Application of ANKRD22 as target in prognosis evaluation and treatment of cervical cancer
By targeting ANKRD22, detecting and inhibiting ANKRD22 expression, and regulating the PI3K/AKT/mTOR signaling pathway, the treatment challenges of advanced cervical cancer have been solved, providing personalized treatment options and achieving effective inhibition of cervical cancer.
Patent Information
- Application Number
- CN202511416777.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2025-12-05
AI Technical Summary
Current technologies have limited effectiveness in treating advanced and recurrent cervical cancer, lacking effective molecular targets and personalized treatment options.
Using ANKRD22 as a target, we will detect ANKRD22 expression levels and reduce ANKRD22 gene expression through gene knockout and gene reduction, develop ANKRD22 inhibitors such as siRNA and shRNA, inhibit the migration and invasion of cervical cancer cells, regulate the PI3K/AKT/mTOR signaling pathway to promote apoptosis and autophagy, and develop drugs targeting ANKRD22.
High expression of ANKRD22 is associated with good clinical prognosis. Knocking down ANKRD22 inhibits the invasion and migration of cervical cancer cells and regulates signaling pathways to achieve tumor suppression, providing new evidence for the treatment of cervical cancer.
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Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of ANKRD22 as a target in prognosis evaluation and treatment of cervical cancer. BACKGROUND
[0002] Cervical cancer (CC) is one of the most common malignant tumors of the female reproductive system, which seriously endangers women's health. Although the cervical cancer vaccine combined with early screening of cervical cancer plays a crucial role in the prevention of cervical cancer, the incidence and mortality of cervical cancer are still on the rise. At present, the treatment methods of cervical cancer include surgery, concurrent radiotherapy and chemotherapy, and immunotherapy, but the treatment effect on advanced and recurrent cervical cancer is limited. Therefore, it is urgent to find new potential molecular targets to provide individualized treatment for cervical cancer patients.
[0003] Ankyrin repeat domains 22 (ANKRD22) belongs to the ankyrin (ANK) family, which is an N-myristoylated hairpin single-channel transmembrane protein synthesized in the endoplasmic reticulum. The protein has four repeated ankyrin motifs and contains 191 amino acids. ANK family proteins are involved in various physiological activities of cells, including transcriptional regulation, cytoskeleton, ion transport, signal transduction, immunity, inflammation and tumorigenesis. Current studies have shown that ANKRD22 is highly expressed in various malignant tumors such as prostate cancer, non-small cell lung cancer and nasopharyngeal carcinoma, and is involved in the occurrence and development of tumors, but the biological function of ANKRD22 has not been clear, and the relationship between ANKRD22 and cervical cancer has not been reported. PI3K / AKT / mTOR has important regulatory effects on physiological activities such as cell proliferation, apoptosis, angiogenesis and energy metabolism in normal cells, and its abnormal activation can inhibit cell apoptosis, promote cell growth and proliferation, promote cell cycle progression, promote angiogenesis and cell metabolism, and is involved in the invasion and metastasis of various tumor cells. Therefore, ANKRD22 may become a potential target for the treatment of cervical cancer by regulating the PI3K / AKT / mTOR signaling pathway. SUMMARY
[0004] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide the application of ANKRD22 as a target in prognosis evaluation and treatment of cervical cancer, which discloses the role of ANKRD22 in the occurrence and development of cervical cancer and its potential molecular mechanism, and provides a new basis for the treatment of cervical cancer.
[0005] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present application: In a first aspect, the present application provides the use of a reagent for detecting the expression level of ANKRD22 in the preparation of a product for evaluating the prognosis of cervical cancer.
[0006] In the above technical solution, the product comprises a reagent or a kit.
[0007] In a second aspect, the application provides use of a substance targeting ANKRD22 in preparation of a drug for treating cervical cancer, wherein the targeting ANKRD22 refers to reducing expression of the ANKRD22 gene by using gene knockout or gene knockdown.
[0008] In the above technical solution, the drug targets ANKRD22 to inhibit migration and invasion of cervical cancer cells.
[0009] In the above technical solution, the drug targets ANKRD22 to promote apoptosis and autophagy through the PI3K / AKT / mTOR signaling pathway, thereby playing a tumor inhibition role in cervical cancer cells.
[0010] In a third aspect, the application provides use of an ANKRD22 gene inhibitor in preparation of a drug for treating cervical cancer.
[0011] In the above technical solution, the ANKRD22 gene inhibitor comprises a reagent for knocking down or silencing ANKRD22.
[0012] In the above technical solution, the reagent for knocking down or silencing ANKRD22 comprises siRNA, shRNA or miRNA.
[0013] In a fourth aspect, the application provides a drug for treating cervical cancer, wherein the drug comprises siRNA with a sequence as shown in SEQ ID NO. 1.
