A lncRNA NUTM2B-AS1-encoded polypeptide and its application in the preparation of chemosensitizing drugs.

Abstract

This invention discloses a lncRNA NUTM2B-AS1-encoded polypeptide and its application in the preparation of chemosensitizing drugs. Based on the principle of endogenous natural peptides inhibiting ERK phosphorylation, this invention designs a novel polypeptide based on the amino acid sequence of the NUTM2B-AS1 binding domain. This polypeptide is designed based on the natural polypeptide sequence and functions by mimicking endogenous physiological regulatory pathways. Compared to natural peptides, this novel polypeptide sequence is shorter, which is beneficial for chemical synthesis. The ERK signaling pathway plays an important role in tumor chemotherapeutic resistance. This invention enhances chemosensitivity by inhibiting the feedback activation of ERK under chemotherapeutic stress, thus preventing secondary tumor resistance.

Description

(I) Technical Field

[0002] This invention belongs to the field of biomedical technology, and specifically relates to a lncRNA NUTM2B-AS1 encoded polypeptide and its application in the preparation of chemotherapeutic drugs. (II) Background Technology

[0004] Gastric cancer is one of the most common and deadliest malignant tumors worldwide. Statistics show that over one million new cases of gastric cancer are diagnosed annually, with more than half occurring in East Asia, particularly China, Japan, and South Korea. Although advancements in early screening and diagnostic treatment strategies have led to better outcomes for some patients through surgery or targeted therapy, treatment options remain limited and prognoses are poor for patients with advanced or recurrent gastric cancer. Chemotherapy, as one of the main treatments for gastric cancer, especially in cases where surgery is not possible or the disease has metastasized, continues to play a crucial role in the comprehensive treatment of gastric cancer. While combination chemotherapy regimens have significantly improved progression-free survival and overall survival in some patients, the overall efficacy of chemotherapy remains limited by various factors, making the further optimization of chemotherapy regimens for gastric cancer an important direction for current research.

[0005] Oxaliplatin, a third-generation platinum compound, has become one of the core chemotherapy drugs for treating gastric cancer due to its good anticancer activity and low toxicity. It induces tumor cell apoptosis by binding to DNA and interfering with DNA replication and transcription. Oxaliplatin is widely used in standard treatment regimens for gastric cancer (such as FOLFOX and SOX regimens), significantly improving progression-free survival and overall survival. However, despite its important role in gastric cancer chemotherapy, its efficacy is often limited by several factors. First, acquired resistance to oxaliplatin often develops in tumor cells during chemotherapy, significantly weakening the effect of chemotherapy. This resistance may be related to multiple mechanisms, including enhanced DNA damage repair, upregulation of anti-apoptotic proteins, and drug efflux. Simultaneously, the complex tumor microenvironment of gastric cancer, including its heterogeneity and immunosuppressive properties, may further weaken the antitumor effect of oxaliplatin.

[0006] Therefore, finding new strategies to enhance the efficacy of oxaliplatin chemotherapy is urgently needed. For example, discovering and applying novel micropeptides to enhance oxaliplatin sensitivity, reverse resistance, or improve its toxic side effects could provide more effective treatment options for patients with gastric cancer and other tumors. These explorations hold the promise of significantly improving the treatment prognosis and quality of life for patients with gastric cancer and other tumors. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a long non-coding RNA (lncRNA) NUTM2B-AS1 encoded polypeptide and its application in the preparation of chemotherapy sensitizing drugs. The aim is to enhance the chemosensitivity of tumors (such as gastric cancer) by inhibiting chemotherapy-induced extracellular signal-regulated kinase (ERK) phosphorylation, thereby promoting the efficacy of chemotherapy for gastric cancer.

[0009] The technical solution adopted in this invention is:

[0010] This invention provides a lncRNA NUTM2B-AS1 encoded polypeptide, the polypeptide comprising one or more of the following:

[0011] (a) A polypeptide with the amino acid sequence HSCIDIKKYLRLGKCQRKKGLVGSHFCSLYRK (SEQ ID NO.1);

[0012] (b) A polypeptide with more than 90% identity to the amino acid sequence shown in (a);

[0013] (c) A polypeptide comprising one or more amino acid sequences, including the amino acid sequence shown in (a), through substitution, deletion, or insertion of one or more amino acids.

