Biomarker of low folate-mediated hpv infection induced cervical lesions and application thereof

Abstract

The application provides a biomarker of low folate-mediated HPV infection causing cervical lesions and an application thereof, the biomarker is IGF2BP3 and miR-27a, and the two can be used as a treatment target and a research tool of low folate-mediated HPV infection causing cervical lesions. Small nucleic acids targeting IGF2BP3 and miR-27a are delivered through folate-modified exosomes in the treatment of cervical lesions, and the delivery efficiency and treatment effect are significantly improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to biomarkers of cervical lesions mediated by low folic acid-induced HPV infection and their applications. Background Technology

[0002] Cervical lesions are a common disease of the female reproductive system, and early screening and precise intervention are crucial to preventing the development of cervical cancer. However, current screening technologies have significant limitations. Existing screening mainly relies on HPV DNA testing and TCT cytology, but both have inherent defects. HPV DNA testing cannot distinguish between transient and persistent infections, easily leading to overdiagnosis; cytology is affected by factors such as sample quality and subjective interpretation, resulting in a false negative rate of up to 30%.

[0003] Currently, traditional treatments for cervical lesions mainly include surgical treatment (such as cervical conization and hysterectomy), physical therapy (such as laser and cryotherapy), and chemotherapy. These treatments have many limitations. For example, surgical treatment can easily cause irreversible damage to the cervical structure and function, increasing the risk of pregnancy complications such as premature birth and miscarriage, affecting the patient's fertility and quality of life; physical therapy has unstable effects and is prone to recurrence; chemotherapy has significant side effects, poor patient tolerance, and long-term use may lead to drug resistance, limiting its clinical application.

[0004] Therefore, developing a safe, effective treatment method that preserves cervical function is of great significance for improving the prognosis and quality of life of patients with cervical lesions. Summary of the Invention

[0005] To address the above problems, this invention provides biomarkers for cervical lesions mediated by low folic acid-induced HPV infection and their applications.

[0006] To achieve the above objectives, the specific technical solution provided by the present invention is as follows:

[0007] The first aspect of the present invention provides an exosome for the treatment of cervical lesions caused by low folic acid-mediated HPV infection, wherein the exosome is a folic acid-modified exosome.

[0008] The folic acid-modified exosomes contain small nucleic acids that target IGF2BP3 and / or miR-27a.

[0009] In this invention, folate-mediated HPV infection leading to cervical lesions refers to the pathological process in which insufficient folate levels in the body, combined with persistent human papillomavirus (HPV) infection, trigger abnormal changes in cervical epithelial cells, resulting in cervical lesions. Cervical intraepithelial neoplasia (CIN) is a precancerous lesion with cancerous potential, formed by abnormal proliferation and differentiation of cervical cells under the influence of factors such as persistent high-risk HPV infection. Based on the severity of the lesion, it is classified into CIN I (mild dysplasia), CIN II (moderate dysplasia), and CIN III (severe dysplasia, with the extreme manifestation being carcinoma in situ). CIN is an essential stage in the development of cervical cancer, and the progression from CIN I to CIN III and then to invasive carcinoma typically takes several to several decades. Early detection and treatment of CIN can significantly reduce the incidence and mortality of cervical cancer.

[0010] In this invention, IGF2BP3, also known as KOC, CT98, IMP3, KOC1, and VICKZ3, refers to the gene encoding insulin-like growth factor 2 mRNA binding protein 3, its transcribed mRNA and its variants, and the translated protein, with a gene ID of 10643.

[0011] In this invention, miR-27a refers to the miRNA with gene ID 407018, located at 19p13.13 on chromosome 19. It belongs to the microRNA (miRNA) family, a class of non-coding single-stranded RNA molecules of 20-24 nucleotides in length, which can regulate the expression of target genes by incompletely pairing with the 3' untranslated region (3'-UTR) of the target gene mRNA. In specific embodiments of this invention, miR-27a includes its precursor and its mature form after processing, such as miR-27a-3p and miR-27a-5p.

[0012] In this invention, the term "small nucleic acid" refers to linear or structured nucleic acid molecules of 15 to 100 nucleotides (or nucleotide analogs) in length, which achieve at least one of the following functions by specifically binding to target nucleic acids or proteins: regulating gene expression (e.g., inhibiting translation, inducing degradation, or splicing regulation); interfering with signaling pathways (e.g., blocking protein-nucleic acid interactions); serving as diagnostic biomarkers or therapeutic molecular carriers; and delivering exogenous nucleic acids (e.g., gene editing tools or vaccine antigens). In the context of this invention, small nucleic acids also encompass natural nucleotides and chemically modified analogs, including but not limited to sugar ring modifications (e.g., 2'-O-alkylation, LNA); phosphodiester bond modifications (e.g., thiophosphate, borate phosphate); base modifications (e.g., 5-methylcytosine, pseudouracil); and terminal modifications (e.g., cholesterol, polyethylene glycol (PEG), or fluorescent group conjugation).

[0013] In some embodiments, the small nucleic acid includes siRNA, saRNA, shRNA, antisense oligonucleotide, nucleic acid aptamer, miRNA inhibitor, miRNA mimics, ribozymes, deoxyribozymes, CpG oligonucleotides, and antibody-nucleic acid conjugates.

[0014] Furthermore, the small nucleic acids targeting IGF2BP3 and / or miR-27a include siRNA targeting IGF2BP3 and / or miRNA inhibitor targeting miR-27a.

[0015] Furthermore, the nucleotide sequence of the siRNA targeting IGF2BP3 is shown in SEQ ID NO:1-2.

[0016] Furthermore, the nucleotide sequence of the miRNA inhibitor targeting miR-27a is shown in SEQ ID NO:3.

[0017] The second aspect of the present invention provides a method for preparing exosomes for the treatment of cervical lesions caused by low folic acid-mediated HPV infection, wherein the exosomes are folic acid-modified exosomes, and the preparation method includes the following steps: 1) mixing DSPE-PEG-FA with Exo and collecting the modified DPF-Exo; 2) loading small nucleic acids into DPF-Exo to obtain folic acid-modified exosomes.

[0018] Furthermore, the small nucleic acid is a small nucleic acid that targets IGF2BP3 and / or miR-27a.

[0019] In some embodiments, the small nucleic acid includes siRNA, saRNA, shRNA, antisense oligonucleotide, nucleic acid aptamer, miRNA inhibitor, miRNA mimics, ribozymes, deoxyribozymes, CpG oligonucleotides, and antibody-nucleic acid conjugates.

[0020] Furthermore, the small nucleic acids targeting IGF2BP3 and / or miR-27a include siRNA targeting IGF2BP3 and / or miRNA inhibitor targeting miR-27a.

[0021] Furthermore, the nucleotide sequence of the siRNA targeting IGF2BP3 is shown in SEQ ID NO:1-2;

[0022] Furthermore, the nucleotide sequence of the miRNA inhibitor targeting miR-27a is shown in SEQ ID NO:3.

[0023] Furthermore, the particle size of the folic acid-modified exosomes is 50~1000 nm.

[0024] Furthermore, the particle size of the folic acid-modified exosomes is 100~500 nm.

