Application of FEN1 as target spot in treatment of cervical small cell neuroendocrine cancer

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to application of FEN1 as a target spot in treatment of cervical small cell neuroendocrine cancer. In order to further explore molecular pathological reasons of the SCNECC and search for proper therapeutic targets or drugs, flap structure specific endonuclease 1 (FEN1) is used as a target gene for research, and the functions of FEN1 in the aspects of SCNECC cell proliferation, clone formation, migration, invasion, cell apoptosis and the like are verified through in-vitro and in-vivo experiments. And the molecular mechanism is preliminarily discussed by means of molecular biology. In addition, the influence of the inhibitor SC13 of FEN1 on cell biological behaviors such as SCNECC cell proliferation, clone formation, migration, invasion, cell apoptosis and the like is also verified. The result of the invention provides a new scientific basis for the molecular pathological reason of SCNECC, and provides a scientific evidence for FEN1 as a new therapeutic target.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the application of FEN1 as a target in the treatment of cervical small cell neuroendocrine carcinoma. Background Art

[0002] Cervical cancer is the fourth most common cancer in terms of morbidity and mortality among women. Small cell neuroendocrine carcinoma (SCNECC) is a rare subtype of cervical cancer, accounting for less than 1.0% of all cervical cancers. However, compared to other cervical cancer types, it has a more aggressive phenotype and rapid metastasis, resulting in a poorer prognosis. High-risk HPV infection is common in SCNECC patients, particularly HPV 18 and 16, suggesting that HPV infection may be a key cause of SCNECC development. However, studies have also shown that HPV-positive neuroendocrine cervical cancer cells are dependent on Myc rather than HPV E6 / E7 viral oncogenes. Furthermore, using modern sequencing technologies, several scientists have identified genetic mutations as a key contributor to SCNECC development, with PIK3CA and TP53 mutations being the most frequent. However, the etiology of SCNECC remains unclear, and guidelines for its treatment are currently unavailable. Due to the lack of information on its molecular etiology and its histopathological similarities to small cell lung cancer, current treatment regimens mimic those used for neuroendocrine lung cancer. Surgery is associated with a better prognosis for patients with stage IB3-IIA2 cancers, but the prognosis for SCNECC remains poor due to its frequent recurrences and high metastatic burden, with 3- and 5-year survival rates of 36.2% and 30.6%, respectively.

[0003] Therefore, further exploring the molecular pathological causes of SCNECC and finding appropriate therapeutic targets or drugs are extremely important for understanding the pathogenesis of SCNECC and improving the treatment outcomes of SCNECC. Summary of the Invention

[0004] In response to the above problems, the present invention uses flap structure-specific endonuclease 1 (FEN1) as the target gene for research. FEN1 is a 45kDa divalent metal-dependent protein with exonuclease and endonuclease activities. It is a core protein of the base excision repair (BER) pathway and is involved in the maturation of Okazaki fragments during DNA replication. The present invention verified the function of FEN1 in SCNECC cell proliferation, clone formation, migration, invasion and apoptosis through in vitro and in vivo experiments, and preliminarily explored its molecular mechanism through molecular biological methods. In addition, we also verified the effect of FEN1 inhibitor SC13 on cell biological behaviors such as SCNECC cell proliferation, clone formation, migration, invasion and apoptosis. The results of the present invention will provide a new scientific basis for the molecular pathological causes of SCNECC and provide scientific evidence for FEN1 as its new therapeutic target.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides the use of FEN1 as a target in the preparation of a drug for treating cervical small cell neuroendocrine carcinoma.

[0007] Furthermore, the drug is used to knock down FEN1.

[0008] Furthermore, knocking down FEN1 inhibits cancer cell proliferation, colony formation, invasion, migration ability and promotes cell apoptosis through PCNA.

[0009] In a second aspect, the present invention provides use of FEN1 siRNA in the preparation of a drug for treating cervical small cell neuroendocrine carcinoma.

[0010] Furthermore, the siRNA inhibits FEN1 expression, and the nucleotide sequence of the FEN1 siRNA is: S: GCCCGUGUAUGUCUUUGAUTT; AS: AUCAAAGACAUACACGGGCTT.

[0011] In a third aspect, the present invention provides a drug for treating cervical small cell neuroendocrine carcinoma, comprising a FEN1 inhibitor or FEN1 siRNA.

[0012] Furthermore, the FEN1 inhibitor is SC13.