[0014] In a fifth aspect, the application provides a drug for treating cervical cancer, wherein the drug comprises siRNA with a sequence as shown in SEQ ID NO. 3.
[0015] The one or more technical solutions in the embodiments of the application have at least the following technical effects or advantages: the application provides application of ANKRD22 as a target in prognosis evaluation and treatment of cervical cancer. It is found in the research that ANKRD22 is highly expressed in human cervical cancer tissues, and high expression level of ANKRD22 in cervical cancer patients is related to better clinical prognosis. Knocking down ANKRD22 in cervical cancer cells can inhibit the invasion and migration ability of the cells. Knocking down ANKRD22 can down-regulate the phosphorylation level of PI3K, AKT and mTOR, and then up-regulate apoptosis and autophagy through the PI3K / AKT / mTOR signaling pathway to inhibit occurrence and development of cervical cancer. It is found in the research that siRNA targeting ANKRD22 shows good effect in inhibiting development of TNBC tumor, and is a potential new drug for treating cervical cancer. BRIEF DESCRIPTION OF DRAWINGS
[0016] Figure 1 TCGA and GTEx database. ***P < 0.001.
[0017] Figure 2 HPA database. ***P < 0.001.
[0018] Figure 3 ROC curve of ANKRD22 in cervical cancer. X axis represents false positive rate, Y axis represents true positive rate.
[0019] Figure 4 The relationship between ANKRD22 expression level and the clinical survival and prognosis of cervical cancer patients. (A) The correlation between ANKRD22 expression level and the overall survival of cervical cancer patients; (B) The correlation between ANKRD22 expression level and the progression-free survival of cervical cancer patients; (C) Forest plot of single-factor prognostic factor analysis; (D) Forest plot of multi-factor prognostic factor analysis.
[0020] Figure 5 The expression level of ANKRD22 in cervical normal tissue and cervical cancer tissue. (A) HE staining results of cervical normal tissue and cervical cancer tissue; (B) Western blot was used to detect the protein expression of ANKRD22 in cervical normal tissue and cervical cancer tissue.
[0021] Figure 6 The expression of ANKRD22 after knockdown in cell lines. (A) Western blot was used to detect the protein expression level of ANKRD22 after transfection of siRNA in Siha; (B) Statistical results of the relative expression amount of ANKRD22 protein in Figure A; (C) Western blot was used to detect the protein expression level of ANKRD22 after transfection of siRNA in Hela; (D) Statistical results of the relative expression amount of ANKRD22 protein in Figure C. N = 3, * P < 0.05, compared with Si-NC group.
[0022] Figure 7: Effect of ANKRD22 on the apoptosis level of cervical cancer cells. (A) The protein expression levels of apoptosis-related proteins Cleaved Caspase3, Caspase3, Bax, Bcl-2 and cytochrome C in each treatment group were detected by Western Blot. The statistical results of the relative expression levels of apoptosis-related proteins in each group: cytochrome C (B), Bax (C), Bcl-2 (D), Caspase3 (E), and Cleaved Caspase3. N = 3, * P < 0.05, compared with the Si-NC group.
[0023] Figure 8 : Effect of knocking down ANKRD22 on the migration ability of Siha cells. (A) The results of scratch test in each group after knocking down ANKRD22; (B) The statistical results of migration rate of scratch test in each group after knocking down ANKRD22. N = 3, * P < 0.05, compared with the Si-NC group.
[0024] Figure 9 : Effect of knocking down ANKRD22 on the migration ability of Hela cells. (A) The results of scratch test in each group after knocking down ANKRD22; (B) The statistical results of migration rate of scratch test in each group after knocking down ANKRD22. N = 3, * P < 0.05, compared with the Si-NC group.
[0025] Figure 10 : Effect of knocking down ANKRD22 on the invasion ability of cervical cancer cell lines Siha and Hela. (A) The results of Transwell test in each group after knocking down ANKRD22; (B) The statistical results of migrated cells of Transwell test in each group after knocking down ANKRD22 in cervical cancer cell line Siha; (C) The statistical results of migrated cells of Transwell test in each group after knocking down ANKRD22 in cervical cancer cell line Hela. N = 3, * P < 0.05, compared with the Si-NC group.
[0026] Figure 11 : Effect of ANKRD22 on the PI3K / AKT / mTOR signaling pathway of cervical cancer cells. (A) The protein expression levels of signaling pathway-related proteins p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR were detected by Western Blot. N = 3, * P < 0.05, compared with the Si-NC group. The statistical results of the relative expression levels of related proteins in each group: p-PI3K, PI3K (B), p-AKT, AKT (C), p-mTOR and mTOR (D). N = 3, * P < 0.05, compared with the Si-NC group. DETAILED DESCRIPTION
[0027] In order to better illustrate the purposes, technical solutions and advantages of the present application, the present application will be further described below in conjunction with specific embodiments. The present application can be implemented in many different forms, and should not be understood as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art, and the present application will be limited only by the claims.
