Micropeptide molecular marker for early gastric cancer screening, detection reagent and application thereof

Abstract

The application discloses a micropeptide molecular marker for early gastric cancer screening, a detection reagent and application, and the micropeptide molecular marker is an OLMLINC gene. The expression of the new type of functional biological macromolecule, i.e. the micropeptide, at a diagnosis target is detected to assist in predicting the progress of a tumor, and simple screening of gastric cancer before endoscopy is realized.

Description

(I) Technical Field

[0001] This invention relates to a micropeptide molecular marker, detection reagent, and application for early gastric cancer screening. (II) Background Technology

[0002] Proteins are the material basis of life, the basic organic components of cells, and the main carriers of life activities. Abnormal expression and translation of some proteins are closely related to tumorigenesis. To date, nearly 20,000 genes in the human genome have been annotated as protein-coding genes, of which approximately 17,000 proteins have been validated at the biochemical level. The remaining 98% of the genome contains a vast portion transcribed as "non-coding RNA." As its name suggests, for a long time, the scientific community generally believed that these RNAs did not directly encode proteins, but rather regulated cellular life activities through different mechanisms. However, with the development of high-throughput technologies and advancements in bioinformatics, our understanding of these non-coding RNAs has been gradually updated. More and more non-coding RNAs have been found to have the ability to encode short, non-classical proteins, namely micropeptides. Existing research has shown that these micropeptides play important functions in multiple regulatory networks, including cell proliferation, metabolic regulation, and immune responses, and play a key role in the progression of some cancers.

[0003] Therefore, the search for differentially expressed micropeptide molecules in gastric cancer is needed to design diagnostic biomarkers for gastric cancer, thereby enabling early screening for gastric cancer. (III) Summary of the Invention

[0004] The purpose of this invention is to provide a micropeptide molecular marker, detection reagent, and application for early gastric cancer screening. Detecting the gene expression level of relevant micropeptides in patients and the expression of micropeptide proteins in tissue sections can assist in the early gastric cancer screening and diagnosis.

[0005] The technical solution adopted in this invention is:

[0006] This invention provides a micropeptide molecular marker for early gastric cancer screening, wherein the micropeptide molecular marker includes one or more of the following genes: AC027045.3, OLMLINC, ZNF436-AS1, or TRHDE-AS1.

[0007] Preferably, the AC027045.3 gene nucleotide sequence is as shown in SEQ ID NO.1 or its homologous sequence, and the amino acid sequence encoding the protein is as shown in SEQ ID NO.2; the OLMLINC gene nucleotide sequence is as shown in SEQ ID NO.3 or its homologous sequence, and the amino acid sequence encoding the protein is as shown in SEQ ID NO.4; the ZNF436-AS1 gene nucleotide sequence is as shown in SEQ ID NO.5 or its homologous sequence, and the amino acid sequence encoding the protein is as shown in SEQ ID NO.6; the TRHDE-AS1 gene nucleotide sequence is as shown in SEQ ID NO.7 or its homologous sequence, and the amino acid sequence encoding the protein is as shown in SEQ ID NO.8.

[0008] The present invention also provides a detection reagent for the micropeptide molecular marker, the detection reagent comprising substances for detecting the levels of the micropeptide molecular marker DNA, mRNA and / or protein in the sample to be tested.

[0009] Preferably, the detection reagent includes reagents for detecting micropeptides using immunohistochemistry, chemiluminescence, radioisotope assays, fluorescence luminescence, enzyme-linked immunosorbent assays (ELISA), colloidal gold assays, real-time quantitative reverse transcription PCR, microarray detection, DNA blotting, RNA blotting in situ hybridization, or protein blotting. The reagent includes probes that specifically recognize micropeptide molecular markers, primers that specifically amplify micropeptide molecular markers, antibodies that specifically detect micropeptides, and gene or protein chips for detecting micropeptides. The reagent also includes detectable markers, such as radioisotopes, fluorescent groups, chemiluminescent components, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions, ligands (e.g., biotin or haptens), or any combination thereof.