[0013] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

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

[0015] This study used proteomic analysis of six pairs of cancerous and adjacent tissues to screen for differentially expressed proteins. The results revealed 2,169 significantly upregulated proteins and 162 significantly downregulated proteins. GO, KEGG, and STRING analyses of the differentially upregulated proteins identified FEN1 as a target protein. Immunohistochemistry confirmed that FEN1 expression was significantly higher in SCNECC cancer tissues than in adjacent tissues. In the SCNECC cell line TC-YIK, FEN1 was shown to regulate PCNA expression. FEN1 / PCNA knockdown and treatment with the FEN1 inhibitor SC13 significantly inhibited TC-YIK cell viability, colony formation, migration, and invasion, while promoting apoptosis. In vivo experiments in nude mice also demonstrated that FEN1 knockdown significantly inhibited SCNECC tumor growth. Both in vivo and in vitro studies demonstrated that FEN1 knockdown significantly reduced the mRNA and protein expression levels of FEN1, BCL-2, RB1, PCNA, and PIK3CA, while increasing the mRNA and protein expression levels of Caspase-9. In summary, FEN1 is highly expressed in SCNECC and can significantly affect the biological behavior of cancer cells through PCNA, promoting the occurrence and development of the disease. FEN1 is a potential therapeutic target for SCNECC. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the result of proteomic analysis of differentially expressed proteins in SCNECC;

[0017] Figure 2 This is a characteristic diagram of protein expression profile in SCNECC;

[0018] Figure 3 This is a diagram showing the detection results of the role of FEN1 in SCNECC;

[0019] Figure 4 This is a diagram verifying the regulatory relationship between FEN1 and PCNA in SCNECC;

[0020] Figure 5 This figure shows the effect of knocking down FEN1 on tumor growth in vivo;

[0021] Figure 6 This figure shows the effect of knocking down FEN1 on the biological functions of TC-YIK cells;

[0022] Figure 7 This figure shows the effect of FEN1 inhibitor SC13 on the occurrence and development of SCNECC. DETAILED DESCRIPTION

[0023] In order to further illustrate the technical solution of the present invention, the present invention is further described below through examples.

[0024] Example 1

[0025] In this embodiment, FEN1 is used as a target in the preparation of a drug for treating cervical small cell neuroendocrine carcinoma. The drug is used to knock down FEN1. Knockdown of FEN1 inhibits cancer cell proliferation, colony formation, invasion, migration ability and promotes cell apoptosis through PCNA.

[0026] Use of FEN1 siRNA in preparing a drug for treating cervical small cell neuroendocrine carcinoma, wherein the siRNA inhibits FEN1 expression.

[0027] A drug for treating cervical small cell neuroendocrine carcinoma includes a FEN1 inhibitor or, wherein the FEN1 inhibitor is SC13 or FEN1 siRNA.

[0028] As a further improvement, a pharmaceutically acceptable carrier is also included.

[0029] Example 2

[0030] 1. Materials and Methods

[0031] 1) Clinical samples, cell lines, and experimental animals

[0032] Six human cervical small cell neuroendocrine carcinoma tissues were obtained from the Affiliated Cancer Hospital of Shanxi Medical University. This study was approved by the Ethics Committee of Shanxi Medical University and informed consent was obtained from the patients.

[0033] Cervical small cell neuroendocrine carcinoma cell line (TC-YIK) was purchased from the China Center for Type Culture Collection, Wuhan University. Cells were cultured in Dulbecco's modified Eagle's medium (Dulbecco's modified Eagle's medium, Cat No. 8122691, Gibco, Thermo Fisher, USA) supplemented with 10% fetal bovine serum (Cat No. 10100147, Gibco, Thermo Fisher, USA) and 1% penicillin and streptomycin. Cells were cultured in an incubator with 5% CO₂ and 37°C.

[0034] Female Balb / cA-nu mice were purchased from the Experimental Animal Center of Shanxi Provincial People's Hospital. Mice were housed in an SPF environment with a temperature of 25 ± 2°C, a 12-h light / dark cycle, and a relative humidity of 50 ± 10%. They had free access to water and food throughout the study.

