Use of ythdf3 in preparation of products for diagnosing and treating cervical cancer

Abstract

The application belongs to the field of biological medicine, and discloses application of YTHDF3 in preparation of products for diagnosing and treating cervical cancer. Specifically disclosed are: application of YTHDF3 as a biomarker in preparation of reagents for diagnosing cervical cancer and lymph node metastasis of cervical cancer; application of YTHDF3 protein in preparation of a prognostic evaluation kit for cervical cancer, the kit taking the above protein as a prognostic evaluation marker; a drug for treating cervical cancer, including any one of a drug capable of specifically inhibiting SREBF1 gene or protein, a drug capable of specifically inhibiting YTHDF3 gene or protein, a drug for inhibiting translation of an m6A modified transcript of LRP6, and a drug for inhibiting proliferation, migration and invasion of cervical cancer cells. The above products are used for diagnosing and treating cervical cancer, and have good specificity. In addition, YTHDF3 can promote lymph node metastasis of cervical cancer, so products taking the molecule as a marker can effectively treat cervical cancer with lymph node metastasis.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to application of YTDHF3 in preparation of products for diagnosing and treating cervical cancer. BACKGROUND

[0002] Cervical cancer is one of the most common gynecological malignancies, and is the second highest incidence tumor next to breast cancer. The main cause of the disease is high-risk human papillomavirus (HPV) infection. At present, for early cervical cancer, the main treatment is surgery, and postoperative adjuvant radiotherapy and chemotherapy are determined according to the risk factors; for advanced cervical cancer, radical concurrent chemoradiotherapy is the main treatment. However, although after standard treatment, the average 5-year survival rate of cervical cancer is about 45%; but the 5-year survival rate of recurrent metastatic cervical cancer after first-line treatment is only 15%. Therefore, it is urgent to improve the early diagnosis efficiency and treatment efficiency of cervical cancer.

[0003] An ideal biomarker not only helps early diagnosis and prognosis of tumors, but also is crucial for developing precise treatment plans for tumors. So far, there are few effective early diagnosis and prognosis methods based on biomarkers for cervical cancer. Therefore, it is of great clinical value to provide an ideal biomarker and prepare products for diagnosing and treating cervical cancer based on the biomarker. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes application of YTHDF3 as a biomarker in preparation of a reagent for diagnosing cervical cancer.

[0005] The present application also proposes application of YTHDF3 as a biomarker in preparation of a reagent for diagnosing lymph node metastasis of cervical cancer.

[0006] The present application also proposes application of YTHDF3 protein in preparation of a reagent for evaluating prognosis of cervical cancer.

[0007] The present application also proposes a drug for treating cervical cancer.

[0008] N6-methyladenosine (m6A) is the most abundant modification in eukaryotic messenger RNA (mRNA) and plays an important role in cell differentiation and tissue development. It regulates multiple RNA life processes: through recognition and selective binding proteins, m6A regulates the RNA life cycle including RNA processing, translation and degradation. YTH domain family proteins as a kind of m6A "reader" protein directly bind and recognize the methylation of m6A in mRNA. YTHDF3, the third member of the YTH family, is a cytoplasmic m6A binding protein that can bind m6A.

[0009] In previous studies, transcriptomic sequencing of cervical cancer cell line SiHa and normal immortalized epithelial cells H8 was performed using RNA sequencing (RNA-seq) technology. MeRIP-seq, employing m6A-specific methylated RNA immunoprecipitation, revealed that multiple mRNA sites in SiHa cells showed m6A-RNA methylation modification compared to H8 cells. KEGG analysis further identified differentially methylated genes primarily associated with the important cancer regulatory pathway, the Wnt signaling pathway. Furthermore, RNA-seq studies of SiHa and YTHDF3-knockout SiHa cells also revealed differentially expressed genes related to the Wnt signaling pathway, such as LRP5, LRP6, and FZD3, through GO and KEGG analyses. Therefore, subsequent analyses in this invention primarily focus on the Wnt signaling pathway. Based on the above research, tissue microarray analysis was performed on 116 clinical samples (19 normal cervical epithelial cells, 13 cervical epithelial neoplasia grade 3 (C1N3), and 84 cervical squamous cell carcinoma samples). The results showed that compared to normal cervical epithelial tissue, the expression of YTHDF3 protein was increased in both C1N3 and cervical cancer tissues, and the abundance of m6A modification was also increased (p < 0.05). Furthermore, YTHDF3 expression was found to be associated with poor patient prognosis (p < 0.05). Currently, the function and role of YTHDF3 in cervical cancer have not been reported, and its upstream and downstream regulatory mechanisms remain unclear, especially its relationship with HPV infection. Based on this, the present invention is proposed.

[0010] According to one aspect of the present invention, the application of YTHDF3 as a biomarker in the preparation of reagents for diagnosing cervical cancer is proposed.

[0011] According to a second aspect of the present invention, the application of YTHDF3 as a biomarker in the preparation of reagents for diagnosing lymph node metastasis in cervical cancer is proposed.

[0012] According to a third aspect of the present invention, the application of YTHDF3 protein in the preparation of a cervical cancer prognostic assessment kit is proposed, wherein the cervical cancer prognostic assessment kit uses the YTHDF3 protein as a prognostic assessment marker.

[0013] In some embodiments of the present invention, the cervical cancer prognostic assessment kit detects the expression of YTHDF3 protein in surgically removed tissue of cervical cancer patients using immunohistochemistry. The degree of expression of YTHDF3 protein within the cancer is negatively correlated with the prognostic survival rate of cervical cancer patients.

[0014] In some embodiments of the present invention, the expression level of the YTHDF3 protein in cancerous tissue is higher than that in adjacent or normal tissue and is positively correlated with the occurrence of cervical cancer.