[0028] The test methods or test methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are all obtained from conventional commercial channels or prepared by conventional methods unless otherwise specified.
[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
[0030] The present application will be further described below in conjunction with the examples.
[0031] Example 1: Expression of ANKRD22 in cervical cancer tissues in TCGA, GTEx and HPA databases The gene expression data (RNA-seq) and clinical data of cervical cancer samples downloaded from UCSC Xena were combined and sorted using R language, wherein the gene expression data of 306 cervical cancer tissues and 3 normal cervical tissues with clinical data were downloaded from TCGA database, and 10 RNA-seq of normal cervical tissues were downloaded from GTEx database. The bioinformatics analysis results of the above data showed that the expression of ANKRD22 in cervical cancer tissues was significantly higher than that in normal cervical tissues Figure 1 ).
[0032] The immunohistochemical staining images of cervical cancer were downloaded from the HPA database to verify the expression level of ANKRD22 in cervical cancer tissues and normal cervical tissues. The results showed that ANKRD22 was lowly expressed in normal cervical tissues, but was more highly expressed in cervical cancer tissues Figure 2 ).
[0033] In addition, we also used R language to make ROC curve graph, and used the area under the ROC curve to evaluate the prediction value of ANKRD22 in cervical cancer tissues and normal cervical tissues. The analysis results showed that the area under the curve (Area Under the Curve, AUC) was 0.95 (95% CI=0.851-1.000), which indicated that the expression level of ANKRD22 in tissues could help the diagnosis of cervical cancer Figure 3 ).
[0034] Example 2: The impact of ANKRD22 expression on the prognosis of cervical cancer patients Kaplan-Meier survival analysis was used to assess the relationship between ANKRD22 expression level and the prognosis of cervical cancer patients. The results showed that patients with high ANKRD22 expression had better overall survival than those with low expression (P=0.023). Figure 4 A). Furthermore, compared to patients in the low ANKRD22 expression group, patients in the high expression group had better progression-free survival (P=0.030). Figure 4 B). Univariate Cox regression analysis showed that FIGO stage (HR=1.473, 95% CI=1.152-1.884, P=0.002) and ANKRD22 expression level (HR=0.423, 95% CI=0.271-0.660, P<0.001) were associated with the prognosis of cervical cancer patients. Figure 4 C). Further multivariate analysis to screen for independent prognostic factors showed that FIGO stage (HR=1.395, 95% CI=1.085-1.792, P=0.009) and ANKRD22 expression level (HR=0.393, 95% CI=0.246-0.629, P<0.001) were also independent prognostic factors for cervical cancer patients. Figure 4 D).
[0035] Example 3: Expression of ANKRD22 in clinical cervical cancer tissue and normal cervical tissue specimens To further analyze and validate the expression of ANKRD22 in cervical cancer tissues, we performed Western blotting analysis on the expression of ANKRD22 protein in fresh normal cervical tissue specimens and cervical cancer tissue specimens. The results showed that the expression of ANKRD22 was significantly increased in cervical cancer tissues compared to normal cervical tissues. Figure 5 ).
[0036] Example 4: ANKRD22 expression after siRNA knockdown of cervical cancer cell lines Siha and Hela The cervical cancer cell lines Siha and Hela were obtained from the ATCC Cell Resource Center. To investigate the biological function of ANKRD22 in cervical cancer, we constructed three different ANKRD22 siRNAs (si-ANKRD22#1, si-ANKRD22#2, and siANKRD22#3) and transfected them into Siha and Hela cells to knock down the expression level of ANKRD22 in both cervical cancer cell lines. The results were then validated by Western blot.
[0037] The nucleotide sequences of the three siRNAs are as follows: si-ANKRD22#1: CGUCGAAGUUAAUGCUACATTUGUAGCAUUAACUUCGACGTT (SEQ ID NO. 1) si-ANKRD22#2: GCUAAGGAAAGCAUUGUAATTUUACAAUGCUUUCCUUAGCTT (SEQ ID NO. 2) siANKRD22#3: GAGAGAACCUGCUUGCAUUTTAAUGCAAGCAGGUUCUCUCTT (SEQ ID NO. 3) Wherein, TT is a overhang structure, U is the base sequence of RNA, the first TT is the sense strand (target sequence), and the second TT is the antisense strand (action strand).
