A long-chain non-coding RNA encoded short peptide and its corresponding monoclonal antibody

Abstract

The application relates to the field of biological medicines, and particularly relates to a long-chain non-coding RNA (lncRNA) encoded short peptide and corresponding monoclonal antibody, the short peptide is encoded by long-chain non-coding RNA LOXL1-AS1 and is named PEPLA, research shows that PEPLA is highly specifically expressed in gallbladder cancer tissues and has a significant difference compared with normal tissues, based on this, a recombinant expression method of PEPLA is provided, and a monoclonal antibody taking PEPLA as an antigen is constructed, the monoclonal antibody can specifically recognize PEPLA, and is further applied to the diagnosis, prognosis evaluation and targeted treatment of gallbladder cancer, the technology provides a new molecular target for early diagnosis and precise treatment of gallbladder cancer, and has a wide clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical research and development technology, specifically to a short peptide encoded by a long non-coding RNA and its corresponding monoclonal antibody, and more specifically to a small peptide PEPLA encoded by the long non-coding RNA LOXL1-AS1, a method for generating an antibody against the small peptide, and the use of the small peptide. Background Technology

[0002] Long non-coding RNA (lncRNA) is a type of non-coding RNA that is longer than 200 base pairs and is abundant in the human body.

[0003] As important gene regulatory molecules, lncRNAs are widely involved in various levels of gene expression regulation, including epigenetic modification, transcriptional regulation, and post-transcriptional regulation, thereby affecting life processes such as cell proliferation, differentiation, apoptosis, and invasion and metastasis. Numerous studies have confirmed that lncRNAs play a crucial role in the development and progression of malignant tumors. In various tumors, lncRNAs exhibit specific differential expression patterns, which are closely related to the malignant biological behavior of tumors, and are therefore considered potential tumor biomarkers and therapeutic targets.

[0004] Previously, lncRNAs were thought not to encode proteins. However, with the development of sequencing technology in recent years, studies have discovered that some lncRNAs contain small open reading frames (sORFs), which can be translated into functional peptides, typically less than 100 amino acids in length, known as micropeptides or short peptides. The discovery of these short peptides encoded by lncRNAs demonstrates the more complex potential of lncRNAs in regulating cellular function, thus their function in tumors and other diseases is receiving increasing attention. For example, lncRNA HOXB-AS3 was found to encode a 53-amino acid short peptide, and this short peptide, rather than the lncRNA itself, plays a tumor-suppressive role in colorectal cancer. This peptide, by binding to the splicing factor hnRNP A1, blocks its regulation of pyruvate kinase M (PKM) gene splicing, reducing the production of the pro-tumorigenic metabolic isoform PKM2, thereby inhibiting the reprogramming of glucose metabolism in tumor cells (the Warburg effect). The short peptide SPAR encoded by lncRNA LINC00961 plays a significant role in skeletal muscle regeneration and cancer cell proliferation. This evidence suggests that short peptides encoded by lncRNAs can influence tumor cell proliferation through multiple mechanisms.

[0005] Gallbladder cancer (GBC) is a highly malignant gastrointestinal tumor with insidious onset and rapid progression. Most patients are diagnosed at an advanced stage, resulting in a very poor prognosis. Current clinical treatments for GBC mainly include surgical resection, chemotherapy, and radiotherapy. However, due to the lack of effective early diagnostic biomarkers and targeted therapies, the 5-year survival rate is less than 20%. Although recent research has made some progress in understanding the molecular mechanisms of tumors, many key issues remain unresolved in the field of GBC, necessitating the development of new diagnostic methods and treatment strategies. In GBC, several lncRNAs have been found to be abnormally expressed and influence the malignant phenotype of the tumor. For example, HOTAIR is significantly upregulated in GBC tissue, promoting tumor cell proliferation and invasion. Its high expression in the serum of GBC patients has also been shown to be useful for early diagnosis. The long non-coding RNA MALAT1 is also highly expressed in GBC, and its elevated expression is closely associated with aggressive phenotypes such as lymph node metastasis and poor prognosis. These studies suggest that lncRNAs play an important role in the development and progression of gallbladder cancer. Further elucidation of their functional mechanisms is expected to provide new insights into the diagnosis and treatment of gallbladder cancer. Therefore, short peptides encoded by lncRNAs may become ideal intervention targets due to their potential multifunctional regulatory properties. To this end, we propose a short peptide encoded by a long non-coding RNA and its corresponding monoclonal antibody. Summary of the Invention

