Micropeptide pamp targeting proline metabolism and application thereof in preparation of lung adenocarcinoma treatment drug
By developing PAMP, a micropeptide encoded by the long non-coding RNA PSMA3-AS1, to inhibit the proline metabolic pathway, a therapeutic drug for lung adenocarcinoma was prepared, overcoming the limitations of existing lung adenocarcinoma treatments, providing new treatment strategies and diagnostic biomarkers, and significantly improving patient prognosis.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-05-07
- Publication Date
- 2026-06-09
AI Technical Summary
Current treatments for lung adenocarcinoma have limitations, including the inability of surgery to completely remove micrometastases, the significant toxic side effects and drug resistance associated with chemotherapy, and the lack of effective molecular targets and treatment strategies.
We developed a micropeptide PAMP encoded by the long non-coding RNA PSMA3-AS1, and prepared injectable or topical formulations by inhibiting the proline metabolic pathway. Combined with gene therapy strategies, we developed a lung adenocarcinoma treatment drug by knocking down PSMA3-AS1 with siRNA or targeting PYCR1.
It effectively inhibits the proliferation of lung adenocarcinoma cells, interferes with proline metabolism, provides a new treatment strategy, and serves as an auxiliary diagnostic marker to improve patient prognosis.
Smart Images
Figure CN122167558A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a micropeptide PAMP that targets proline metabolism and its application in the preparation of drugs for the treatment of lung cancer and adenocarcinoma. Furthermore, this invention relates to the preparation method of the micropeptide and its functional truncated form, functional verification, and pharmaceutical compositions containing the micropeptide. Background Technology
[0002] Lung cancer is a leading cause of cancer-related deaths worldwide, with lung adenocarcinoma being the most common subtype, accounting for approximately 35%-40% of all lung cancer cases. Traditional treatments for lung adenocarcinoma include surgical resection and chemotherapy, but these methods have significant limitations. For example, surgery may not completely remove micrometastases or residual cancer cells; chemotherapy drugs often have strong toxic side effects, and long-term use can easily lead to drug resistance. Furthermore, because early symptoms of lung adenocarcinoma are often subtle, most patients are diagnosed at an advanced stage, greatly increasing the difficulty of treatment. Although advances in targeted therapy and immunotherapy in recent years have significantly improved the prognosis for some patients, the overall survival rate for lung adenocarcinoma patients remains low, necessitating the development of new molecular targets and more effective treatment strategies.
[0003] Long non-coding RNAs (lncRNAs) have traditionally been considered to lack protein-coding capabilities. However, recent studies have shown that many lncRNAs contain small open reading frames (sORFs) capable of encoding functional micropeptides (typically less than 100 amino acids in length). These micropeptides play a crucial role in tumorigenesis and development by regulating key biological processes such as cell metabolism, proliferation, and apoptosis. For example, the MIAC micropeptide encoded by lncRNA LINC00473 can inhibit renal cell carcinoma progression by binding to the AQP2 protein; the Mtln micropeptide encoded by lncRNA LINC00116 participates in regulating mitochondrial function and affecting cell metabolism. However, little is currently known about the function of lncRNA-encoded micropeptides in lung adenocarcinoma and their regulatory mechanisms in tumor metabolic reprogramming, requiring further investigation. Notably, tumor cells undergo significant metabolic reprogramming to meet the demands of rapid proliferation, with abnormal proline metabolism being a key characteristic. Proline, a crucial raw material for cell proliferation, is primarily synthesized by pyrroline-5-carboxylic acid reductase 1 (PYCR1). Clinical data show that PYCR1 is highly expressed in lung adenocarcinoma, and its expression level is closely related to tumor invasiveness and poor patient prognosis. Therefore, targeting and inhibiting PYCR1 activity or interfering with the proline metabolism pathway has become a potential new strategy for the treatment of lung adenocarcinoma.