[0035] 2) Sample preparation and mass spectrometry analysis

[0036] Pathological specimens of SCNECC were preserved as formaldehyde-fixed paraffin-embedded (FFPE) tissue blocks. Protein extraction was performed using FFPE samples from 6 patients. First, the samples were cut into 5-μm-thick sections using a microtome and dried at 44°C for 16 hours. After dewaxing and drying the slides at room temperature for 1 hour, each sample was suspended in 100 μL of 0.1 mol / L NH4HCO3 solution containing 30% (v / v) acetonitrile and homogenized three times using a lysis and homogenization system (Bertin Technologies SAS, France) (speed: 7800 rpm, cycle: 3×20 seconds, pause: 30 seconds). Subsequently, centrifugation was performed at 10,000 g for 1 minute at 4°C. To remove formaldehyde cross-links, the samples were heated at 95°C for 90 minutes and centrifuged again at 10,000 g for 1 minute at 4°C. Protein concentration was estimated using a Qubit 2.0 fluorometer (Qubit Protein Assay kit, Thermo Fisher Scientific, Waltham, MA).

[0037] For high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis, peptides were separated using a Thermo-Dionex Ultimate 3000 HPLC system directly connected to a Thermo Orbitrap Fusion mass spectrometer at a flow rate of 0.300 μL / min using a 120-minute gradient elution. The analytical column was a homemade fused-silica capillary column (75 μm inner diameter, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å, 5 μm; Varian, Lexington, MA). Mobile phase A consisted of 0.1% formic acid, and mobile phase B consisted of 100% acetonitrile and 0.1% formic acid. The Orbitrap Fusion mass spectrometer was operated in data-dependent acquisition mode using Xcalibur 3.0 software. A single full-scan mass spectra (350-1550 m / z, 120,000 resolution) was acquired in the Orbitrap, followed by a 3-second data-dependent MS / MS scan at 30% normalized collision energy (high-energy collisional dissociation) in an ion routing multipole. The MS / MS spectra from each LC-MS / MS run were searched against the selected database using the Proteome Discovery search algorithm (version 1.4).

[0038] 3) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis

[0039] GO was used for enrichment analysis, which encompasses the cellular components, biological processes, and molecular functions of selected proteins. KEGG is a database that describes the functions and signaling pathways of selected proteins. GO and KEGG pathway analyses were performed using RStudio software using the "clusterProfiler" package. Data visualization was performed using the "GOplot" package.

[0040] 4) Immunohistochemistry and scoring analysis

[0041] Sections (4 μm) of SCNECC tissue and adjacent normal cervical tissue were deparaffinized and hydrated, followed by antigen retrieval (citrate buffer, pH 6.0; heating at 95°C for 15 minutes). For Ki67, P16, AE1 / AE3, SYN, and FEN1 staining, sections were immersed in 3% H2O2 for 15 minutes and incubated overnight at 4°C with rabbit antibodies (Ki67, Catalog No. 27309-1-AP, dilution 1:16,000; P16, Catalog No. 10883-1-AP, dilution 1:2,000; AE1 / AE3, Catalog No. 26411-1-AP, dilution 1:3,000; SYN, Catalog No. 17785-1-AP, dilution 1:2,000; FEN1, Catalog No. 14768-1-AP, dilution 1:2,000; Proteintech, USA). IgG was used as a negative control. Subsequently, sections were treated with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (PV-6001; Zymed Laboratories, China) and incubated at 37°C for 20 minutes. Proteins were visualized using 3,3'-diaminobenzidine tetrahydrochloride and counterstained with hematoxylin. Two observers, blinded to the patients' clinical outcomes, scored the stained sections according to the following criteria: <10% stained cells, scored 1; 10-49% stained cells, scored 2; 50-74% stained cells, scored 3; and 75-100% stained cells, scored 4. Staining color: No staining was scored 0, pale yellow granules, scored 1, brownish yellow granules, scored 2, and brown granules, scored 3. The final score was the number of staining points multiplied by the color of the staining points. Interobserver variability was less than 10% in the examined sections, and consensus was reached after further review.

[0042] 5) Cell transfection

[0043] (1) One day before transfection, the cell culture medium should be discarded and 2 mL of fresh culture medium without double antibody but containing fetal bovine serum should be added. When the cell growth density reaches about 80%, the transfection operation can be started.

[0044] (2) Preparation of transfection solution: Prepare two 5 mL centrifuge tubes and mark them as A and B. Add 250 μL of Opti-MEM medium to each centrifuge tube, add 5 μL of siRNA to tube A, and add 5 μL of Lipofectamine 2000 to tube B. Let them stand for about 5 minutes.

[0045] (3) Slowly add Solution B to Solution A and mix gently to avoid bubbles. Let the mixed transfection solution stand at room temperature for 25-30 minutes to ensure that the siRNA and transfection reagent are fully combined and ready for subsequent transfection operations.