[0010] Preferably, the reagent for real-time quantitative reverse transcription PCR detection of micropeptides includes specific primers, wherein the primers for detecting AC027045.3 are shown in SEQ ID NO. 9 and SEQ ID NO. 10; the primers for detecting OLMLINC are shown in SEQ ID NO. 11 and SEQ ID NO. 12; the primers for detecting ZNF436-AS1 are shown in SEQ ID NO. 13 and SEQ ID NO. 14; and the primers for detecting TRHDE-AS1 are shown in SEQ ID NO. 15 and SEQ ID NO. 16.

[0011] Preferably, the reagents for immunohistochemical detection of micropeptides include blocking buffer, primary antibody, HRP goat anti-rabbit secondary antibody, DAB substrate buffer, and DAB chromogenic solution; the blocking buffer is PBS containing 10% goat serum; the primary antibodies are respectively anti-human AC027045.3 rabbit polyclonal antibody (synthesized by GenScript, antigenic epitope MDSHKTPAKERDVG), anti-human OLMLINC rabbit polyclonal antibody (synthesized by GenScript, antigenic epitope EKEIKGIQLGKEEV), anti-human ZNF436-AS1 rabbit polyclonal antibody (synthesized by GenScript, antigenic epitope PPTAKFGYKNKIDL), and anti-human TRHDE-AS1 rabbit polyclonal antibody (synthesized by GenScript, antigenic epitope MSKHVTEEDVQMAK).

[0012] The sample to be tested is obtained from fresh ex vivo tissue or cell samples from mammals, formalin-fixed samples, or paraffin-embedded samples. Preferably, the sample is obtained from non-tumor tissues and / or cells (such as adjacent normal tissues and / or cells), tumor tissues and / or cells, for example selected from: tissues and / or cells suspected of having tumors, tissues and / or cells that have already developed tumors, tumor tissues and / or cells suppressed by the tumor immune microenvironment, or combinations thereof, such as tumor cells, dendritic cells, macrophages, fibroblasts, and T cells.

[0013] This invention also provides the application of the aforementioned micropeptide molecular marker in the preparation of products for assisting in the diagnosis of gastric cancer. The products include kits, test strips, test cards, detection instruments, or any combination thereof for detecting micropeptides. The products may also contain one or more substances selected from the group consisting of: containers, buffers, auxiliaries, solvents, positive controls, negative controls, and instructions for use.

[0014] The aforementioned auxiliary gastric cancer diagnostic product predicts the risk of gastric cancer in the sample tissue by detecting the content of micropeptides in the sample. Combined with existing detection methods, it further improves the detection efficiency and accuracy of existing methods. Specifically, if real-time quantitative reverse transcription PCR is used, the Ct value is used to determine the risk. If any one of the following is met relative to GAPDH calibration: ZNF436-AS1 CT < 27, OLMLINC CT < 25, TRHDE-AS1 CT < 27, or AC027045.3 CT > 28, it indicates that the patient has a high risk of gastric cancer and endoscopic examination is recommended.

[0015] Compared with existing technologies, the main advantages of this invention are as follows: This invention provides a micropeptide molecular marker for early gastric cancer screening, wherein the micropeptide molecular marker includes one or more combinations of the AC027045.3 gene, OLMLINC gene, ZNF436-AS1 gene, or TRHDE-AS1 gene. By detecting the expression of this novel functional biomolecule, micropeptide, at diagnostic targets, it can help predict tumor progression and achieve convenient pre-endoscopic screening for gastric cancer. (iv) Description of the attached drawings

[0016] Figure 1 A reference database of human micropeptide information constructed for the human reference genome and transcripts; A represents a partial human whole transcript micropeptide database; B represents a diagram of the start codons of human whole transcript micropeptides.

[0017] Figure 2 This is a mass-charge ratio spectrum of peptide fragments derived from clinical samples of gastric cancer; A represents peptide ID: ENST00000456120_120761840_120762226_387, with the sequence CDSASVSRASLTLTYFCQMQWK; B represents peptide ID: ENST00000668145_75741828_75741986_159, with the sequence NLGIHSLVC. The peptide is PCVLCILHKM; C represents the peptide ID: ENST00000662515_67578457_67578996_540, with the sequence FVSKCPLTMALGYSRTVLK; D represents the peptide ID: ENST00000664629_135498552_135498815_264, with the sequence ITNPMRTELSCSTFRSK.