[0025] Furthermore, the potential of the folic acid-modified exosomes is -140 to -10 mV.

[0026] Furthermore, the folic acid-modified exosome surface proteins CD63 and Flotillin-1 were positive.

[0027] In some embodiments, the loading is performed via any one of electroporation, viral transduction, liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymeric complexes, dendritic polymers, nanoparticles, natural endocytosis or phagocytosis pathways, cell-penetrating peptides, microinjection, microneedle delivery, and particle bombardment.

[0028] Furthermore, the loading is performed using the electroporation method.

[0029] The third aspect of the present invention provides the application of exosomes as described in the first aspect of the present invention or the preparation method as described in the second aspect of the present invention in the preparation of a treatment product for cervical lesions caused by low folic acid-mediated HPV infection.

[0030] Furthermore, the products include pharmaceuticals and reagent kits.

[0031] In some embodiments, the drug may be used alone or in the form of a pharmaceutical composition.

[0032] In this invention, the term "pharmaceutical composition" refers to a substance containing at least one bioactive substance (such as the folic acid-modified exosomes provided by this invention). The pharmaceutical compositions of this invention can be administered orally, non-gastrointestinally, via inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted drug delivery device. The pharmaceutical compositions of this invention may contain any commonly used non-toxic pharmaceutically acceptable carrier, excipient, or excipient. In some cases, pharmaceutical acids, bases, or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form. The term "non-gastrointestinal" as used in this invention includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can be administered to the receptor via any route, provided the target tissue can be reached.

[0033] Furthermore, the pharmaceutical composition also includes pharmaceutically acceptable excipients.

[0034] In this invention, pharmaceutically acceptable excipients include carriers, diluents, or excipients, including but not limited to any adjuvants, carriers, excipients, gliding agents, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, surfactants, or emulsifiers that have been approved by the U.S. Food and Drug Administration for use in humans or livestock. Excipients should be biologically acceptable, meaning they are compatible with other components in the formulation and harmless to the patient, and do not elicit adverse reactions (e.g., immune responses) when administered to the host.

[0035] In some embodiments, the pharmaceutically acceptable excipients include one or more of fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH adjusters, antioxidants, or antibacterial agents.

[0036] In some embodiments, the dosage forms of the drug and the drug composition include drops, tablets, capsules, granules, films, gels, powders, emulsions, pills, suppositories, patches, solutions, ointments, or creams.

[0037] The fourth aspect of the present invention provides the application of reagents for detecting the expression levels of IGF2BP3 and / or miR-27a in the preparation of early screening products for cervical lesions caused by low folate-mediated HPV infection, wherein the reagents include primers and probes that specifically bind to IGF2BP3 and / or miR-27a.

[0038] The fifth aspect of this invention provides a computer-aided drug screening method based on IGF2BP3 and miR-27a, wherein the drug is used to treat cervical lesions caused by low folic acid-mediated HPV infection. The method specifically includes: acquiring IGF2BP3 gene data and miR-27a gene data; selecting the sequence information and spatial structure of the IGF2BP3 gene and the sequence information and spatial structure of the miR-27a gene to determine the binding sites of the two as the binding sites of the targeted drug; and using a computer-aided drug screening method to obtain candidate drugs targeting the binding sites.

[0039] Furthermore, the computer-aided drug screening method comprises: obtaining the binding sites of the IGF2BP3 gene and the miR-27a gene; screening compounds with similar sequences and structures in a molecular database based on the sequence information and spatial structure of the binding sites of the IGF2BP3 gene and the miR-27a gene; performing molecular docking with the IGF2BP3 gene on the screened compounds to calculate the binding energy score of the target compounds; and sorting the compounds according to the scores to obtain candidate drugs.

[0040] In some embodiments, the compound includes protein analogs, antibodies, DNA, and RNA.

[0041] Furthermore, the compounds include various pharmaceutically acceptable salt forms.

[0042] Furthermore, the DNA includes single-stranded DNA, closed circular DNA, and linked DNA.

[0043] Furthermore, the RNA includes mRNA, tRNA, rRNA, snRNA, hRNA, antisense RNA, tCRNA, dsRNA, SCRNA, catalytically active RNA, and various viral RNAs.

[0044] Furthermore, the DNA includes DNA of various conformations, specifically including type A, type B, type C, type D, type E, type H, type L, type P, and type Z.

[0045] Furthermore, the antibody includes dAb, Fab, Fab', scFv, Fv, disulfide-bonded Fv, or contains a single immunoglobulin variable domain.

[0046] Furthermore, the protein analogs include proteins artificially synthesized through protein engineering.

[0047] In some embodiments, the compounds include, but are not limited to, oxides, acids, bases, salts, and organic compounds. In some embodiments, the oxide is a compound composed of two elements, one of which is oxygen. In some embodiments, the acid is a compound that produces only hydrogen ions as cations upon ionization. In some embodiments, the base is a compound that produces only hydroxide ions as anions upon ionization. In some embodiments, the salt is a compound that produces a metal cation (or ammonium ion) and an acid radical upon ionization. In some embodiments, the organic compound is a general term for carbon-containing compounds (excluding carbon monoxide, carbon dioxide, carbonates, metal carbides, and cyanides) or hydrocarbons and their derivatives.

[0048] Advantages and beneficial effects of the present invention: The present invention provides biomarkers and therapeutic targets for cervical lesions caused by low folate-mediated HPV infection, a computer-aided drug screening method based on the interaction of biomarkers, and provides a folate-modified exosome for treating cervical lesions caused by low folate-mediated HPV infection. It has high cellular uptake rate, good targeting, and extremely low cytotoxicity, and is an effective drug for the treatment of cervical lesions. Attached Figure Description

[0049] Figure 1 This is a graph showing the results of a prospective cohort study on the negative impact of folic acid concentration on the risk of cervical intraepithelial neoplasia (CIN). In the graph, A represents serum folic acid concentration, B represents erythrocyte folic acid concentration, and C represents dietary folic acid concentration.

[0050] Figure 2 For m 6 Expression profiles of enzyme A in folate-deficient HPV(+) patients, where A is the tSNE plot of cells from control and folate-deficient HPV(+) patients, B is the UMAP showing the expression of IGF2BP3 and METTL14 genes in control and folate-deficient HPV(+) patients, and CD are the expression profiles of enzyme A in various cell types of folate-deficient HPV(+) patients. 6 A. Heatmap of enzyme expression intensity (C) and expression level (D); E. Differential gene analysis of cell types in control individuals and low-folate HPV (+) patients; F. Heatmap of cell communication intensity and ligand-receptor interaction between epithelial cells and adjacent cells; G. m 6 A comparison of the cumulative probabilities of IGF2BP3 markers in positive and negative epithelial cells, with H representing the significantly upregulated frequency of IGF2BP3 in epithelial cells after HPV infection.

