Polypeptide for inhibiting lung adenocarcinoma metastasis and application thereof
By designing the specific polypeptide sequence X-RHTQIRPTMFPPY to block the methylation of arginine at position 389 of NSUN2, the problem of high metastasis in lung adenocarcinoma cells was solved, and the migration and invasion abilities of lung adenocarcinoma cells were significantly inhibited.
Patent Information
- Application Number
- CN202511589564.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-03
- Publication Date
- 2026-02-17
AI Technical Summary
Lung adenocarcinoma cells are highly metastatic, and current technologies struggle to effectively inhibit the methylation of NSUN2 to block its migration and invasion, thus increasing the difficulty of treatment.
The designed and synthesized polypeptide sequence X-RHTQIRPTMFPPY, containing a modifying group and a membrane-penetrating peptide, can enter the cell to block the methylation of arginine 389 at NSUN2 and inhibit its function.
It significantly inhibits the migration and invasion of lung adenocarcinoma cells, reduces the formation of lung adenocarcinoma metastases, and demonstrates good safety and biocompatibility.
Smart Images
Figure CN121537482A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and more particularly to a polypeptide for inhibiting lung adenocarcinoma metastasis and its applications. Background Technology
[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide, posing a serious threat to human life and health. Among the many pathological types of lung cancer, lung adenocarcinoma is particularly challenging due to its high metastatic potential. Lung adenocarcinoma cells have extremely strong invasive and migratory abilities, rapidly spreading to other organs beyond the lungs, such as the brain, bones, and liver, leading to a rapid deterioration of the disease and significantly increasing the difficulty of treatment.
[0003] NSUN2 (NOP2 / Sun RNA methyltransferase) is an RNA m 5 C-modified methyltransferases utilize S-adenosylmethionine (SAM) as a methyl donor to catalyze the transfer of methyl groups to RNA cytosine. They play crucial roles in maintaining tissue homeostasis, early embryonic development, and cell differentiation. In tumor research, NSUN2 has been reported to promote gastric cancer cell proliferation by inhibiting p57kip2 expression. Furthermore, it has been found to participate in glucose-sensing regulation in tumorigenesis and immunotherapy resistance. Additionally, non-small cell lung cancer patients with high NSUN2 expression are more likely to develop resistance to gefitinib treatment.
[0004] Previous studies have found that NSUN2 is highly expressed in lung adenocarcinoma tissues, and its expression level is closely related to the metastatic potential of lung adenocarcinoma. In vitro and in vivo experimental results show that high expression of NSUN2 in lung adenocarcinoma cells significantly enhances their migration and invasion abilities. Furthermore, our research revealed that the high expression of NSUN2 in lung adenocarcinoma tissues is due to asymmetric methylation modification at arginine position 389 of the protein. Therefore, it is essential to develop a peptide that inhibits NSUN2 methylation modification to suppress lung adenocarcinoma metastasis. Summary of the Invention
[0005] The purpose of this invention is to provide a polypeptide for inhibiting lung adenocarcinoma metastasis and its application, which can inhibit methylation modification of NSUN2, thereby inhibiting lung adenocarcinoma metastasis.
[0006] To achieve the above objectives, the present invention is implemented using the following technical solution:
[0007] In a first aspect, the present invention provides a polypeptide for inhibiting lung adenocarcinoma metastasis, wherein the amino acid sequence of the polypeptide for inhibiting lung adenocarcinoma metastasis is shown as in the general formula X-RHTQIRPTMFPPY; in the general formula, the X at the N-terminus represents a modifying group, and the Y at the tail represents a membrane-penetrating peptide.
