Micropeptide miPEP060 encoded by linc00060 gene and use thereof

Abstract

The application relates to the technical field of biological medicine, and discloses a micropeptide miPEP060 coded by a LINC00060 gene and application thereof; the application provides the following application of LINC00060 and the micropeptide miPEP060 coded thereby: preparing a product for predicting tumor chemotherapy efficacy, preparing a product for predicting tumor prognosis; preparing a tumor treatment product, preparing a product for enhancing tumor chemotherapy efficacy, preparing a product for overcoming tumor chemotherapy drug resistance, preparing a product for inhibiting PARP1 enzyme activity or PARylation modification, and preparing a product for inhibiting replication stress response and replication fork stability; the LINC00060 and the micropeptide miPEP060 coded thereby provided by the application can significantly enhance the chemotherapy killing effect of tumor cells or tumor tissues and reduce tumor volume.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the micropeptide miPEP060 encoded by the LINC00060 gene and its uses. Background Technology

[0002] PARP inhibitors (PARPi) are the first small-molecule targeted therapies approved for cancer treatment that utilize a "synthetic lethality" effect, significantly prolonging the survival of cancer patients. The tumor-killing effect of PARPi depends on its inhibition of the catalytic activity of poly-ADP polymerase PARP1, thereby triggering replication stress and DNA double-strand breaks (DSB) damage. BRCA Patients with gene mutations are highly sensitive to PARPi because they cannot effectively repair their homologous recombination defects (HRD) and cannot effectively repair their duplicated stem cells (DSBs). However, PARPi resistance severely restricts its clinical efficacy. The development of PARPi resistance is largely due to the restoration of replication fork stability. Therefore, it is urgent to discover new regulatory factors of replication fork stability to improve the efficacy of PARPi and provide effective targets and intervention strategies to overcome PARPi resistance.

[0003] Long non-coding RNAs (lncRNAs) were initially defined as RNA molecules longer than 200 nucleotides that lacked protein-coding capabilities. However, an increasing number of lncRNAs have been found to encode micropeptides of 100 amino acids or less, thereby regulating tumor progression and treatment. Currently, there are few reports on whether lncRNA-encoded micropeptides regulate replication fork stability and PARPi resistance. TAT is a positively charged cell-penetrating peptide (CPP) that can serve as a highly efficient transport carrier to carry macromolecules such as proteins and DNA across cell membranes and nuclear membranes. TAT has no strict limitations on the size of its conjugates and possesses extremely strong cell penetration and very low cytotoxicity, thus it is widely used in oncology, diabetes, and other fields, offering broad prospects for clinical drug development and application. Summary of the Invention

[0004] The purpose of this invention is to disclose the micropeptide miPEP060 encoded by the LINC00060 gene and its uses. This invention combines a coding ability prediction database and in vivo and in vitro biological experiments to identify novel lncRNA-encoded micropeptides, clarify their regulatory role in replication fork stability and PARPi sensitivity, provide an effective biomarker for predicting the efficacy of PARPi in tumor treatment, and combine CPP technology to evaluate the effect of micropeptide-TAT peptide sensitizing PARPi, providing a novel intervention method for tumor chemotherapy treatment.

[0005] This invention is achieved through the following technical solution: The LINC00060 gene encodes the micropeptide miPEP060, and the nucleotide sequence of the micropeptide miPEP060 is shown in SEQ ID No. 2.

[0006] The nucleotide sequence of the LINC00060 gene is shown in SEQ ID No. 1.

[0007] The miPEP060 and / or LINC00060 genes are used for the preparation or screening of tumor therapeutic drugs.

[0008] Furthermore, the aforementioned uses include two aspects: first, directly applying the miPEP060 and / or LINC00060 genes to the preparation of tumor therapeutic drugs or formulations; second, using the miPEP060 and / or LINC00060 genes as targets for drugs or formulations targeting tumor cells in the screening of tumor therapeutic drugs or formulations.

[0009] Furthermore, when directly applied to the preparation of tumor therapeutic drugs or formulations, the miPEP060 and / or LINC00060 genes are used to prepare products that inhibit tumor progression, or to prepare products that enhance the efficacy of tumor chemotherapy, or to prepare products that overcome tumor chemotherapy resistance.

[0010] The micropeptide miPEP060 and / or LINC00060 are used as biomarkers to assess or assist in assessing the sensitivity of cancer patients to chemotherapy drugs and to predict the prognostic status of cancer patients after chemotherapy.

[0011] Furthermore, when the micropeptide miPEP060 and / or LINC00060 are used as markers, they contain a monoclonal antibody against the micropeptide miPEP060.

[0012] Products having any of the following functions are used in at least one of the following 1)-8): a. Promotes the expression of the LINC00060 gene; b. Promote the biological functions of LINC00060 and the encoded micropeptide miPEP060; c. Promote the biological function of fusion proteins containing miPEP060; d. Promotes the biological function of complexes containing miPEP060; 1) Preparation of tumor treatment products; 2) To prepare products that reduce tumor chemotherapy resistance; 3) To prepare products that improve the sensitivity of tumors to chemotherapy; 4) Prepare products that inhibit tumor drug resistance caused by abnormal PARP1 enzyme activity or PARylation; 5) To prepare products that inhibit tumor drug resistance caused by the restoration of replication fork stability; 6) Prepare products that suppress replication stress response and replication fork stability; 7) Prepare products that inhibit PARP1 enzyme activity or are modified by PARylation; 8) Preparation of products for use in combination with other antitumor drugs or methods; The nucleotide sequence of the LINC00060 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the micropeptide miPEP060 is shown in SEQ ID No. 2.