[0015] According to a fourth aspect of the present invention, a medicament for treating cervical cancer is provided, said medicament comprising any one of the following A to D:

[0016] A. Drugs that can specifically inhibit the SREBF1 gene or protein;

[0017] B. Drugs that can specifically inhibit the YTHDF3 gene or protein;

[0018] C. Drugs used to inhibit the translation of LRP6 m6A-modified transcripts;

[0019] D. Drugs used to inhibit the proliferation, migration, and invasion of cervical cancer cells.

[0020] In some embodiments of the present invention, the drug is an inhibitor that interferes with the transcription and translation of YTHDF3.

[0021] In some embodiments of the present invention, the drug is an SREBF1 inhibitor used to inhibit YTHDF3 gene transcription or protein expression to treat the cervical cancer.

[0022] SREBF1, sterol regulatory element-binding protein 1, is a key transcription factor in the sterol pathway, regulating the expression of a series of important lipid synthesis genes by sensing the concentration of environmental sterols. The inventors predicted and experimentally verified that SREBF1 is an upstream regulator of YTHDF3 molecule expression using ATAC-seq and ChIP-seq technologies. Therefore, the drug, by inhibiting SREBF1, can inhibit the expression of the YTHDF3 gene or protein, and thus can be used to treat cervical cancer.

[0023] In some embodiments of the present invention, the drug is a drug that specifically inhibits the activity of the YTHDF3 gene or protein.

[0024] Specifically, drugs that specifically inhibit the activity of the YTHDF3 gene or protein include at least one of a knockdown drug, a knockout drug, or a receptor blocking drug targeting the YTHDF3 protein.

[0025] In some embodiments of the present invention, the knockdown drug includes any one of shRNA, siRNA, or CRISPR / Cas drugs that target the YTHDF3 gene.

[0026] In some embodiments of the present invention, the knockout drug is a drug comprising the Cre / LoxP system, used to specifically knock out the YTHDF3 gene.

[0027] In some embodiments of the present invention, the receptor blocking drug comprises any one of small molecule compounds or polypeptides.

[0028] The receptor-blocking drug exerts its effect by specifically binding to the YTHDF3 protein, thereby blocking the YTHDF3 protein from exerting its corresponding action.

[0029] In some embodiments of the present invention, the drug is a YTHDF3 inhibitor used to inhibit the proliferation, migration and invasion of the cervical cancer cells in order to treat the cervical cancer.

[0030] Specifically, knocking out the YTHDF3 protein reduced the proliferation, migration, and invasion of the cervical cancer cells.

[0031] In some embodiments of the present invention, the drug is a YTHDF3 inhibitor used to inhibit the translation of the m6A-modified transcript of LRP6, thereby inhibiting the expression of LRP6 and treating the cervical cancer.

[0032] LRP6, low-density lipoprotein (LDL) receptor-associated protein 6, is a member of the LDL receptor gene family. LDL receptors are transmembrane cell surface proteins involved in receptor-mediated endocytosis of lipoproteins and protein ligands. LRP6 acts as a receptor for Wnt or, together with frizzled, as a co-receptor for Wnt, thereby transmitting the typical Wnt / β-catenin signaling cascade. Through its interaction with the Wnt / β-catenin signaling cascade, the LRP6 gene plays a role in regulating cell differentiation, proliferation, migration, and the development of various cancer types.

[0033] Specifically, YTHDF3 affects LRP6 protein expression by promoting LRP6 translation efficiency. Therefore, the drug specifically inhibits YTHDF3 to inhibit LRP6 m6A, further inhibiting the translation of LRP6 m6A-modified transcripts, thus inhibiting LRP6 translation efficiency and consequently inhibiting LRP6 protein expression, thereby achieving the treatment of cervical cancer.

[0034] More specifically, advanced cervical cancer often metastasizes to lymph nodes, exhibiting high malignancy and a very low cure rate. This invention has found that YTHDF3 can promote lymph node metastasis and lymphatic canal formation. Therefore, by using the aforementioned drug to inhibit YTHDF3, the expression of LRP6 protein can be suppressed, thereby inhibiting the proliferation, migration, and invasion of cervical cancer cells, as well as suppressing lymph node metastasis and lymphatic canal formation, thus achieving a therapeutic effect on cervical cancer.

[0035] In some embodiments of the present invention, the drug is an LRP6 inhibitor used to inhibit the proliferation, migration and invasion of the cervical cancer cells, thereby treating the cervical cancer.

[0036] According to a preferred embodiment of the present invention, at least the following beneficial effects are achieved:

[0037] The products prepared using YTHDF3 as a biomarker proposed in this invention can be used for the diagnosis, treatment, and prognostic assessment of cervical cancer with high specificity, as well as for the diagnosis of cervical cancer lymph node metastasis. Reagents prepared using YTHDF3 as a biomarker can rapidly and effectively diagnose cervical cancer and cervical cancer lymph node metastasis at an early stage, facilitating medical professionals to develop precise treatment plans for cervical cancer patients or those with lymph node metastasis, thereby achieving early detection and treatment and improving survival rates. Kits prepared using YTHDF3 protein as a prognostic assessment marker play an important role in determining the prognosis of cervical cancer patients and provide important guidance for postoperative follow-up and sequential treatment of cervical cancer patients. Furthermore, the drug proposed in this invention can specifically inhibit SRERF1 or YTHDF3, thereby inhibiting LRP6 m6A, further inhibiting the translation of LRP6 m6A-modified transcripts, inhibiting LRP6 translation efficiency, and thus inhibiting LRP6 protein expression, thereby inhibiting the proliferation, migration, and invasion of cervical cancer cells, achieving the treatment of cervical cancer. This drug has high specificity and can effectively treat cervical cancer. Attached Figure Description