[0051] Figure 3 For epithelial cells m of patients with low folate and HPV (+) 6The diagram shows the results of persistent HPV infection caused by receptor-ligand dysregulation due to specific upregulation of the IGF2BP3 enzyme A in cervical lesions. In this diagram, A represents a key gene for folate metabolism in cervical epithelial cells and an important gene associated with cervical lesions, along with m... 6 A. Correlation analysis of regulatory factor expression; B. Scatter plot showing a negative correlation between IGF2BP3 expression and the folate metabolism gene MTFHD2L; C. WGCNA identified 12 modules with related gene expression patterns in epithelial cells of low-folate HPV(+) patients; network diagram showing 20 genes highly correlated with module M8 in the IGF2BP3-positive epithelial cell group; D. GO enrichment analysis of the 20 related genes; E. Key pathways in epithelial cells and m 6 A is a correlation analysis of regulatory factor expression; F is a comparison of the expression levels of IGF2BP3 in epithelial cells of normal individuals and patients with low folate and HPV (+) with other cell ligand-receptor expression levels; G is a scatter plot showing that IGF2BP3 expression is negatively correlated with the folate metabolic transport pathway and positively correlated with epithelial-mesenchymal transition (EMT).

[0052] Figure 4 m in EP3 subset of epithelial cells with low folate and HPV (+) 6 The diagram shows the activation of the cervical epithelial EMT-related pathway caused by persistent HPV infection due to specific dysregulation of enzyme A IGF2BP3. A shows the CNV expression distribution of each subgroup in cervical epithelial cells using InferCNV, with red indicating copy number amplification and blue indicating copy number loss; the red box represents the CNV distribution of EP3. B shows the gene clustering distribution of each subgroup in cervical epithelial cells using ClusterVis, including differentially expressed genes in each cluster and the functional enrichment results of the top 5 genes in each cluster. C shows the distribution of re-clustered subgroups in epithelial cells. D shows the distribution of 23 m... 6 Enzyme A was scored, and the m values ​​in the normal group were compared with those in the diseased group. 6 A. Score, differential expression of IGF2BP3 and folate metabolism gene MTHFD2L (red indicates high expression, blue indicates low expression); E represents the eight major significant m... 6 A represents the key gene involved in the main function of cervical lesions; F shows the correlation analysis of cervical lesion-related pathways, with red indicating positive correlation and blue indicating negative correlation; G shows the high and low expression groups of IGF2BP3, and the scoring of genes in the folic acid metabolism pathway and one-carbon unit metabolism, respectively showing the difference in IGF2BP3 expression between the high and low groups in the two groups.

[0053] Figure 5The results of various cell lines, tumorigenesis experiments, and folic acid metabolism disorder models were used to verify the effects of folic acid on cervical lesions. Figure A shows the cell proliferation ability at different folic acid concentrations; Figure B shows the cell migration ability at different folic acid concentrations as detected by the scratch assay; and Figure C shows the cell invasion ability at different folic acid concentrations as detected by the Transwell assay. To verify that both folic acid and methylation inhibitors inhibit the migration ability of HPV cells, cell lines were divided into a control group (NC group), a folic acid group (Folate group), a 5-aza group (5-aza group), and a combined rescue group (Folate+5-aza group). After 24 hours of intervention, the migration ability was monitored using a live cell workstation. D shows the scratch assay results of HeLa and Siha cell lines in the NC, Folate, 5-aza, and Folate+5-aza groups. E shows the migration ability of HeLa and Siha cell lines in each group monitored by the live cell workstation. F shows the difference in proliferation ability among the Ect1, E6 / E7, Siha, and HeLa cell lines. G shows the tumor size of each group after 5 weeks of tumor formation in nude mice, after which the mice were sacrificed. The difference in tumor size among the groups is statistically significant, and the tumor sections from each group were stained with HE to observe the tissue necrosis.

[0054] Figure 6 Folic acid inhibits the overall and IGF2BP3 activity in HPV(+) cells. 6 Figure A shows the modification level, and its concentration is negatively correlated with IGF2BP3 expression. A represents the migration rate measured by a 24-hour scratch assay; B represents the cell proliferation rate measured by cell number in a Transwell chamber after 24 hours; C represents the IGF2BP3 m value measured by MeRIP after 48 hours of folic acid (IC50 = 220 nM) intervention in Ect1 E6 / E7 cells, divided into control and folic acid groups. 6 A shows the modification changes; D shows the Q-PCR determination of IGF2BP3 expression in 90 HPV(+) patients and the analysis of its correlation with serum folic acid concentration; E shows the mRNA expression of all cells in Ect1E6 / E7 cells after folic acid intervention. 6 A is the modification measurement, divided into control group, folic acid intervention group, and folic acid intervention replenishment (Folate+5-aza) group; F is the representative map of IGF2BP3 expression measured by immunohistochemistry in HeLa cells to induce tumors in nude mice; G is the expression level statistical map.

[0055] Figure 7 Folic acid inhibits the m of miR-27a in HPV(+) cells 6A. Abnormal modification, miR-27a and IGF2BP3 targeting correlation results. A shows the invasion rate of each group calculated using cell number after 48 hours in a Transwell chamber, divided into mimics group (upregulated expression group), NC group, IN group (downregulated control group), and inhibitor group (downregulated expression group); B shows the migration rate of each group measured by a live-cell workstation scratch assay after plasmid transfection; C shows the miR-27a m+ expression after 48 hours of folic acid (IC50-220 nM) intervention in Ect1 E6 / E7 cells, divided into control and folic acid groups, with MeRIP measurement of miR-27a m+ expression after folic acid intervention. 6 A represents the modification changes; D shows the proliferation rate, migration rate, and invasion rate of Siha cells transfected with miR-27a-3p mimics after the initial number of cells were the same, measured by a live-cell workstation; E shows the cell clustering ability of each group measured by a live-cell workstation combined with 3D spherical culture plates; FG shows the dual-luciferase reporter assay, where 1 μl of oligo was selected based on oligo efficiency evaluation, with a cell transfection efficiency of approximately 70% or higher; IGF2BP3 WT represents the IGF2BP3 wild-type sequence constructed into the pmiRGLO vector group, and IGF2BP3 MUT represents the IGF2BP3 mutant sequence constructed into the pmiRGLO vector group; PC represents the positive control group.

[0056] Figure 8The results of persistent HPV infection in the cervical epithelium caused by IGF2BP3 / miR-27a dysregulation are shown in Figure A, a heatmap illustrating differentially expressed miRNAs in HPV(+) patients; Figure B shows a significant correlation between miR-27a-related genes through miRNA-pathway-network enrichment analysis; Figure C shows a negative correlation between miR-27a expression and serum folate levels in 90 subjects as determined by Q-PCR; Figure D shows a positive correlation between miR-27a and IGF2BP3 expression; and Figure E verifies HPV16 / 18 after transfection of Siha cells with the miR-27a-3p mimics / inhibitor. E6 expression was analyzed using β-actin as a reference, with the grayscale ratio of expression in a bar chart. miR-IN was a negative control for transfection with miR-27a-inhibitor; miR-27i was transfection with miR-27a-inhibitor; miR-NC was a negative control for transfection with miR-27a-mimics; miR-27m was transfection with miR-27a-mimics; F was the in vivo experiment involving subcutaneous tumorigenesis injection, with tumor size measured in each group; immunohistochemical experiments were performed on tumor sections, and the expression of HPV16 / 18 E6 was detected using the OD values ​​of each group; G was the verification of IGF2BP3 and HPV E7 expression after transfection of HeLa cells with miR-27a-3p mimics / inhibitor.

