Voltage-gated potassium channel kv10.1 binding peptides and uses thereof

Abstract

This invention discloses a voltage-gated potassium channel Kv10.1 binding peptide and its applications, relating to the fields of biopharmaceutical and genetic engineering technologies. The amino acid sequence of the peptide provided by this invention is shown in SEQ ID NO:5. This peptide can specifically bind to the Kv10.1 protein and inhibit its channel current, showing promise for application in the treatment of tumors and inflammatory diseases associated with Kv10.1 high expression.

Description

Cross-reference to related applications

[0001] This application is a divisional application of the invention patent application filed on May 6, 2023, with application number 2023105070495 and invention title "Voltage-gated potassium channel Kv10.1 binding peptide and its application". Technical Field

[0002] This invention relates to the fields of biopharmaceutical and genetic engineering technologies, and more specifically, to a voltage-gated potassium channel Kv10.1 binding peptide and its applications. Background Technology

[0003] Ion channels are pore-forming proteins on the cell membrane. Their structure and function are fundamental to maintaining life processes, and their genetic variations and functional disorders are associated with the occurrence and development of many diseases. Ion channels can regulate tumor evolution through various possible pathways, such as altering membrane potential, promoting calcium ion influx, and changing cell osmosis processes.

[0004] Currently, more than 30 types of dysfunctional ion channels have been identified as being associated with the development of more than 20 types of tumors. Potassium ion channels are the most widely distributed ion channels in organisms and have the most discovered subtypes. They play a key role in many physiological activities such as neurotransmitter release, neuronal excitability, epithelial cell electrolyte transport, and cell volume regulation.

[0005] There are 12 families and 78 members of potassium channels. The Kv10.1 channel is one of these members, encoded by the oncogene EAG1 (ether-à-go-go-1) located on chromosome 1q32-41. It is a voltage-gated potassium channel and the first channel discovered to have oncogenic effects. Studies have shown that the EAG1 gene exhibits abnormally elevated expression levels in more than 70% of tumor cells, including brain, lung, gastric, breast, and colorectal cancers, which is closely related to tumor evolution. Downregulating Kv10.1 expression levels using siRNA interference or reducing Kv10.1 channel activity with specific inhibitors can effectively inhibit the proliferation rate of tumor cells. Due to the pathological effects of Eag1 and its proven potential for various cancers, efforts have been made to develop specific inhibitors. However, most Eag1 channel inhibitors exhibit poor selectivity within the EAG family, particularly against HERG channels, which pose a cardiac risk. This makes drug development for Eag1 channels still challenging.

[0006] Therefore, screening for an inhibitor that can effectively inhibit Kv10.1 is of great importance for the research on Kv10.1 and the treatment of Kv10.1-related diseases.

[0007] In view of this, the present invention is proposed. Summary of the Invention

[0008] The purpose of this invention is to provide a voltage-gated potassium channel Kv10.1 binding peptide and its applications.

[0009] This invention is implemented as follows: In a first aspect, embodiments of the present invention provide a polypeptide or an acceptable salt thereof, the amino acid sequence of which is shown as X1-X2-AG-X3-P-X4-ILML-X5; wherein X1 is selected from any one of C, R, D and N, X2 is selected from F or I, X3 is selected from T or W, X4 is selected from S or V, and X5 is selected from any one of A, V and L.

[0010] Secondly, embodiments of the present invention provide a nucleic acid that encodes the polypeptide described in the foregoing embodiments.

[0011] Thirdly, embodiments of the present invention provide a fusion protein comprising the polypeptide described in the foregoing embodiments or an acceptable salt thereof.

[0012] Fourthly, embodiments of the present invention provide a composition whose active ingredient includes: the polypeptide described in the foregoing embodiments or an acceptable salt thereof, or the nucleic acid described in the foregoing embodiments, or the fusion protein described in the foregoing embodiments.

[0013] Fifthly, embodiments of the present invention provide a pharmaceutical formulation whose active ingredient includes: the polypeptide described in the foregoing embodiments or an acceptable salt thereof, or the nucleic acid described in the foregoing embodiments, or the fusion protein described in the foregoing embodiments, or the composition described in the foregoing embodiments.