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0039] Figure 1 This is a tissue microarray detection image of YTHDF3 protein expression in normal cervical epithelial tissue and cervical squamous cell carcinoma tissue in Example 1 of the present invention; wherein, the top is a chemical staining image of the tissue microarray, the second row is a magnified view of a portion of the first row, the magnified portion is the part circled by a rectangle; the bottom is a quantitative image of YTHDF3 protein in the tissue microarray, Normal - normal cervical epithelial tissue, CCa - cervical squamous cell carcinoma tissue;

[0040] Figure 2 This is a survival curve diagram showing the effect of YTHDF3 protein expression on the survival of cervical cancer patients in Example 1 of the present invention; the left side is the survival curve diagram of 273 cervical cancer patients, and the right side is the survival curve diagram of 98 cervical cancer patients.

[0041] Figure 3 This is a Western blotting detection diagram of Example 2 of the present invention; wherein, WT - cervical cancer cells without YTHDF3 knockout, KO Y3 - cervical cancer cells with YTHDF3 knockout, shNC - human cervical cancer epithelial cells without YTHDF3 knockdown, shY3 - human cervical cancer epithelial cells with YTHDF3 knockdown, the same below, GAPDH - internal reference gene;

[0042] Figure 4 This is a graph showing the effect of YTHDF3 on the proliferation of SiHa and Caski cells detected by the CCK-8 assay in Example 2 of this invention.

[0043] Figure 5 The following graphs show the effects of YTHDF3 on the number of SiHa and Caski cell clones in Example 2 of this invention; where A is the effect on SiHa cell clones, B is a statistical graph of the number of cell clones in A, C is the effect on Caski cell clones, and D is a statistical graph of the number of cell clones in C.

[0044] Figure 6 The graphs show the effects of YTHDF3 on the migration of SiHa and Caski cells in Example 2 of this invention; where A is the graph showing the effect on SiHa cell migration, B is a statistical graph of cell migration rate in A, C is the graph showing the effect on Caski cell migration, and D is a statistical graph of cell migration rate in C; the scale bar is 250 μm.

[0045] Figure 7 The graphs show the effects of YTHDF3 on SiHa and Caski cell invasion in Example 2 of this invention; where A is the graph showing the effect on SiHa cell invasion, B is a graph showing the number of cells in each field of view of A, C is the graph showing the effect on Caski cell invasion, and D is a graph showing the number of cells in each field of view of C; the scale bar is 100 μm.

[0046] Figure 8 This is a diagram illustrating the effect of YTHDF3 on metastasis of the plantar-popliteal lymph nodes in mice in Example 2 of this invention; the left image is an in vivo imaging image, and the right image is a photograph.

[0047] Figure 9 This is an immunohistochemical image showing the effect of YTHDF3 on the formation of lymphatic tubules in mice in Example 2 of this invention;

[0048] Figure 10 This is a detection image of the effect of YTHDF3 on lymphatic tubule formation in Example 2 of the present invention; the left image is a microscopic image, and the right image is a quantitative image of lymphatic tubule length; the scale bar is 250 μm;

[0049] Figure 11 The images show the results of ChIP-qPCR and dual-luciferase reporter assays in Example 3 of this invention; the left image shows the results of ChIP-qPCR, and the right image shows the results of the dual-luciferase reporter assay; anti-IgG is the sample precipitated with IgG antibody, and anti-SREBF1 is the sample precipitated with SREBF1 antibody;

[0050] Figure 12This is a Western blot image showing the effect of SREBF1 on YTHDF3 protein in Example 3 of the present invention; si-NC is the control group, and si-SREBF1#2 and si-SREBF1#3 are two samples with SREBF1 knocked out, respectively.

[0051] Figure 13 This is a diagram showing the relative expression results of YTHDF3 and LRP6 at the RNA level in SiHa and Caski cells in Example 3 of this invention;

[0052] Figure 14 This is a diagram showing the protein expression results of YTHDF3 and LRP6 in SiHa and Caski cells in Example 3 of this invention;

[0053] Figure 15 This is a statistical graph of the results of MeRIP-qPCR in Example 3 of the present invention; where the left side shows the relative expression level of LRP6 mRNA in SiHa and Caski cells; and the right side shows the relative abundance of LRP6 mRNA in SiHa cells.

[0054] Figure 16 This is a Western blot image of YTHDF3 protein expression detected by RIP-qPCR in Example 3 of the present invention.

[0055] Figure 17 This is a WB image showing the LRP6 protein expression detection results after DAA treatment of SiHa and Caski cells in Example 3 of the present invention.

[0056] Figure 18 This is a statistical graph showing the expression levels of LRP6 mRNA in SiHa cells of the WT and KO Y3 groups after treatment with actinomycin-D in Example 3 of the present invention.

[0057] Figure 19 This is a WB image showing the expression of LRP6 and YTHDF3 proteins in SiHa cells of the WT and KO Y3 groups after treatment with actinomycinone in Example 3 of the present invention.

[0058] Figure 20 This is a detection diagram of LRP6 protein expression in normal cervical epithelial tissue and cervical squamous cell carcinoma tissue in Example 4 of the present invention; wherein, the upper part is an immunohistochemical staining diagram; the lower part is a quantitative diagram of LRP6 protein in the immunohistochemical staining diagram, Normal - normal cervical epithelial tissue, CCa - cervical squamous cell carcinoma tissue;

[0059] Figure 21 This is a Western blotting detection image of Example 4 of the present invention; where shNC - LRP6 not knocked out, shLRP6 - LRP6 knocked out, the same below, GAPDH - internal reference gene;

[0060] Figure 22 This is a graph showing the effect of LRP6 on the proliferation of SiHa and Caski cells detected by the CCK-8 assay in Example 4 of this invention.