[0046] (4) Aspirate the culture medium in the wells, add 500 μL of the mixed solution to the wells, and supplement with 1 mL of pure RPMI 1640 culture medium.

[0047] (5) After 6 h, the medium was changed to complete culture medium and the gene expression was detected after culturing at 37°C for 24-48 h.

[0048] 6) Detection of cell proliferation

[0049] (1) Normal control group (NC group) and FEN1 siRNA group. Three replicate wells were set up in each group to facilitate parallel experiments and data analysis. NC sequence (S: UUCUCCGAACGUGUCACGUTT; AS: ACGUGACACGUUCGGAGAATT); FEN1 siRNA sequence (S: GCCCGUGUAU GUCUUUGAUTT; AS: AUCAAAGACAUACACGGGCTT).

[0050] (2) Cell counting was performed. TC-YIK cells in the NC group and the FEN1 siRNA group were seeded at a density of 5000 cells per well in a 96-well plate. To prevent the culture medium from evaporating during the culture process, 100 μL of PBS was added to the wells around the 96-well plate.

[0051] (3) The 96-well plates of the NC group and the FEN1 siRNA group were placed in a 5% CO2, 37°C incubator and cultured for 0 h, 24 h, 48 h, and 72 h.

[0052] (4) Add 10 μL of CCK-8 reagent to each well and incubate in a 37°C incubator in the dark for 90 min.

[0053] (5) Detect the OD value using a 450 nm wavelength in an enzyme reader and draw a graph.

[0054] 7) Detection of cell migration and invasion ability

[0055] (1) Experimental groups: NC group and FEN1 siRNA group, with 3 replicate wells in each group.

[0056] (2) Add 600 μL of DMEM medium containing 10% FBS to the bottom of the Transwell chamber, and add 200 μL of pure DMEM medium to the top of the Transwell chamber. We selected cells in the logarithmic growth phase for digestion and counting. After resuspending the cells with pure 1 mL DMEM medium, 5 × 10 4 Cells were added on top of the Transwell chamber.

[0057] (3) After the Transwell chamber containing cells was placed in a cell culture incubator and cultured for 48 to 72 hours, the Transwell chamber was removed, the excess culture medium was aspirated, and the chamber was washed with PBS, repeated three times, and then the cells in the chamber were fixed with 4% paraformaldehyde for 30 minutes.

[0058] (4) After washing with PBS solution three times, the chamber was stained with crystal violet dye for 30 minutes and then washed with PBS solution three times again to remove excess dye.

[0059] (5) After the chamber was dried naturally, we observed and photographed it under a microscope and counted and analyzed the cells.

[0060] 8) Detection of cell invasion ability

[0061] (1) Dilute Matrigel with DMEM medium at a ratio of 1:6, add 50 μL of Matrigel to each well above the Transwell chamber, and then incubate in a 37°C incubator for 4 h until the Matrigel solidifies.

[0062] (2) Experimental groups: divided into NC group and FEN1 siRNA group, with 3 replicate wells in each group.

[0063] (3) Add 600 μL of DMEM medium containing 10% FBS to the bottom of the Transwell chamber, and add 200 μL of pure DMEM medium to the top of the Transwell chamber. We selected cells in the logarithmic growth phase for digestion and counting. After resuspending the cells with pure 1 mL DMEM medium, 5 × 10 4 Cells were added on top of the Transwell chamber.

[0064] (4) After the Transwell chamber containing cells was placed in a cell culture incubator and cultured for 48 to 72 hours, the Transwell chamber was removed, the excess culture medium was aspirated, and the chamber was washed with PBS, repeated three times, and then the cells in the chamber were fixed with 4% paraformaldehyde for 30 minutes.

[0065] (5) After washing with PBS solution three times, the chamber was stained with crystal violet dye for 30 minutes and then washed with PBS solution three times again to remove excess dye.

[0066] (6) After the chamber was dried naturally, we observed and photographed it under a microscope and counted and analyzed the cells.

[0067] 9) Clone formation assay

[0068] (1) The experiments were divided into two groups: NC group and FEN1 siRNA group, with three replicate wells in each group.

[0069] (2) Cell counting was performed. TC-YIK cells in the NC group and the FEN1 siRNA group were seeded at 5000 cells / well in a six-well plate, with 100 μL per well, and 2 mL of complete culture medium was added.

[0070] (3) The cells of different groups were placed in an incubator containing 5% CO2 and 37°C for culture.

[0071] (4) Observe cell growth every other day and replace the culture medium as appropriate.