[0013] Furthermore, the tumors include, but are not limited to, ovarian cancer, breast cancer, lung cancer, nasopharyngeal carcinoma, gastric cancer, colorectal cancer, liver cancer, esophageal cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, ovarian cancer, bladder cancer, thyroid cancer, and skin cancer.

[0014] Furthermore, the amino acid sequence of the micropeptide miPEP060-TAT is shown in SEQ ID No. 3.

[0015] Furthermore, its transmembrane peptide sequence includes, but is not limited to, the TAT sequence.

[0016] Furthermore, the inhibitors of the micropeptide miPEP060 and / or LINC00060 include, but are not limited to, shRNA, the target sequence of which is shown in SEQ ID No. 4.

[0017] The beneficial effects of this invention compared to the prior art are as follows: This invention focuses on tumor chemotherapy and develops a novel tumor marker and drug target, specifically the long non-coding RNA LINC00060 and its encoded micropeptide miPEP060. This micropeptide can significantly improve the efficacy of PARPi and other chemotherapeutic drugs in tumor treatment and has a direct regulatory effect on PARP1 enzyme activity and PARylation modification. An antibody obtained by immunopurification of the micropeptide miPEP060 can detect the endogenous expression of this micropeptide, and is expected to provide new tumor markers and detection methods for clinical use. Coupled with a TAT membrane-penetrating peptide, this micropeptide, in combination with PARPi, can significantly improve the efficacy of PARPi and inhibit tumor growth both in vivo and in vitro, providing a new combination therapy strategy for improving the efficacy of PARPi and other chemotherapeutic drugs in clinical practice. Attached Figure Description

[0018] Figure 1The prediction and identification of the coding ability of LINC00060 were as follows: (A) Bioinformatics predicted that there were 3 potential open reading frames (ORF1-3) in the positive strand of LINC00060; (B) Immunoprecipitation combined with mass spectrometry confirmed the sequence specificity of the peptide ORF1.

[0019] Figure 2 LINC00060 encodes the functional peptide miPEP060; (A) Western blot detection of GFP-miPEP060 fusion protein expression; (B) Immunoprecipitation assay using miPEP060-specific antibody to confirm endogenous expression of miPEP060 in cells; (CD) Immunofluorescence assay using GFP antibody (C) and miPEP060 antibody (D) to show that miPEP060 is located in the nucleus and cytoplasm.

[0020] Figure 3 LINC00060 and its encoded peptide miPEP060 regulate PARPi sensitivity; (A) In control and stable knockdown of LINC00060 SKOV3 cells, empty vector, LINC00060 and miPEP060 were rotated back, and the sensitivity of cells to Olaparib was detected by CCK8 assay; (B) qPCR and Western blot experiments were used to detect the knockdown, rotation effect and miPEP060 expression of LINC00060 in (A); (CE) Tumor formation experiment in nude mice overexpressing LINC00060 and its encoded peptide miPEP060; Under the treatment of saline and Olaparib, the size of the transplanted tumor in the control group, the LINC00060 overexpression group and the miPEP060 overexpression group were compared (C), tumor volume (D) and mouse weight (E).

[0021] Figure 4LINC00060 regulates the sensitivity of chemotherapy drugs and replication stressors. Specifically, (AC) CCK8 assays detected the sensitivity of SKOV3 cells to the chemotherapy drugs Olaparib (A), ICRF-193 (B), and the replication stressor HU (C); (D) qPCR assays detected LINC00060 expression in (AC); (EG) CCK8 assays detected the sensitivity of A2780 cells to the chemotherapy drugs Olaparib (E), CPT (F), and the replication stressor HU (G); (H) qPCR assays detected LINC00060 expression in (EG); (IK) CCK8 assays detected the sensitivity of HO8910 cells to the chemotherapy drugs Olaparib (I), CPT (J), and the replication stressor HU (K); and (L) qPCR assays detected LINC00060 expression in (IK).

[0022] Figure 5 This is a schematic diagram of the miPEP060-TAT peptide design.

[0023] Figure 6 The miPEP060-TAT peptide regulates PARPi sensitivity; (A) miPEP060-TAT peptide (5 μM) was added to SKOV3 cells, and the CCK8 assay was used to detect the sensitivity of cells to olaparib; (B) a clonogenic assay was used to detect the sensitivity of SKOV3 cells to olaparib with miPEP060-TAT peptide (2 μM); (CE) female mice with tumors were intraperitoneally injected with saline, olaparib, miPEP060-TAT peptide, and olaparib combined with miPEP060-TAT peptide, respectively. The size of the transplanted tumor (C), tumor volume (D), and mouse body weight (E) were measured.

[0024] Figure 7 The miPEP060-TAT peptide regulates the sensitivity of cells to chemotherapeutic drugs and replication stressors. In the case of (AD), miPEP060-TAT peptide (5 μM) was added to SKOV3 cells, and the sensitivity of cells to chemotherapeutic drugs CPT (A), CDDP (B), and ETO (C) and replication stressor HU (D) was detected by CCK8 assay.

[0025] Figure 8LINC00060 and its encoded peptide miPEP060 inhibit PARylation modification, leading to replication fork instability. Specifically, (A-B) DNA fiber experiments were performed in control and stably knocked-down SKOV3 cells after transduction with empty vector, LINC00060, and miPEP060, respectively, to detect the effects of LINC00060 and its encoded peptide miPEP060 on replication fork restart (A) and nascent DNA degradation (B). (C) SKOV3 cells knocked down with hydrogen peroxide (H2O2), overexpressing LINC00060, and miPEP060 were treated, and the effect on PARylation modification was detected by Western blot. (D) The effect of miPEP060-TAT peptide on PARylation modification in SKOV3 cells was detected by Western blot. (E) The effect of miPEP060 on PARP1 enzyme activity was detected by in vitro PARylation assay.