[0014] (d) A polypeptide comprising one or more amino acid sequences modified by acetylation, phosphorylation, glycosylation, succinylation, or ubiquitination of the amino acid sequence shown in (a).

[0015] Furthermore, the amino acid sequence of the polypeptide is shown in SEQ ID NO.1.

[0016] The present invention also provides the application of the polypeptide in the preparation of chemotherapeutic drugs.

[0017] Furthermore, the tumors targeted by the chemotherapy are selected from: gastric cancer, lung cancer (such as non-small cell lung cancer), melanoma, breast cancer, ovarian cancer, prostate cancer, liver cancer, kidney cancer, colorectal cancer, head and neck cancer, skin cancer, bladder cancer, and pancreatic cancer.

[0018] Furthermore, the drug includes drugs that inhibit ERK phosphorylation.

[0019] Furthermore, the chemotherapy sensitizers include drugs used to enhance the efficacy of oxaliplatin, a chemotherapy drug for gastric cancer.

[0020] Preferably, the formulation of the drug includes: solution, emulsion, and suspension.

[0021] The inhibitors described in this invention, or drugs containing said inhibitors, can be used in combination with known chemotherapy or targeted therapies in the art. Such combinations include: simultaneous, sequential, separate, or individual administration of the substances or products of this invention, as well as other known drugs or therapies.

[0022] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0023] (1) This invention is based on the principle of endogenous natural peptides inhibiting ERK phosphorylation. A novel polypeptide was designed based on the amino acid sequence of the NUTM2B-AS1 binding domain. This polypeptide is designed based on the natural polypeptide sequence and functions by mimicking the endogenous physiological regulatory pathway. Compared with natural peptides, the sequence of this novel polypeptide is shorter, which is conducive to chemical synthesis.

[0024] (2) The ERK signaling pathway plays an important role in tumor chemotherapy resistance. This invention avoids secondary tumor resistance and enhances chemotherapy sensitivity by inhibiting the feedback activation of ERK under chemotherapy stress. (iv) Description of the attached drawings

[0026] Figure 1 This is a laser confocal microscopy image of FITC-MIST-p entering human gastric cancer cells in Example 1.

[0027] Figure 2 This is a gel image showing how FITC-MIST-p reduced ERK phosphorylation in human gastric cancer cells in Example 2.

[0028] Figure 3 This is the IC50 curve of MIST-p reducing the effect of oxaliplatin on human gastric cancer cells in Example 2.

[0029] Figure 4 This is a statistical chart showing the reduction in tumor volume growth in the MIST in vivo experiment of Example 3.

[0030] Figure 5 This is a statistical chart showing the slowing of tumor growth in the MIST in vivo experiment in Example 3. (V) Detailed Implementation Methods

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] The human gastric cancer cell line AGS used in this embodiment of the invention was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0034] Example 1: Design of MIST-p, a peptide derived from MIST, and its entry into cells.

[0035] 1. Design and synthesis of peptide MIST-p

[0036] Based on the endogenous micropeptide MIST encoded by the NUTM2B-AS1 transcript (amino acid sequence shown in SEQ ID NO.2), and its core functional region inhibiting ERK phosphorylation (amino acids 20-51 of SEQ ID NO.2), we extracted this effective binding sequence to design a shorter novel peptide, MIST-p (amino acid sequence shown in SEQ ID NO.1: HSCIDIKKYLRLGKCQRKKGLVGSHFCSLYRK). This sequence length is moderate, making it more suitable for in vitro chemical synthesis. To facilitate intracellular tracking, we labeled the N-terminus of MIST-p with the fluorescent group FITC to synthesize FITC-MIST-p.

[0037] SEQ ID NO.2:

[0038] VYKSLIKLLFSILWGIVLGHSCIDIKKYLRLGKCQRKKGLVGSHFCSLYRKHSSICFQGGFRKLPIMMEGKVGAGMSHGKSQSKRGSGEVLHTFKQSNLVRTHNLDDS

[0039] 2. The entry of FITC-MIST-p into human gastric cancer cells.