[0072] (5) Stop the culture when visible clones are observed in about two weeks.

[0073] (6) Discard the culture medium and add 1-2 mL of 4% paraformaldehyde for fixation for 30 min.

[0074] (7) Aspirate 4% paraformaldehyde, add 1-2 mL of crystal violet dye, and stain for 30 minutes.

[0075] (8) Gently wash off the staining solution with running water and dry at room temperature.

[0076] (9) Invert the plate and count the number of colonies under a microscope.

[0077] 10) Flow cytometry analysis

[0078] Apoptosis was analyzed by flow cytometry using the Alexa Fluor 488 annexin V / Dead Cell Apoptosis Kit (Invitrogen). Samples contained 5 μL of Alexa Fluor 488 annexin V and 1 μL of 100 μg / mL propidium iodide (PI) to detect phosphatidylserine (PS) exposure on the outer layer of the plasma membrane. After incubation for 15 minutes in the dark, samples were quantified by flow cytometry (BD Biosciences, USA). Each treatment was repeated in duplicate, and the entire experiment was repeated three times.

[0079] For the transfected TC-YIK cells, first, they were gently centrifuged at 20g for 5 minutes at 25°C. Then, the cells were resuspended in phosphate buffered saline (PBS) to a cell density of 1×10 5 After being thoroughly mixed with 75% ethanol, the cells were incubated at 4°C for 2 hours. Afterwards, the cells were washed with PBS and centrifuged at 20g for 3 minutes. TM The cells were resuspended in 400 μL of cell cycle detection reagent in the cell cycle kit (Thermo Fisher Scientific) and then subjected to further flow cytometric analysis.

[0080] 11) Real-time quantitative PCR (qRT-PCR)

[0081] First, RNA was extracted using TRIzol reagent (Cat. No. 15596026, Invitrogen, USA), and cDNA was synthesized from approximately 2 μg of total RNA using Moloney murine leukemia virus reverse transcriptase (MMLV, TaKaRa Bio, Japan) and oligo-(dT)18 primers. qRT-PCR experiments were performed on a StepOnePlus TM Real-time PCR was performed on an Applied Biosystems, Thermo Fisher Scientific, USA, and SYBR Premix Ex Taq II (TaKaRa Bio, Japan) was used as a fluorescent dye. GAPDH was used as an internal reference gene to normalize the expression level of mRNA. The thermal cycling conditions were as follows: an initial activation cycle of 95°C for 30 seconds, followed by 40 cycles, each cycle consisting of 95°C for 10 seconds and 60°C for 30 seconds. The primer sequences were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., as shown in the following table. Each sample was analyzed in triplicate. 2 -ΔΔCt Methods Relative RNA quantification analysis was performed.

[0082] Table 1 Primer sequences

[0083]

[0084]

[0085] 12) Western blotting

[0086] RIPA lysis and extraction buffer (Cat No. 89900, Thermo Scientific, USA) was used. Protein concentration was determined using a BCA protein assay kit (Cat No. 23227, Thermo Scientific, USA). Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (PVDF, Millipore, Billerica, MA, USA). The membranes were blocked in 5% (w / v) skim milk for 1 h at room temperature and incubated with antibodies against FEN1 (Cat No. 14768-1-AP; 1:2000, Proteintech, USA), PCNA (Cat No. 10205-2-AP; 1:10000, Proteintech, USA), Caspase-9 (Cat No. 10380-1-AP; 1:3000, Proteintech, USA), BCL-2 (Cat No. 12789-1-AP; 1:900, Proteintech, USA), RB1 (Cat No. 10048-2-Ig; 1:10000, Proteintech, USA), PIK3CA (Cat No. 10380-1-AP; 1:3000, Proteintech, USA), and BCL-2 (Cat No. 12789-1-AP; 1:900, Proteintech, USA). Bands were visualized using an enhanced chemiluminescence kit (Cat No. 67071-1-Ig; 1:10,000, Proteintech, USA) and an enhanced chemiluminescence kit (Cat No. AR1171, Boster Biotechnology Co., Ltd., China). Finally, automatic exposure was performed using a chemiluminescence gel imaging system (FluorChem E, ProteinSimple, USA). The grayscale values ​​of the exposed bands were quantified and analyzed using ImageJ software (Java 1.8.0_345, http: / / imagej.org, National Institutes of Health, USA). Each sample was analyzed in triplicate.