[0018] Figure 3 This diagram illustrates candidate micropeptides that regulate the proliferation of gastric cancer cells, obtained by screening using the open reading frame of a targeted micropeptide and its upstream sgRNA library.

[0019] Figure 4Cell proliferation activity and clone number of AGS gastric cancer cells after overexpression of micropeptide were used as controls and indicators. EV represents control, OV represents micropeptide; A represents cell viability curve of micropeptide AC027045.3; B represents cell viability curve of TRHDE-AS1; C represents cell viability curve of OLMLINC; D represents cell viability curve of ZNF436-AS1; E represents crystal violet staining and clone number of micropeptide AC027045.3; F represents crystal violet staining and clone number of micropeptide TRHDE-AS1; G represents crystal violet staining and clone number of micropeptide OLMLINC; H represents crystal violet staining and clone number of micropeptide ZNF436-AS1.

[0020] Figure 5 Micrographs showing the levels of micropeptides in tissue samples from clinical patients with gastric cancer detected by immunohistochemistry. A represents the protein level of OLMLINC in cancer and adjacent normal tissue samples; B represents the protein level of ZNF436-AS1 in cancer and adjacent normal tissue samples; C represents the protein level of TRHDE-AS1 in cancer and adjacent normal tissue samples; and D represents the protein level of AC027045.3 in cancer and adjacent normal tissue samples.

[0021] Figure 6 The expression level of micropeptide in AGS-overexpressing cell lines was detected using a micropeptide gene detection kit. A represents the gene expression level of AC027045.3 in the control and AC027045.3 AGS-overexpressing cell lines; B represents the gene expression level of TRHDE-AS1 in the control TRHDE-AS1 AGS-overexpressing cell line; C represents the gene expression level of ZNF436-AS1 in the control and ZNF436-AS1 AGS-overexpressing cell lines; and D represents the gene expression level of OLMLINC in the control and OLMLINC AGS-overexpressing cell lines.

[0022] Figure 7 This is a graph showing the correlation between the levels of micropeptide genes in RNA samples from clinical patients with gastric cancer detected by qPCR and patient survival time. A represents the gene level of AC027045.3 in cancer and adjacent normal samples; B represents the survival time of patients with high and low expression of AC027045.3; C represents the gene level of TRHDE-AS1 in cancer and adjacent normal samples; D represents the survival time of patients with high and low expression of TRHDE-AS1; E represents the gene level of OLMLINC in cancer and adjacent normal samples; F represents the survival time of patients with high and low expression of OLMLINC; G represents the gene level of ZNF436-AS1 in cancer and adjacent normal samples; and H represents the survival time of patients with high and low expression of ZNF436-AS1. (V) Detailed Implementation Methods

[0023] 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:

[0024] Example 1: Screening of micropeptides for early gastric cancer diagnosis

[0025] Using Ribotricer (v1.3.2) software, all possible transcribed peptides were predicted across the entire genome of the human reference genome version hg38. The amino acid and nucleotide sequence information of all peptides with start codons ATG / CTG / GTG / TTG and lengths between 10 and 250 were extracted, totaling 11,931,202 peptides. A peptide information reference library was constructed based on this. Figure 1 ).

[0026] Six pairs of clinical gastric cancer tissue samples (cancer and adjacent normal tissue) were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from both patients and the ethics committee for all samples. After tissue lysis, micropeptides were enriched using 30KD and 10KD ultrafiltration tubes. The filtrate from the 10KD ultrafiltration tube was then used for mass spectrometry analysis. The obtained peptide information was matched against a micropeptide reference library using Mascot software (Matrix Science, Boston, MA) to identify the micropeptides. Mass charge ratio spectra of some micropeptide fragments from the gastric cancer clinical samples are shown below. Figure 2 As shown, A represents a micropeptide (ID: ENST00000456120_120761840_120762226_387, with the sequence CDSASVSRASLTLTYFCQMQWK); B represents a micropeptide (ID: ENST00000668145_75741828_75741986_159, with the sequence NLGIHSLVCPCVLCILHKM); C represents a micropeptide (ID: ENST00000662515_67578457_67578996_540, with the sequence FVSKCPLTMALGYSRTVLK); and D represents a micropeptide (ID: ENST00000664629_135498552_135498815_264, with the sequence ITNPMRTELSCSTFRSK).