[0061] Figure 23 The graph shows the effect of LRP6 on the migration of SiHa and Caski cells in Example 4 of this invention; where A is the effect on SiHa cell migration, B is the effect on Caski cell migration, and C is a statistical graph of cell migration rates in A and B; the scale bar is 250 μm.

[0062] Figure 24 The image shows the effect of LRP6 on the invasion of SiHa and Caski cells in Example 4 of this invention; the left image shows the effect on the invasion of SiHa and Caski cells, and the right image shows the statistical graph of the number of cells in each field of view; the scale bar is 100 μm. Detailed Implementation

[0063] The embodiments of the present invention are described in detail below. These embodiments are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0064] In the description of this invention, unless otherwise explicitly defined, terms such as "culture" and "sequencing" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0065] In the description of this invention, references to terms such as "one embodiment," "some embodiments," etc., indicate that a specific feature, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, materials, or characteristics described may be combined in any suitable manner in one or more embodiments.

[0066] Unless otherwise specified, the experimental methods used in the examples are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0067] In previous studies, transcriptomic sequencing of cervical cancer cell line SiHa and normal immortalized epithelial cells H8 was performed using RNA sequencing (RNA-seq) technology. MeRIP-seq, employing m6A-specific methylated RNA immunoprecipitation, revealed that multiple mRNA sites in SiHa cells showed m6A-RNA methylation modification compared to H8 cells. KEGG analysis further identified differentially methylated genes primarily associated with the important cancer regulatory pathway, the Wnt signaling pathway. Furthermore, RNA-seq studies of SiHa and YTHDF3-knockout SiHa cells also revealed differentially expressed genes related to the Wnt signaling pathway, such as LRP5, LRP6, and FZD3, through GO and KEGG analyses. Therefore, subsequent analyses in this invention primarily focus on the Wnt signaling pathway. Based on the above research, tissue microarray analysis was performed on 116 clinical samples (19 normal cervical epithelial tissues, 13 cervical epithelial neoplasia grade 3 (C1N3) tissues, and 84 cervical squamous cell carcinoma samples). The results showed that compared with normal cervical epithelial tissues, the expression of YTHDF3 protein was increased in C1N3 and cervical cancer tissues, and the abundance of m6A modification was increased (p < 0.05). In addition, YTHDF3 expression was found to be associated with poor patient prognosis (p < 0.05).

[0068] Example 1

[0069] This embodiment tested the expression of YTHDF3 protein in cervical cancer tissue and the relationship between this protein and the prognosis of cervical cancer. The specific process is as follows:

[0070] 1. Expression: Normal cervical epithelium and clinical samples of cervical squamous cell carcinoma were collected. Tissue samples from both groups were paraffin-embedded to create paraffin-embedded tissue microarrays. Sections were incubated with an antibody against YTHDF3 protein and then stained with horseradish peroxidase-labeled secondary antibody to detect the expression of YTHDF3 protein in cervical cancer tissues. The results are as follows: Figure 1 As shown. Figure 1 The image above represents a chemical staining pattern of the tissue microarray. Figure 1 The image below shows the quantitative distribution of YTHDF3 protein in a tissue microarray. The results indicate that the expression level of YTHDF3 protein is very low in normal cervical cancer epithelial tissue, while the expression level is significantly higher in cervical squamous cell carcinoma tissue than in normal cervical epithelial tissue. The quantitative image also shows the same results. This suggests that YTHDF3 protein is highly expressed in cervical cancer tissue, and therefore it can serve as a biomarker for the diagnosis, detection, and treatment of cervical cancer.

[0071] 2. Prognosis: Based on case follow-up, patients corresponding to the above-mentioned clinical samples of cervical squamous cell carcinoma were interviewed to calculate survival rates and plot survival curves, such as...Figure 2 As shown. Figure 2 The left image shows the survival analysis results of 273 cervical cancer patients using TCGA data, while the right image shows the survival analysis results of 98 cervical cancer patients based on tissue microarray data. The results show that patients with low YTHDF3 expression (Low, L) had significantly higher survival rates than those with high YTHDF3 expression (High, H), indicating a negative correlation between the expression level of YTHDF3 within the cancer and the prognostic survival rate of cervical cancer patients. This suggests that YTHDF3 protein can serve as a prognostic marker for cervical cancer, and a prognostic assessment kit for cervical cancer can be prepared based on this.

[0072] Example 2

[0073] This embodiment investigated the effects of YTHDF3 on cervical cancer cells, lymph node metastasis, and lymphatic canaliculus formation. The specific process was as follows:

[0074] 1. Knockout / knockdown of YTHDF3 protein in cervical cancer cell line (SiHa) and human cervical cancer epithelial cells (Caski).

[0075] SiHa cells (two groups: cervical cancer cells without YTHDF3 knockout (WT) and cervical cancer cells with YTHDF3 knockout (KOY3)) and Caski cells (two groups: human cervical cancer epithelial cells without YTHDF3 knockdown (shNC) and human cervical cancer epithelial cells with YTHDF3 knockdown (shY3); similar abbreviations below refer to these cell types) were seeded into different culture dishes and cultured with DMEM high-glucose medium + 10% fetal bovine serum + 1% penicillin / streptomycin. After the cells reached confluence with the bottom of the culture dish, the cells were harvested, and the corresponding total protein was extracted. The total protein was prepared into samples and subjected to Western blotting (WB) to detect the expression of YTHDF3 protein. The results are shown below. Figure 3 As shown. Figure 3 The results showed that, using GAPDH as an internal reference gene, the expression level of YTHDF3 protein in the knockout / knockdown group was significantly reduced compared with that in the non-knockout / non-knockdown group, indicating that the knockout / knockdown of YTHDF3 protein in KO Y3 and shY3 cells was successful.