[0087] 13) Animal experiments

[0088] Four-week-old female BALB / c nude mice were purchased from Weitonglihua Laboratory Animal Company and housed in a specific pathogen-free (SPF) isolation environment. A 1 ml disposable sterile syringe was used to extract the cell suspension, and 300 μl of TC-YIK cell suspension was injected subcutaneously into each nude mouse. The time of tumor formation was recorded. When the tumor volume reached 50 mm, the tumor was induced. 3 When the NC group and FEN1siRNA group were injected with small interfering RNA (in vivo siRNA), the amount of siRNA at each injection point was 1 nM (50 mm 3 -100 mm 3 1nM; 100mm 3 -250 mm 32 nanomoles of drug at a time; greater than 250 mm 3 The tumor volume was recorded. After 12 days, the tumor weight was recorded and used to extract tumor RNA and protein.

[0089] 14) FEN1 inhibitor treatment

[0090] TC-YIK cells were seeded into 24-well plates. A FEN1 inhibitor (SC13) (at a concentration of 40 μM) was added to the cell culture medium and culture continued. After 0, 24, and 48 hours of culture, the migration and invasion abilities of the cells were assessed. TC-YIK cells were cultured in 96-well plates and supplemented with a FEN1 inhibitor (SC13) (at a concentration of 40 μM) for 0, 24, 48, and 72 hours, respectively. TC-YIK cells were seeded into 6-well plates at a cell density of 5000 cells / mL, and a FEN1 inhibitor (SC13) (at a concentration of 40 μM) was added and cultured for 7-9 days. The culture medium was changed every other day to ensure that the concentration of the FEN1 inhibitor was maintained at 40 μM, and the cell colony formation ability was assessed. TC-YIK cells were seeded into 6-well plates. When the cell density reached 80%, a FEN1 inhibitor (SC13) (at a concentration of 40 μM) was added for 48 hours. Cells were collected to assess apoptosis ability and cell cycle changes.

[0091] 15) Statistical analysis

[0092] GraphPad Prism (version 9.0.0, GraphPad Software) was used for statistical analysis of the experimental data. The measurement data were statistically described using x±s. The two groups were compared using two independent sample t-tests. * indicates P < 0.05, indicating statistically significant differences.

[0093] 2. Results

[0094] 1) Proteomic analysis of differentially expressed proteins in SCNECC

[0095] In order to perform proteomic analysis of differentially expressed proteins in SCNECC, we first used immunohistochemistry to detect the expression of SCNECC's signature proteins AE1 / AE3, SYN, Ki67, and P16. The experimental results showed that the staining of AE1 / AE3, SYN, Ki67, and P16 in cancer tissues was significantly deeper than that in adjacent tissues ( Figure 1 Middle A).

[0096] Furthermore, we performed a scoring analysis on the staining depth, and the results showed that the scores of AE1 / AE3, SYN, Ki67, and P16 in cancer tissues were 6, 6.3, 6, and 9, respectively, while the expression scores in adjacent tissues were 1, 0.67, 0.67, and 1. The expression levels of AE1 / AE3, SYN, Ki67, and P16 in SCNECC were significantly higher than those in adjacent tissues ( Figure 1 Middle B).

[0097] Comparative proteomic analysis of adjacent and cancerous tissue specimens from 6 SCNECC patients was performed, with FC>1.5 and P<0.05 as the criteria for screening. The results showed that compared with adjacent tissues, 2169 proteins were upregulated and 162 proteins were downregulated in cancer tissues ( Figure 1 Middle C).

[0098] 2) FEN1 is significantly overexpressed in SCNECC tissues

[0099] To further analyze the protein expression profile characteristics in SCNECC, we selected the majority of up-regulated genes for GO and KEGG analysis. BP analysis results showed that differentially expressed proteins were mainly concentrated in mRNA splicing, viaspliceosome, RNA splicing, mRNA processing, translational initiation, etc. CC analysis results showed that differentially expressed proteins were mainly concentrated in nucleoplasm, nucleus, catalytic step 2spliceosome, membrane, spliceosomal complex, etc. MF analysis results showed that differentially expressed genes were mainly concentrated in RNA binding, mRNA binding, RNA helicase activity, nucleic acid binding, DNA binding, etc. ( Figure 2 Middle A). KEGG analysis showed that the differentially expressed proteins were mainly concentrated in Spliceosome, RNA transport, DNA replication, Base excision repair, Ribosome, etc. ( Figure 2 Middle B).