[0026] Figure 9 The study aimed to detect the expression levels of LINC00060 and its encoded peptide miPEP060 in clinical samples; (A) qPCR assay was used to detect the expression of LINC00060 in adjacent and cancerous tissues of 10 ovarian cancer patients; (B) immunohistochemical staining with miPEP060-specific antibody was used to quantitatively assess the proportion of miPEP060-positive cells in normal ovarian tissue and ovarian tumor tissue. Detailed Implementation

[0027] To make the technical problems to be solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. The technical solutions of this invention are described in detail below with reference to the embodiments and accompanying drawings, but the scope of protection is not limited thereto. Example 1

[0028] In this embodiment, the human long non-coding RNA LINC00060 encodes the micropeptide miPEP060; 1. Method 1.1 Analyzing the potential of long non-coding RNA LINC00060 to encode micropeptides Using ORFfinder (https: / / www.ncbi.nlm.nih.gov / orffinder / ), three open reading frames were found in the LINC00060 justice chain. Further analysis using the GWIPS-vi database (https: / / gwips.ucc.ie / index.html) showed that open reading frames 1-3 contained ribosome binding signals, suggesting that they have encoding potential.

[0029] 1.2 Antibody Preparation Based on the results of ribosome profiling analysis, open reading frame 1 was selected as the research target. According to its amino acid sequence, the antigen sequence that produces miPEPE060 antibody was determined. Then, experimental-grade white rabbits were immunized to obtain affinity-purified antibody anti-miPEP060.

[0030] 1.3 Construction of eukaryotic expression plasmids For pEGFP*-miPEP060: Green fluorescent protein EGFP (EGFP*) with the start codon and a subsequent triplet codon deleted was added to the end of the predicted SEQ ID No. 2 sequence. Primers were designed according to the restriction enzyme digestion sequences of BamHI and EcoRI. The target fragment was amplified by PCR. The target fragment and plasmid vector were digested (37℃) for 4 h, recovered by gel extraction, ligated overnight at 4℃ with T4 ligase, and transformed to obtain the pEGFP*-miPEP060 plasmid. For the construction of the pcDNA3.1-LINC00060 plasmid, the above steps were followed, using EcoRI and HindIII as restriction sites to design primers. The LINC00060 gene was amplified by PCR and cloned into the single cloning site of the pcDNA3.1 vector to construct the pcDNA3.1-LINC00060 eukaryotic expression plasmid.

[0031] 1.4 Plasmid transfection One day before transfection, SKOV3 cells were seeded into 35 mm dishes at a seeding density of 65%. The next day, the cells were transfected using VigoFect transfection reagent, and the pEGFP*-miPEP060 plasmid was transfected into the cells. After 6-8 hours of transfection, the medium was replaced with fresh complete medium, and the cells were collected 48 hours after transfection.

[0032] 1.5 miPEP060 immunoprecipitation (IP) Prepare 6 10 cm dishes containing HEK293T cells. Wash twice with PBS, centrifuge at 1500 rpm and discard the supernatant. Add an appropriate amount of HEPES lysis buffer containing protease inhibitors and lyse at 4°C for 1 h. Add anti-miPEP060 antibody to the supernatant and incubate overnight at 4°C. The next day, mix the prepared GFP beads with the antibody mixture and incubate at 4°C for 4 h. Wash 5 times with HEPES lysis buffer, centrifuge, discard the supernatant, add an equal volume of 2×SDS loading buffer to the beads, and boil in a metal bath at 95°C for 15 min to obtain antibody IP samples for subsequent Western blot experiments.

[0033] 1.6 Mass Spectrometry Analysis HEK293T cells overexpressing pEGFP*-miPEP060 were collected, and proteins were extracted by lysis. Immunoprecipitation was performed on the protein extracts using an anti-GFP antibody. The precipitated proteins were separated by 10% SDS-PAGE gel extraction, and specific target gel bands were excised. These bands underwent in-gel reduction, alkylation, and enzymatic digestion. The peptides obtained from the enzymatic digestion were analyzed using a nanoElute ultra-high performance liquid chromatography (UPLC) system coupled with a temps-to-free (tTOF) mass spectrometer (Bruker). Raw mass spectrometry data were processed using MaxQuant software, and the ZJK database was used for searching. The search parameters were set as follows: trypsin digestion was used, allowing a maximum of two missed cleavage sites; the fixed modification was carboxymethylation of cysteine; the variable modifications were methionine oxidation and N-terminal acetylation of protein. The precursor ion mass tolerance was set to 20 ppm, and the fragment ion mass tolerance was set to 0.1 Da. Peptide and protein level identification results were screened based on a false discovery rate (FDR) <0.01. Protein abundance was determined using an intensity-based absolute quantification (iBAQ) method.

[0034] 1.7 Endogenous expression of miPEP060 1) Western blot: Transfected SKOV3 ovarian cancer cells or untransfected SKOV3 cells were washed twice with PBS, and then the protein was collected with an appropriate amount of 1×SDS loading buffer. The cells were lysed in a metal bath at 95 ℃ for 15 min, centrifuged, and stored at -20 ℃ for subsequent Western blot experiments.