[0076] 2. CCK-8 assay for detecting the proliferation of cervical cancer cells.

[0077] The two cell groups constructed in step 1, SiHa (WT, KO Y3) and Caski cells (shNC, shY3), were cultured until they reached confluence with the bottom of the culture dish. The cells were then digested and seeded into 96-well plates. CCK-8 reagent was added at 24h, 48h, and 72h post-seeding. OD values ​​were measured at 450nm after 2h. The results are as follows: Figure 4 As shown. Figure 4 The results showed that both groups of cells were in a proliferative state during the period from 0 to 72 hours after inoculation; however, starting from 24 hours, the OD value of the SiHa KO Y3 group was significantly lower than that of the WT group; and the OD value of the Caski shY3 group was significantly lower than that of the shNC group. This indicates that knocking out or reducing the YTHDF3 protein in cervical cancer cells reduces the proliferation of cervical cancer cells, meaning that YTHDF3 promotes the proliferation of cervical cancer cells.

[0078] 3. Detect the proliferation of cervical cancer cells using a clonogenic assay.

[0079] Two groups of cells (SiHa (WT, KO Y3) and Caski cells (shNC, shY3)) in logarithmic growth phase were digested with trypsin, added to complete culture medium, resuspended into cell suspensions, and counted. The two groups of cells were seeded into 6-well plates, 400–1000 cells per well. Culture was continued until 14 days or the cell count in most individual clones exceeded 50, with medium changes and cell status observation every 3 days. After cloning, the cells were photographed under a microscope, washed once with PBS, fixed with 1 mL of 4% paraformaldehyde per well for 30–60 min, washed once with PBS, stained with 1 mL of crystal violet solution per well for 10–20 min, washed several times with PBS, air-dried, and photographed with a digital camera. The results are shown below. Figure 5 As shown. Figure 5 In the diagram, A represents the effect on SiHa cell clones, B represents the statistical graph of cell clone number in A, C represents the effect on Caski cell clones, and D represents the statistical graph of cell clone number in C. The results show that in the SiHa cell group, compared with the WT group, knocking out YTHDF3 protein significantly reduced the number of cell clones; in the Caski cell group, compared with the shNC group, knocking down YTHDF3 protein also significantly reduced the number of cell clones. This indicates that knocking out or knocking down YTHDF3 protein in cervical cancer cells inhibits cervical cancer cell clonal expansion, i.e., inhibits cervical cancer cell proliferation.

[0080] 4. Detect the migration of cervical cancer cells using a cell scratch assay.

[0081] First, using a marker pen, draw evenly spaced horizontal lines on the back of a 6-well plate, approximately every 0.5–1 cm, passing through each well. Seed two cell groups (SiHa (WT, KO Y3) and Caski cells (shNC, shY3)) in the 6-well plates and culture overnight. The next day, when cell confluence reaches 100%, use a pipette tip or sterile toothpick perpendicular to the cell plane to make incisions along the horizontal lines on the back of the plate. After incision, wash the cells three times with sterile PBS to remove non-adherent cells (those drawn during incision), ensuring the incision gaps are clearly visible. Then replace with fresh serum-free medium. Incubate the cells at 37°C in a 5% CO2 incubator. Observe and measure the width of the incisions under a microscope at 0h and 24h, and take photographs. Use ImageJ software to extract 6–8 horizontal lines and calculate the mean intercellular distance. The results are shown below. Figure 6 As shown. Figure 6 In the diagram, A represents the effect on SiHa cell migration, B represents the statistical graph of cell migration rate in A, C represents the effect on Caski cell migration, and D represents the statistical graph of cell migration rate in C. The results show that in the SiHa cell group, after 24 hours of culture, the reduction in scratch width of WT group cells was significantly less than that of KO Y3 group, indicating that the migration rate of WT group cells was higher than that of KO Y3 group cells, consistent with the statistical graph results. Similarly, in the Caski cell group, after 24 hours of culture, the reduction in scratch width of shNC group cells was significantly less than that of shY3 group, indicating that shNC group cells migrated faster than shY3 group cells, consistent with the statistical graph results. This indicates that knocking out or downregulating YTHDF3 protein in cervical cancer cells inhibits cervical cancer cell migration.

[0082] 5. The invasive ability of cervical cancer cells was detected by the transwell assay.

[0083] The upper surface of the bottom membrane of the transwell chamber was coated with Matrigel and incubated at 37°C for 30 min to allow Matrigel to polymerize into a gel. Cells were digested, and after digestion, the culture medium was discarded by centrifugation. The cells were washed 1-2 times with PBS and resuspended in serum-free medium containing BSA. The cell suspension was added to the transwell chamber, and culture medium was added to the lower chamber. After culture, the transwell chamber was removed, the culture medium in the wells was discarded, and the cells were washed twice with calcium-free PBS, fixed with methanol for 30 min, and then air-dried appropriately. The cells were stained with 0.1% crystal violet for 20 min, and the unmigrated cells on the upper layer were gently wiped away with a cotton swab. The cells were washed 3 times with PBS. Five fields of view were randomly selected under a microscope to observe the cells, photographed, counted, and statistically analyzed. The results are as follows: Figure 7 As shown. Figure 7In the diagram, A represents the effect on SiHa cell invasion, B represents the cell count in each field of view of A, C represents the effect on Caski cell invasion, and D represents the cell count in each field of view of C. The results show that in the SiHa cell group, compared with the WT group, knocking out YTHDF3 protein significantly reduced the cell number, and the number of cells observed in each field of view was also significantly reduced. In the Caski cell group, compared with the shNC group, knocking down YTHDF3 protein also significantly reduced the cell number, and the number of cells observed in each field of view was also significantly reduced. This indicates that knocking out or knocking down YTHDF3 protein in cervical cancer cells inhibits the invasive ability of cervical cancer cells.