[0006] This invention provides a short peptide PEPLA encoded by the long non-coding RNA LOXL1-AS1, the amino acid sequence of which is shown in SEQ ID NO: 1. This short peptide is specifically and highly expressed in gallbladder cancer tissue and can serve as a diagnostic marker and therapeutic target for gallbladder cancer.

[0007] The present invention also provides a monoclonal antibody that specifically binds to PEPLA, the antibody comprising a heavy chain variable region and a light chain variable region;

[0008] The heavy chain variable region includes HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5;

[0009] The light chain variable region includes LCDR1 as shown in SEQ ID NO: 6, LCDR2 as shown in SEQ ID NO: 7, and LCDR3 as shown in SEQ ID NO: 8.

[0010] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody may be selected from SEQ ID NO: 9, 10, 11, 12 or 13; and the amino acid sequence of the light chain variable region may be selected from SEQ ID NO: 14, 15, 16, 17 or 18.

[0011] The present invention also provides a nucleic acid molecule encoding the heavy chain variable region and / or light chain variable region of the above-mentioned monoclonal antibody, a recombinant expression vector containing the nucleic acid molecule, and a host cell.

[0012] The present invention further provides a method for preparing the monoclonal antibody, comprising the following steps:

[0013] a) Immunize animals with PEPLA fusion protein as an immunogen;

[0014] b) Isolate antigen-specific B cells and obtain antibody genes;

[0015] c) Introduce the antibody gene into host cells for expression to obtain monoclonal antibodies.

[0016] Compared with the prior art, the beneficial effects of the present invention by adopting the above technical solution are as follows:

[0017] 1. This invention reveals that the long non-coding RNA LOXL1-AS1 and its encoded short peptide PEPLA are specifically and highly expressed in gallbladder cancer tissues, showing significant differences from normal tissues. This provides a novel and promising molecular target for the development of highly specific diagnostic reagents and targeted therapeutic drugs for gallbladder cancer.

[0018] 2. The monoclonal antibody prepared by the short peptide PEPLA in this invention has both high affinity and high specificity. It can be widely used in various pathological diagnosis and basic research technology platforms such as immunohistochemistry, immunofluorescence, ELISA, and Western blotting. It provides a reliable means for the detection of tumor neoantigens and can meet the diversified needs of clinical diagnosis, prognostic assessment and efficacy monitoring of gallbladder cancer. It has extremely high clinical translational value and market application potential. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the recombinant protein sequence in an embodiment of the present invention;

[0020] Figure 2 This is a schematic diagram of the recombinant antigen protein in an embodiment of the present invention;

[0021] Figure 3 This is a schematic diagram of the WB verification results in an embodiment of the present invention;

[0022] Figure 4 This is a schematic diagram of single-cell sequencing results of gallbladder cancer in an embodiment of the present invention;

[0023] Figure 5 This is a comparison chart of the experimental results of LOXL1-AS1 in the embodiments of the present invention;

[0024] Figure 6This is a schematic diagram of the detection results of LOXL1-AS1 in an embodiment of the present invention;

[0025] Figure 7 This is a schematic diagram illustrating the interference with the detection results of gallbladder cancer cells in an embodiment of the present invention;

[0026] Figure 8 This is a comparison chart of the results of the nude mouse experiment in the embodiments of the present invention;

[0027] Figure 9 This is a schematic diagram illustrating the effect of pepla on gallbladder cancer tissue in an embodiment of the present invention. Detailed Implementation

[0028] The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted that the description of these embodiments is for the purpose of helping to understand the present invention, but does not constitute a limitation of the present invention.