[0014] Sixthly, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments or their acceptable salts, or the nucleic acids described in the foregoing embodiments, or the fusion proteins described in the foregoing embodiments, or the compositions described in the foregoing embodiments, in the preparation of binding reagents for the targeted voltage-gated potassium channel Kv10.1.

[0015] In a seventh aspect, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments or their acceptable salts, or the nucleic acids described in the foregoing embodiments, or the fusion proteins described in the foregoing embodiments, or the compositions described in the foregoing embodiments in the preparation of reagents having activity of the Kv10.1 protein with a targeted voltage-gated potassium channel.

[0016] Eighthly, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments or acceptable salts thereof, or the nucleic acids described in the foregoing embodiments, or the fusion proteins described in the foregoing embodiments, or the compositions described in the foregoing embodiments in the preparation of medicaments for treating or adjuvant treatment of diseases or clinical manifestations related to voltage-gated potassium channel Kv10.1 channel abnormalities.

[0017] Ninthly, embodiments of the present invention provide the use of the polypeptides described in the foregoing embodiments or acceptable salts thereof, or the nucleic acids described in the foregoing embodiments, or the fusion proteins described in the foregoing embodiments, or the compositions described in the foregoing embodiments, in the preparation of reagents for inhibiting tumor cell proliferation.

[0018] The present invention has the following beneficial effects: The polypeptide provided by this invention can specifically bind to Kv10.1 protein and effectively inhibit its channel current, thus effectively inhibiting the proliferation of tumor cells that highly express Kv10.1. It has broad application prospects in the treatment of tumors and inflammatory diseases related to high expression of Kv10.1. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The patch-clamp assay was used to identify whole-cell currents of recombinant human Kv10.1 protein on the membrane of HEK-293T cells; Figure 2 The results show positive clones of phages that bind to HEK-293T-Kv10.1, which were recovered through screening of the phage peptide library. Figure 3 The patch-clamp experiment demonstrates the suppression effect of KvPt1.1 on the channel current of Kv10.1; Figure 4 The MTT assay showed the inhibitory effect of KvPt1.1 on the activity of the HepG2 hepatocellular carcinoma cell line that highly expresses Kv10.1. Figure 5 Analysis of molecular docking to determine the binding sites of KvPt1.1 and Kv10.1 proteins; Figure 6 This demonstrates the optimized design and patch-clamp validation of KvPt1.1 based on computer-aided drug design. Figure 7 This demonstrates the optimized design and patch-clamp validation of KvPt1.2 based on computer-aided drug design. Figure 8 The optimized Kv10.1 binding peptide was shown to inhibit the proliferation of tumor cell lines that highly express Kv10.1. Figure 9 This figure shows the results of the KvPt1.5 inhibition experiment on the HepG2 tumor model in BALB / c mice. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] The inventors of this application experimentally obtained a Kv10.1 peptide inhibitor molecule with good anti-tumor application potential, and optimized it with computer-aided drug design to obtain some peptides with higher activity (specifically including the amino acid sequence shown in X1-FAG-X2-PSILMLA). The inventors' experimental studies show that these peptides can not only bind to the Kv10.1 channel and inhibit its channel current in a concentration-dependent manner, but also, at the cellular level, inhibit the proliferation of tumor cells (such as liver cancer cells) that highly express the Kv10.1 channel, without significantly inhibiting other cancer cells that do not express or have low expression of the Kv10.1 channel. These results indicate that the voltage-gated potassium channel Kv10.1 binding peptide of this invention has potential application prospects for treating tumors and inflammatory diseases associated with high Kv10.1 expression.

[0023] Specific technical solutions On one hand, embodiments of the present invention provide a polypeptide or an acceptable salt thereof, the amino acid sequence of which is shown as X1-X2-AG-X3-P-X4-ILML-X5; wherein X1 is selected from any one of C, R, D and N, X2 is selected from F or I, X3 is selected from T or W, X4 is selected from S or V, and X5 is selected from any one of A, V and L.