[0035] Prepare a 10% SDS-PAGE gel. Add 15 μl of each of the denatured proteins to the corresponding gel lanes. Run the stacking gel at 80 V for 20 min, then run the separating gel at 120 V for approximately 1 h. Transfer the proteins to a 0.22 μm PVDF membrane using a 300 mA current for 2 h. After transfer, block with 5% skim milk powder for 1 h, then incubate with primary antibodies (anti-miPEP060 and anti-GFP) overnight at 4 °C. The next day, wash the membrane with TBST buffer, then incubate with the corresponding secondary antibodies at room temperature for 1 h. After washing 10 times with TBST buffer, expose for imaging.

[0036] 2) Immunofluorescence: SKOV3 cells were seeded on slides at a density of 60%-80%. The next day, the culture medium was aspirated, and the cells were washed three times with PBS. Pre-absorption was performed for 2 min with PBS solution containing 0.2% Triton X-100, followed by two PBS washes. The cells were then fixed with 4% paraformaldehyde solution at room temperature for 15 min. The fixative was discarded, and the cells were gently washed three times with PBS. Permeabilization was then performed for 10 min at room temperature with PBS solution containing 5% Triton X-100. Blocking was then performed for 1 h at room temperature with 5% BSA blocking buffer containing 3% goat serum. After blocking, the cells were incubated overnight at 4°C with the primary antibody (anti-miPEP060). The next day, the cells were washed five times for 5 min each time with PBST wash buffer (containing 0.2% Tween in PBS) on a horizontal shaker at room temperature. The cells were then incubated with the corresponding secondary antibody at room temperature for 1 h, washed five times with PBST wash buffer on a shaker at room temperature, mounted with DAPI-containing mounting medium, and then subjected to fluorescence imaging as soon as possible. After transfection, the cells were washed three times with PBS, fixed with 4% paraformaldehyde, washed with PBS, and then mounted and photographed directly using mounting medium containing DAPI.

[0037] 2. Results 2.1 Ribosome mapping analysis: The open reading frame of long non-coding RNA LINC00060 has the potential to encode micropeptides. Figure 1 A), ORF1 was selected as the research target and named miPEP060. Mass spectrometry analysis yielded peptide spectra that matched the predicted amino acid sequence of miPEP060. Figure 1 B).

[0038] 2.2 Western blot experiments revealed that the anti-miPEP060 antibody produced a band at the same location as the antibody targeting the GFP tag protein, indicating the effectiveness of the anti-miPEP060 antibody targeting the micropeptide. Figure 2 A). miPEP060 was found in SKOV3 cells after antibody IP, demonstrating the endogenous expression of miPEP060. Figure 2 B).

[0039] 2.3 Immunofluorescence assays showed that in cells transfected with pEGFP*-miPEP060, miPEP060 was able to initiate translation, express the fusion protein, and localize it in the cytoplasm and nucleus. Figure 2 C); In SKOV3 cells, immunofluorescence assay of miPEP060 antibody showed that it was localized in the cytoplasm and nucleus (C). Figure 2 D). Example 2

[0040] This embodiment demonstrates that LINC00060 and micropeptide miPEP060 can improve the efficacy of tumor chemotherapy drugs; 1. Method LINC00060 was knocked down using shRNA technology, and the effect of LINC00060 knockdown on the sensitivity of chemotherapeutic drugs olaparib, camptothecin (CPT), ICRF-193, and the replication stressor hydroxyurea (HU) was examined using a CCK8 assay. After knockdown of LINC00060, the full-length LINC00060 or the micropeptide miPEP060 was reversed, and the survival of ovarian cancer cells after olaparib treatment was examined. A nude mouse tumorigenesis assay was conducted to investigate the role of LINC00060 and miPEP060 in regulating the efficacy of PARPi in vivo.

[0041] 1.1 Construction of shLINC00060 knockdown plasmid shRNA coding sequences targeting LINC00060 were designed and synthesized, with the target sequence for shLINC00060 being AGGTGGTCCGTGATAATTAAA (SEQ ID No. 4). The target sequence for shNC is GCAACAAGATGAAGAGCACCAA (SEQ ID No. 5). These sequences were ligated into the pLKO.1 vector. The specific steps were as follows: The synthesized primers were prepared into an annealing reaction system using the following composition: 2 μl of positive sense nucleotide (100 μM), 2 μl of antisense oligonucleotide (100 μM), 1 μl of 10×T4 DNA ligase buffer, and 15 μl of ddH2O. The reaction system was incubated at 95℃ for 4 min, and then allowed to cool naturally to room temperature. Age I and EcoRI were used as restriction enzyme sites to digest the vector plasmid. After digestion, the annealed products were ligated and transformed to obtain the shRNA viral plasmid.

[0042] 1.2 shRNA Lentiviral Packaging and Infection One day before transfection, HEK293T cells were seeded in 6 cm dishes at a seeding density of 70%, so that the cell density would be 90% at transfection the next day. Before transfection, the culture medium was replaced with fresh complete medium. Following the manufacturer's instructions, the viral packaging plasmid and shRNA plasmid were transfected into HEK293T cells using VigoFect. The medium was changed 12 h after transfection. After 48 or 72 h, the cell culture supernatant was collected and filtered through a 0.22 μm filter to obtain the corresponding lentivirus. The collected lentivirus was used to infect adherent SKOV3 cells in good condition. Polybrene was added to the fresh culture medium at a final concentration of 10 μg / mL. The medium was replaced with fresh medium 12 h after infection. The cell knockdown efficiency was detected by qPCR after 72 h of cell infection.