[0029] Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0030] Please see the appendix Figure 1 - Appendix Figure 9 This invention discloses a short peptide encoded by a long non-coding RNA, comprising constructing a fusion protein. The amino acid sequence of the short peptide PEPLA is recombinantly expressed using genetic engineering methods, and then fused with a glutathione S-transferase tag to construct a GST-PEPLA fusion protein. The specific preparation steps are as follows:

[0031] Step 1: Digest the target fragment of the cloned plasmid PATX-GH with BamHI and EcoRI restriction endonucleases, and then ligate the target gene fragment into the expression vector using DNA ligase to form a recombinant plasmid.

[0032] Step 2: Transform the recombinant plasmid into Stbl3 competent cells, plate them on plates containing ampicillin, select positive clones for amplification, and verify the correct plasmid construction by sequencing.

[0033] Step 3: The GST-PEPLA fusion protein was induced to express using the E. coli expression system. SDS-PAGE electrophoresis results confirmed the appearance of a clear target band at approximately 32.94 kDa, indicating that the fusion protein was successfully expressed.

[0034] In one embodiment of the present invention: *E. coli* is used to express a recombinant protein (GST-PEPLA) with a dual tag, as shown in the appendix. Figure 1 As shown, the recombinant antigen protein was subjected to reducing SDS-PAGE (2 μg loading), and the structure is shown in the attached figure. Figure 2 As shown.

[0035] In one embodiment of the present invention: the specificity and titer of polyclonal antibodies were verified by using pre-immunization serum as a negative control and PBS as a blank control. The results of indirect ELISA are shown in Table 1.

[0036] Table 1

[0037] WB verification results:

[0038] The antibody dilution ratio was 1:64000, and the protein loading amount was 0.2 ug / lane, as shown in the attached document. Figure 3 As shown.

[0039] In one embodiment of the present invention: antigen protein labeling:

[0040] The synthesized antigen protein was conjugated with FITC / APC to construct protein-FITC / APC, which was then used in subsequent flow cytometry screening experiments.

[0041] In one embodiment of the present invention: spleen cell separation:

[0042] After verifying the serum immunoassay results, the spleen was harvested for cell separation. The spleen was placed in a culture dish containing cell culture medium, and the spleen tissue was gently ground to release the cells. The ground cell solution was filtered through a cell sieve to remove tissue fragments and connective tissue, obtaining a single-cell suspension. The cell suspension was treated with erythrocyte lysis buffer to remove erythrocyte impurities.

[0043] In one embodiment of the present invention: positive B cell screening:

[0044] Double-positive screening using membrane-type IgG and antigen-positive cells was employed. Single B cells (membrane-type IgG positive and antigen-positive cells) were obtained by flow cytometry sorting and cultured. At least 960 single B cell (membrane-type IgG positive and antigen-positive) clones were selected (i.e., 10 96-well plates, enriched with antigen). Finally, the cell supernatant was used to screen out positive clones of B cells that specifically recognize the antigen using ELISA (coated with GST-PEPLA fusion protein and GST tag protein, respectively).

[0045] In one embodiment of the present invention: positive clone amplification and antibody recombinant expression verification:

[0046] The constant region of the antibody is rabbit IgG / kappa. Based on the ELISA results, five positive clones of B cells with better results were selected. The antibody gene sequence was obtained by single-cell sequencing, as shown in Table 2. Genes of heavy chain and light chain sequences were synthesized and co-transfected into CHO cells. The binding energy between cell supernatant and antigen protein was detected by ELISA.

[0047]

[0048] Table 2

[0049]

[0050] Table 3

[0051]

[0052] Table 4

[0053] Bioinformatics analysis identified an sORF encoding a short peptide of 77 amino acids in the LOXL1-AS1 transcript, named PEPLA, whose amino acid sequence is shown in SEQ ID NO: 1 and whose encoding nucleic acid sequence is shown in SEQ ID NO: 2.

[0054] Immunogen preparation: The PEPLA coding sequence was cloned into the pGEX-4T-1 vector, and the GST-PEPLA fusion protein was expressed and purified.

[0055] Animal immunization: New Zealand white rabbits were immunized with the fusion protein to obtain high-titer antiserum.

[0056] B cell screening: Antigen-specific memory B cells were sorted using flow cytometry.