[0004] Peptide drugs, as an emerging direction in the field of anti-tumor therapy, possess the following unique advantages: Compared to small-molecule chemical drugs, peptide drugs have higher target specificity, precisely binding to tumor-related molecules (such as receptors, enzymes, or signaling molecules), significantly reducing off-target toxicity; compared to monoclonal antibodies and other biomacromolecules, peptide drugs have smaller molecular weights (typically <50 amino acids), relatively simpler structures, are easier to chemically synthesize and modify, have lower production costs, and exhibit better stability; furthermore, peptide drugs can be administered via multiple routes, including injection, oral administration, and transdermal delivery, offering high flexibility in clinical application. These characteristics make them highly competitive candidate molecules in anti-tumor therapy, providing safer and more effective treatment options for patients with lung adenocarcinoma.
[0005] In summary, further exploration of the functions of lncRNA-encoded micropeptides in lung adenocarcinoma, especially their role in proline metabolism regulation, combined with the advantages of peptide drug development, holds promise for providing new targets and strategies for the precision treatment of lung adenocarcinoma. Summary of the Invention
[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a proline-associated micropeptide (PAMP) encoded by the long non-coding RNA (lncRNA) PSMA3-AS1, clarifying its anti-tumor function and mechanism of action in lung adenocarcinoma (LUAD), and developing PAMP-based therapeutic drugs for lung adenocarcinoma.
[0007] The objective of this invention is achieved through the following technical solution: This invention provides a micropeptide PAMP, the amino acid sequence of which is shown in SEQ ID NO.1 and is encoded by the open reading frame ORF of lncRNA PSMA3-AS1.
[0008] The present invention also provides a nucleic acid molecule capable of encoding a truncated sequence of the functional fragment of the micropeptide PAMP.
[0009] Furthermore, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.
[0010] The present invention also provides a gene expression vector containing the nucleic acid molecule, wherein the gene expression vector is a prokaryotic or eukaryotic expression vector.
[0011] Furthermore, the gene expression vector is a PLVX-CMV-FLAG-Puro series vector.
[0012] The present invention also provides a host cell containing the gene expression vector, wherein the host cell is Escherichia coli, HEK293T cells or A549 cells.
[0013] The present invention also provides a method for preparing a truncated sequence of the functional fragment of the micropeptide PAMP, wherein PAMP and its functional truncated form are directly chemically synthesized by solid-phase micropeptide synthesis technology.
[0014] The present invention also provides the application of the micropeptide PAMP and its functional truncated form in the preparation of a drug for treating lung adenocarcinoma, the drug exerting its effect by inhibiting the proliferation of lung adenocarcinoma cells and interfering with proline metabolism.
[0015] Furthermore, the drug is an injectable formulation or a topical formulation, the active ingredient of which is micropeptide PAMP and its functional truncated form or pharmaceutically acceptable modified derivative thereof, including PEGylation modification or D-type amino acid modification.
[0016] The present invention also provides a combination of biomarkers for the auxiliary diagnosis of lung adenocarcinoma, which is used to detect the expression level of micropeptide PAMP in the tissues of lung adenocarcinoma patients, wherein the expression of micropeptide PAMP below a threshold indicates an increased risk of lung adenocarcinoma or a poor prognosis.
[0017] The present invention has the following beneficial effects: This invention is the first to discover a novel functional peptide, PAMP, encoded by the long non-coding RNA PSMA3-AS1, and confirms its anti-cancer effect in lung adenocarcinoma, filling a gap in the functional research of this lncRNA-encoded product and providing new insights into the pathogenesis of lung adenocarcinoma. Simultaneously, this invention elucidates the molecular mechanism by which PAMP exerts its anti-cancer activity by inhibiting the proliferation of lung adenocarcinoma cells and interfering with proline metabolism, providing a new target and theoretical basis for anti-tumor strategies targeting proline metabolism. Based on PAMP and its functional truncated form, injectable or topical formulations can be further prepared, and their pharmacokinetic properties can be optimized through PEGylation or D-type amino acid modification, thereby developing candidate peptide drugs for anti-lung adenocarcinoma with clinical application potential. Furthermore, this invention found that PAMP expression levels below a threshold in lung adenocarcinoma tissue are significantly associated with increased cancer risk and poor patient prognosis, suggesting that PAMP can serve as a molecular marker for auxiliary diagnosis and prognosis, possessing significant clinical translational value. Attached Figure Description
[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention 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 the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0019] Figure 1 The graph shows the verification results of PAMP's coding capabilities.