[0027] 9954 micropeptides were obtained using the above method. For the micropeptides encoded by lncRNAs, eight sgRNAs were designed using CRISPick (https: / / portals.broadinstitute.org / gppx / crispick / public) based on the open reading frames of the targeted micropeptides and their upstream sequences, forming an sgRNA library. Following the method in Example 2, the sgRNA library was packaged into lentivirus and used to infect human gastric cancer (AGS) cell lines (purchased from the American College of Cell Culture, ATCC). Cells were harvested at 7 and 35 days post-infection, and the genome was extracted to detect changes in the relative abundance of each sgRNA. Based on the fold change of sgRNA abundance in the upstream target sequence being less than 2 and the fold change of sgRNA abundance in the target open reading frame being greater than 2, 1164 micropeptide candidates with the function of regulating tumor cell proliferation were screened. These included the AC027045.3 gene-encoded protein, which inhibits tumor cell proliferation, and the OLMLINC, ZNF436-AS1, and TRHDE-AS1 gene-encoded proteins, which promote tumor proliferation. Figure 3 ).

[0028] The nucleotide sequence of the AC027045.3 gene (Gene ID: 27261635) is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2; the nucleotide sequence of the OLMLINC gene (Gene ID: 90271) is shown in SEQ ID NO.3, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.4; the nucleotide sequence of the ZNF436-AS1 gene (Gene ID: 148898) is shown in SEQ ID NO.5, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.6; the nucleotide sequence of the TRHDE-AS1 gene (Gene ID: 283392) is shown in SEQ ID NO.7, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.8.

[0029] AC027045.3 Gene sequence (SEQ ID NO.1): ATGAAGATGGCTACCAAGGCATCCACCCTGAGCCAGTGGAACCCAGGGCTGCGACAACTCACTCTGGAGCCCAGGAGGATGGCGCTTGTCAGTATCAGAACGATATCCAGGACCTCGCCCCCGATGGACAGCCACAAAACACCTGCCAAAGAACGGGATGTTGGAAACGCTGGACACTTC.

[0030] AC027045.3 Amino acid sequence (SEQ ID NO.2): MKMATKASTLSQWNPGLRQLTLEPRRMALVSIRTISRTSPPMDSHKTPAKERDVGNAGHF.

[0031] OLMLINC gene sequence (SEQ ID NO.3): ATGCAGAAGACCTTTGACAAAATTCAACAACGCTTCATGCTAAAAACTCTCAATAAATTAGGTATTGATGGGACGTATCTCAAAATAATAAGAGCTATCTATGACAAACCCACAGCCAATATCATACTGAATGGAGAAAAACTGGAAGCATTCCTTTTGAAATCTGGCACAAGACAGGGATGCCCTCTCTCACCACTCCTAGTCAACATAGTGTTGGAAGTTCTGGCCAGGGCAATCAGGCAGGAGAAGGAAATAAAGGGTATTCAATTAGGAAAAGAGGAAGTCAAATTGTCCCTGTTTGCAGATGACATGATTGTATATCTAGAAAACCCCATTGTCTCAGCCCAAAATCTCCTTAAGCTGATAGGCAACTTCAGCAAAGTCTCAGGATACAAAATCAATGTGCGAAAATCACAAGCATTCCTATACGCCAATAACAGACAAACAGAGAGCCAAATCATGAGTGAACTCCCATTCACAACTGCTTCAAAGAGAATAAAATACCTAGGAACCCAACTTACAAGGGAAGTGAAGGACCTCTTCAAGGAGAACTACAAACCACTGCTCAATGACATAAAAGAGGATACAAACAAATGGAAGAAAATTCCATGCTCA.