[0043] 1.3 Restoration of the full-length LINC00060 and its encoding peptide miPEP060 After transiently knocking down LINC00060 cells with lentivirus for 24 hours, pcDNA3.1-LINC00060 or pEGFP*-miPEP060 plasmids were transfected with VigoFect transfection reagent. The culture medium was replaced with fresh medium 8 hours after transfection. Then, shNC, shLINC00060, shLINC00060+LINC00060 and shLINC00060+miPEP060 cells were seeded in 96-well or 12-well plates for CCK8 assays and colony formation assays.

[0044] 1.4 Verification of Knockdown and Rotation Effects The knockdown and rotation effects of LINC00060 cells were detected using qPCR. The specific steps were as follows: Knockdown or rotation cells were washed three times with PBS. 1 ml of Trizol was added to a dish, mixed, and allowed to stand for 5 min. Cells were then fully lysed and transferred to an RNase-free centrifuge tube. 200 μl of chloroform was added, and the mixture was vigorously vortexed and allowed to stand at room temperature for 3 min. The tube was then centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was collected and transferred to a new centrifuge tube. An equal volume of isopropanol was added, and the tube was precipitated at room temperature for half an hour. The precipitated RNA-isopropanol mixture was centrifuged at 4°C for 10 min. A white RNA precipitate was observed at the bottom of the EP tube. The supernatant was carefully discarded using an RNase-free pipette tip. 1 ml of 75% ethanol prepared with DEPC water was added, mixed, and centrifuged at 1000 rpm / min for 5 min at 4°C. The supernatant was discarded. This process was repeated twice. After the precipitate was air-dried at room temperature, an appropriate amount of DEPC water was added to elute the precipitate, and the mixture was eluted with a pipette tip to obtain RNA. The RNA was reverse transcribed into cDNA according to the instructions of the reverse transcription kit (YEASEN) and stored at -20℃ for subsequent qPCR experiments. The reverse-transcribed cDNA was diluted 5-fold, and 100 ng of cDNA, qPCR mix, and primers were added, with the remainder made up to 20 μl with ddH2O. GAPDH was used as the internal control. The reaction program was as follows: Step 1: 94℃ pre-denaturation for 30 sec; Step 2: 94℃ denaturation for 5 sec, 50-60℃ annealing for 30 sec, and 72℃ extension for 10 sec (40-45 cycles). Ct values ​​were obtained after the reaction, and the relative expression level of the target gene was calculated using the 2-ΔΔCt method. The primer sequence used for LINC00060 was: LINC00060-F: ATCCAAGTGGTAATGGGCGAAG (SEQ ID No. 6); LINC00060-R: AATAGGTGTCCTGATAACTTCC (SEQ ID No. 7) Western blot was used to detect miPEP060 expression. Knockdown and rotated cells were collected using 1×SDS loading buffer. The protein sample was lysed in a metal bath at 95°C for 15 min, centrifuged, and stored at -20°C for subsequent Western blot experiments. GFP expression was detected to reflect miPEP060 expression, following the detailed Western blot procedure outlined above.

[0045] 1.5 CCK8 Experiment The cells in good growth condition were seeded into 96-well culture plates. Before seeding, the cell suspension was counted to ensure that the number of cells seeded in each well was approximately 1000. The plates were then incubated at 37°C. After the cells had fully adhered, different concentrations of Olaparib, ICRF-193, HU, and CPT were added to the 96 wells and incubated at 37°C for 72 h (Olaparib) and 48 h (ICRF-193, HU, and CPT), respectively. After the drug treatment was completed, 100 μl of 10% CCK8 solution was added to each well of the 96-well plate, and the plates were incubated for 2 h before the OD450 value was measured using a microplate reader.

[0046] 1.6 Xenograft Tumor Model Experiment The plasmids plvx-GFP, plvx-GFP-LINC00060, and plvx-GFP-miPEP060 were constructed according to the above plasmid construction method, with EcoRI and BamHI restriction sites selected. These plasmids were then transfected into well-growing SKOV3 cells, and purine mold was added to a final concentration of 1 μg / ml. white Drug screening was performed until stable cell lines expressing GFP, LINC00060, or miPEP060 were obtained. These stable cell lines were then amplified, digested with trypsin, centrifuged, and resuspended in a small amount of pre-chilled PBS. An appropriate amount of cell suspension was aspirated, diluted, and the cells were counted to adjust the cell density to 7 × 10⁶ cells / mL. 6 Cells / ml. 100 μl of cell suspension was inoculated subcutaneously into 4-6 week old female BALB / c nude mice using a 1 ml medical syringe. Inoculation was performed when the tumor volume reached 100 mm². 3 Mice were randomly divided into two groups: ① Control group: intraperitoneal injection of saline; ② Drug group: intraperitoneal injection of Olaparib (50 mg / kg). Tumor size, mouse weight, and survival time were recorded daily. After the experiment, the nude mice were sacrificed, and the tumors were collected and weighed.

[0047] 2. Results 2.1 CCK8 assay showed that knockdown of LINC00060 significantly reduced the sensitivity of SKOV3 cells to Olaparib. After knockdown of LINC00060, reversal to LINC00060 or miPEP060 restored the chemosensitivity of Olaparib. Figure 3 A).

[0048] 2.2 Knockdown and rotation effects of LINC00060, and the effect of miPEP060 are shown in the figure ( Figure 3 B).

[0049] 2.3 Nude mouse tumorigenesis experiments showed that mice overexpressing LINC00060 and the micropeptide miPEP060 exhibited significantly reduced tumor volume after Olaparib treatment, while mouse body weight remained largely unchanged. Figure 3 CE).