[0100] DNA replication is the cellular event with the highest enrichment of differentially expressed proteins in SCNECC. In our study, we found that 30 differentially expressed proteins were enriched in DNA replication. Further analysis of these proteins using the STRING database revealed that FEN1 / PCNA had the highest interaction score ( Figure 2 Finally, we used immunohistochemistry to verify the expression of FEN1 in SCNECC. The results showed that in SCNECC, the FEN1 staining in cancer tissues was significantly darker than that in adjacent tissues. The average score of cancer tissues was 8 points, while the average score of adjacent tissues was 1 point, which was a statistically significant difference ( Figure 2 Middle D).

[0101] 3) The role of FEN1 in SCNECC

[0102] To examine the role of FEN1 in the development and progression of SCNECC, we first used gene silencing technology to construct a FEN1 knockdown cell model. After transfection of NC and FEN1 siRNA into TC-YIK cells, qRT-PCR demonstrated that the FEN1 mRNA level in the FEN1 siRNA group was significantly decreased compared with that in the NC group ( Figure 3 Middle A), Western blotting demonstrated that the FEN1 protein level in the FEN1 siRNA group was significantly decreased ( Figure 3 The results of CCK-8 assay showed that 48 hours after transfection, the proliferation activity of the FEN1 siRNA group was significantly lower than that of the NC group. At 72 hours, the gap between the two groups further widened. Figure 3 D); Cell colony formation experiments showed that compared with the NC group, the number of cell clones in the FEN1 siRNA group was significantly reduced, and the decrease was statistically significant ( Figure 3 E); The results of cell migration and invasion experiments showed that 48 hours after transfection, the number of cells that could complete migration and invasion in the FEN1 siRNA group was significantly reduced compared with the NC group ( Figure 3 F); Flow cytometry results showed that compared with the NC group, the apoptosis rate of TC-YIK cells increased significantly after knocking down FEN1 ( Figure 3 In the FEN1 siRNA group, the proportion of cells in the G0 / G1 phase did not change significantly, but the proportion of cells in the S phase and G2 / M phase decreased significantly ( Figure 3 Middle H).

[0103] 4) FEN1 regulates the biological behavior of TC-YIK cells through PCNA

[0104] To verify the regulatory relationship between FEN1 and PCNA in SCNECC, we used qRT-PCR and Western blotting to detect changes in PCNA mRNA and protein levels in the FEN1 knockdown TC-YIK cell model. The results showed that compared with the NC group, the expression levels of PCNA mRNA and protein in the FEN1 knockdown TC-YIK cells were significantly reduced ( Figure 4 (A~C).

[0105] To verify whether PCNA and FEN1 have the same effect on the biological behavior of SCNECC cells, we used CCK-8, cell colony formation, migration and invasion assays, and flow cytometry to evaluate the effects of PCNA knockdown on cell viability, cell motility, and cell cycle in the SCNECC cell line TC-YIK. The results showed that after transfection of NC and PCNA siRNA into TC-YIK cells, the cell viability of the PCNA siRNA group was significantly inhibited at 72 hours compared with the NC group ( Figure 4 Middle D); The number of cell clones in the PCNA siRNA group was significantly reduced ( Figure 4 E); The migration and invasion abilities of cells in the PCNA siRNA group were significantly weakened ( Figure 4 F), the number of cells entering the apoptotic phase in the PCNA siRNA group increased significantly ( Figure 4 In the PCNA siRNA group, the proportion of cells in the G0 / G1 phase increased significantly, the proportion of cells in the S phase decreased significantly, and the proportion of cells in the G2 / M phase did not change ( Figure 4 Middle H).

[0106] 5) In vivo experiments demonstrated that knockdown of FEN1 could inhibit tumor growth in SCNECC

[0107] To examine the effect of FEN1 knockdown on tumor growth in vivo, we first subcutaneously injected TC-YIK cells into nude mice to form tumors. When the tumor volume reached 50 mm 3 After that, in vivo NC and in vivo FEN1 siRNA were injected respectively ( Figure 5 The results showed that compared with the invivo NC group, the mRNA and protein levels of FEN1 and PCNA in the invivo FEN1 siRNA group were significantly decreased (P < 0.05, Figure 5 C, D), the body weight of mice did not change significantly (P>0.05, Figure 5 E), the tumor weight was significantly reduced (P < 0.05, Figure 5 F), and the tumor volume was significantly reduced (P < 0.05, Figure 5 Middle G).