[0084] In summary, knocking out the YTHDF3 protein can inhibit the proliferation, migration, and invasion of cervical cancer cells. Therefore, the development of drugs that specifically inhibit YTHDF3 gene transcription or protein expression, or drugs that specifically inhibit the activity of the YTHDF3 gene or protein, can suppress the proliferation, migration, and invasion of cervical cancer cells and can be used for targeted therapy of cervical cancer.

[0085] 6. Effects of YTHDF3 on lymph node metastasis and lymphatic canal formation.

[0086] Advanced cervical cancer often metastasizes to lymph nodes, exhibiting high malignancy and a low cure rate. Therefore, this study investigated the impact of YTHDF3 protein knockout on lymph node metastasis and lymphatic canaliculus formation. First, Cox regression analysis was performed based on tissue microarray data, revealing that lymph node metastasis is a risk factor for cervical cancer. Second, a plantar-popliteal lymph node metastasis model was established in nude mice using fluorescently labeled non-YTHDF3-knockout cervical cancer cells (SiHa) and YTHDF3-knockout cervical cancer cells (KO Y3-SiHa). In vivo imaging was used to observe lymph node metastasis. The results are as follows: Figure 8 As shown, Figure 8 The left image is an in vivo imaging study, and the right image is a photograph. It can be seen that in mice transfected with SiHa cells, plantar-popliteal lymph node metastasis occurred. Knocking out the YTHDF3 protein reduced lymph node metastasis, with the metastasis concentrated in the plantar surface. Immunohistochemistry was used to observe the formation of lymphatic tubules, and the results are as follows: Figure 9 As shown, the number of lymphatic tubules in tissues with YTHDF3 protein knocked out was significantly lower than that in the non-knockout group. Finally, lymphoendothelial cells were co-cultured in vitro using the cell supernatant of cultured SiHa / KO Y3-SiHa to conduct tubule formation experiments and observe changes in the tubule-forming ability of lymphoendothelial cells. The results are as follows: Figure 10 As shown, Figure 10The results showed that knocking out YTHDF3 reduced the tubulogenic capacity of lymphoendothelial cells and decreased the formation of lymphatic tubules. This indicates that YTHDF3 can promote lymph node metastasis and lymphatic tubule formation.

[0087] Example 3

[0088] This embodiment investigated the molecular mechanism by which YTHDF3 affects cervical cancer. The specific process is as follows:

[0089] First, upstream regulators of YTHDF were detected. SREBF1 was predicted to be an upstream regulator of YTHDF3 using chromatin open sequencing (ATAC-seq) and chromatin immunoprecipitation-sequencing (ChIP-seq) techniques. Next, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) and dual-luciferase reporter assays were performed. The results are as follows: Figure 11 As shown, Figure 11 The left image shows the ChIP-qPCR results. It can be seen that in the control group (si-NC), the expression level of YTHDF3 was higher in samples precipitated with anti-SREBF1 antibody; while after SREBF1 knockout (si-SREBF1), the expression level of YTHDF3 in samples precipitated with anti-SREBF1 antibody was significantly reduced. The right image shows the dual-luciferase reporter assay results. It can be seen that compared with the SiHa group with only YTHDF3, the simultaneous addition of YTHDF3 and SREBF1 significantly increased the expression level of YTHDF3. This indicates that SREBF1 upregulates the expression of YTHDF3 mRNA. Western blot results are shown below. Figure 12 As shown, compared with SiHa cells without SREBF1 knockout, the expression of YTHDF3 protein in SiHa cells with SREBF1 knockout was significantly reduced, indicating that downregulation of SREBF1 leads to a decrease in YTHDF3 protein expression. These experiments demonstrate that SREBF1 is an upstream regulator of YTHDF3.

[0090] Secondly, two groups of SiHa cells were constructed: SiHa (WT) without YTHDF3 knockout and SiHa (KOY3) with YTHDF3 knockout. RIP-seq, RNA-seq, and Ribo-seq sequencing were performed on these cells, and comprehensive analysis predicted the following target genes: FZD3, LRP6, and LRP5. Real-time quantitative PCR and Western blotting (WB) were then performed on the predicted target genes, revealing that only LRP6 protein expression was significantly downregulated after YTHDF3 knockout.

[0091] 1. Real-time quantitative PCR (Q-PCR) detection of the effect of YTHDF3 on LRP6 RNA expression.

[0092] RNA was extracted from two groups of cells (SiHa (WT, KO Y3) and Caski cells (shNC, shY3)), and cDNA was obtained by reverse transcription. A Q-PCR reaction system was prepared, and the prepared reaction system was placed in a real-time PCR instrument. The Q-PCR reaction was performed, and the data were analyzed and statistically analyzed after the reaction. The statistical results are shown below. Figure 13 As shown. Figure 13 The results showed that in the SiHa cell group, compared with WT cells, the expression of YTHDF3 mRNA in KO Y3 cells was significantly reduced, while the expression level of LRP6 mRNA was not significantly different from that in WT cells. Similarly, in the Caski cell group, compared with shNC cells, the expression of YTHDF3 mRNA in shY3 cells was significantly reduced, while the expression level of LRP6 mRNA was not significantly different from that in shNC cells. This indicates that knocking out or knocking down YTHDF3 in cervical cancer cells has no significant effect on LRP6 RNA expression.