[0032] OLMLINC amino acid sequence (SEQ ID NO.4): MQKTFDKIQQRFMLKTLNKLGIDGTYLKIIRAIYDKPTANIILNGEKLEAFLLKSGTRQGCPLSPLLVNIVLEVLARAIRQEKEIKGIQLGKEEVKLSLFADDMIVYLENPIVSAQNLLKLIGNFSKVSGYKINVRKSQAFLYANNRQTESQIMSELPFTTASKRIKYLGTQLTREVKDLFKENYKPLLNDIKEDTNKWKKIPCS.

[0033] ZNF436-AS1 gene sequence (SEQ ID NO.5): ATGATTAAATTACTGAAGTGTCTAAAAAGAACAGAAGGACCCACCCCAGCCATAATGCAGACTTTCCCATTCCTTCTCTTTCCACCCGCAACTGTGGGGCTAAGAATTCTTAAGATGTCACAGAATTATACCATCCTACCTCATCTAAAGTCGATCCGAGGCTGGTCCGATGGTAGTGGGTTACCAGAACTTAATAACATTAATAACTCTAAAGTTAGTATACAACCCCCCACTGCTAAATTTGGCTATAAAAATAAAATTGATCTGAGCACCATA.

[0034] ZNF436-AS1 amino acid sequence (SEQ ID NO.6): MIKLLKCLKRTEGPTPAIMQTFPFLLFPPATVGLRILKMSQNYTILPHLKSIRGWSDGSGLPELNNINNSKVSIQPPTAKFGYKNKIDLSTI.

[0035] TRHDE-AS1 gene sequence (SEQ ID NO.7): ATGAGCAAACATGTCACTGAAGAGGATGTACAGATGGCAAAAATGCATGTAAAAAGATTTCAACTTCATAAACCGTTAGGGAAATGTGAATTAAGCAACCAATCAGAATGGTTAAAATGTTTAAAAAATAGTAACACCACCAAATATTGGCAAGGATATAAAGGAATCGAATCACTCATGCATTGC.

[0036] TRHDE-AS1 amino acid sequence (SEQ ID NO.8):

[0037] MSKHVTEEDVQMAKMHVKRFQLHKPLGKCELSNQSEWLKCLKNSNTTKYWQGYKGIESLMHC.

[0038] Example 2: Manipulating micropeptide gene expression to regulate gastric cancer cell proliferation

[0039] 1. Cell lines

[0040] Human gastric cancer cell line AGS was purchased from the American Standard Cell Culture Center (ATCC) and cultured in Gibco medium containing 10% FBS (Bovogen) DMEM / F12 at 37°C in a 5% CO2 incubator. MycoAlert kit (Lonza) showed no mycoplasma contamination or cross-contamination in the cells.

[0041] 2. Cloning and expression of biomarkers

[0042] Using cDNA from the human gastric cancer cell line AGS as a template, PCR amplification was performed with primers 1 and 2, and the resulting plasmid PCDNA3.1 (purchased from Addgene, https: / / www.addgene.org / ) was ligated with NotI and SFaAI-digested vector to construct the expression plasmid PCDNA3.1-AC027045.3-Flag-His.

[0043] Primer 1 (SEQ ID NO.19):

[0044] F- CTAGCGCCACCATGGCGGCCATGAAGATGGCTACCAAGGCATC;

[0045] Primer 2 (SEQ ID NO.20):

[0046] R-ACCGGTAAGCTTTGCGATCGCGAAGTGTCCAGCGTTTCCAACAT.

[0047] Using cDNA from the human gastric cancer cell line AGS as a template, PCR amplification was performed with primers 3 and 4, and the resulting plasmid was ligated with the PCDNA3.1 vector digested with NotI and SSaAI to construct the expression plasmid PCDNA3.1-OLMLINC-Flag-His.

[0048] Primer 3 (SEQ ID NO.21):

[0049] F- CTAGCGCCACCATGGCGGCCATGCAGAAGACCTTTGACAAAAT;

[0050] Primer 4 (SEQ ID NO.22):

[0051] R-ACCGGTAAGCTTTGCGATCGCTGAGCATGGAATTTTCTTCATT.