[0020] Figure 2 The graph shows the results of low PAMP expression in lung adenocarcinoma and its positive correlation with good patient prognosis.
[0021] Figure 3 The figure shows the results of PAMP's functional verification of inhibiting LUAD cell proliferation and tumorigenesis in vitro and in vivo.
[0022] Figure 4 Figure showing the results of the investigation into the mechanism by which PAMP interacts with PYCR1 and regulates proline metabolism.
[0023] Figure 5 The figure shows the results of PAMP synthesized in vitro as a micropeptide drug inhibiting proline synthesis and lung adenocarcinoma cell proliferation. Detailed Implementation
[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to the accompanying drawings and examples. It should be understood that the specific examples described herein are merely illustrative and not intended to limit the invention.
[0025] This invention provides a micropeptide PAMP targeting proline metabolism. The PAMP is a 48-amino acid peptide, the amino acid sequence of which is shown in SEQ ID NO.1 (the specific sequence is determined based on the PAMP sequence identified in the study; example: SEQ ID NO.1: MFLARRRERSFHQVDWFLCGRPPPFPPGQHLVVAGGVSELSESHALSPG). PAMP is encoded by the open reading frame (ORF) of lncRNA PSMA3-AS1, the nucleotide sequence of which is shown in SEQ ID NO.2: atgtttctgg cgagaaggga acggagtttt catcaggtag attggttttt gtgcggccgt cctccaccgtttcctccagg acagcaccta gtcgtggccg gaggagtctc agagctgtca gaaagtcacg ctctgtcgccaggctga.
[0026] Micropeptide PAMP can be directly prepared using solid-phase micropeptide synthesis technology. After purification, the purity (≥95%) and amino acid sequence of the synthesized product are verified by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS).
[0027] In vitro experiments showed that knocking down the micropeptide PAMP promoted the proliferation of lung adenocarcinoma cells, while the exogenous addition of synthetic micropeptide PAMP inhibited the proliferation of lung adenocarcinoma cells (detected by CCK-8 assay).
[0028] In a nude mouse subcutaneous xenograft model, mice injected with PAMP-knockout A549 cells exhibited significantly larger tumor volumes than the control group. Conversely, in a well-established xenograft model, the treatment group receiving synthetic PAMP showed significantly smaller tumor volumes than the control group. In a nude mouse tail vein model, treatment with synthetic PAMP significantly delayed the progression of lung adenocarcinoma.
[0029] Mechanistic studies have shown that the micropeptide PAMP reduces intracellular proline levels by directly binding to PYCR1 and inhibiting its enzymatic activity.
[0030] Micropeptide PAMP and its functional truncated forms or pharmaceutically acceptable modified derivatives (such as PEGylation or D-amino acid modification) can be used as active ingredients in the preparation of drugs for the treatment of lung adenocarcinoma (e.g., injectable formulations, oral formulations, etc.).
[0031] Detecting the expression level of micropeptide PAMP in lung adenocarcinoma patient tissues can serve as a biomarker for prognostic assessment (low expression of micropeptide PAMP indicates a poor prognosis).
[0032] Using micropeptide PAMP or its encoding gene PSMA3-AS1 as a target, gene therapy strategies (such as using siRNA to knock down the antisense of PSMA3-AS1) or drugs targeting PYCR1 (such as PAMP analogs) can be developed.
[0033] Specific embodiments of the present invention are as follows: Example 1: Validation of PAMP's encoding capability and expression ORF screening of lncRNA PSMA3-AS1: Ribosomal profiling analysis revealed that the ORF of PSMA3-AS1 (nucleotide position: chr14:58,295,790-58,298,079; GRCh38 / hg38) has potential coding ability (see results). Figure 1 (A in the middle).