[0093] 2. Western blot analysis of the effect of YTHDF3 on LRP6 protein expression.

[0094] Total protein was extracted from two cell groups (SiHa (WT, KO Y3) and Caski cells (shNC, shY3)). The total protein was formulated into samples and subjected to Western blotting (WB) to detect the expression of YTHDF3 and LRP6 proteins. The results are shown below. Figure 14 As shown. Figure 14 The results showed that, compared with WT cells or shNC cells, the expression of YTHDF3 and LRP6 proteins was significantly reduced in KO Y3 cells or shY3 cells in both the SiHa and Caski cell groups. This indicates that knocking out or downregulating YTHDF3 in cervical cancer cells significantly downregulated LRP6 protein expression.

[0095] Because m6A regulates multiple RNA life processes—including RNA processing, translation, and degradation—by recognizing selectively binding proteins, m6A regulates the RNA life cycle. YTH domain family proteins, acting as "reader" proteins for m6A, directly bind to and recognize m6A methylation in mRNA; therefore, it is hypothesized that YTHDF3 affects m6A modification of LRP6. To verify this hypothesis, the following experiments were conducted:

[0096] 3. Treatment of cervical cancer cells with RNA methylation immunoprecipitation-qPCR (MeRIP-qPCR), RNA-binding protein immunoprecipitation-qPCR (RIP-qPCR), and the methylation inhibitor 3-deadenosine (DAA) was used to detect the effect of YTHDF3 on the m6A modification of LRP6.

[0097] MeRIP-qPCR: RNA was extracted from two cell groups (SiHa and Caski), and fragmented by sonication. 50 μL of RNA was used as the input sample, and the remaining 400 μL was used for immunoprecipitation (IP). The IP sample was divided into two aliquots: one aliquot was added with 20 μL IP buffer and 4 μg m6A antibody, and the other aliquot was added with 20 μL IP buffer and 4 μg IgG antibody. Both aliquots were incubated at 4°C for 4 h on a vertical mixer. 25–50 μL of magnetic bead suspension was transferred to centrifuge tubes, and 200 μL of binding buffer was added. The centrifuge tubes were placed on a magnetic rack and allowed to stand for 1 min until the solution became clear. The supernatant was discarded, and this process was repeated twice. 200 μL of elution buffer was added, and the RNA was extracted together with the input sample using phenol-chloroform. An equal volume of RNA was used for reverse transcription, and qPCR was performed for validation. The results were statistically analyzed, and the statistical graph is shown below. Figure 15 As shown. Figure 15 The left side shows the relative expression levels of LRP6 mRNA in the two cell groups. The results represent the proportion of LRP after normalizing the input. The results show that, in both the SiHa cell group and the Caski cell group, LRP6 mRNA was significantly expressed in the IP group precipitated with m6A antibody compared to the IP group precipitated with IgG antibody. Figure 15 The right side shows the relative abundance of LRP6 mRNA in the SiHa cell group. The results show that the relative abundance of LRP6 protein in the IP group precipitated with m6A antibody was significantly higher than that in the IP group precipitated with IgG antibody in the SiHa cell group. This indicates that LRP6 is modified by m6A.

[0098] RIP-qPCR: Two groups of cells (SiHa and Caski) were collected, cell nuclei were isolated, and the resuspended nuclei were divided into two 500 μL portions. Chromatin was mechanically sheared, centrifuged, and the supernatant was collected. One portion was used as the input sample, and the remainder was used for IP. IgG antibody and m6A antibody were added to the supernatant, and the mixture was incubated at 4°C for 2 h. Protein A / G magnetic beads were added, and the mixture was incubated at 4°C for 1 h. The magnetic beads were washed, resuspended in RIP buffer, and this process was repeated twice. The mixture was then washed once in PBS to remove the protein complex from the magnetic beads. Western blotting was performed to detect the expression of YTHDF3 protein. The results are shown below. Figure 16 As shown. Figure 16 The results showed that in SiHa and Caski cells, the IP group precipitated with IgG antibody showed almost no expression of YTHDF3 protein, while the IP group precipitated with m6A antibody showed expression of YTHDF3 protein. This indicates that YTHDF3 can bind to the m6A of LRP6.

[0099] Treatment with the methylation inhibitor 3-deadenosine (DAA): When culturing SiHa and Caski cells, each group was divided into three subgroups, and different concentrations (0 μM, 100 μM, 200 μM) of DAA were added to inhibit methylation, specifically m6A. After cell culture, total protein was extracted from each group and subjected to Western blotting (WB) to detect LRP6 protein expression. The results are as follows: Figure 17 As shown. Figure 17 The results showed that in SiHa and Caski cells, LRP6 expression significantly decreased with increasing DAA concentration, and was almost inhibited at 200 μM. This indicates that cervical cancer is indeed related to LRP6 m6A modification. Furthermore, it suggests that YTHDF3 affects LRP6 m6A modification in cervical cancer cells.

[0100] 4. Studies have reported that YTHDF3 can both promote the translation and degradation of m6A-modified target transcripts. Therefore, we will use actinomycin-D and actinomycinone (CHX) to verify whether YTHDF3 affects LRP6 mRNA stability or protein stability in cervical cancer cells.

[0101] Actinomycin-D treatment of cervical cancer cells: SiHa cells from WT and KO Y3 groups were cultured, and actinomycin-D was added at different time points (0h, 6h, 9h) during culture. The LRP6 mRNA content was measured, and the results are as follows: Figure 18 As shown. Figure 18 The results showed that the expression of LRP6 mRNA in cells of both the WT group and the KO Y3 group decreased with the extension of culture time, but there was no significant difference between the two, indicating that YTHDF3 has no effect on the stability of LRP6 mRNA.