[0057] Antibody gene acquisition and expression: The antibody variable region gene was obtained by single-cell sequencing, with the heavy chain variable region sequence shown in SEQ ID NO: 9-13 and the light chain variable region sequence shown in SEQ ID NO: 14-18; it was cloned into an expression vector and transfected into CHO cells for expression.

[0058] Antibody validation: ELISA and Western Blot confirmed that the antibody specifically recognizes PEPLA and does not cross-react with the GST tag.

[0059] Immunohistochemical staining of gallbladder cancer tissue sections using the antibody of this invention showed that PEPLA was highly expressed in the cancer tissue, and the expression level was significantly correlated with TNM stage, liver metastasis and patient prognosis, indicating that the antibody can be used for pathological diagnosis and prognostic assessment of gallbladder cancer.

[0060] The ELISA detection method established using this antibody can specifically detect PEPLA in serum or cell lysates. It has high sensitivity, good repeatability, and is suitable for high-throughput screening of clinical samples.

[0061] In vitro experiments showed that the antibody could inhibit the proliferation and migration of PEPLA-expressing gallbladder cancer cells; in animal models, tumor growth was significantly inhibited in the antibody treatment group, suggesting its potential for targeted therapy.

[0062]

[0063]

[0064] Table 5

[0065] The antibody concentration gradient settings are shown in Table 3.

[0066]

[0067] Table 6

[0068]

[0069] Table 7

[0070] The ELISA results are shown in Tables 4 and 5.

[0071] Western blotting assay

[0072] (1) Gel preparation: Mix 3.75 mL of lower layer gel solution, 3.75 mL of lower layer gel buffer and 100 μL of 10% ammonium persulfate coagulant evenly, and inject it into the lower layer of the 1.5 mm gel preparation glass plate. Then, quickly and slowly inject 1 mL of anhydrous ethanol for liquid sealing. After the lower layer gel solidifies (about 15 min), discard the upper layer ethanol. Mix 2 mL of upper layer gel solution, 2 mL of colored upper layer gel buffer and 30 μL of 10% ammonium persulfate coagulant evenly, and inject it into the upper layer of the gel preparation glass plate. Insert the comb teeth of 15 wells and let it stand for the upper layer gel to solidify (about 15 min).

[0073] (2) Electrophoresis: Load the prepared gel into the electrophoresis tank, slowly remove the comb teeth, add 15 μg of the protein sample to be tested to each well and add at least 5 μL of pre-stained protein marker at the appropriate position. After correctly connecting the positive and negative electrodes of the electrophoresis tank, turn on the power supply and maintain a constant voltage of 90V for electrophoresis. Observe the separation state of the marker and terminate the electrophoresis as needed.

[0074] (3) Transfer: Take out the gel after electrophoresis, use a gel remover to cut off the excess gel, put the PVDF membrane cut to the appropriate size into methanol for 1 min to activate it for later use. With the black side of the transfer clamp facing down, place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in order from bottom to top, ensuring that there are no air bubbles between the gel and the PVDF membrane. Place the transfer clamp into the transfer core of the electrophoresis tank, fill the electrophoresis tank with transfer solution and place it in the matching ice box, correctly connect the positive and negative electrodes of the electrophoresis tank, turn on the power and transfer the membrane at a constant voltage of 80V for 120 min.

[0075] (4) Blocking: Prepare a blocking solution containing 5% skim milk powder using skim milk powder and PBST solution. Use tweezers to pick up the PVDF membrane and immerse it in the blocking solution with the side in contact with the gel facing up. Place it on a shaker and shake it slowly. Block at room temperature for 1 hour.

[0076] (5) Immunohistochemistry: The blocked PVDF membrane was washed three times with PBST solution for 5 min each time, and then immersed in an antibody incubation box containing primary antibody solution. It was incubated overnight at 4°C and 50 rpm on a shaker. The next day, the PVDF membrane was washed three times with PBST for 10 min each time. Then, it was immersed in an antibody incubation box containing HRP-labeled goat anti-mouse or rabbit IgG (secondary antibody) solution (secondary antibody diluted 10,000 times) and incubated at room temperature for 1 h. Then, the PVDF membrane was washed five times with PBST for 10 min each time.