[0008] This invention is based on the molecular mechanism by which the high expression of NSUN2 in lung adenocarcinoma tissue is maintained by asymmetric methylation at arginine 389. A polypeptide specifically synthesizes to inhibit methylation of NSUN2. The core of the polypeptide for inhibiting lung adenocarcinoma metastasis is the amino acid sequence shown in SEQ ID.1. The N-terminal modification and the transmembrane peptide at the tail can be replaced with similar compounds. The N-terminal modification group in the amino acid sequence is used to detect the intracellular localization of the polypeptide, and the transmembrane peptide at the tail assists the polypeptide core sequence in entering the cell. In vitro and in vivo experiments have confirmed that the polypeptide designed in this invention can effectively block methylation at arginine 389 of NSUN2, significantly inhibiting the migration and invasion of lung adenocarcinoma cells. In animal models, this polypeptide significantly reduces the formation of lung adenocarcinoma metastases without significant toxicity, demonstrating good safety and biocompatibility.
[0009] Furthermore, the membrane-penetrating peptide includes one of octaarginine and TAT peptide.
[0010] In the above technical solution, octarginine Arg8 and TAT peptide YGRKKRRQRRR are cell-penetrating peptides with strong cell penetration ability. They can cross the cell membrane and enter the cell nucleus, and can effectively assist in inhibiting the core sequence of lung adenocarcinoma metastasis to enter the target location and exert its effect.
[0011] Furthermore, the modifying group is a fluorescent labeling group;
[0012] The fluorescent labeling group includes either fluorescein isothiocyanate or NHS ester dyes.
[0013] In the above technical solution, fluorescent labeling groups are used for modification, which makes it easy to detect the localization of peptides in cells. Fluorescein isothiocyanate is abbreviated as FITC.
[0014] Furthermore, the specific amino acid sequence of the fluorescent labeling group, fluorescein isothiocyanate, is shown in SEQ ID.1 and SEQ ID.2;
[0015] The specific amino acid sequences of the fluorescent labeling group, which is an NHS ester dye, are shown in SEQ ID.3 and SEQ ID.4.
[0016] Secondly, the present invention provides the application of a polypeptide for inhibiting lung adenocarcinoma metastasis in the preparation of a small molecule formulation that weakens the migration and invasion ability of lung adenocarcinoma cells.
[0017] Thirdly, the present invention provides the application of a polypeptide for inhibiting lung adenocarcinoma metastasis in the preparation of a small molecule formulation that inhibits methylation modification at the 389 site of NSUN2 arginine.
[0018] Fourthly, the present invention provides the application of a polypeptide for inhibiting lung adenocarcinoma metastasis in the preparation of a small molecule formulation that promotes the degradation of NSUN2 protein.
[0019] Fifthly, the present invention provides the application of a polypeptide for inhibiting lung adenocarcinoma metastasis in the preparation of a pharmaceutical formulation for inhibiting lung adenocarcinoma metastasis.
[0020] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0021] This invention is based on the molecular mechanism that maintains high expression of NSUN2 in lung adenocarcinoma tissue through asymmetric methylation modification at arginine 389. A polypeptide that specifically inhibits NSUN2 methylation modification was synthesized. Lung adenocarcinoma cells A549 and H1299 were treated with the designed CIP-NR389-1 polypeptide, and the distribution of the polypeptide in the cells was detected. The results showed that the polypeptide could successfully reach the cell nucleus. A549 and H1299 cells were treated with CIP-NR389-1, respectively, and the methylation level at NSUN2 arginine 389 was detected by immunoprecipitation. The results showed that methylation at this site was significantly inhibited. The half-life of NSUN2 protein was significantly reduced in the CIP-NR389-1 treatment group; in addition, the stability of NSUN2 protein was detected by CIP-NR389-1 treatment, and the half-life of NSUN2 protein was significantly shortened in the CIP-NR389-1 treatment group; A549 and H1299 cells were treated with CIP-NR389-1 respectively, and the changes in cell migration and invasion ability were detected by Transwell technology. It was found that the migration and invasion ability of lung adenocarcinoma cells after CIP-NR389-1 treatment was significantly reduced; using a lung cancer nude mouse metastasis model, the effect of CIP-NR389-1 on the in vivo metastasis of lung adenocarcinoma cells was detected, and it was found that CIP-NR389-1 significantly inhibited the in vivo metastasis ability of lung adenocarcinoma cells;
[0022] Therefore, in vitro and in vivo experiments confirmed that the polypeptide sequence used to inhibit lung adenocarcinoma metastasis can effectively block the methylation of arginine 389 at NSUN2, significantly inhibiting the migration and invasion ability of lung adenocarcinoma cells. In animal models, the polypeptide can significantly reduce the formation of lung adenocarcinoma metastases without obvious toxic reactions, demonstrating good safety and biocompatibility. Attached Figure Description
[0023] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0024] Figure 1This is a CIP-NR389-1 polypeptide sequence mass spectrum of some embodiments of the polypeptide provided by this invention for inhibiting lung adenocarcinoma metastasis;
[0025] Figure 2 This is a schematic diagram showing the intracellular distribution of FITC fluorescence signals of some embodiments of the polypeptides provided by the present invention for inhibiting lung adenocarcinoma metastasis;
[0026] Figure 3 This is a schematic diagram showing the arginine methylation level and degradation rate of NSUN2 protein after CIP-NR389-1 treatment of A549 and H1299, representing some embodiments of the polypeptide for inhibiting lung adenocarcinoma metastasis provided by the present invention.