[0057] Figure 9 This diagram illustrates the preparation process and characterization results of folic acid-modified exosomes (DPF-Exo). FA-Exo represents the DPF-Exo complex. In the diagram, A shows the preparation process of DPF-Exo; B shows the particle size detected by NTA; C shows the zeta potential of Exo; D shows the particle size of DPF-Exo detected by NTA; E shows the zeta potential of DPF-Exo; F shows the morphology of Exo as captured by TEM; G shows the morphology of DPF-Exo as captured by TEM; H shows the expression levels of exosomal marker proteins of 293T cells, Exo, and DPF-Exo as detected by Western blotting; and I is a statistical graph showing the cytotoxicity of Lipo2000, Exo, and DPF-Exo on 293T cells as detected by CCK8.

[0058] Figure 10Figure shows the in vitro interference effect validation results of delivering small nucleic acids to folic acid-modified exosomes (DPF-Exo). In the figure, FA-Exo represents the DPF-Exo complex. A shows the efficiency of DPF-Exo / Cy3-siRNA uptake by Siha cells as displayed by fluorescence imaging; B shows the efficiency of DPF-Exo / Cy3-siRNA uptake by Siha cells as detected by flow cytometry; C shows the efficiency of DPF-Exo / Cy3-siRNA uptake by Siha cells as displayed by flow cytometry statistical results; D shows the efficiency of DPF-Exo / Cy3-siRNA uptake by HeLa cells as displayed by fluorescence imaging; E shows the efficiency of DPF-Exo / Cy3-siRNA uptake by HeLa cells as detected by flow cytometry; F shows the efficiency of DPF-Exo / Cy3-siRNA uptake by HeLa cells as displayed by flow cytometry statistical results; G shows the interference effect of DPF-Exo delivery of si-IGF2BP3 in Siha cells as detected by Q-PCR; H shows the interference effect of DPF-Exo delivery of siIGF2BP3 in HeLa cells as detected by Q-PCR; I shows the interference effect of DPF-Exo delivery of miR-27a-3p in HeLa cells as detected by Q-PCR. The interference effect of the inhibitor in Siha cells; J is the Q-PCR detection of the interference effect of DPF-Exo delivery miR-27a-3p inhibitor in HeLa cells.

[0059] Figure 11 Folic acid deficiency leads to IGF2BP3 dysregulation via miR-27a m 6 A schematic diagram illustrating how abnormal modification can lead to persistent HPV infection. Detailed Implementation

[0060] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0061] Example

[0062] I. Experimental Materials

[0063] 1. Study Subjects: Gynecological patients who visited the Second Hospital of Shanxi Medical University from January to December 2013 were selected. Cervical exfoliated cells were detected using TCT to exclude patients with abnormal cervical glandular cells. On a voluntary basis, patients with TCT results of ASCUS or higher underwent further colposcopy-guided biopsy and cervical histopathological examination. HPV type testing and serum folic acid testing were also performed. After clinical pathological diagnosis of HPV, 90 HPV(+) patients were randomly selected as experimental samples. Serum folic acid levels were measured in these patients, and the expression level of IGF2BP3 in the 90 HPV(+) patients was determined by Q-PCR, and its correlation with serum folic acid concentration was analyzed.

[0064] Patients meeting any of the following criteria should be excluded: ① Pregnant women. ② Those with a history of hysterectomy. ③ Those with a history of treatment for cervical and vaginal lesions. ④ Patients with other malignant tumors. ⑤ Patients with hematologic or digestive system diseases. ⑥ Recent (within three months) users of B vitamins. This study was approved by the Ethics Committee of the Second Affiliated Hospital of Shanxi Medical University, and informed consent was obtained from all participants. Five cervical cancer tissue samples with serum folate-sufficient HPV16 infection (age 47.40±2.41 years) and five cervical cancer tissue samples with serum folate-deficient HPV16 infection (age 48.40±1.82 years) were selected. All cervical cancer patients had moderately differentiated squamous cell carcinoma histologically, and were classified as Figo 20091 clinical stage Ia-IIa. miRNA microarray screening was performed, and there was no statistically significant age difference between the two groups (P>0.05).

[0065] 2. Data Collection:

[0066] (1) Collection of cervical cancer-related factors: A standardized structured questionnaire was designed, and trained investigators conducted face-to-face interviews with the research subjects. The survey content mainly included demographic characteristics such as age, occupation, education level, and annual income; marital and reproductive information such as menstrual history, marital history, pregnancy and childbirth history, sexual behavior history, and contraception history; vitamin usage history, past medical history, and family history of cancer.

[0067] (2) Specimen Collection: ① Serum Specimen: Collect 5ml of fasting venous blood from all subjects in the morning, separate the serum and blood clots, and store at -80℃ for later testing. ② Cervical Exfoliated Cells: All subjects were required to have abstained from sexual activity, vaginal irrigation, and medication within 48 hours. After exposing the cervix, cervical exfoliated cells were collected by rotating the cervical cell brush 5 times. The sampler was rinsed 10 times in preservation solution, capped, and stored at 4℃ for testing within one week. ③ Cervical Tissue Specimen: Colposcopy was performed by a colpologist with more than 2 years of experience. For patients initially suspected of having cervical cancer, two additional tissue samples (approximately 5mm each) were taken without affecting routine pathological diagnosis. After the fresh cervical tissue specimens were removed from the body, they were cleaned of blood with physiological saline, placed in pre-cooled cryovials, and promptly (<10min) placed in an insulated container filled with liquid nitrogen. After the specimen has been placed in the liquid nitrogen container for 1 hour, tighten the cap of the cryopreservation tube and place it in the cryopreservation box as required, then store it in a -80°C freezer.

[0068] 3. Main reagents and instruments: The fully automated cell detection and preparation instrument (ThinPrep2000 System), slide preparation and staining system consumables and staining agents were purchased from Beijing Hologic Medical Technology Co., Ltd.; the TC-96 / G / H(6) nucleic acid amplification analyzer, the HMM-2 medical nucleic acid molecular rapid hybridization instrument, and the HPV 21 genotype typing detection kit (National Medical Device Registration Certificate 20143402188) were all purchased from Guangdong Kaipu Biotechnology Co., Ltd.; the Beckman Coulter ACCESS 2 fully automated microparticle chemiluminescence immunoassay system, folic acid assay kit, substrates and buffers were purchased from Beckman Coulter, Inc., USA; the live cell workstation system was from BioTek, USA.

[0069] Shanghai Jima Biotechnology Co., Ltd. was commissioned to synthesize nucleic acid sequences, including constructing plasmid vectors for silencing miRNAs and siRNA. The target protein genes of miRNA-27a mimics / inhibitor, inhibitor NC, NC, and siRNA, as well as primer sequences such as siRNA-HPV E6 / E7 and siRNA-IGF2BP3, were synthesized. The sequences involved in this application are shown in Table 1.

[0070] Table 1. Sequence List

[0071]

[0072] Note: In the table, dT represents a chemically modified 3' dideoxythymidine dimer, used to replace the UU ending in natural RNA to enhance siRNA stability, reduce immunogenicity, and improve gene silencing efficiency.