[0077] (6) Chemiluminescence: Equal volumes of ECL luminescent liquid A and B are mixed to prepare a developing solution. An appropriate amount of developing solution is dropped onto the membrane and then placed in an ultra-sensitive multifunctional imager for development.

[0078] Multiplex immunofluorescence assay for cells:

[0079] (1) Preparation of cell spread sheets: The cells were seeded into 6-well plates at a density of 30,000 cells / well and cultured overnight in a cell culture incubator;

[0080] (2) The next day, the culture medium was aspirated, the cells were washed three times with PBS, and then fixed in 4% paraformaldehyde for 30 min.

[0081] (3) Wash cells 3 times with PBS and permeabilize with 0.5% Triton-X for 30 min;

[0082] (4) Block with 3% BSA for 30 min;

[0083] (5) Incubate the primary antibody for the target purpose, cover the surface of the glass slide, and incubate overnight at 4°C;

[0084] (6) The next day, wash the cells three times with PBS for 5 minutes each time, and incubate with the corresponding HRP secondary antibody for 50 minutes;

[0085] (7) Wash the cells with PBS 3 times for 5 min each time, add TYR dye for 15 min, wash the cells with PBS 3 times for 5 min each time, and observe the fluorescence intensity under a microscope after staining to evaluate the staining status.

[0086] (8) Add primary / secondary antibody removal solution, 37℃, 50 rpm, 30 min;

[0087] (9) Repeat steps 3-8, rinsing the cells with PBS 3 times, 5 min each time;

[0088] (10) Mount the slides with an anti-fluorescence quenching mounting medium containing DAPI dye;

[0089] (11) Take pictures using an inverted microscope.

[0090] CCK-8 cell proliferation assay:

[0091] (1) Cell seeding: One day in advance, take cells in the logarithmic growth phase, count them, and seed them in a 96-well plate at a density of about 1×104 cells / well to keep the total number of cells in each well consistent;

[0092] (2) After cell ASO transfection and other treatments, cell adhesion or drug stimulation is completed, the culture medium is discarded and replaced with 100µL of complete culture medium. At least 3 replicates are set for each group.

[0093] (3) Add CCK-8 reagent: Add 10 µL CCK-8 reagent to 100 µL of culture medium in each well, incubate at 37°C and 5% CO2 for 2 hours, and set blank wells (no cells, only culture medium and CCK-8 added) for background subtraction;

[0094] (4) Microplate reader reading: Gently shake the plate and use the microplate reader to read the absorbance (OD value) at a wavelength of 450nm.

[0095] (5) Growth curve plotting and analysis.

[0096] Example 1: Expression and prognostic value analysis of LOXL1-AS1 in gallbladder cancer

[0097] To verify the clinical relevance of LOXL1-AS1 in gallbladder cancer, the following experiment was conducted:

[0098] Single-cell sequencing analysis: Single-cell sequencing of 9 gallbladder cancer tissues showed that LOXL1-AS1 was specifically highly expressed in malignant gallbladder epithelial cells;

[0099] RNA fluorescence in situ hybridization: Tissue sections from 127 gallbladder cancer patients with complete case information were analyzed. The average fluorescence intensity of LOXL1-AS1 was calculated, and patients were divided into a high-expression group and a low-expression group of LOXL1-AS1, as shown in the appendix. Figure 4 As shown;

[0100] Survival analysis: Kaplan-Meier survival curve analysis showed that patients in the LOXL1-AS1 high expression group had significantly shorter overall survival, as shown in the attached figure. Figure 5 -B is shown;

[0101] Clinicopathological correlation analysis:

[0102] One-way ANOVA analysis showed that LOXL1-AS1 expression levels were significantly correlated with TNM stage, as shown in the attached figure. Figure 5 -C is shown;

[0103] Unpaired t-test showed that LOXL1-AS1 was expressed at higher levels in patients with liver metastases, as shown in the attached figure. Figure 5 -D is shown;

[0104] Conclusion: LOXL1-AS1 is specifically highly expressed in gallbladder cancer, and its high expression is significantly associated with poor prognosis, showing potential as a diagnostic biomarker and prognostic indicator.