[0027] Figure 4 This is a schematic diagram showing the changes in cell migration and invasion ability of A549 and H1299 cells after CIP-NR389-1, an embodiment of the polypeptide provided by this invention for inhibiting lung adenocarcinoma metastasis, was treated with respectively.
[0028] Figure 5 This is a schematic diagram showing the formation of lung metastases after treatment with CIP-NR389-1 polypeptide via tail vein injection, based on some embodiments of the polypeptide provided by this invention for inhibiting lung adenocarcinoma metastasis. Detailed Implementation
[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.
[0030] It should be understood that all experimental procedures not detailed in the experiment are routine experimental procedures well known to those skilled in the art.
[0031] Example 1
[0032] This embodiment provides a polypeptide for inhibiting lung adenocarcinoma metastasis.
[0033] Based on the high expression of NSUN2 in lung adenocarcinoma tissue, a polypeptide for inhibiting lung adenocarcinoma metastasis was invented. The amino acid sequence of the polypeptide is shown in Table 1. It was synthesized by Nanjing Genscript using solid-phase polypeptide synthesis method.
[0034] When the N-terminus is modified with FITC and the C-terminus contains eight consecutive arginine residues (octaarginine), the amino acid sequence of the peptide is SEQ ID.1 (CIP-NR389-1): FITC-RHTQIRPTMFPP RRRRRRRR. The mass spectrum of SEQ ID.1 is shown below. Figure 1 As shown;
[0035] When the N-terminus is modified with FITC, the amino acid sequence of the polypeptide containing the TAT peptide at the C-terminus is SEQ ID.2 (CIP-NR389-2): FITC-RHTQIRPTMFPPYGRKKRRQRRR;
[0036] When the N-terminus is modified with NHS ester dye and the C-terminus contains 8 consecutive arginine sequences (octaarginine), the amino acid sequence of the polypeptide is SEQ ID.3 (CIP-NR389-3): NHS ester dye-RHTQIRPTMFPPRRRRRRRR;
[0037] When the N-terminus is modified with NHS ester dye and the C-terminus contains TAT peptide, the amino acid sequence of the polypeptide is SEQ ID.4 (CIP-NR389-4): NHS ester dye-RHTQIRPTMFPPYGRKKRRQRRR;
[0038] Figure 1 In the figure, the function of the amino acid sequence is to inhibit the methylation modification of arginine at position 389 in the NSUN2 protein. The R (arginine) specified by the sequence label 389 in the figure is used to perform this function.
[0039] Example 2
[0040] This embodiment provides a method for preparing the working solution of the polypeptide CIP-NR389-1 (SEQ ID.1) for inhibiting lung adenocarcinoma metastasis obtained in Example 1.
[0041] Stock solution preparation (in vivo experiments): 1 mg / tube of peptide (CIP-NR389-1), centrifuged at 4℃, 12000 rpm, 2 min; dissolved in 1 mL of PBS buffer to prepare a stock solution of 1 mg / mL (0.56 mol / L), aliquoted and stored at -80℃ protected from light.