[0050] 2.4 Knockdown of LINC00060 significantly reduced the sensitivity of ovarian cancer cells (SKOV3, A2780, HO8910) to the chemotherapy drugs Olaparib, CPT, ICRF-193, and the replication stressor HU. Figure 4 ). Example 3

[0051] This example demonstrates that the miPEP060-TAT polypeptide has a significant effect in improving the sensitivity of tumors to chemotherapy. 1. Method Synthesis of miPEP060-TAT peptide A TAT transmembrane sequence (YGRKKRRORRR) with strong cell membrane penetration capability was selected and coupled to the C-terminus of the miPEP060 sequence to synthesize a miPEP060-TAT polypeptide that can directly act on cells. The synthesized powder was centrifuged at low temperature and then prepared into a stock solution with an appropriate amount of PBS to a final concentration of 20 mM.

[0052] CCK8 Experiment Ovarian cancer SKOV3 cells in good growth condition were seeded into 96-well plates at a density of 2000 cells per well. After cell attachment, miPEP060-TAT peptide at a final concentration of 5 μM was added to the cells for 24 h. Different concentrations of olaparib, cisplatin (CDDP), etoposide (ETO), and replication stressors HU and CPT were then added. The cells were incubated at 37°C for 48 h (CDDP, ETO, HU, and CPT) and 72 h (olaparib), respectively. After drug treatment, 100 μl of 10% CCK8 solution was added to each well of the 96-well plate, and the cells were incubated for 2 h before the OD450 value was measured using a microplate reader.

[0053] 1.3 Cloning Experiment Ovarian cancer SKOV3 cells were seeded into 12-well plates at a density of 200 cells per well and cultured in a cell culture incubator. The next day, after cell attachment, 2.5 μM miPEP060-TAT peptide was added to the plate for 24 h. After 24 h of treatment, 0, 10 μM Olaparib was added. The medium was changed every three days. After 10 days, the 12-well plates were removed, the culture medium was aspirated, and the plates were washed with ddH2O. 600 μL of methanol was added for fixation, and the plates were incubated at room temperature for 30 min. After aspirating the methanol fixative, 600 μL of 0.1% crystal violet solution was added, and the plates were incubated at room temperature for 20 min. The crystal violet solution was then recovered, and the plates were washed with ddH2O. After air drying, data were recorded using a mobile phone.

[0054] 1.4 Xenograft Tumor Model Experiment SKOV3 cells in good growth condition were expanded, digested with trypsin, centrifuged, and resuspended in a small amount of pre-chilled PBS. An appropriate amount of cell suspension was aspirated and the cell density was adjusted to 7 × 10⁶ cells / mL. 6 Cells / ml. 100 μl of cell suspension was injected subcutaneously into 4-6 week old female BALB / c nude mice using a 1 ml medical syringe. Inoculation was performed when the tumor volume reached 100 mm². 3 Mice were randomly divided into four groups: ① Control group: intraperitoneal injection of saline; ② Olaparib group: intraperitoneal injection of Olaparib (50 mg / kg); ③ miPEP060-TAT peptide group: intraperitoneal injection of miPEP060-TAT peptide (5 mg / kg); ④ miPEP060-TAT peptide combined with Olaparib group: intraperitoneal injection of miPEP060-TAT peptide (5 mg / kg) and Olaparib (50 mg / kg). Tumor size, mouse weight, and survival time were recorded daily. After the experiment, nude mice were sacrificed, and tumors were collected and weighed.

[0055] 2. Results 2.1 Schematic diagram of mi-PEP060 micropeptide conjugated with TAT membrane-penetrating peptide to form mi-PEP060-TAT polypeptide ( Figure 5 ) 2.2 CCK8 assay results showed that miPEP060-TAT peptide significantly improved the chemosensitivity of tumor cells to Olaparib ( Figure 6 A).

[0056] 2.3 Cloning experiments showed that the number of clones in the combination of miPEP060-TAT peptide and Olaparib was significantly lower than that in the Olaparib monotherapy group. Figure 6 B).

[0057] 2.4 Tumor formation experiments in nude mice showed that the combined treatment of miPEP060-TAT peptide and Olaparib significantly reduced tumor volume, while the body weight of mice did not change significantly. Figure 6 CE).

[0058] 2.5 The results of the CCK8 experiment showed that the miPEP060-TAT peptide significantly improved the sensitivity of tumor cells to the chemotherapeutic drugs cisplatin (CDDP), etoposide (ETO), camptothecin (CPT), and the replication stressor HU. Figure 7 ). Example 4

[0059] This embodiment demonstrates that LINC00060 and its encoded peptide miPEP060 have the effect of inhibiting PARylation modification, which leads to replication fork instability. 1. Method 1.1 DNA fiber experiment The knockdown of LINC00060, the full-length transcription, and the rotation method encoding the micropeptide miPEP060 were consistent with those described in Example 2. SKOV3 cells of shNC, shLINC00060, shLINC00060+LINC00060, and shLINC00060+miPEP060 were seeded in 35 mm dishes at a seeding density of approximately 70%. After 24 hours, the initiation and degradation of the replication fork were detected using different thymidine analogs (IdU and CldU). The cell treatment method for replication fork initiation was as follows: first, label with IdU (0.1 mM) for 20 min; after labeling, wash twice with pre-warmed PBS; then treat with HU (2 mM) for 1 h; finally, wash twice with PBS and label with CldU for 30 min. The treatment method for the degradation of newly synthesized DNA was as follows: IdU (0.1 mM) was labeled for 20 min, washed twice with PBS, then labeled with CldU (0.1 mM) for 30 min, and finally washed twice with PBS and treated with HU (5 mM) for 5 h.