[0105] Example 2: Bioinformatics Prediction and Identification of the Short Peptide Pepla

[0106] sORF identification: Through bioinformatics analysis, a small open reading frame (sORF) of 284 bp was identified in the LOXL1-AS1 transcript. This sORF encodes a short peptide consisting of 77 amino acids, named Pepla.

[0107] Physicochemical property prediction:

[0108] Theoretical isoelectric point (pI): 9.30;

[0109] Theoretical molecular weight: 8.5 kDa;

[0110] Structural prediction: AlphaFold3 prediction shows that Pepla exhibits a hairpin-like structure, as shown in the attached figure. Figure 6 -B is shown;

[0111] Domain analysis:

[0112] DeepTMHMM predictions show no transmembrane domains, as shown in the attached figure. Figure 6 -C is shown;

[0113] Signal 6.0 analysis showed no signal peptide, as shown in the attached image. Figure 6 -D is shown;

[0114] Immunogen selection: Based on antigenicity score and hydrophilicity analysis, amino acids 35-77 of Pepla were selected as the immunogen region;

[0115] In the appendix Figure 6 In the diagram, A represents the genomic location of LOXL1-AS1 and the location of the potential sORF;

[0116] B indicates that AlphaFold3 predicts the pepla structure;

[0117] C represents the predicted transmembrane region of pepla by DeepTMHMM;

[0118] D indicates that the analysis of the PEPLA signal peptide was performed using Signal 6.0;

[0119] E indicates the recombinant protein expression sequence, GST-[Pro35-Ser77]-6×his;

[0120] F indicates Coomassie Brilliant Blue staining for recombinant protein expression.

[0121] Example 3: Expression and purification of GST-Pepla fusion protein

[0122] Vector construction: The DNA sequence encoding Pepla[35-77] was cloned into the pGEX-4T-1 vector to construct the GST-Pepla fusion expression vector;

[0123] Protein expression: The recombinant plasmid was transformed into Stbl3 competent cells, plated on LB plates containing ampicillin, positive clones were picked, and after being verified by sequencing, expression was induced using Escherichia coli BL21(DE3);

[0124] Protein purification and validation: The GST-Pepla fusion protein was purified using glutathione agarose beads. A clear band appeared at approximately 32.94 kDa on reduced SDS-PAGE (2 μg loading), confirming successful expression of the fusion protein.

[0125] Example 4: Preparation and Validation of Rabbit-Derived Monoclonal Antibodies

[0126] Animal immunization: New Zealand white rabbits were immunized with purified GST-Pepla fusion protein, for a total of 4 immunizations;

[0127] Potency assessment and purification: Blood was collected after the 3rd and 4th immunizations. The antiserum titer was >1:32000 by indirect ELISA, as shown in Table 1. Antigen-specific affinity purification was used to remove the anti-GST tag antibody.

[0128] Specificity verification: Western blot analysis showed that the purified antibody, even at a dilution of 1:64000, still specifically recognized GST-Pepla (32.94 kDa) but did not react with the GST tag (28.38 kDa), as shown in the attached figure. Figure 3 As shown;

[0129] B-cell screening:

[0130] Single-cell suspensions were prepared from the spleen of immunized rabbits.

[0131] Flow cytometry was used to screen membrane surface IgG and antigen double-positive memory B cells, and a total of 960 B cell clones were screened.

[0132] Five positive clones that specifically recognize Pepla were identified by ELISA, as shown in Tables 4 and 5.

[0133] Antibody gene cloning and expression:

[0134] The gene sequences of the heavy and light chain variable regions were obtained by single-cell sequencing, as shown in Table 2.

[0135] The antibody gene was cloned into an expression vector and co-transfected into CHO cells for recombinant expression.

[0136] ELISA validation showed that the recombinant antibody could still effectively bind to Peplea at a concentration as low as 0.01563 μg / mL.