[0052] Using cDNA from the human gastric cancer cell line AGS as a template, PCR amplification was performed with primers 5 and 6, and the resulting plasmid was ligated with the PCDNA3.1 vector digested with NotI and SSaAI to construct the expression plasmid PCDNA3.1-ZNF436-AS1-Flag-His.

[0053] Primer 5 (SEQ ID NO. 23): F-CTAGCGCCACCATGGCGGCCATGATTAAATTACTGAAGTGTCT;

[0054] Primer 6 (SEQ ID NO.24): R-ACCGGTAAGCTTTGCGATCGCTATGGTGCTCAGATCAATTTTAT.

[0055] Using cDNA from the human gastric cancer cell line AGS as a template, PCR amplification was performed with primers 7 and 8, and the resulting plasmid PCDNA3.1, digested with NotI and SFaAI, was ligated to construct the expression plasmid PCDNA3.1-TRHDE-AS1-Flag-His.

[0056] Primer 7 (SEQ ID NO.25):

[0057] F- CTAGCGCCACCATGGCGGCCATGAGCAAACATGTCACTGAAGA;

[0058] Primer 8 (SEQ ID NO.26):

[0059] R-ACCGGTAAGCTTTGCGATCGCGCAATGCATGAGTGATTCGATTC.

[0060] 3. Plasmid transfection

[0061] Using the ViraPower™ Lentiviral Expression System (Thermo Scientific), the plasmids constructed in step 2 were packaged into lentiviruses and used to infect the gastric cancer cell line AGS, constructing four cell lines with different micropeptide overexpressions: OV-AC027045.3; OV-OLMLINC; OV-ZNF436-AS1; and OV-TRHDE-AS1. Simultaneously, a cell line (EV) overexpressing the PCDNA3.1 plasmid vector was constructed and designated as the control cell line.

[0062] 4. Cell viability detection

[0063] The cell lines constructed in step 3 were seeded into DMEM / F12 medium containing 10% FBS and cultured at 37°C in a 5% CO2 incubator for 12, 24, 36, 48, 60, and 72 hours. Cell viability was detected using a cell viability assay kit (CCK-8), and colony formation experiments were conducted. After one week of culture in 6-well plates, the cells were stained with crystal violet and the colonies were counted to evaluate the regulatory effect of the micropeptides on cell proliferation.

[0064] The results are as follows Figure 4 As shown, overexpression of the micropeptides OLMLINC, TRHDE-AS1, and ZNF436-AS1 significantly enhanced the cell proliferation activity of the gastric cancer cell line AGS, while overexpression of the micropeptide AC027045.3 significantly inhibited the cell proliferation activity of the gastric cancer cell line AGS. This indicates that the above micropeptides play a key role in the proliferation of gastric cancer cells and can serve as marker molecules for gastric cancer detection.

[0065] Example 3: Primer design for micropeptide detection

[0066] Based on the micropeptides screened in Example 1 that play an important regulatory role in gastric cancer, relevant qPCR detection primers were designed.

[0067] Design micropeptide molecular detection primers and internal control primers (GAPDH-F, GAPDH-R) based on NCBI BLAST:

[0068] AC027045.3 -F (SEQ ID NO.9): 5'-CTACCAAGGCATCCACCCTG-3',

[0069] AC027045.3 -R (SEQ ID NO. 10): 5'-GCGTTTCCAACATCCCGTTC-3';

[0070] OLMLINC-F (SEQ ID NO.11): 5'-CTGAGAGTTCAGGCCTGGTTAC-3'

[0071] OLMLINC -R (SEQ ID NO.12): 5'-ATCGAATGTTGGACTTCTTAGTCT-3'

[0072] ZNF436-AS1 -F (SEQ ID NO.13): 5'-TCCAAAACCTGAGAGACCGC-3'

[0073] ZNF436-AS1 -R (SEQ ID NO.14): 5'-AAGAGGGTGGGAGTCCCTAA-3'

[0074] TRHDE-AS1 -F (SEQ ID NO.15): 5'-TTGGAAAGCGTCTTGCTTGC-3'

[0075] TRHDE-AS1 -R (SEQ ID NO.16): 5'-GCGAAAGCCCTAACTTCCCT-3'

[0076] GAPDH F (SEQ ID NO.17): 5'-CAGGGCTGCTTTTAACTCTGGTA-3',

[0077] GAPDH R (SEQ ID NO. 18): 5'-CATGGGTGGAATCATATTGGAAC-3'.