[0024] The polypeptide or its acceptable salt provided by the present invention can bind to Kv10.1, also known as a Kv10.1-targeting binding peptide, which has the ability to specifically bind to Kv10.1 and inhibit its channel current through concentration gradient, as well as the ability to effectively inhibit the proliferation of tumor cells that highly express Kv10.1.

[0025] In some embodiments, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:1 to 9, as shown in Table 1.

[0026]

[0027] On the other hand, embodiments of the present invention also provide the application of the polypeptide or an acceptable salt thereof described in any of the foregoing embodiments as a target Kv10.1 binding peptide.

[0028] On the other hand, embodiments of the present invention provide a nucleic acid that encodes the polypeptide described in any of the foregoing embodiments.

[0029] On the other hand, embodiments of the present invention provide a vector containing the nucleic acid described in any of the foregoing embodiments. The vector includes a cloning vector and an expression vector.

[0030] On the other hand, embodiments of the present invention also provide a recombinant cell containing the vector described in any of the foregoing embodiments.

[0031] Based on the amino acid sequence of the polypeptide disclosed in this invention, those skilled in the art will readily conceive of preparing the polypeptide using genetic engineering or other techniques (chemical synthesis, recombinant expression), such as isolating and purifying the polypeptide from the culture product of recombinant cells capable of recombinantly expressing the polypeptide described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the antibody or its functional fragment of this invention, it falls within the protection scope of this invention.

[0032] On the other hand, embodiments of the present invention provide a fusion protein comprising the polypeptide described in any of the foregoing embodiments or an acceptable salt thereof.

[0033] In some embodiments, in addition to the portion containing the polypeptide or an acceptable salt thereof, the fusion protein may also include one or more additional domains that provide desirable properties, such as improved pharmacokinetics, easier purification, targeting of specific tissues, etc. These domains can enhance any one or more of the following: in vivo stability, in vivo half-life, uptake or absorption, tissue localization or distribution, protein complex formation, and protein complex purification.

[0034] In some embodiments, the fusion protein further includes an unstructured linker located between the polypeptide or an acceptable salt thereof and a domain that provides additional desirable properties. The linker may be rich in glycine and proline residues, or may contain a simple sequence of threonine / serine and glycine or a repeating sequence of threonine / serine and glycine (e.g., a singlet or repeating sequence of TG4 or SG4).

[0035] On the other hand, embodiments of the present invention provide a composition whose active ingredients include: the polypeptide described in any of the foregoing embodiments or an acceptable salt thereof, or the nucleic acid described in any of the foregoing embodiments, or the fusion protein described in any of the foregoing embodiments.

[0036] In some embodiments, the composition is a pharmaceutical composition.

[0037] In some embodiments, the polypeptide disclosed herein or an acceptable salt thereof may be used in combination with or sequentially with one or more other therapeutic agents. The pharmaceutical composition may also include other therapeutic agents, which may be agents for inhibiting the activity of voltage-gated potassium channels Kv10.1, or therapeutic agents for treating tumors and inflammatory diseases associated with Kv10.1 high expression.

[0038] In some embodiments, the composition further includes a delivery system for delivering the peptide or an acceptable salt thereof. The delivery carrier includes, but is not limited to, systems such as emulsions, liposomes, microspheres, and nanoparticles.

[0039] In some embodiments, the active ingredient includes any one or more of the polypeptides with amino acid sequences such as SEQ ID NO:1~9.

[0040] On the other hand, embodiments of the present invention provide a pharmaceutical formulation whose active ingredient includes: the polypeptide described in any of the foregoing embodiments or an acceptable salt thereof, or the nucleic acid described in any of the foregoing embodiments, or the fusion protein described in any of the foregoing embodiments, or the composition described in any of the foregoing embodiments.