[0038] The results suggest that in two cell lines, the expression of ANKRD22 protein in the three knockdown groups is significantly lower than that in the control group, and the knockdown effect of si-ANKRD22#1 and siANKRD22#3 groups is more significant Figure 6 Therefore, si-ANKRD22#1 and si-ANKRD22#3 are selected for subsequent research on cell migration and invasion and related signal pathways.
[0039] Example 5: Effect of knocking down ANKRD22 on apoptosis of cervical cancer cells si-ANKRD22#1 and si-ANKRD22#3 were selected to detect the expression of apoptosis-related proteins Caspase3, Cleaved caspase3, Bax, Bcl-2, and cytochrome C in Siha cells by Western Blot. The experimental results show that after knocking down ANKRD22 in Siha cells, the expression level of pro-apoptotic protein Bax is significantly up-regulated, while the expression level of anti-apoptotic protein Bcl-2 is significantly decreased. In addition, the expression levels of cytochrome C and Cleaved caspase3 are significantly increased Figure 7 .
[0040] Example 6: Effect of knocking down ANKRD22 on migration and invasion of cervical cancer cells To further investigate the effect of ANKRD22 on the invasion and migration ability of cervical cancer cells, we first performed a scratch test. The results show that the migration speed of Siha and Hela cells in the si-ANKRD22 group is significantly lower than that in the si-NC group Figure 8 , Figure 9). Then we carried out Transwell test, and the results showed that the number of cells penetrating the membrane in si-ANKRD22 group was lower than that in si-NC group Figure 10 ].
[0041] Example 7: Effect of knocking down ANKRD22 on PI3K / AKT / mTOR signaling pathway in cervical cancer cells PI3K / AKT / mTOR is one of the important intracellular signaling pathways, which plays an important role in physiological activities such as cell proliferation, apoptosis, angiogenesis and energy metabolism in normal cells. Its abnormal activation can inhibit cell apoptosis, promote cell growth and proliferation, promote cell cycle progression, promote angiogenesis and cell metabolism, and is involved in tumor cell invasion and metastasis, and plays a key role in many diseases, especially malignant tumors. After knocking down Siha cell ANKRD22, the expression levels of p-PI3K, p-AKT and p-mTOR were significantly reduced, and the PI3K / AKT / mTOR pathway was inhibited Figure 11 ].
[0042] The present application finds that ANKRD22 is highly expressed in human cervical cancer tissue by bioinformatics analysis, and high expression level of ANKRD22 in cervical cancer patients is related to better clinical prognosis. Subsequent in vitro experiments at the cellular level found that knocking down ANKRD22 in cervical cancer cells can inhibit the invasion and migration ability of cells. To further explore the mechanism of ANKRD22 in cervical cancer cells, it is found that ANKRD22 is related to PI3K / AKT / mTOR signaling pathway by knocking down the expression of ANKRD22 by SiRNA, and it is proved by experiments that knocking down ANKRD22 can down-regulate the phosphorylation level of PI3K, AKT and mTOR, and then up-regulate cell apoptosis and autophagy through PI3K / AKT / mTOR signaling pathway to inhibit the occurrence and development of cervical cancer.
[0043] Obviously, the above examples are only examples for clearly illustrating, and not limit the embodiments. For those skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.
Claims
1. Use of a reagent for detecting the expression level of ANKRD22 in the preparation of a product for evaluating the prognosis of cervical cancer.
2. Use according to claim 1, characterized in that: The product comprises a reagent or a kit.
3. Use of a substance targeting ANKRD22 in the manufacture of a medicament for the treatment of cervical cancer, characterized in that: The target of ANKRD22 refers to the use of gene knockout, gene knockdown to reduce the expression of ANKRD22 gene.
4. Use according to claim 3, characterized in that: The drug targets ANKRD22 to inhibit the migration and invasion of cervical cancer cells.
5. Use according to claim 3, characterized in that: The drug targets ANKRD22 to promote apoptosis and autophagy through the PI3K / AKT / mTOR signaling pathway and thus plays an anti-tumor role in cervical cancer cells.
6. Use of an ANKRD22 gene inhibitor in the preparation of a drug for treating cervical cancer.
7. Use according to claim 6, characterized in that: The ANKRD22 gene inhibitor includes a reagent for knocking down or silencing ANKRD22.
8. Use according to claim 7, characterized in that: The reagent for knocking down or silencing ANKRD22 includes siRNA, shRNA or miRNA.
9. A drug for treating cervical cancer, characterized in that: The drug includes an siRNA with a sequence as shown in SEQ ID NO.
1.
10. A medicament for treating cervical cancer, characterized by comprising: (a) a compound of the formula (I) or a pharmaceutically acceptable salt thereof; and (b) a platinum compound. The drug includes an siRNA with a sequence as shown in SEQ ID NO. 3.