[0073] II. Experimental Methods

[0074] 1. Cervical exfoliated cell test: Thinprepcytologic test (TCT) of the cervix, and the results are reported using the TBS (The Bethesda system).

[0075] 2. HPV Genotyping: The KAP HPV 21 Genotyping Kit was used for flow hybridization detection. It can detect 21 HPV subtypes, including 15 high-risk HPV types (16,18,31,33,35,39,45,51,52,53,56,58,59,66,68) and 6 low-risk HPV types (6,11,41,42,44,CP8304).

[0076] 3. Serum folic acid detection: The folic acid content in the serum of the study subjects was detected using a chemiluminescence method. The World Health Organization Technical Advisory Group on Folic Acid and Vitamin B12 Deficiency has defined folic acid deficiency as a folic acid concentration of less than 4 ng / ml (10 mol / L).

[0077] 4. miRNA microarray detection:

[0078] (1) Total RNA extraction from cervical tissue: In a mortar with liquid nitrogen, cut cervical tissue into small pieces and grind it into powder. Take 50 mg of the powder and add it to an EP tube (containing 1 ml of Trizol solution), mix well. Incubate at room temperature for 5 min, add chloroform (200 μl), seal the EP tube tightly, and shake vigorously for 15 s. Centrifuge at 12000 rpm (10 min), transfer the upper aqueous phase to another EP tube, add isopropanol (500 μl), and mix gently. Incubate at room temperature for 10 min, centrifuge at 12000 rpm (10 min). Discard the supernatant, add 75% ethanol (1 ml), vortex to mix, and centrifuge at 12000 rpm (5 min) at 4℃. Repeat the operation once. Discard the supernatant and dry at room temperature or under vacuum (5-10 min). Dissolve the RNA in DEPC water, and if necessary, incubate in a warm water bath for 10 min. Transfer the supernatant to a centrifuge tube, add chloroform (1 / 5 volume), mix well, incubate for 3 min, and centrifuge at 12000 rpm for 15 min at 4°C; repeat once. Transfer the supernatant to a centrifuge tube, add anhydrous ethanol (1.5 volume), and mix well. Transfer the above solution to an adsorption column, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and return it to the collection tube. Add RWT (350 μl) to the adsorption column, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and then return it to the collection tube. Add DNAseI (80 μl) to the adsorption column, digest DNA at 25°C for 15 min, and mix gently; then add RWT (350 μl) to the adsorption column, centrifuge at 8000 rpm for 1 min, discard the waste liquid, and then return it to the collection tube. Wash once with RPE washing buffer (500 μl), centrifuge at 8000 rpm for 1 min. Discard the waste liquid in the collection tube, and then centrifuge at 12000 rpm for 2 min. Transfer the adsorption column to the collection tube, add 15-20 μl of preheated (50°C) RNase-free water, incubate at room temperature for 2 min, and centrifuge at 12000 rpm for 2 min to obtain the RNA solution.

[0079] (2) miRNA microarray detection steps: ① Poly(A) Tailing: Add 1 μg of total RNA to an RNase-free PCR tube, add RNase-free water to 8 μl, and incubate on ice. Add 2 μl of RNA Spike Control Oligos and mix well. Dilute ATP Mix with 1 mM Tris, dilution factor = 1:500. Add 5 μl of master mix to the PCR tube, mix well and centrifuge for 5 s. The master mix components include 1.5 μl of 10×Reaction, 1.5 μl of 25 mM MnCl2, 1 μl of Diluted ATP Mix, and 1 μl of PAP Enzyme. Incubate in a PCR instrument at 37℃ for 15 min, then centrifuge for 5 s and place on ice. ② Flash Tag Biotin HSR Ligation: Add 4 μl of 5×Flash Tag Biotin HSR ligation mixture, add 2 μl of T4 ligase, mix gently, centrifuge for 5 s, incubate at 25℃ for 30 min, and centrifuge for 5 s. Add 2.5 μl of HSR Stop Solution, mix well, centrifuge for 5 seconds, then aspirate 2 μl for ELOSA QC Assay testing; store the remainder at -20℃. ③ Chip hybridization: Remove the chip from the refrigerator and place it at room temperature; add 5 μl of 20 × Eukaryotic Hybridization controls to an RNase-free PCR tube and incubate at 65℃ for 5 min on a PCR instrument; prepare 103.2 μl of hybridization solution, including 21.5 μl of biotin-labeled sample, 50 μl of 2×Hybridization, 15 μl of 27.5% Formamide, 10 μl of DMSO, 5 μl of 20×Eukaryotic Hybridization controls, and 1.7 μl of Control Oligonucleotide B2. Centrifuge for 5 seconds and incubate on a PCR instrument. Centrifuge the above liquid for 5 seconds, then inject 100 μl into the chip and seal the sample opening with a sticker. Place in a hybridization oven at 48℃, 60 rpm for 16 h.④ Chip washing and scanning: After hybridization, aspirate the hybridization solution and add 100 μl of array holding buffer; in the Affymetrix GeneChipCommand Console (AGCC), scan the chip barcode, register the sample information, and generate the corresponding chip ARR file; place the chip washing buffer in the appropriate position and run the AGCCFutions Control software; after initialization, add 600 ml of stain Cocktail 1, 600 ml of stain Cocktail 2, and 800 ml of array holding buffer to three RNase-free centrifuge tubes (1.5 ml), respectively, and place them in the appropriate positions (1, 2, 3) of the elution station, then insert the chip into the elution station slot; scan the chip barcode in the elution station, select the chip type, click run, and wash for 1 hour; place the chip in the Affymetrix scanner, click AGCCScan Control, and generate chip scan data.

[0080] 5. Preparation of DPF-Exo:

[0081] (1) Preparation of exosomes by extrusion method: 1) Cell culture and collection: 293T cells in good growth condition were expanded and cultured. When the cells reached 70%-80% confluence, they were harvested, centrifuged, and washed with PBS. 2) Extrusion: The cells were continuously extruded using a handheld liposome extruder to break them and obtain extruded exosomes. 3) Exosome collection: Cell debris was removed by centrifugation at 10,000g, followed by filtration through 0.22μm, and finally exosomes were collected by centrifugation at 100,000g and resuspended in PBS. 4) DSPE-PEG-FA modification: DSPE-PEG-FA and exosomes were mixed in PBS and incubated at room temperature for 30 min. Excess DSPE-PEG-FA was removed by ultrafiltration, and the modified DPF-Exo was collected by ultracentrifugation.

[0082] (2) Physicochemical characterization of exosomes: 1) The particle size and zeta potential of freshly prepared Exo and DPF-Exo were detected by NTA detector. 2) The morphology of Exo and DPF-Exo was observed and photographed by transmission electron microscopy.

[0083] (3) Detection of exosome markers: 1) Proteins from 293T cells, Exo, and DPF-Exo were extracted using Ripa lysis buffer. Specifically, Ripa lysis buffer and Exo or DPF-Exo resuspended in PBS were mixed at a 1:1 ratio, incubated on ice for 1 h, and the cells were sonicated to disrupt the cells and extract total protein from the exosomes. 2) The positive marker proteins CD63 and Flotillin of native exosomes and the negative marker protein Calnexin were detected.