[0102] Cervical cancer cells were treated with actinomycin D (CHX): SiHa cells from the WT and KO Y3 groups were cultured, and CHX was added at different time points (0h, 3h, 6h, 12h). Cells were harvested, proteins were extracted, and Western blotting was performed to detect the expression levels of LRP6 and YTHDF3 proteins. The results are as follows: Figure 19 As shown. Figure 19 The results showed that the expression level of LRP6 protein was significantly reduced in KO Y3 group cells due to the knockout of YTHDF3; however, there was no significant difference in the expression of LRP6 protein between WT and KO Y3 cells, indicating that YTHDF3 had no effect on the stability of LRP6 protein.

[0103] The above experiments demonstrate that YTHDF3 affects LRP6 protein expression in cervical cancer cells by influencing the translation efficiency of LRP6 protein. Therefore, a drug is proposed that specifically inhibits YTHDF3 to suppress the translation of the m6A-modified transcript of LRP6, thereby inhibiting LRP6 translation efficiency and ultimately suppressing LRP6 protein expression, thus achieving therapeutic effects against cervical cancer.

[0104] Example 4

[0105] This embodiment investigated the effect of LRP6 on cervical cancer cells. The specific process was as follows:

[0106] 1. The expression of LRP6 protein in cervical cancer tissue was detected. The method was the same as in Example 1, except that immunohistochemistry was performed using an anti-LRP6 antibody. The results are as follows: Figure 20 As shown. Figure 20 The image above represents an immunohistochemical staining pattern. Figure 20 The following represents the... Figure 20 The quantitative map of LRP6 protein in the upper part shows that the expression level of LRP6 protein is very low in normal cervical cancer epithelial tissue, while the expression level of LRP6 protein in cervical squamous cell carcinoma tissue is significantly higher than that in normal cervical epithelial tissue; the quantitative map also shows the same result. This indicates that LRP6 protein is highly expressed in cervical cancer tissue.

[0107] 2. Knock out LRP6 in SiHa and Caski cell lines.

[0108] LRP6 knockout cell lines were constructed using lentiviruses, resulting in two cell groups: SiHa (cervical cancer cells without LRP6 knockout (shNC) and cervical cancer cells without LRP6 knockout (shLRP6)) and Caski (human cervical cancer epithelial cells without LRP6 knockout (shNC) and human cervical cancer epithelial cells without LRP6 knockout (shLRP6)). Similar abbreviations will be used to refer to these cell types in the following text.

[0109] Similar to the first part of Example 2, cells were cultured first, then total protein was extracted and subjected to Western blotting to detect the expression of LRP6 protein. The results are as follows: Figure 21 As shown. Figure 21 The results showed that, compared with the shNC group, the expression level of LRP6 protein in the shLRP6 group was significantly reduced in both SiHa cells and Caski cells, indicating that the LRP6 protein knockout in shLRP6 cells was successful.

[0110] 3. Using the same method as in Part 2 of Example 2, the effect of LRP6 on the proliferation of cervical cancer cells was detected by the CCK-8 assay. The results are as follows: Figure 22 As shown. Figure 22The results showed that both groups of cells were in a proliferative state during the period from 0 to 72 hours after inoculation; however, starting from 24 hours (Caski) or 48 hours (SiHa), the OD value of the shLRP6 group was significantly lower than that of the shNC group. This indicates that knocking out the LRP6 protein in cervical cancer cells reduces the proliferation of cervical cancer cells, meaning that LRP6 promotes the proliferation of cervical cancer cells.

[0111] 4. Using the same method as in Part 4 of Example 2, the effect of LRP6 on the migration of cervical cancer cells was detected by a cell scratch assay. The results are as follows: Figure 23 As shown. Figure 23 In the diagram, A represents the effect on SiHa cell migration, B represents the effect on Caski cell migration, and C is a statistical graph of cell migration rates in A and B. The results show that in the SiHa cell group, after 24 hours of culture, the reduction in scratch width of shNC cells was significantly less than that of the shLRP6 group, indicating that the migration rate of shNC cells was higher than that of the shLRP6 group, consistent with the statistical graph. Similarly, in the Caski cell group, after 24 hours of culture, the reduction in scratch width of shNC cells was significantly less than that of the shLRP6 group, indicating that the migration of shNC cells was faster than that of the shLRP6 group, consistent with the statistical graph. This indicates that knocking out LRP6 protein in cervical cancer cells inhibits cervical cancer cell migration.

[0112] 5. Using the same method as in Part 5 of Example 2, the effect of LRP6 on cervical cancer cell migration was detected by transwell assay, and the results are as follows: Figure 24 As shown. Figure 24 The left image shows the effect of LRP6 knockout on the invasion of SiHa and Caski cells, while the right image shows the cell count in each field of view. The results show that, compared to the shNC group, knockout of LRP6 significantly reduced the number of cells in both the SiHa and Caski cell groups, and the number of cells observed in each field of view was also significantly reduced. This indicates that knockout of LRP6 protein in cervical cancer cells inhibits their invasive ability.

[0113] In summary, knocking out the LRP6 protein can inhibit the proliferation, migration, and invasion of cervical cancer cells. Therefore, a drug is proposed that specifically inhibits YTHDF3 to suppress the translation of the m6A-modified transcript of LRP6, i.e., inhibiting the translation efficiency of LRP6, thereby suppressing the expression of the LRP6 protein and thus inhibiting the proliferation, migration, and invasion of cervical cancer cells, thereby achieving the treatment of cervical cancer.

[0114] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of reagents for detecting YTHDF3 expression levels in the preparation of kits for diagnosing lymph node metastasis in cervical cancer.

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