[0040] Human gastric cancer cell line AGS was seeded in confocal microscopy dishes, with 800 μL of RMPI-1640 medium (Gibco) containing 10% FBS (Bovogen) added. The cells were incubated at 37°C in a 5% CO2 incubator until they adhered and reached approximately 70% confluence. Then, 10 μM of in vitro synthesized FITC-MIST-p peptide was added to the culture dish. After incubation at 37°C in a 5% CO2 incubator for 12 hours, the medium was aspirated, and the cells were washed three times with PBS. Subsequently, the cells were treated with proteinase K for 1 minute to remove peptides bound to the cell membrane that failed to enter the cells. Finally, the cell nuclei were stained with DAPI to prepare fluorescent slides. The entry of FITC-MIST-p peptide into the cells was detected using laser confocal microscopy. Figure 1 As shown, green fluorescence (FITC) is abundant in the cytoplasm of gastric cancer cells, demonstrating that the synthesized polypeptide MIST-p has good cell penetration ability and can directly enter the tumor cells to exert its effects.

[0041] Example 2: Peptide MIST-p inhibits ERK phosphorylation and promotes chemosensitivity in gastric cancer cells.

[0042] 1. MIST-p reduces chemotherapy-induced ERK phosphorylation levels.

[0043] When gastric cancer cells are subjected to chemotherapy drugs such as oxaliplatin, they often produce anti-apoptotic effects by feedback activation of the ERK signaling pathway (manifested as increased ERK phosphorylation level), thereby leading to drug resistance.

[0044] Human gastric cancer cell line AGS was seeded in six-well plates, and 2 mL of RMPI-1640 medium containing 10% FBS was added. The cells were incubated at 37°C in a 5% CO2 incubator. When the cells adhered and grew to approximately 70% confluence, the AGS cells were divided into three groups: a control group (4 μL DMSO + 2 μL sterile water), an oxaliplatin (OXA) treatment group (2 μL 5 mM oxaliplatin aqueous solution + 4 μL sterile water, final oxaliplatin concentration 5 μM), and a combined treatment group (2 μL 5 mM oxaliplatin aqueous solution + 4 μL 5 mM MIST-p DMSO solution, i.e., final concentration 5 μM oxaliplatin + final concentration 10 μM MIST-p). After 24 hours of incubation, total protein was extracted from the cells in each group. Protein expression was detected using gel electrophoresis immunoblotting with specific antibodies against p-ERK (phosphorylated ERK, Huabio ET1610-13), total ERK (Huabio ET1601-29), and the internal control vinculin (Huabio ET1705-94). Figure 2 As shown, treatment with oxaliplatin alone significantly upregulated p-ERK levels in gastric cancer cells; however, the addition of MIST-p and oxaliplatin strongly inhibited p-ERK expression, while total ERK levels remained unchanged. This indicates that MIST-p can effectively block chemotherapy-induced abnormal activation of the ERK signaling pathway.

[0045] 2. MIST-p reduces the IC50 of oxaliplatin in human gastric cancer cells.

[0046] To further verify the effect of MIST-p on promoting the chemosensitivity of gastric cancer cells in vitro, cell viability was detected using the CCK-8 assay.

[0047] AGS cells were seeded in 96-well plates, with 100 μL of RMPI-1640 medium containing 10% FBS added to each well. The plates were incubated at 37°C in a 5% CO2 incubator. Once the cells adhered and reached approximately 70% confluence, they were divided into a chemotherapy-only group and a combined peptide group. The chemotherapy-only group received oxaliplatin at progressively increasing concentrations (0, 1, 2, 3, 4, 5, 6, 7, 8 μM, dissolved in sterile water). The combined peptide group received oxaliplatin at the same concentration gradient, plus a fixed concentration (10 μM) of MIST-p in each well. After culturing for 24 hours at 37°C in a 5% CO2 incubator, CCK-8 reagent (Yeasen) was added, followed by incubation for 1 hour. The absorbance (OD value) was measured at 450 nm using a microplate reader. Cell viability was calculated, dose-response curves were plotted, and the IC50 was calculated.

[0048] like Figure 3 As shown in the IC50 curves, the dose-response curve shifted significantly to the left after the combined addition of oxaliplatin and MIST-p, indicating a substantial decrease in the half-maximal inhibitory concentration (IC50) of oxaliplatin in gastric cancer cells. This result fully demonstrates that MIST-p effectively reverses the resistance of gastric cancer cells to chemotherapy drugs by inhibiting ERK phosphorylation, thereby enhancing the cytotoxic killing effect of oxaliplatin.

[0049] Example 3: NUTM2B-AS1-encoded peptide MIST promotes the efficacy of chemotherapy in a nude mouse xenograft model of gastric cancer.