[0084] (4) CCK8 assay for the biocompatibility of DPF-Exo: 1) Logarithmic growth phase 293T cells were seeded at an appropriate density in 96-well plates. 2) After 24 h, concentrations of Exo or DPF-Exo of 0, 3.125, 6.25, 12.5, 25, 50 and 100 μg / ml were added, while the control group was treated with the same concentration of commercially available transfection reagent Lipo2000. 3) After culturing for another 24 h, cell viability was detected using CCK8 assay to reflect the cytotoxicity of different concentrations of Lipo2000, Exo and DPF-Exo.

[0085] (5) Detection of the ability of DPF-Exo to deliver small nucleic acids by fluorescence imaging and flow cytometry: 1) Take cells in the logarithmic growth phase and seed HeLa and Siha cells at an appropriate density in 24-well plates. After 24 hours of adhesion, they are used for subsequent experiments. 2) Load fluorescently labeled small nucleic acids (siRNA or miRNA inhibitor) into Exo or DPF-Exo using electroporation and add them to Siha or HeLa cells. The positive control uses the commercially available transfection reagent Lipo2000. 3) After 24 hours of treatment, observe the red fluorescence in Siha or HeLa cells by fluorescence imaging. 4) Detect the uptake efficiency and average fluorescence intensity of Siha and HeLa cells by flow cytometry.

[0086] (6) Q-PCR detection of DPF-Exo-mediated gene silencing efficiency: 1) Cell treatment: HeLa and Siha cells in logarithmic growth phase were seeded at appropriate densities in 6-well plates and allowed to adhere for 24 hours before subsequent experiments. 2) Therapeutic small nucleic acids (IGF2BP3 siRNA or miR-27a-3p inhibitor) were loaded into DPF-Exo via electroporation and added to Siha or HeLa cells. The positive control used commercially available transfection reagent Lipo2000. 3) After 48 hours, total RNA or miRNA was extracted as needed, and its concentration was detected using a NanoDrop2000 instrument. cDNA or stem-loop miRNA was prepared by reverse transcription, followed by Q-PCR amplification and real-time quantitative analysis.

[0087] 7. Automated Live-Cell Imaging Viability Assay: Viability was assessed using a density of 30,000 cells per well. Cells were cultured for 24 hours in fresh, folic acid-free RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) containing 0, 200, 500, 1000, or 2000 nM folic acid (Sigma-Aldrich; Merck, KGaA, Germany). Three technical replicate wells were used for each experiment under each condition, and imaging was performed using a Lionheart™ FX (BioTek Instruments, USA). Phase difference and fluorescence images were acquired at each time point using a 10x objective lens. Images were automatically acquired hourly for a total of 48 hours.

[0088] 8. Wound Healing Assay: HeLa and SiHa cells were seeded in fully cultured 96-well plates. Once the cells reached 90% confluence, a scratch was made using the tip of a 2 mm wide plastic pipette. After washing three times with phosphate-buffered saline (PBS), the cells were cultured for 24 hours in folic acid-free RPMI 1640 medium containing different folic acid concentrations (0, 200, 500, 1000, 2000 nM). Subsequently, cell migration to the blank area was observed using a Lionheart™ FX automated live-cell imaging system (BioTek Instruments, Flinklin Lake, NJ, USA). The migration rate was calculated as: (0-hour scratch distance - 24-hour scratch distance) / 0-hour scratch distance. The migration index was calculated as: (0-hour scratch distance - 24-hour scratch distance) / 0-hour scratch distance. All experiments were repeated at least three times.

[0089] 9. Transwell Invasion Assay: The Matrigel invasion assay was used. 24-well plates (Sigma-Aldrich, Merck Kgaa, Germany) were inserted into Matrigel-containing membranes (BDBiosciences, Franklin Lake, New Jersey, USA) with 8-micron pores. 300 cells per well were suspended in 100 µL of RPMI 1640 medium containing different folic acid concentrations (0, 200, 500, 1000, 2000 nM). The upper chamber was added, and the plates were incubated at 37°C for 24 hours. 500 µL of RPMI 1640 medium containing 30% fetal bovine serum (FBS) was added to each lower chamber. After incubation, uninvaded cells on the upper surface were gently removed with a cotton swab. The cells were then fixed with 4% formaldehyde (for 20 minutes) or without, stained with 0.1% crystal violet at room temperature for 5 minutes, and finally, invaded cells on the lower surface were observed using a Lionheart™ FX automated live-cell imaging system. All assays were performed in triplicate, and the average number of invaded cells was calculated.

[0090] 10. RT-qPCR: Total RNA was extracted from HeLa and SiHa cells using TRIzol reagent (Invitrogen, Carlsbad, California, USA) according to the manufacturer's instructions. Real-time quantitative PCR was performed using the SYBR Green PCR kit (Takara Biotech, Dalian, China). U6 was used as an internal control for miR-27a-3p and target genes. The PCR was performed on an ABIStep One Plus real-time PCR system (Applied Biosystems, Foster City, USA).

[0091] 11. Genomics:

[0092] (1) Spatial transcriptomics and single-cell sequencing: 1) Sample collection: Serum folate and erythrocyte folate were first detected by peripheral blood samples. Cervical tissue was collected from those that met the requirements (control group: 340-1540 nmol / L; experimental group <340 nmol / L) and lysed with cell lysis buffer. 2) Dialysis: The collected samples were dialyzed using dialysis tubes. 3) Protein concentration determination: The protein concentration of the samples was detected using a BCA kit. 4) Biotin labeling: ① 100 μl of 1×PBS was added to the tube to prepare the labeling reagent. ② After adding the labeling reagent, the tube was incubated on a shaker at room temperature for 30 min. ③ Unbound biotin was removed using a stop solution. ④ Dialysis was performed at 4℃. The solution was changed every 3 h. 5) Blocking: ① BSA blocking solution was added and incubated on a shaker at room temperature for 30 min. ② Part of the blocking solution was removed, the sample was added, and the tube was shaken overnight at 4℃. ③ The slides were washed. ④ Add Cy3-streptavidin for secondary antibody incubation, and incubate at room temperature with shaking in the dark for 2 hours. ⑤ Clean the slides. 6) Fluorescence detection: Use a laser scanner such as Axon GenePix to scan the signal, using the Cy3 or green channel (excitation frequency = 532nm). Analyze the data using software.

[0093] (2) Multi-omics data analysis: Gene expression data of control and HPV(+) tissues were analyzed using R software (version 4.0). 1) Spatial and single-cell data integration: The data were converted into Seurat objects using the Seurat R package (version 4.2.0), and integrated using the "FindIntegrationAnchors" and "IntegrateData" functions in the Seurat package. 2) Pseudo-bμlk labeling of cell number: The MetaCell method used the K-nn graph algorithm to divide the scRNA-seq dataset into disjoint and homogeneous cell groups (metacells), and performed independent analysis on epithelial cells, endothelial cells, myeloid cells, smooth muscle cells, and fibroblasts. Based on the inter-cell similarity matrix, two balanced K-nn similarity graphs for different cells were constructed using the parameter K (the number of neighbors of each cell is limited by K). 3) WGCNA was used to examine key pathways related to cell function: Single-nuclear data were analyzed using high-dimensional weighted gene co-expression network analysis (hdWGCNA). Modules were defined based on gene expression in the metacellular. The first major component of a module, called the module intrinsic gene, is associated with diagnosis and other variables. Hub genes were defined using the module intra-module connectivity (kME) parameter. Gene set enrichment analysis was performed using EnrichR. 4) A communication network via ligand-receptor interactions was constructed using CellPhoneDB software (version 2.1.4).