[0078] Example 4: Micropeptide Protein Immunohistochemical Detection Kit

[0079] The detection kit is prepared according to the following composition and is suitable for detecting the expression of micropeptides in biological samples by immunohistochemistry:

[0080] Reagent a: Blocking solution, 10% goat serum, 1 bottle, ready to use;

[0081] Reagent b: Anti-human AC027045.3 rabbit polyclonal antibody (synthesized by GenScript, with the antigenic epitope being MDSHKTPAKERDVG), 1 vial, 200× concentrated solution;

[0082] Reagent c: Anti-human OLMLINC rabbit polyclonal antibody (synthesized by GenScript, epitope EKEIKGIQLGKEEV), 1 vial, 200× concentrated solution.

[0083] Reagent d: Anti-human ZNF436-AS1 rabbit polyclonal antibody (synthesized by GenScript, epitope: PPTAKFGYKNKIDL), 1 vial, 200× concentrated solution.

[0084] Reagent e: Anti-human TRHDE-AS1 rabbit polyclonal antibody (synthesized by GenScript, epitope MSKHVTEEDVQMAK), 1 vial, 200× concentrated solution.

[0085] Reagent f: HRP goat anti-rabbit secondary antibody (purchased from Jackson Immuno Research, catalog number 115-035-003), 1 vial, ready to use;

[0086] Reagent g: DAB substrate buffer, 1 vial, 20× concentrated solution;

[0087] Reagent h: DAB colorimetric solution, 1 vial, 20× concentrated solution;

[0088] And the container containing the above reagents and the instruction manual.

[0089] Example 5: Clinical Relevance of Micropeptide Levels in Gastric Cancer

[0090] Ninety-two clinical samples of gastric cancer were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from both patients and the ethics committee before sample collection. Immunohistochemistry was used, employing the micropeptide immunohistochemical detection kit described in Example 4, to stain adjacent and tumor tissues of these 92 gastric cancer clinical samples. The expression of the aforementioned micropeptides in cancer and adjacent tissues was analyzed, and the positive rate and staining intensity of micropeptides in cancer and adjacent tissues were observed. The results are as follows: Figure 5 The results indicate that the expression levels of micropeptides ZNF436-AS1, TRHDE-AS1, and OLMINC are relatively high in gastric cancer tissues, while the expression level of micropeptide AC027045.3 is relatively high in adjacent normal tissues, suggesting that the above micropeptides have the ability to serve as diagnostic markers for gastric cancer.

[0091] Example 6: Micropeptide Protein RT-qPCR Detection Kit

[0092] The micropeptide protein RT-qPCR detection kit consists of the primers and PCR reaction solution described in Example 3. The PCR reaction solution comprises: a 20 μL system containing 10 μL of ChamQ SYBR qPCR Master Mix (High ROX Premixed), 7 μL of water, 1 μL each of forward and reverse primers, and 50 ng / μL-1 μL of sample cDNA. The samples were selected from the micropeptide overexpression cell lines OV-AC027045.3, OV-OLMLINC, OV-ZNF436-AS1, and OV-TRHDE-AS1 constructed in step 3 of Example 2, as well as the control cell line EV.

[0093] RT-qPCR reaction program: ① 95℃-30s; ② 95℃-10s, 60℃-30s, 40 cycles.

[0094] The above reaction system and primers can accurately detect the expression of each micropeptide gene in the overexpression stable cell line constructed in Example 2. Figure 6 ).