[0034] Obtaining the PAMP encoding gene: Primers were designed based on the PSMA3-AS1 ORF sequence (SEQ ID NO.2) (upstream: SEQ ID NO.3: 5'-ATGTTTCTGGCGAGAAGGGA-3', downstream: SEQ ID NO.4: 5'-GCCTGGCGACAGAGCGTGACT-3'), and the PSMA3-AS1 ORF fragment (containing the start codon ATG) was amplified by PCR. It was then cloned into the PLVX-CMV-GFP-Puro lentiviral vector (the GFP tag was used for expression detection) to construct the PLVX-CMV-PAMP-GFP-Puro plasmid.
[0035] Cell transfection and validation: The constructed PLVX-CMV-PAMP-GFP-Puro plasmid was transfected into A549 cells. Cell lysates were collected 24 hours after transfection and Western blot was performed to detect GFP tag expression. Figure 1 The B result shows that the start codon of this ORF has the ability to initiate translation, confirming that PAMP can be expressed.
[0036] Immunoprecipitation enrichment of PAMP-GFP fusion protein expressed in A549 cells was performed using GFP antibody, and PAMP-specific peptides were identified by mass spectrometry, further confirming that PAMP can be translated (see results). Figure 1 C in Figure 1 (D in the middle).
[0037] Example 2: Application of PAMP as a diagnostic biomarker Immunohistochemical (IHC) analysis was performed on 74 pairs of lung adenocarcinoma tissues and their paired adjacent normal tissues. The results showed that the expression level of PAMP in cancerous tissues was significantly lower than that in adjacent normal tissues. Figure 2 (A) Kaplan-Meier survival curve analysis showed that PAMP expression level was significantly positively correlated with the survival of patients with lung adenocarcinoma. Figure 2 In the B group, low PAMP expression indicates a poor prognosis and a higher risk of death.
[0038] Example 3: Effects of PAMP on LUAD cell proliferation and tumorigenesis in vivo Cell model construction: Construction of A549 cell line with PAMP knockdown ( Figure 3 A in the middle) and A549 cell line overexpressing PAMP ( Figure 3 (C in the middle).
[0039] Cell proliferation assay (CCK-8 assay): Cells were cultured at a density of 1 × 10⁶ cells per well. 3 Each well was seeded in a 96-well plate with 5 replicates per group. After culturing for 0, 24, 48, 72, and 96 hours, 10 μL of CCK-8 reagent (final volume 110 μL) was added to each well, and the plate was incubated at 37°C for 1 hour. The absorbance (OD) at 450 nm was then measured. Figure 3 Results B showed that knockdown of PAMP significantly promoted the proliferation of A549 cells; Figure 3 The results showed that overexpression of PAMP significantly inhibited the proliferation of A549 cells.
[0040] Subcutaneous tumorigenesis experiment in nude mice: Five-week-old female BALB / c nude mice (n=5 per group) were used, and each mouse was subcutaneously injected with 150 μL of cell suspension (containing approximately 1×10⁻⁶ cells). 7(Number of cells). Tumor volume was measured every 5 days after inoculation (calculation formula: volume = major axis × minor axis). 2 (× 0.5), mice were sacrificed on day 25 after inoculation, and tumor tissue was isolated and photographed for record. Figure 3 E and Figure 3 The results showed that knocking down PAMP significantly promoted the tumorigenicity of lung adenocarcinoma cells in nude mice.
[0041] Example 4: Mechanism of PAMP regulation of proline metabolism in lung adenocarcinoma cells Protein-protein interaction verification: Co-immunoprecipitation (Co-IP) experiments confirmed a direct interaction between PAMP and PYCR1, a key enzyme in proline synthesis. Figure 4 (A in the middle).
[0042] PYCR1 function validation: Knockdown of PYCR1 in lung adenocarcinoma cells significantly inhibited cell proliferation. Figure 4 B in Figure 4 (C in the middle).