[0108] 6) Knockdown of FEN1 altered the expression of genes such as BCL-2, RB1, PIK3CA, and Caspase-9 in vivo and in vitro experiments

[0109] To further verify the effect of FEN1 knockdown on the biological functions of TC-YIK cells at the molecular level, we verified the expression changes of some star genes related to cell biological functions. After TC-YIK cells were transfected with NC or FEN1 siRNA, qRT-PCR experiments showed that the mRNA expression levels of BCL-2, RB1, and PIK3CA in the FEN1 siRNA group were significantly reduced compared with the NC group, while the mRNA expression level of Caspase-9 gene was significantly increased (P < 0.05, Figure 6 At 48 h, Western blotting experiments showed that the protein expression levels of BCL-2, RB1, and PIK3CA in the FEN1 siRNA group were significantly decreased, while the protein expression level of Caspase-9 was significantly increased (P < 0.05, Figure 6 (B, C).

[0110] TC-YIK cells were subcutaneously grown in nude mice to form tumors. Compared with the in vivo NC group, the mRNA expression levels of BCL-2, RB1, and PIK3CA in the invivo FEN1 si-RNA group were significantly decreased, and the mRNA expression level of Caspase-9 gene was significantly increased (P < 0.05, Figure 6 In the in vivo FEN1 si-RNA group, the protein expression levels of BCL-2, RB1, and PIK3CA were significantly decreased, and the protein expression level of Caspase-9 gene was significantly increased (P<0.05, Figure 6 (E, F).

[0111] 7) FEN1 inhibitor SC13 can inhibit the proliferation, colony formation, migration and invasion of TC-YIK cells and promote apoptosis

[0112] To investigate whether FEN1 inhibitors can affect the development and progression of SCNECC, we treated TC-YIK cells with a FEN1 inhibitor (SC13) and examined changes in cell biological behavior. Compared with the control group, the FEN1 inhibitor SC13 significantly reduced the viability of TC-YIK cells at 48 and 72 hours ( Figure 7 A); it can also significantly reduce the number of TC-YIK cell clones ( Figure 7 Middle B) and the migration and invasion abilities were significantly weakened ( Figure 7 C), but resulted in a significant increase in TC-YIK cell apoptosis ( Figure 7 Among them, the proportion of cells in G0 / G1 and S phases did not change significantly, while the proportion of cells in G2 / M phase decreased ( Figure 7 Middle F).

[0113] To verify the effect of FEN1 inhibitor SC13 on the biological function of TC-YIK cells at the molecular level, we used qRT-PCR experiments to show that compared with the control group, FEN1 inhibitor SC13 can significantly reduce the mRNA levels of FEN1, PCNA, BCL-2, RB1, and PIK3CA in TC-YIK cells at 24 hours, while increasing the mRNA expression level of Caspase-9 gene ( Figure 7 F); WB experiments showed that compared with the control group, the FEN1 inhibitor SC13 could significantly reduce the protein expression levels of FEN1, PCNA, BCL-2, RB1 and PIK3CA in TC-YIK cells at 48 h, while increasing the protein expression level of Caspase-9 gene ( Figure 7 (G, H).

[0114] In summary, FEN1 is highly expressed in SCNECC tissues. Knockdown of FEN1 can inhibit cancer cell proliferation, colony formation, invasion, and migration, and promote apoptosis through PCNA. The FEN1 inhibitor SC13 also inhibits cancer cell proliferation, colony formation, invasion, and migration, and promotes apoptosis. Overexpression of FEN1 is an important factor in promoting the development and progression of cervical small cell neuroendocrine carcinoma and is expected to become a potential therapeutic target for cervical small cell neuroendocrine carcinoma.

Claims

1. Application of FEN1 as a target in the preparation of drugs for the treatment of cervical small cell neuroendocrine carcinoma.

2. The use according to claim 1, characterized in that: The drug is used to knock down FEN1.

3. The use according to claim 2, characterized in that: The knockdown of FEN1 inhibits cancer cell proliferation, clone formation, invasion, migration ability and promotes cell apoptosis through PCNA.

4. Application of FEN1 siRNA in the preparation of drugs for the treatment of cervical small cell neuroendocrine carcinoma.

5. The use according to claim 4, characterized in that: The siRNA inhibits FEN1 expression.

6. A drug for treating cervical small cell neuroendocrine carcinoma, characterized in that: These include inhibitors of FEN1 or siRNA of FEN1.

7. A drug for treating cervical small cell neuroendocrine carcinoma according to claim 6, characterized in that: The FEN1 inhibitor is SC13.

8. The drug for treating cervical small cell neuroendocrine carcinoma according to claim 6, characterized in that: Also included are pharmaceutically acceptable carriers.

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