[0095] In conjunction with the expression data of various micropeptide genes in patients in Example 5, if any of the following criteria are met: ZNF436-AS1 CT < 27, OLMLINC CT < 25, TRHDE-AS1 < 27, or AC027045.3 > 28 (all relative to GAPDH calibration), it indicates a higher risk of gastric cancer, and further endoscopic examination is recommended. It is worth noting that the expression of each micropeptide gene varies significantly among different patients; therefore, the above values ​​are for reference only and require specific analysis for each individual case.

[0096] Example 7: The impact of micropeptide expression levels on patient clinical prognosis

[0097] Eighty clinical samples of breast cancer were collected from the Second Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from both patients and the ethics committee before sample collection. The micropeptide protein RT-qPCR detection kit described in Example 6 was used to detect the cancer cells in these 80 clinical samples. Figure 7 (T) and adjacent cancer cells (T) Figure 7 Gene expression of the above-mentioned micropeptides was detected using N)RNA, and the mean values ​​were taken. The gene expression of these micropeptides in different samples from cancer and adjacent normal tissues was analyzed. Simultaneously, using the median CT number in cancer tissue as a cutoff line, the micropeptide expression was divided into high-expression and low-expression groups for prognostic correlation analysis. Results are as follows: Figure 7This example illustrates that in gastric cancer tissue, the gene expression levels of micropeptides ZNF436-AS1, TRHDE-AS1, and OLMINC are relatively high, while the gene expression level of micropeptide AC027045.3 is relatively high in adjacent normal tissue. Furthermore, patients with high expression of ZNF436-AS1, high expression of TRHDE-AS1, and high expression of OLMINC have shorter overall survival, while patients with low expression of AC027045.3 have shorter overall survival. High expression of ZNF436-AS1, TRHDE-AS1, and OLMINC, and low expression of AC027045.3 are associated with poor patient prognosis.

[0098] It should be noted that the above experiments are only some specific examples of the present invention. Obviously, there are many variations of the present invention. All variations that can be directly derived or conceived by those skilled in the art from the content disclosed in the present invention should be considered to be within the scope of protection of the present invention.

Claims

1. A micropeptide molecular marker for early gastric cancer screening, characterized in that, The micropeptide molecular marker is the protein encoded by the OLMLINC gene; the nucleotide sequence of the OLMLINC gene is shown in SEQ ID NO.

3.

2. The micropeptide molecular marker as described in claim 1, characterized in that, The amino acid sequence of the protein encoded by the OLMLINC gene is shown in SEQ ID NO.

4.

3. A detection reagent for micropeptide molecular markers as described in claim 1, characterized in that, The detection reagent includes substances for detecting the levels of the micropeptide molecular marker DNA, mRNA, and / or protein in the sample to be tested.

4. The detection reagent as described in claim 3, characterized in that, The detection reagents include those for detecting micropeptides using immunohistochemistry, chemiluminescence, radioisotope assay, fluorescence luminescence assay, enzyme-linked immunosorbent assay (ELISA), colloidal gold assay, real-time quantitative reverse transcription PCR, microarray assay, DNA blotting, RNA blotting in situ hybridization, or protein blotting.

5. The detection reagent as described in claim 4, characterized in that, The reagents for real-time quantitative reverse transcription PCR detection of micropeptides include specific primers, and the primers for detecting OLMLINC are shown in SEQ ID NO.11 and SEQ ID NO.

12.

6. The detection reagent as described in claim 4, characterized in that, The reagents for immunohistochemical detection of micropeptides include blocking buffer, primary antibody, HRP goat anti-rabbit secondary antibody, DAB substrate buffer, and DAB chromogenic solution; the blocking buffer is PBS containing 10% goat serum; the primary antibody is anti-human OLMLINC rabbit polyclonal antibody.

7. The detection reagent as described in claim 3, characterized in that, The test samples were obtained from non-tumor tissues and / or cells, and tumor tissues and / or cells.

8. The application of the micropeptide molecular marker of claim 1 in the preparation of products for assisting in the diagnosis of gastric cancer.

9. The application as described in claim 8, characterized in that, The products include kits, test strips, test cards, detection instruments, or any combination thereof for detecting micropeptide molecular markers.

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