[0137] Example 5: In vitro functional validation of LOXL1-AS1 / Pepla

[0138] Gene knockout validation: After interfering with LOXL1-AS1 using ASO, Western blotting confirmed a significant decrease in Pepla expression, as shown in the attached figure. Figure 7 As shown in -A, B;

[0139] Proliferation assay: CCK-8 assay showed that knockdown of LOXL1-AS1 significantly inhibited the proliferation of NOZ and GBC-SD cells, as shown in the attached figure. Figure 7 -As shown in C,D;

[0140] Migration and invasion experiments:

[0141] (1) Cell seeding: After cell adhesion or drug stimulation, digest and count the cells, then resuspend them in serum-free medium at an appropriate density (total volume of 200µL; migration assay: 2×10⁶ cells per well). 4 cell;

[0142] Invasion test: 5 × 10⁻⁶ per well 4 Cells (with a consistent total number of cells per well) are seeded into the upper chamber of a 24-well Transwell plate. In the invasion assay, the upper chamber of the Transwell plate needs to be pre-coated with 50µL-100µL of matrix gel and incubated at 4℃ for 1-2 hours. After solidification, cell suspension is added. 600µL of complete culture medium containing 10% FBS is added to the lower chamber. At least 3 replicates are set up for each group.

[0143] (2) Incubation: Place the cell culture plate in a cell culture incubator at 37°C and 5% CO2 for 12-24 hours (migration experiment) or 24-48 hours (invasion experiment).

[0144] (3) Fixation: After incubation, remove the Transwell chamber, gently wash it 2-3 times with PBS, immerse the chamber in 4% paraformaldehyde for 30 minutes, and gently wipe the surface of the upper chamber membrane with a cotton swab to remove cells that have not passed through the membrane.

[0145] (4) Staining: Stain with 0.1% crystal violet for 15 min, incubate at room temperature in the dark, and wash with PBS 2-3 times after staining to remove the background;

[0146] (5) Microscopic observation and counting;

[0147] Transwell assays showed that knockdown of LOXL1-AS1 significantly inhibited the migration and invasion of gallbladder cancer cells, as shown in the attached figure. Figure 7 As shown in -E,F;

[0148] In the appendix Figure 7 In the text, A and B indicate that when ASO interferes with LOXL1-AS1, the expression of pepla in gallbladder cancer cells is reduced.

[0149] C indicates the effect of CCK-8 assay on the proliferation of NOZ cells by knockdown of LOXL1AS1;

[0150] D indicates the effect of CCK-8 assay on the proliferation ability of LOXL1-AS1 on GBC-SD cells;

[0151] E indicates the effect of LOXL1-AS1 knockdown on the migration and invasion ability of NOZ cells as detected by the Transwell assay;

[0152] F indicates the effect of LOXL1-AS1 knockdown on the migration and invasion ability of GBC-SD cells as detected by the Transwell assay.

[0153] CCK-8 is the Cell Counting Kit 8 cell counting reagent.

[0154] Example 6: In vivo tumorigenesis experiment of LOXL1-AS1 / Pepla

[0155] Subcutaneous tumor formation experiment in nude mice (1) Male BALB / c nude mice aged 4-6 weeks were selected for in vivo tumor growth experiments. All experiments were conducted in accordance with the regulations for the management of laboratory animals and were approved by the relevant ethics committee. Tumor cells (after transfection treatment) were injected at a rate of 1×10⁻⁶. 6 Cells at a concentration of 100 μL were injected subcutaneously into the right axilla of nude mice.

[0156] (2) Measurement of tumor volume (using the following formula): Tumor volume (mm3) = π / 6 × length × width 2 Starting from the third day after tumor inoculation, the length and width of the tumor were measured regularly using calipers, and the tumor volume was calculated until the end of the experiment.

[0157] (3) Resection and analysis of tumor tissue: At the end of the experiment, the mice were euthanized and the tumors were removed for analysis. The tumor tissue samples were fixed in 10% neutral formalin solution and subjected to routine paraffin embedding and sectioning. The sections were observed by HE staining to assess the histological morphology and proliferation of the tumor.

[0158] (4) Statistical analysis: All experimental data are expressed as mean ± standard deviation (mean ± SD). Tumor volume data were analyzed using GraphPadPrism software. One-way ANOVA was used for intergroup comparisons. p < 0.05 was considered statistically significant.

[0159] Knockout experiment: GBC-SD cells treated with ASO-2 were inoculated into nude mice. Compared with the control group, the tumor volume in the experimental group was significantly reduced (P<0.05).