[0042] Preparation of working solution: Before the experiment, thaw the stock solution on ice and dilute it with PBS to 1 mmol / L to obtain CIP-NR389-1 working solution.
[0043] Example 3
[0044] This embodiment provides a method and results for detecting the distribution of CIP-NR389-1 in lung adenocarcinoma cells.
[0045] A549 and H1299 cells were seeded onto cell slides, and after the cells recovered, the peptide concentration was diluted to 1 µmol / L with cell culture medium and the cells were treated for 24 h.
[0046] The supernatant was removed, and the cells were treated with cell fixative for 30 min, followed by staining of the nuclei with the fluorescent dye DAPI. Finally, FITC and DAPI signals were detected using confocal microscopy.
[0047] Lung adenocarcinoma cells A549 and H1299 were treated with CIP-NR389-1 peptide, and the distribution of the peptide in the cells was detected. Figure 2 As shown, the polypeptide can successfully reach the cell nucleus.
[0048] Example 4
[0049] This embodiment provides a method and results for detecting the effect of CIP-NR389-1 on NSUN2 methylation modification.
[0050] Healthy A549 and H1299 cells were transferred to 60 mm cell culture dishes. When the cell density reached 60%, they were treated with CIP-NR389-1 (1 µmol / L) for 48 h. PBS treatment served as the control group.
[0051] The cells were collected and lysed with 500 µl of cell lysis buffer (containing protease inhibitors). The supernatant was collected for immunoprecipitation experiments using anti-IgG and anti-NSUN2 antibodies. Finally, the immunoprecipitated products were detected using methylated antibodies.
[0052] like Figure 3 As shown, A549 and H1299 cells were treated with the peptide CIP-NR389-1, respectively. Immunoprecipitation analysis of the methylation level at the NSUN2 arginine 389 site revealed a significant decrease in methylation at this site. Figure 3 A is a schematic diagram showing the methylation level of NSUN2 arginine after CIP-NR389-1 treatment with A549 and H1299, as detected by immunoprecipitation technique.
[0053] Example 5
[0054] This embodiment provides a detection method and results for detecting the impact of CIP-NR389-1 on the stability of NSUN2.
[0055] Healthy A549 and H1299 cells were passaged into 12-well plates. When the cell density reached 60%, the cells were treated with CIP-NR389-1 (1 µmol / L) for 48 h. PBS treatment served as the control group.
[0056] The cells were treated with the protein synthesis inhibitor CHX at time points (0, 2, 4, 6 h) and the corresponding proteins were collected. The half-life of NSUN2 protein was detected by Western blot.
[0057] like Figure 3 As shown, stability testing of the NSUN2 protein revealed that the half-life of the NSUN2 protein was significantly shortened in the CIP-NR389-1 treatment group. Figure 3 middle, Figure 3 B is a schematic diagram showing the degradation rate of NSUN2 protein detected by Western blot after A549 and H1299 cells were treated with CIP-NR389-1 and then treated with the protein synthesis inhibitor CHX at the time points shown in the figure. The PBS treatment group served as the control.
[0058] Example 6
[0059] This embodiment provides a method and results for detecting the effect of CIP-NR389-1 on the migration and invasion of lung adenocarcinoma cells.
[0060] Healthy A549 and H1299 cells were passaged into 6-well plates. When the cell density reached 60%, they were treated with CIP-NR389-1 (1 µmol / L) for 48 h. PBS treatment served as the negative control and MS023 treatment served as the positive control.
[0061] The effects of the above-mentioned cell migration and invasion were detected using Transwell technology.
[0062] A549 and H1299 cells were treated with CIP-NR389-1, and the changes in cell migration and invasion abilities were detected using Transwell assay. The results are as follows: Figure 4 As shown, the results indicate that the migration and invasion abilities of lung adenocarcinoma cells treated with CIP-NR389-1 were significantly reduced. Figure 4 PBS treatment was used as a negative control, and MS023, a protein arginine methylation inhibitor, was used as a positive control.