[0041] In this invention, "pharmaceutical formulation" and "pharmaceutical composition" can be used interchangeably. The pharmaceutical composition includes, in addition to the active ingredients of this invention, a pharmaceutically acceptable carrier. The carrier includes, but is not limited to, diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, and adsorbents.

[0042] The polypeptides disclosed in this application or their acceptable salts can be delivered locally or systemically (e.g., intravenously, intra-arterially, or subcutaneously).

[0043] On the other hand, embodiments of the present invention provide the use of the polypeptides or acceptable salts thereof described in any of the foregoing embodiments, or the nucleic acids, or the fusion proteins, or the compositions described in any of the foregoing embodiments, in the preparation of binding reagents for the targeted voltage-gated potassium channel Kv10.1.

[0044] On the other hand, embodiments of the present invention provide the use of the polypeptides described in any of the foregoing embodiments or their acceptable salts, or the nucleic acids described in any of the foregoing embodiments, or the fusion proteins described in any of the foregoing embodiments, or the compositions described in any of the foregoing embodiments, in the preparation of reagents having activity of the Kv10.1 protein with a targeted voltage-gated potassium channel.

[0045] On the other hand, embodiments of the present invention provide the use of the polypeptides or acceptable salts thereof described in any of the foregoing embodiments, or the nucleic acids, or the fusion proteins, or the compositions described in any of the foregoing embodiments, in the preparation of medicaments for treating or adjuvant treatment of diseases or clinical manifestations related to voltage-gated potassium channel Kv10.1 channel abnormalities.

[0046] In some implementations, an abnormality in the voltage-gated potassium channel Kv10.1 can be caused by abnormal proliferation of the voltage-gated potassium channel Kv10.1.

[0047] In some implementations, the relevant diseases include any one or more of the following: arrhythmia, neuropsychiatric disorders, tumors, and inflammation; In some embodiments, the tumor includes any one or more of liver cancer, breast cancer, and cervical cancer.

[0048] The term "treatment" in this invention includes preventing or alleviating a condition, slowing the onset or development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing a complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0049] For cancer, "treatment" can refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0050] Furthermore, embodiments of the present invention provide the use of the polypeptides described in any of the foregoing embodiments or their acceptable salts, or the nucleic acids described in any of the foregoing embodiments, or the fusion proteins described in any of the foregoing embodiments, or the compositions described in any of the foregoing embodiments in the preparation of reagents for inhibiting tumor cell proliferation; In some embodiments, the tumor cell proliferation includes tumor cell proliferation associated with voltage-gated potassium channel Kv10.1 channel abnormalities; In some embodiments, the tumor cells include any one or more of liver cancer, breast cancer, and cervical cancer cells.

[0051] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0052] Example 1 Phage display peptide technology was used to screen for peptides targeting Kv10.1 binding. 1. Patch-clamp assay to identify Kv10.1 current in HEK-293T-Kv10.1 cell line HEK-293T-Kv10.1 cells are the tool cells used in this invention to screen phage display peptide libraries and identify the effects of Kv10.1-binding peptides. To verify the expression of Kv10.1 channel protein on the HEK-293T cell membrane, patch-clamp experiments were performed. Imipramine was used as a positive inhibitor for detecting Kv10.1 currents. Patch-clamp experiments were used to record the currents of negative cells (293T), positive cells (293T-Kv10.1), and the positive cell currents after imipramine inhibition. The results showed that negative cells did not contain Kv10.1, and positive cells did contain Kv10.1, satisfying the experimental requirements.

[0053] Figure 1 The patch-clamp assay was used to identify whole-cell currents of recombinant human Kv10.1 on the membrane of HEK-293T cells.

[0054] 2. Phage display peptide library screening bacteriophage (10) 11 PFU was added to HEK-293T and HEK-293T-Kv10.1 cells (1×10⁻⁶ PFU). 6 Screening was performed in suspension. First, the phages were incubated with negative control HEK-293T cells at 4°C with 360° shaking for 1 hour. Then, after centrifugation with the organic and aqueous phases mixed, the phage supernatant that had not bound to HEK-293T cells was transferred and incubated with HEK-293T-Kv10.1 target cells at 4°C with 360° shaking for 2 hours. Next, after centrifugation with the organic and aqueous phases mixed, the phage bound to HEK-293T-Kv10.1 cells was transferred and amplified in E. coli ER2738 culture. The recovered phages were repeated for the next round of in vitro screening (a total of four rounds of screening experiments were performed).