[0094] 12. Subcutaneous tumorigenesis experiment in animals: Nude mice were randomly divided into groups of 6. The established folic acid deficiency and methylation disorder cell model was subcutaneously injected. 2 × 10⁻⁶ mg / L of the drug was used. 6 The cells from each of the above-mentioned groups, cultured in vitro, were injected weekly into the axillae of nude mice to establish xenograft tumor models. The mice were cultured for 4-6 weeks, and tumor formation was monitored. After euthanizing the mice, tumors and metastatic tissues were removed for relevant analysis.

[0095] 13. Other detection methods:

[0096] (1) m 6 A. Quantitative detection: Total RNA was extracted from HPV(+) samples, m 6 A-RNA methylation quantitative kit measures m in total RNA. 6 A. Modification level. RNA was incubated at 37°C for 90 min, and antibodies, α-antibody, and β-enhancer were added for detection. Finally, chromogenic buffer was added for signal detection using a microplate reader. 6 A-dot hybridization analysis was performed according to the instructions, using an RNA purification kit to isolate poly(A) RNA from total RNA. Serially diluted RNA dots were then visualized on an N-membrane. The membrane was then UV cross-linked and blocked, and m... 6After incubation with antibody A as the primary antibody, followed by incubation with secondary antibody, ECL luminescent solution is added, and the mixture is detected by an instrument.

[0097] (2) Dual-luciferase reporter assay: After determining m 6 IGF2BP3 enzyme A exhibits cell specificity in low-folate HPV(+) cervical epithelium. The binding of miR-27a to IGF2BP3 was detected by dual-luciferase reporter assay, and the binding level was also measured before and after folic acid treatment.

[0098] (3) Proliferation assay to detect cell proliferation capacity: Logarithmic growth phase cells were taken, and Ect1 E6 / 7, HeLa and Siha cells were seeded at appropriate densities in 96-well plates, 1×10⁶ cells / well. 4 Cells / well. Cell proliferation rate was calculated using the built-in software of the BioTek live cell workstation for 24-hour dynamic monitoring. The experiment was repeated three times.

[0099] (4) Flow cytometry detection of apoptosis: The apoptosis detection kit of Annexin V-FITC was used. Log-phase cells were collected, resuspended in Annexin V-FITC binding solution, and incubated at room temperature in the dark. After centrifugation, the supernatant was discarded, the cells were resuspended in Annexin V-FITC binding solution, and PI staining solution was added for flow cytometry detection.

[0100] (5) Scratch test to detect cell migration ability: Logarithmic growth phase cells were seeded into 96-well plates, adhered to form a monolayer, and grown to 100% of the plate area. Scratches were made on the monolayer cells with a 10 μl pipette tip and then cultured. The area change was detected using a live cell workstation over 24 hours, and the built-in software was used for measurement and analysis. The experiment was repeated 3 times.

[0101] (6) Transwell assay to detect cell invasion ability: Matrigel dilution was used to coat the bottom layer of the Transwell chamber, and logarithmic growth phase cells were taken to prepare a number of 1×10⁶ cells. 5 Cell suspensions were prepared by adding high-concentration serum-containing culture medium outside the transwell chamber and serum-free cell suspension inside the chamber. The number of invading cells was observed at the bottom of the transwell chamber 24 hours a day using a live-cell workstation, and the data was measured and analyzed using the built-in software. The experiment was repeated three times.

[0102] (7) MeRIP detection: MeRIP was used to detect the m-cell ... 6 A modifies the level. Determine m. 6 IGF2BP3 exhibits cell specificity in folate-deficient HPV-positive cervical epithelium. Detection of IGF2BP3 and miR-27a levels after folate intervention showed that, following miR-27a silencing, the levels of miR-27a and IGF2BP3 were significantly reduced. 6A. Changes in modification levels; preliminary basic findings showed that low folic acid significantly upregulated miR-27a in HPV(+) cervical epithelium. MeRIP assays showed that miR-27a levels after folic acid intervention were... 6 A modifies the level.

[0103] III. Experimental Results

[0104] A cross-sectional study of 14,000 individuals found that the incidence of cervical lesions was higher in people with low folic acid levels.

[0105] A cross-sectional study was conducted on baseline cervical lesion data from a prospective cohort study of 2304 women screened for cervical lesions out of 40,000 women. The results showed a negative correlation between the concentrations of serum folic acid, erythrocyte folic acid, and dietary folic acid and the risk of cervical lesions. Figure 1 ).

[0106] 2. Using multi-omics sequencing results combined with bioinformatics, we mapped the m-cell structure of cervical epithelial cells with persistent HPV infection and folic acid deficiency. 6 A modifies the abnormal graph.

[0107] 1) Data analysis of scRNA-segments from 3 patients with low folate (<340 nM) and HPV(+) cervical lesions and 3 healthy individuals with normal folate levels was performed, presenting the regulatory profile of various cells in patients with low folate and HPV(+). Compared with the control group, the m... 6 IGF-BP3 enzyme A specifically regulates folic acid metabolism and tumor-related pathways, affecting other cellular dysfunctions and inducing HPV susceptibility. Figure 2 ).

[0108] 2) By each cell type m 6 Specific regulation of enzyme A, determining m 6 IGF2BP3 is specifically upregulated in epithelial cells. IGF2BP3 specifically regulates cervical lesion-related functions and negatively regulates folate metabolism. WGCNA verification showed that IGF2BP3 also abnormally regulates cervical lesion pathways in low-folate HPV(+) cervical epithelial cells, leading to epithelial-mesenchymal transition (EMT). Figure 3 ).

[0109] 3) Determine m 6 The enzyme IGF2BP3 specifically regulates EMT in folate-deficient HPV(+) cervical epithelium. This invention, through subgrouping of diseased cells and combining copy number variation (CNV) analysis, discovered an independent pathogenic cell subpopulation EP3 within epithelial cells. EP3 expresses IGF2BP3 that primarily regulates cell cycle abnormalities, DNA damage, folate metabolism abnormalities, and m... 6Aberrant modification and epithelial EMT. Further evidence shows that low folic acid levels impair cervical epithelial cell m... 6 Aberrant modification of IGF2BP3 leads to specific dysregulation of IGF2BP3, resulting in persistent HPV infection. Figure 4 ).

[0110] 3. Preliminary establishment of a cell model of folic acid deficiency and methylation metabolic disorders.