[0063] Example 7
[0064] This embodiment provides a method and results for detecting the effect of CIP-NR389-1 on the in vivo metastasis of lung adenocarcinoma cells.
[0065] A lung cancer metastasis model was constructed using A549 cells and 4-week-old female nude mice (BALB / c).
[0066] The constructed model was randomly divided into 3 groups (control group, CIP-NR389-1 treatment group, and MS023 treatment group), with 5 animals in each group. Treatment was performed every 4 days. The control group was treated with normal saline; CIP-NR389-1 was administered at a dose of 0.1 µg via tail vein injection; MS023 was administered at a dose of 1.6 mg via intraperitoneal injection.
[0067] Eight weeks later, the nude mice were euthanized, and lung metastases were observed and counted. The expression of NSUN2 was detected by immunohistochemistry.
[0068] Using a lung cancer nude mouse metastasis model, the effect of peptide CIP-NR389-1 on the in vivo metastasis of lung adenocarcinoma cells was examined. The results are as follows: Figure 5 As shown, the results indicate that CIP-NR389-1 significantly inhibits the metastatic ability of lung adenocarcinoma cells in vivo. The Control group in the figure was treated with saline, and MS023 served as a positive control.
[0069] As known from Examples 1 to 7, the polypeptide designed and prepared in this invention for inhibiting lung adenocarcinoma metastasis can successfully reach the cell nucleus, significantly reduce the methylation level of NSUN2 arginine 389, significantly shorten the half-life of NSUN2 protein, significantly reduce the migration and invasion ability of lung adenocarcinoma cells, and significantly inhibit the metastatic ability of lung adenocarcinoma cells in vivo.
[0070] Therefore, the polypeptide sequence used to inhibit lung adenocarcinoma metastasis can effectively block the methylation of arginine 389 at NSUN2, and significantly inhibit the migration and invasion ability of lung adenocarcinoma cells. When used in animal models, this polypeptide can significantly reduce the formation of lung adenocarcinoma metastases, and no obvious toxic reactions were observed, showing good safety and biocompatibility.
[0071] Table 1: Peptide sequences for inhibiting lung adenocarcinoma metastasis
[0072]
Claims
1. A polypeptide for inhibiting lung adenocarcinoma metastasis, characterized in that, The amino acid sequence of the polypeptide for inhibiting lung adenocarcinoma metastasis is shown in general formula X-RHTQIRPTMFPPY; wherein X at the N-terminal of the general formula represents a modification group, and Y at the tail represents a transmembrane peptide.
2. The polypeptide for inhibiting lung adenocarcinoma metastasis according to claim 1, characterized in that, The transmembrane peptide comprises one of octa-arginine and TAT peptide.
3. The polypeptide for inhibiting lung adenocarcinoma metastasis according to claim 1, wherein, The modification group is a fluorescent labeling group. The fluorescent labeling group comprises one of fluorescein isothiocyanate and NHS ester dye.
4. The polypeptide for inhibiting lung adenocarcinoma metastasis according to claim 3, wherein, The specific amino acid sequence of the fluorescent labeling group being fluorescein isothiocyanate is shown in SEQ ID. 1 and SEQ ID.
2. The specific amino acid sequence of the fluorescent labeling group being NHS ester dye is shown in SEQ ID. 3 and SEQ ID.
4.
5. Use of the polypeptide for inhibiting lung adenocarcinoma metastasis according to any one of claims 1-4 in the preparation of a small molecule preparation for weakening the migration and invasion ability of lung adenocarcinoma cells.
6. Use of the polypeptide for inhibiting lung adenocarcinoma metastasis according to any one of claims 1-4 in the preparation of a small molecule preparation for inhibiting methylation modification of NSUN2 at the 389th arginine site.
7. Use of the polypeptide for inhibiting lung adenocarcinoma metastasis according to any one of claims 1-4 in the preparation of a small molecule preparation for promoting degradation of NSUN2 protein.
8. Use of the polypeptide for inhibiting lung adenocarcinoma metastasis according to any one of claims 1-4 in the preparation of a pharmaceutical preparation for inhibiting lung adenocarcinoma metastasis.