[0055] ① Digest HEK-293T cells with pre-chilled PBS containing 5 mM EDTA and collect the cells. Wash the cells with serum-free medium and then incubate them with 1×10⁻⁶ ppm. 6 Cells were resuspended at 10⁶ / mL in serum-free DMEM medium containing 5% BSA and then mixed with phages (10⁶ / mL) in 1.5 mL Eppendorf tubes. 11 Mix with PFU, incubate at 4°C on a 360° shaker for 1 hour; ② Gently transfer the cell-phage suspension to the top of the organic phase and centrifuge at 10000g for 10 min; the organic phase composition is dibutyl phthalate:cyclohexane (9:1; [v:v]). ③ Transfer the unbound phages remaining in the aqueous phase (supernatant) to a fresh test tube and mix with 1 × 10⁻⁶ ppm. 6 Co-incubate HEK-293T-Kv10.1 cells at 4°C on a 360° shaker for 1 hour; ④The mixture is then centrifuged through the organic phase. The cell complex is separated by centrifugation through the organic phase; ⑤ Quick-freeze the test tube in liquid nitrogen, cut off the bottom of the test tube, and transfer the cell-phage precipitate to a new test tube; obtain phage from the particles by infecting logarithmic-phase Escherichia coli ER2738.

[0056] The above screening steps are repeated for 4 rounds.

[0057] After four rounds of biological screening, a portion of the phage library that had undergone four rounds of screening and amplification was subjected to next-generation sequencing to select peptide sequences with high frequency of occurrence.

[0058] Phage-positive clones that bound Kv10.1 and were enriched and recovered through screening of the phage 12 peptide library are shown below. Figure 2 .

[0059] 3. Patch-clamp experiments were used to verify the inhibitory effect of Kv10.1-binding peptide KvPt1.1 on channel current.

[0060] The inhibitory effects of different concentrations of KvPt1.1 peptide on Kv10.1 current were recorded, and the results (see...) Figure 3 The results showed that KvPt1.1 could suppress the Kv10.1 channel current in a concentration-dependent manner, with an IC50 value of 62.9 ± 9.2 µM for the suppression current.

[0061] 4. MTT assay was used to detect the inhibitory effect of KvPt1.1 on the activity of the HepG2 liver cancer cell line that highly expresses Kv10.1.

[0062] The effects of different concentrations of KvPt1.1 on the proliferation of Lo2 (normal liver tissue cells) and A375 (melanoma cells) cells, which do not express Kv10.1, and on the proliferation of HepG2 liver cancer cell line, which highly expresses Kv10.1, were investigated. Results ( Figure 4 The results showed that KvPt1.1 had no significant inhibitory effect on Lo2 and A375 cells, but exhibited a concentration-dependent inhibitory effect on the proliferation of HepG2 cells.

[0063] 5. Molecular docking determined the binding sites of KvPt1.1 and Kv10.1 proteins.

[0064] Based on the existing cryo-electron microscopy structure of Kv10.1, the initial structure of the human Kv10.1 channel was obtained through homology modeling. Molecular docking was performed using AutoDock 4.2, and the binding sites of KvPt1.1 and Kv10.1 were predicted using the implemented empirical free energy function and Lamarck's genetic algorithm (LGA). During global docking, the entire extracellular domain of the Kv10.1 protein was contained within a grid box. The number of independent docking runs for each simulation was set to 100. Precise docking was then performed at locations with a high concentration of the binding peptide KvPt1.1. Ultimately, it was determined that the binding peptide KvPt1.1 binds between TM4 and TM5 of the voltage sensor domain, exhibiting a "7"-shaped insertion between the TM4 and TM5 domains. (See figure). Figure 5 .