[0160] Overexpression experiment: NOZ cells stably overexpressing Pepla were inoculated into nude mice. Compared with the control group, the terminal tumor volume of the overexpression group was significantly larger (P<0.01).

[0161] Conclusion: In vivo experiments confirmed that LOXL1-AS1 / Pepla promotes gallbladder cancer tumor growth.

[0162] Example 7: Analysis of the clinicopathological significance of Pepla

[0163] Immunohistochemical staining: Pepla immunohistochemical staining of gallbladder cancer tissue sections showed high expression of Pepla in the cancer tissue, as shown in the attached figure. Figure 9 -A is shown;

[0164] Survival analysis: Kaplan-Meier analysis showed that patients with high Pepla expression had a significantly worse prognosis (P<0.001), as shown in the attached figure. Figure 9 -B is shown;

[0165] Clinical Relevance: Pepla expression levels were significantly correlated with TNM stage, and were even higher in patients with liver metastases, as shown in the attached figure. Figure 9 -As shown in C,D;

[0166] In the appendix Figure 9 In the image, A represents a representative image of PEPLA in gallbladder cancer tissue using fluorescence immunohistochemistry.

[0167] B indicates that Kaplan-Meier survival curve analysis was performed based on the grouping of pepla expression levels;

[0168] C indicates that the relationship between TNM stage and pepla expression was tested using one-way ANOVA, and multiple comparisons were performed using the Bonferroni test.

[0169] D indicates that the relationship between the presence or absence of liver metastasis and the expression level of pepla was determined using an unpaired t-test (two-tailed test). "ns" indicates no significant difference. * indicates P<0.05 and ** indicates P<0.001.

[0170] As can be seen from the above embodiments, monoclonal antibodies against PEPLA can be used for pathological detection methods such as immunohistochemistry and immunofluorescence staining, as well as for immunological experiments such as ELISA and Western Blot, which can meet the wide range of applications in clinical diagnosis, prognosis and basic medical experiments.

[0171] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Any variations and modifications can be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, any modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention, without departing from the scope of the invention, fall within the protection scope defined by the claims of the present invention.

Claims

1. A short peptide encoded by long non-coding RNA LOXL1-AS1, named PEPLA, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A nucleic acid molecule encoding the short peptide PEPLA of claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

2.

3. A monoclonal antibody that specifically recognizes the short peptide PEPLA of claim 1, characterized in that, The antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region contains amino acid sequences such as HCDR1 as shown in SEQ ID NO: 3, HCDR2 as shown in SEQ ID NO: 4, and HCDR3 as shown in SEQ ID NO: 5; The light chain variable region comprises the amino acid sequences LCDR1 as shown in SEQ ID NO: 6, LCDR2 as shown in SEQ ID NO: 7, and LCDR3 as shown in SEQ ID NO: 8; The amino acid sequence of the framework region of the heavy chain variable region is selected from SEQ ID NO: 9, 10, 11, 12 or 13; and / or the amino acid sequence of the framework region of the light chain variable region is selected from SEQ ID NO: 14, 15, 16, 17 or 18.

4. A nucleic acid molecule encoding the heavy chain variable region and / or the light chain variable region of the monoclonal antibody of claim 3.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the recombinant expression vector of claim 5.

7. A method for preparing the monoclonal antibody according to claim 3, characterized in that, Includes the following steps: a) Immunize animals with an immunogen containing the short peptide shown in SEQ ID NO: 1; b) Isolate antigen-specific B cells from immunized animals and obtain antibody-encoding genes; c) The antibody encoding gene is introduced into a host cell for expression to obtain the monoclonal antibody.

8. The method according to claim 7, characterized in that, The immunogen described in step a) is a fusion protein of the short peptide shown in SEQ ID NO: 1 and the carrier protein.

9. A kit for the diagnosis or prognostic assessment of gallbladder cancer, characterized in that, It includes the monoclonal antibody as described in claim 3.

10. The use of the monoclonal antibody of claim 3 in the preparation of a medicament for the diagnosis, prognostic assessment or treatment of gallbladder cancer.

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