[0060] After cell treatment, the cells were digested with trypsin and centrifuged. After resuspending the cells in PBS, they were counted to ensure the cell suspension density was 1 × 10⁻⁶. 6Cells were counted per ml, and then the labeled cells were diluted with unlabeled cell suspension at a ratio of 4:1 (unlabeled cells:labeled cells). 5 μl of the diluted cell suspension was added to an adhesive slide, followed by 15 μl of lysis buffer (0.5% SDS + 0.2 M Tris-HCl + 50 mM EDTA). The mixture was gently blown around to mix, and then allowed to lyse for 7 min. After lysis, the slide was tilted at approximately 15° to allow DNA to flow slowly along the slide and form DNA fibers. The slide was then thoroughly air-dried and fixed with freshly prepared methanol-acetic acid (3:1, v / v) fixative for 5 min. The fixed slide was activated with methanol for 5 min, washed three times with PBS, and denatured at 37°C with 2.5 M hydrochloric acid (HCl) for 2 h. The slide was then washed three times with PBS and blocked with 5% BSA at 37°C for 1 h. After blocking, the slides were incubated with primary antibodies IdU (1:200) and CldU (1:400) (dissolved in 1% BSA) at 37°C for 2 h. Then, the slides were washed three times with PBST (0.1% Triton X-100 dissolved in PBS), and incubated with secondary antibodies (Alexa Fluor 488 AffiniPure Donkey Anti-Rat IgG (H+L) (1:150) and Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L) (1:150)) (dissolved in 1% BSA) at 37°C for 1 h. After incubation, the slides were washed three times with PBST, thoroughly air-dried, mounted with mounting medium, and stored at room temperature. The slides were observed and photographed using a confocal microscope (63×), and fiber length was calculated using ImageJ software.

[0061] 1.2 Western blot experiment The following protein samples were collected: ① shNC, shLINC00060, shLINC00060+LINC00060, and shLINC00060+miPEP060 cells were treated with 1 mM H2O2 at 4 ℃ in the dark for 30 min to induce parylation modification. After lysis with an appropriate amount of 1×SDS loading buffer, the samples were boiled in a metal bath at 95 ℃ for 15 min to obtain protein samples. ② SKOV3 cells were treated with different concentrations of miPEP060 transmembrane peptides (0, 20, 40, 60, 80 μM) for 24 h, and protein samples were collected. The parylation modification level in the collected protein samples was detected according to the above Western blot experimental method.

[0062] 1.3 In vitro PARation experiment The synthesized miPEP060 was mixed with Olaparib and recombinant PARP1 enzyme and reacted at room temperature for 10 min. Then, the PARP1 mixture was incubated with double-stranded DNA (75 nM) and NAD+ (200 mM) in reaction buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, and 1.5 mM DTT) at room temperature for 6 min. After the reaction was complete, the reaction was quickly terminated with 2×SDS loading buffer and boiled at 95°C for 15 min to obtain protein samples. The level of parylation modification was detected by Western blot.

[0063] 2. Results 2.1 Knockdown of LINC00060 significantly increased the length of CldU-labeled DNA and the CldU / IdU ratio, suggesting that knockdown of LINC00060 can protect nascent DNA strands from degradation and promote replication fork restart. Reversing the full-length LINC00060 or using the micropeptide miPEP060 significantly reduced the length of CldU-labeled DNA and the CldU / IdU ratio in LINC00060 knockdown cells, indicating that the micropeptide imPEP060 mediates the restoration of replication fork stability induced by LINC00060 knockdown. Figure 8 AB).

[0064] 2.2 Western blot experiments showed that knockdown of LINC00060 significantly increased the level of parylation modification. Reversing the full-length LINC00060 or the micropeptide miPEP060 could reverse the parylation inhibition caused by LINC00060 knockdown, indicating that the micropeptide miPEP060 mediates the reduction in parylation modification induced by LINC00060. Figure 8 C).

[0065] 2.3 Western blot experiments showed that miPEP060-TAT peptide significantly reduced PARylation modification in SKOV3 ovarian cancer cells. Figure 8 D).

[0066] 2.4 In vitro PARylation experiments showed that miPEP060 inhibited PARP1-catalyzed PARylation modification, and the higher the concentration of miPEP060, the stronger the inhibitory effect on PARylation modification, indicating that the peptide miPEP060 directly inhibits the catalytic activity of PARP1 enzyme. Figure 8 E). Example 5

[0067] This embodiment proposes the detection of LINC00060 and its encoded micropeptide miPEP060 in clinical samples; method Real-time quantitative PCR experiment Ten ovarian cancer patients had their tissue samples embedded in paraffin-embedded blocks. Each patient's tissue included both normal and cancerous tissue. RNA was extracted from adjacent normal and cancerous tissues using a total RNA extraction kit for paraffin-embedded tissue sections, following the kit's instructions. The extracted RNA was then reverse transcribed into cDNA using reverse transcriptase. Real-time quantitative PCR was then used to detect the expression of LINC00060 in both adjacent normal and cancerous tissue samples.

[0068] Immunohistochemistry Prepared paraffin tissue sections (including 10 normal tissues and 19 cancer tissues) were dewaxed with xylene, hydrated with graded ethanol, and then subjected to microwave antigen retrieval using sodium citrate buffer (pH 6.0) at 95°C for 15 minutes. After cooling, they were incubated with 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase activity, followed by blocking with 5% goat serum at room temperature for 30 minutes. Primary antibody (anti-miPEP060, diluted 1:50) was added and incubated overnight at 4°C. The next day, horseradish peroxidase (HRP)-labeled secondary antibody was added and incubated at room temperature for 30 minutes. Color development was performed using diaminobenzidine (DAB) chromogenic solution, with the development time controlled under a microscope until a positive signal appeared and the background was clear. Cell nuclei were counterstained with hematoxylin, dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin.