[0050] 1. Establishment of a mouse tumor model

[0051] Twenty-four 4-6 week old male BALB / c nude mice were purchased. Human gastric cancer cell line AGS was used, and the cells were cultured in RMPI-1640 medium containing 10% FBS at 37°C in a 5% CO2 incubator. MycoAlert kit (Lonza) assays showed no mycoplasma contamination or cross-contamination.

[0052] Under aseptic conditions, a suspension of AGS cells in the logarithmic growth phase (concentration 5 × 10⁻⁶) was prepared. 6 The MIST-p peptide (0.2 mL / mL) was subcutaneously injected into the right back of nude mice. After injection, the tumor formation in the nude mice was observed periodically. The major and minor diameters of the tumors formed in each mouse were measured using calipers, and the tumor volume was calculated using the formula: Tumor volume (mm³) = 0.5 × major diameter (mm) × minor diameter (mm)². When the tumor volume grew to approximately 100 mm³, a mouse gastric cancer xenograft model was established to verify the sensitizing effect of the peptide MIST-p on gastric cancer chemotherapy.

[0053] 2. Grouping and medication methods

[0054] On day 6 post-injection, 24 nude mice with tumors were randomly divided into 4 groups of 6 mice each. The mice were labeled and their initial tumor size before medication was recorded. The mice in each group were then treated according to the following protocol:

[0055] (1) Control group (WT): intraperitoneal injection of normal saline;

[0056] (2) Oxaliplatin monotherapy group (OXA): intraperitoneal injection of oxaliplatin (5 mg / kg, dissolved in sterile water), twice a week;

[0057] (3) MIST monotherapy group (MIST): Intraperitoneal injection of MIST-p peptide (10 mg / kg, dissolved in sterile water, once every 2 days).

[0058] (4) Combination therapy group (OXA + MIST): intraperitoneal injection of oxaliplatin (5 mg / kg, dissolved in sterile water, twice a week) and intraperitoneal injection of MIST peptide (10 mg / kg, dissolved in sterile water, once every 2 days).

[0059] Administer the medication continuously for 15 days.

[0060] 3. Observation and Experimental Results

[0061] The long and short axes of the tumors in nude mice were measured and the volume was calculated every 3 days. After 15 days of drug administration, the mice were sacrificed, the tumor tissue was completely dissected, and the weight was recorded.

[0062] Figure 4 The graph shows the statistical distribution of tumor volume growth in each group of mice after treatment. Figure 5 The chart shows the statistical distribution of tumor quality after dissection in each group of mice. Figure 4 and Figure 5 The results showed that, compared with the control group, tumor growth was inhibited to some extent in the oxaliplatin monotherapy group; while the tumor volume and tumor mass in the combination therapy group (OXA + MIST) were significantly lower than those in the oxaliplatin monotherapy group. These in vivo experimental results indicate that the peptide MIST-p can significantly enhance the inhibitory effect of oxaliplatin on gastric cancer and promote the efficacy of chemotherapy.

Claims

1. A polypeptide encoded by lncRNA NUTM2B-AS1, characterized in that, The polypeptide includes one or more of the following: (a) A polypeptide with the amino acid sequence shown in SEQ ID NO.1; (b) A polypeptide with more than 90% identity to the amino acid sequence shown in (a); (c) A polypeptide comprising one or more amino acid sequences, including the amino acid sequence shown in (a), through substitution, deletion, or insertion of one or more amino acids. (d) A polypeptide comprising one or more amino acid sequences modified by acetylation, phosphorylation, glycosylation, succinylation, or ubiquitination of the amino acid sequence shown in (a).

2. The polypeptide according to claim 1, characterized in that, The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

3. The use of the polypeptide of claim 1 in the preparation of a chemotherapy sensitizer.

4. The application as described in claim 3, characterized in that, The tumors targeted by the chemotherapy are selected from: gastric cancer, lung cancer, melanoma, breast cancer, ovarian cancer, prostate cancer, liver cancer, kidney cancer, intestinal cancer, head and neck cancer, skin cancer, bladder cancer, and pancreatic cancer.

5. The application as described in claim 3, characterized in that, The drugs include those that inhibit ERK phosphorylation.

6. The application as described in claim 3, characterized in that, The chemotherapy sensitizers include drugs used to enhance the efficacy of oxaliplatin, a chemotherapy drug for gastric cancer.

7. The application as described in claim 3, characterized in that, The formulations of the drug include: solutions, emulsions, and suspensions.

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