 knockdown did significantly increase DNA double-strand breaks in SiHa cells ( Figure 4 B). These results indicated that p62 could affect DNA double-strand breaks in cervical cancer cells.

[0112] 3.5 Knockdown of p62 mainly affects the NHEJ pathway in DNA damage

[0113] To detect how p62 affects DNA damage in cervical cancer cells, we used western blot to detect the changes in related genes in the HR and NHEJ pathways in SiHa cells after knocking down p62. As Figure 5 shown in Figure 5 Figure A, after knocking down p62, the protein levels of ATM and ATR in the HR pathway did not change significantly, while the protein level of DNA-PKcs (a DNA damage repair factor) in the NHEJ pathway increased significantly (

[0114] Figure B). This suggests that p62 may affect the biological functions of cervical cancer cells by regulating DNA-PKcs.

[0115] To detect whether the effect of DNA-PKcs on cervical cancer is similar to that of p62, we detected the expression of DNA-PKcs in cervical cancer cell lines. Western blot results showed that the protein level of DNA-PKcs was significantly increased in cervical cancer cells SiHa and HeLa compared with the immortalized normal cervical epithelial cell line H8 ( Figure 5 Figure C).

[0116] 3.7 Poor prognosis in cervical cancer patients with high expression of DNA-PK

[0117] We performed survival analysis on 146 cervical cancer patients with low expression of DNA-PK and 146 cervical cancer patients with high expression of DNA-PK using the GEPIA online website. The analysis results showed that the overall survival (OS) of cervical cancer patients with high expression of DNA-PK was shorter than that of patients with low expression, that is, high expression of DNA-PK was positively correlated with poor prognosis of cervical cancer patients, and p62 was highly correlated with DNA-PK ( Figure 6 Figure).

[0118] 3.8 Inhibition of DNA-PKcs inhibits the proliferation of cervical cancer cells

[0119] Next, we detected the effects of inhibiting the function of DNA-PKcs on the proliferation, migration and invasion of cervical cancer cells. The CCK-8 assay showed that the growth rate of SiHa cells in the DNA-PKcs inhibition group (the DNA-PKcs inhibitor was NU 7026, from MCE, catalog number: HY-15719) was significantly inhibited compared with the control group ( Figure 7 Figure).

[0120] 3.9 Knocking down p62 significantly inhibits the growth of subcutaneous xenografts of SiHa cells in nude mice

[0121] To further verify the promoting effect of p62 on the occurrence and development of cervical cancer, we established a subcutaneous xenograft tumor model in nude mice by subcutaneous injection to observe the effect of knocking down p62 on the growth of cervical cancer SiHa cells. The experiment was divided into a control group and a shp62 group. The control group was injected with SiHa cells carrying an empty vector, and the shp62 group was injected with SiHa cells stably knocking down p62. As Figure 8 can be seen, knocking down p62 significantly inhibited the size, weight, and growth rate of subcutaneous xenograft tumors.

[0122] 3.10 HE staining was used to verify the effect of knocking down p62 on the formation of subcutaneous xenograft tumors in nude mice

[0123] Paraffin sections of subcutaneous xenograft tumors in nude mice were subjected to HE staining experiments ( Figure 9 A) showed that in the control group, the morphological structure of tumor cells was disordered, with different sizes, a relatively large nuclear-cytoplasmic ratio, and darkly stained nuclei; in the p62 knockdown group, the cell structure was relatively intact, the proportion of nuclei was relatively small, and the nuclear staining was lighter. The IHC results ( Figure 9 B) indicated that the expression of Ki-67, a marker of tumor cell proliferation, was significantly downregulated in the p62 knockdown group.

Claims

1. Use of a reagent for detecting the expression of the biomarker p62 in the preparation of a product for auxiliary diagnosis or diagnosis of cervical cancer subjects.

2. The use according to claim 1, characterized in that: The diagnostic method comprises: obtaining a test sample from a subject suspected of having cervical cancer, determining the expression level of p62 in the test sample, and if p62 is highly expressed compared to a normal control, it indicates that the subject is a high-risk group for cervical cancer or a cervical cancer patient.

3. Use of a reagent for detecting the expression of a biomarker in the preparation of a product for prognosis of a subject with cervical cancer, characterized in that: The biomarker is p62, or p62 is combined with DNA damage repair factor DNA-PKcs as a prognostic biomarker.

4. The use according to claim 3, characterized in that: The prognosis method comprises: obtaining a test sample from a subject suffering from cervical cancer, and determining the expression level of p62 or p62 and DNA-PKcs in the test sample. High expression of p62 or p62 and DNA-PKcs indicates a poor prognosis for the patient.

5. Application of p62 alone or p62 combined with DNA-PKcs as targets in screening drugs for the treatment of cervical cancer.

6. The use according to claim 5, characterized in that: The drug inhibits the expression of p62 or inhibits the expression of p62 and DNA-PKcs, thereby inhibiting the vitality, proliferation, invasion and migration of cervical cancer cells; p62 affects the biological functions of cervical cancer cells by regulating DNA-PKcs in the NHEJ pathway and modulating DNA double-strand breaks.

7. Application of p62 expression inhibitors in the preparation of drugs for treating cervical cancer.

8. The use according to claim 7, characterized in that: The expression inhibitors include nucleic acid molecules, proteins, and compounds.

9. The use according to claim 8, characterized in that: The nucleic acid molecule includes shRNA.

10. The use according to claim 9, characterized in that: The shRNA is shp62-1 or shp62-3 or shp62-4, The sense strand sequence of shp62-1 is: 5′-CGAGGAATTGACAATGGCCAT-3′; The antisense sequence of shp62-1 is: 5′-ATGGCCATTGTCAATTCCTCG-3′; The sense strand sequence of shp62-3 is: 5'-GCAGATGAGAAAGATCGCCTT-3' The antisense sequence of shp62-3 is: 5′-AAGGCGATCTTTCTCATCTGC-3′; The sense strand sequence of shp62-4 is: 5'-CCGAATCTACATTAAAGAGAA-3' The antisense sequence of shp62-4 is: 5'-TTCTCTTTAATGTAGATTCGG-3'.

Images