[0043] Proline content was determined using the acidic ninhydrin colorimetric method. Figure 4 D and Figure 4 The results of the study showed that knocking down PAMP upregulated intracellular proline synthesis, while knocking down PYCR1 inhibited it.
[0044] Example 5: In vitro synthesis and efficacy verification of PAMP Solid-phase synthesis: An Fmoc / t-Bu strategy was employed, using solid-phase micropeptide synthesis technology to sequentially couple amino acids (PAMP sequence: SEQ ID NO.1: MFLARRRERSFHQVDWFLCGRPPPFPPGQHLVVAGGVSELSESHALSPG). The synthesized product was verified for amino acid sequence and purity (≥95%) by liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC). Figure 5 A in Figure 5 (B in the middle).
[0045] In vitro cell experiments: Different concentrations of synthetic PAMP were added to A549 cell culture systems, and Western blot was used to detect the uptake or binding of PAMP by cells. Figure 5 (C in the text). Functional experiments showed that the synthesized PAMP could effectively inhibit the proliferation of lung adenocarcinoma cells (C). Figure 5 D in the middle) and the synthesis of intracellular proline ( Figure 5 (E in the text).
[0046] In vivo efficacy experiments: a) Subcutaneous model: Nude mice were subcutaneously inoculated with A549 cells (1×10⁻⁶). 7 Each mouse was randomly assigned to a group (n=5 / group) after the tumor became palpable. The control group received intraperitoneal injection of PBS, while the treatment group received intraperitoneal injection of synthetic PAMP (dose: 500 μg / time, administered every two days). Tumor volume and mouse weight changes were monitored regularly. Figure 5 (F in the middle).
[0047] b) Metastasis model: Nude mice were injected with luciferase-labeled A549 cells via the tail vein and randomly divided into a control group and a PAMP treatment group. Twenty-eight days after injection, bioluminescence imaging was used to detect tumor colonization in the lungs of the nude mice and to assess tumor proliferation. Figure 5 (G in the middle).
[0048] The above embodiments are used to explain and illustrate the present invention, but not to limit the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims shall fall within the protection scope of the present invention.
Claims
1. A micropeptide PAMP, characterized in that, The amino acid sequence of the micropeptide PAMP is shown in SEQ ID NO.1 and is encoded by the open reading frame ORF of lncRNA PSMA3-AS1.
2. A nucleic acid molecule capable of encoding a truncated sequence of the functional fragment of the micropeptide PAMP of claim 1.
3. The nucleic acid molecule according to claim 2, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.
2.
4. A gene expression vector containing the nucleic acid molecule of claim 2, characterized in that, The gene expression vector is a prokaryotic or eukaryotic expression vector.
5. The gene expression vector according to claim 3, characterized in that, The gene expression vector is a PLVX-CMV-FLAG-Puro series vector.
6. A host cell containing the gene expression vector of claim 4, characterized in that, The host cells are Escherichia coli, HEK293T cells, or A549 cells.
7. A method for preparing a truncated sequence based on the micropeptide PAMP functional fragment of claim 2, characterized in that, PAMP and its functional truncated form were directly chemically synthesized using solid-phase micropeptide synthesis technology.
8. An application of the micropeptide PAMP and its functional truncated form as described in claim 7 in the preparation of a drug for treating lung adenocarcinoma, characterized in that, The drug works by inhibiting the proliferation of lung adenocarcinoma cells and interfering with proline metabolism.
9. The application according to claim 8, characterized in that, The drug is an injectable or topical formulation, the active ingredient of which is micropeptide PAMP and its functional truncated form or pharmaceutically acceptable modified derivative thereof, including PEGylation or D-amino acid modification.
10. A combination of biomarkers for the auxiliary diagnosis of lung adenocarcinoma, characterized in that, This biomarker combination is used to detect the expression level of micropeptide PAMP in the tissues of patients with lung adenocarcinoma, where expression of micropeptide PAMP below a threshold indicates an increased risk of lung adenocarcinoma or a poor prognosis.