[0111] HPV-positive cervical immortalized epithelial cell lines (Ect1 E6 / E7) and cervical lesion cell lines (HeLa, Siha) were selected as cell models to analyze and verify folic acid deficiency and folic acid-methylation metabolic disorders. This invention demonstrates that the proliferation, migration, and invasion abilities of HPV-positive cervical cells decrease with increasing folic acid concentration. The correlation between folic acid-methylation and cell function in cervical lesion cells was dynamically explored. Scratch assay results showed that the migration abilities of Ect1 E6 / E7, HeLa, and Siha cells in the Folate group were significantly reduced compared to the NC group; the migration abilities of Ect1 E6 / E7, HeLa, and Siha cells in the 5-aza (5-azacytidine, a demethylating drug) group were significantly reduced compared to the NC group; and the migration abilities of Ect1 E6 / E7, HeLa, and Siha cells in the Folate+5-aza group were significantly reduced compared to the 5-aza group. The cell model confirmed that folic acid deficiency can affect methylation in cervical lesion cells. Further animal studies validated the relationship between folic acid deficiency and methylation in cervical lesions. Results showed statistically significant differences in tumor size among the NC group, Folate group, 5-aza group, and Folate+5-aza group, consistent with the cell experiments. Figure 5 ).

[0112] 4. Low folic acid levels can lead to an imbalance of IGF2BP3 / miR-27a in cervical tissue with persistent HPV infection, causing further HPV infection of cervical epithelial cells and resulting in cervical lesions.

[0113] 1) Low folic acid makes m 6 Enzyme A, IGF2BP3, specifically regulates viral susceptibility in the cervical epithelium of HPV-positive patients, leading to epithelial cell EMT. Using siRNA to silence IGF2BP3 and examining the cellular function of HPV-positive cells, it was found that IGF2BP3 promotes the proliferation, migration, and invasion of HPV-positive cells. Further analysis of IGF2BP3 levels in HPV-infected ECT1 cells after folic acid concentration IC50 intervention revealed that... 6 A modification level revealed that folic acid inhibited IGF2BP3 m 6A modification level was determined and validated using a folate metabolism disorder model. A negative correlation was found between serum folate levels and IGF2BP3 expression in HPV(+) patients, which was further validated by immunohistochemistry using an animal model of folate metabolism disorder. Figure 6 ).

[0114] 2) Using the Affymetrix GeneChip miRNA 4.0 Array microarray, the expression characteristics of different miRNAs were determined in 5 healthy individuals with normal serum folate levels and 5 patients with folate deficiency and HPV (+), and miR-27a was screened out. After transfection with miR-27a-3p mimics using plasmids, it was found that high expression of miR-27a promoted the proliferation, migration, invasion, and clustering of HPV (+) cells; it was clarified that folate can inhibit the expression of miR-27a. 6 A modification was reduced; and IGF2BP3 WT and IGF2BP3 MUT gene fragments were designed and synthesized (the mutant sequence is a mutant sequence that causes IGF2BP3 to lose its original function and is only used to verify function). Dual-luciferase reporter assay showed that miR-27a targets and is associated with IGF2BP3. Figure 7 ).

[0115] 3) Microarray screening identified 15 miRNAs associated with folic acid deficiency, revealing upregulated expression of miR-27a. Expanding the sample size of miR-27a expression in different cervical tissues, PCR analysis showed a negative correlation between miR-27a expression levels and serum folic acid levels; however, a positive correlation was found between miR-27a expression and IGF2BP3 expression. Further, cellular, molecular, and animal experiments validated the role of folic acid deficiency in miR-27a expression. 6 Aberrant modification leading to dysregulation of IGF2BP3 / miR-27a can promote the expression of HPV-related pathways, resulting in cervical epithelial cell EMT (emergency metastasis). Figure 8 ).

[0116] 5. Construct a folic acid-modified engineered Exo carrying siRNA DPF-Exo / miR-27a-3p-inhibitor combined with DPF-Exo / IGF2BP3-siRNA complex for targeted treatment of HPV-infected cervical lesions.

[0117] 1) Exosomes were collected from 293T cells. DSPE-PEG-FA (DPF) and Exo were mixed, and the modified DPF-Exo was collected. NTA and transmission electron microscopy were used for quality control of freshly prepared Exo and DPF-Exo. Western blotting was used to detect the positive marker proteins CD63 and Flotillin-1 of native exosomes, and cell viability was assessed to reflect the biocompatibility and cytotoxicity of DPF-Exo. Figure 9 ).

[0118] 2) Fluorescently labeled small nucleic acids (siRNA or miRNA inhibitor) were loaded into Exo or DPF-Exo via electroporation. DPF-Exo / siRNA was then added to Siha or HeLa cells. Confocal microscopy and flow cytometry were used to detect the uptake efficiency of DPF-Exo / miR-27a-3p-inhibitor and DPF-Exo / IGF2BP3-siRNA by HPV(+) cervical cells. After 48 hours, the inhibitory effect of DPF-Exo / siRNA on relevant genes in Siha and HeLa cells was analyzed by real-time quantitative PCR. Figure 10 The results showed that folic acid-modified exosomes DPF-Exo were more efficient at delivering small nucleic acid drugs to HPV(+) cervical cells, exhibiting better inhibitory effects and improved safety. This indicates that DPF-Exo / miR-27a-3p-inhibitor and DPF-Exo / IGF2BP3-siRNA may be used as drugs for treating cervical lesions caused by low folic acid-mediated HPV infection.

[0119] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An exosome for the treatment of cervical lesions induced by low folic acid-mediated HPV infection, characterized in that, The exosomes are folic acid-modified exosomes and small nucleic acids targeting IGF2BP3 and / or miR-27a loaded therein; The small nucleic acid is a siRNA targeting IGF2BP3 and / or a miRNA inhibitor targeting miR-27a; The nucleotide sequence of the siRNA targeting IGF2BP3 is shown in SEQ ID NO:1-2; The nucleotide sequence of the miRNA inhibitor targeting miR-27a is shown in SEQ ID NO:3; The folic acid-modified exosome surface proteins CD63 and Flotillin-1 were positive.

2. A method for preparing exosomes as described in claim 1, characterized in that, The exosomes are folic acid-modified exosomes and small nucleic acids targeting IGF2BP3 and / or miR-27a loaded therein, and the preparation method includes the following steps: 1) Mix DSPE-PEG-FA with Exo and collect the modified DPF-Exo; 2) Load small nucleic acids targeting IGF2BP3 and / or miR-27a into DPF-Exo to obtain folic acid-modified exosomes.

3. The preparation method according to claim 2, characterized in that, The particle size of the folic acid-modified exosomes is 50~1000 nm.

4. The preparation method according to claim 3, characterized in that, The particle size of the folic acid-modified exosomes is 100~500 nm.

5. The preparation method according to claim 2, characterized in that, The loading is performed using the electroporation method.

6. The use of the exosomes as described in claim 1 or the preparation method as described in any one of claims 2-5 in the preparation of a drug or kit for the treatment of cervical lesions caused by low folic acid-mediated HPV infection.

7. The application according to claim 6, characterized in that, The drug can be used alone or in the form of a pharmaceutical composition.

8. The application according to claim 7, characterized in that, The pharmaceutical composition also includes pharmaceutically acceptable excipients.

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