[0069] 2. Results 2.1 Among 10 patients with ovarian cancer, LINC00060 expression was significantly decreased in 6 patients, significantly upregulated in 2 patients, and showed no significant difference in LINC00060 expression in 2 patients. Figure 9 A).

[0070] 2.1 Immunohistochemical results showed that miPEP060 was significantly underexpressed in ovarian cancer patients ( Figure 9 B).

[0071] sequence list SEQ ID No.1 cacattcggg aagcgtcggg attaggtgaa agtacgtagt tgtctttcgt aagttaaaatgaaattggg ccgaaactta ctgccttacc taaaaggcag cgcagtcagg atattggtag gtcgggggcggctttggaaa cccttaagtt tacaagcatg cgcggacttggcatgcct aggtgg gcgtccacgtgcagccctgg accctgaacc ccggcgtgcg tgggccgtgg gccctcgggg aaaggttccg tgcactcggggactccggtg aagcctgttc agccgtctgt gtcatgtggc catcttgagt ctactctgtc gctcttgtgccctagccgcgcgcgaggc caggagcgc ctgagctgaa cacattacga tggatgatggaaacataaga ctatcaagaa atccaagtgg taatgggcga agtttattca gcatccggca atggacttatcgtagttggg gaaacgggtg ttccgaataa tatcctggaa gttatcagga cacctatttt aatataggcctgaatttgtaagttaagttagttagttagtta tttagtaga tgcttaattc atgtggctaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaaa SEQ ID No.2 atggatgatggaaacataagactatcaagaaatccaagtggtaatgggcgaagtttattcagcatccggcaatgacttatcgtagttggggaaacgggtgttccgaataa SEQ ID No.3 MDDGNIRLSRNPSGNGRSLFSIRQWTYRSWGNGCSE-GGS-{YGRKKRRORRR} Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The micropeptide miPEP060 encoded by the LINC00060 gene, characterized in that, The nucleotide sequence of the micropeptide miPEP060 is shown in SEQ ID No.

2.

2. The micropeptide miPEP060 encoded by the LINC00060 gene according to claim 1, characterized in that, The nucleotide sequence of the LINC00060 gene is shown in SEQ ID No.

1.

3. The use of the micropeptide miPEP060 and / or LINC00060 gene according to claim 1 or 2 for the preparation or screening of tumor therapeutic drugs.

4. The use according to claim 3, characterized in that, This includes two aspects: first, directly applying the miPEP060 and / or LINC00060 genes to the preparation of tumor therapeutic drugs or formulations; second, using the miPEP060 and / or LINC00060 genes as targets for drugs or formulations targeting tumor cells in the screening of tumor therapeutic drugs or formulations.

5. The use according to claim 4, characterized in that, When directly applied to the preparation of tumor therapeutic drugs or formulations, the miPEP060 and / or LINC00060 genes are used to prepare products that inhibit tumor progression, or to prepare products that enhance the efficacy of tumor chemotherapy, or to prepare products that overcome tumor chemotherapy resistance.

6. The application of the micropeptide miPEP060 and / or LINC00060 according to claim 1 or 2 in the preparation of detection reagents, characterized in that, The micropeptide miPEP060 and / or LINC00060 serve as biomarkers, containing a monoclonal antibody against the micropeptide miPEP060, and are used to assess or assist in assessing the sensitivity of cancer patients to chemotherapy drugs and predict the prognostic status of cancer patients after chemotherapy.

7. A product having any of the functions listed below is used in at least one of the following: 1)-8) a. Promotes the expression of the LINC00060 gene; b. Promote the biological functions of LINC00060 and the encoded micropeptide miPEP060; c. Promote the biological function of fusion proteins containing miPEP060; d. Promotes the biological function of complexes containing miPEP060; 1) Preparation of tumor treatment products; 2) To prepare products that reduce tumor chemotherapy resistance; 3) To prepare products that improve the sensitivity of tumors to chemotherapy; 4) Prepare products that inhibit tumor drug resistance caused by abnormal PARP1 enzyme activity or PARylation; 5) To prepare products that inhibit tumor drug resistance caused by the restoration of replication fork stability; 6) Prepare products that suppress replication stress response and replication fork stability; 7) Prepare products that inhibit PARP1 enzyme activity or are modified by PARylation; 8) Preparation of products for use in combination with other antitumor drugs or methods; The nucleotide sequence of the LINC00060 gene is shown in SEQ ID No. 1, and the nucleotide sequence of the micropeptide miPEP060 is shown in SEQ ID No.

2.

8. The use according to claim 7, characterized in that, The tumors mentioned include, but are not limited to, ovarian cancer, breast cancer, lung cancer, nasopharyngeal carcinoma, stomach cancer, colorectal cancer, liver cancer, esophageal cancer, pancreatic cancer, breast cancer, cervical cancer, prostate cancer, ovarian cancer, bladder cancer, thyroid cancer, and skin cancer.

9. The micropeptide miPEP060 according to claim 1, characterized in that, The amino acid sequence of the micropeptide miPEP060-TAT polypeptide is shown in SEQ ID No. 3; the transmembrane peptide sequence of micropeptide miPEP060 includes, but is not limited to, the TAT sequence.

10. The inhibitor of the micropeptide miPEP060 and / or LINC00060 according to claim 1 or 2 comprises, but is not limited to, shRNA, the target sequence of which is shown in SEQ ID No. 4.

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