[0065] 6. Optimization design and patch-clamp validation of KvPt1.1 based on computer-aided drug design.

[0066] Based on simulations, a large cavity was found in the KvPt1.1 region, located between the TM1 and TM4 domains. Therefore, the threonine residue in this region of KvPt1.1 was first replaced with other amino acids. The results showed that tryptophan, due to its larger side chain, exhibited higher binding affinity. Simultaneously, patch-clamp experiments confirmed that KvPt1.2 had better inhibitory efficacy than KvPt1.1. Figure 6 .

[0067] 7. Optimization design and patch-clamp validation of KvPt1.2 based on computer-aided drug design.

[0068] Based on KvPt1.2, the binding fragment of the extracellular loop region of Kv10.1 was optimized. The results showed that the substitution of the polar, uncharged amino acid asparagine further enhanced the inhibitory effect on the Kv10.1 channel current. (See...) Figure 7 .

[0069] 8. The MTT assay was used to detect the inhibitory effect of the optimized Kv10.1 binding peptide on the activity of the HepG2 liver cancer cell line that highly expresses Kv10.1.

[0070] MTT assays revealed that, compared to KvPt1.1 peptide, the optimized KvPt1.2 peptide further enhanced the inhibitory effect on the activity of the HepG2 liver cancer cell line. Based on the optimization of KvPt1.2, the inhibitory effects of KvPt1.3 and KvPt1.4 on HepG2 cells were reduced, while the anti-cancer efficacy of KvPt1.5 was further enhanced. The inhibitory effect of KvPt1.7 on HepG2 cells was significantly reduced, indicating that serine plays a key role in the Kv10.1 binding peptide. Meanwhile, the KvPt1.5 peptide showed similar inhibitory effects on the activity of the breast cancer cell line MCF-7 and the cervical cancer cell line HeLa. Figure 8 .

[0071] 9. Inhibition experiment of KvPt1.5 on HepG2 tumor model in BALB / c mice.

[0072] Based on the good in vitro inhibitory effect of KvPt1.5, this embodiment conducted an in vivo anti-tumor experiment in a liver cancer tumor model. The tumor line used in this experiment was human liver cancer cell line HepG2. The experiment was divided into two groups, with three mice in each group. The drug was administered via peritumoral injection at a concentration of 5 mg / kg. The specific groupings were: PBS group and KvPt1.5 group. Drug administration began one week after tumor cell injection, once daily. Mice were sacrificed on day 21, and the tumors were weighed. The tumor model mouse experiment showed that, compared with the control group, the KvPt1.5 group significantly inhibited tumor growth in mice in vivo, with no obvious side effects. Figure 9 .

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO:

5.

2. A nucleic acid, characterized in that, It encodes the polypeptide described in claim 1.

3. A composition, characterized in that, Its active ingredients include: the polypeptide of claim 1 or the nucleic acid of claim 2.

4. A pharmaceutical preparation, characterized in that, Its active ingredients include: the polypeptide of claim 1, the nucleic acid of claim 2, or the composition of claim 3.

5. The use of the polypeptide of claim 1, the nucleic acid of claim 2, or the composition of claim 3 in the preparation of a medicament for the treatment or adjuvant treatment of tumors associated with abnormalities in voltage-gated potassium channels Kv10.

1.

6. The application according to claim 5, characterized in that, The tumors include any one or more of the following: liver cancer, breast cancer, and cervical cancer.

7. The use of the polypeptide of claim 1, the nucleic acid of claim 2, or the composition of claim 3 in the preparation of a reagent for inhibiting the proliferation of tumor cells that highly express Kv10.

1.

8. The application according to claim 7, characterized in that, The tumor cells include any one or more of liver cancer cells, breast cancer cells, and cervical cancer cells.

9. The application according to claim 8, characterized in that, The liver cancer cells were HepG2 cells.

10. The application according to claim 8, characterized in that, The breast cancer cells were the breast cancer cell line MCF-7. Optionally, the cervical cancer cells are the HeLa cervical cancer cell line.

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