A novel micropeptide MP36 and application thereof

Abstract

A novel micropeptide, MP36, and its applications are provided. This MP36 micropeptide can be used to prepare reagents or drugs for the detection, prevention, or treatment of tumors, including one or more of lung cancer, breast cancer, colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer, and sarcoma. In tumors, the MP36 micropeptide can inhibit tumor cell growth and / or metastasis by specifically inhibiting MP36 expression via small interfering RNA; or by overexpressing MP36 to inhibit tumor cell growth and / or metastasis; or by conjugating a membrane-penetrating peptide to inhibit tumor growth and / or metastasis. It has significant value for new drug development and tumor detection.

Description

A novel micropeptide MP36 and its application Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a micropeptide MP36 with a completely new structure and its application. Background Art

[0002] Long noncoding RNAs (lncRNAs) are RNA molecules with transcripts longer than 200 nucleotides (nt). They lack specific, complete open reading frames (ORFs) and have no protein-coding function. They account for a significant proportion of all noncoding RNAs (ncRNAs). Initially, lncRNAs were considered "noise" in genomic transcription, devoid of biological function. However, recent studies have shown that lncRNAs, acting as RNAs, regulate gene expression at multiple levels, including epigenetic, transcriptional, and post-transcriptional regulation. However, it was still widely believed that lncRNAs did not encode proteins. In 2011, American scientist Jonathan S. Weissman used ribosome display technology to discover that a large number of lincRNAs were bound to ribosomes, indicating that they could be translated. In 2015, Eric Olson and colleagues at the University of Texas Southwestern Medical Center discovered a lncRNA specifically expressed in skeletal muscle and confirmed that this lncRNA encodes a 46-amino acid micropeptide, which they named myoregulin. In the past two years, there have been few international reports on lncRNAs encoding micropeptides, with research primarily focused on muscle differentiation and skeletal muscle development. Therefore, discovering and identifying new lncRNA-encoded micropeptides and exploring new applications are crucial for unlocking the potential of non-coding RNAs.

[0003] The long noncoding RNA C5ORF66-AS1, also known as Epist, is located on human chromosome 5. Studies have reported that C5ORF66-AS1 expression is significantly downregulated in human esophageal squamous cell carcinoma and pituitary adenomas, effectively inhibiting tumor malignant progression and invasion. However, whether C5ORF66-AS1 plays a role in other malignancies or is associated with other diseases remains unknown. Malignant tumors are a leading cause of death in humans. Uncontrolled growth, invasion, and metastasis are hallmarks of malignancy and the main causes of treatment failure and death. Currently, drug therapy remains one of the mainstays of clinical treatment for cancer patients. However, the high metastatic potential of tumors and the heterogeneity of tumor cells severely limit their effectiveness. Continuous research into the causes of tumors, the development of treatment and prevention strategies, and the development of new and effective drugs are key to clinical cancer treatment. Summary of the Invention

[0004] The first purpose of the present invention is to provide a novel micropeptide MP36, which is a polypeptide with a completely new structure discovered for the first time.

[0005] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0006] A micropeptide, wherein the amino acid sequence of the micropeptide is any one of the following:

[0007] (a) the amino acid sequence shown in SEQ ID NO. 1;

[0008] (b) an amino acid sequence having at least 85% homology to the amino acid sequence shown in SEQ ID NO. 1;

[0009] (c) An amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.1.

[0010] The second object of the present invention is to provide a nucleotide sequence encoding the above-mentioned micropeptide.

[0011] The third object of the present invention is to provide a recombinant vector containing the above nucleotide sequence.

[0012] The fourth object of the present invention is to provide the use of the above-mentioned micropeptide in the preparation of a reagent or drug for detecting, preventing or treating tumors. The micropeptide is used in the form of amino acids or nucleotides.

[0013] As a preferred embodiment, the tumor is one or more of human lung cancer, breast cancer, colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer, and sarcoma.

[0014] As a preferred embodiment, the tumor treatment is achieved by overexpressing the nucleotide sequence shown in SEQ ID NO. 2 in the tumor; or using a small interfering RNA specific for the nucleotide sequence shown in SEQ ID NO. 2; or coupling the micropeptide with a cell-penetrating peptide. Specifically, for breast cancer and lung cancer, the tumor is treated by inhibiting MP36 expression using a small interfering RNA specific for the nucleotide sequence shown in SEQ ID NO. 2; for colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer, and sarcoma tissue, the tumor is treated by overexpressing the nucleotide sequence shown in SEQ ID NO. 2 in the tumor.

[0015] As a preferred embodiment, the nucleotide sequence of the specific small interfering RNA is shown in SEQ ID NO: 5.

[0016] As a preferred embodiment, the prevention or treatment of tumors includes inhibiting tumor cell growth and / or metastasis.

[0017] As a preferred embodiment, the tumor cells include: head and neck cancer cancer cells: CAL27 and / or KB; colon cancer cancer cells: HT29 and / or SW480; brain glioma cancer cells: U87-MG; bladder cancer cancer cells: T24 and / or EJ; gastric cancer cancer cells: MGC-803 and / or BGC-823; sarcoma cancer cells: MG63 and / or HT-1080; breast cancer cancer cells: MDA-MB-231 and / or MCF7; lung cancer cancer cells: A459.

[0018] The fifth object of the present invention is to provide a pharmaceutical composition containing the above-mentioned micropeptide or a specific small interfering RNA of the micropeptide or a molecule that promotes the degradation of the micropeptide and a pharmaceutically acceptable carrier thereof. Specifically, for breast cancer and lung cancer, the expression of MP36 is inhibited by a pharmaceutical composition containing a specific small interfering RNA of the micropeptide and a pharmaceutically acceptable carrier thereof, or MP36 is degraded by a pharmaceutical composition containing or promoting the degradation of the micropeptide and a pharmaceutically acceptable carrier thereof to reduce its expression level, thereby achieving the prevention or treatment of tumors; for colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer and sarcoma tissue, the prevention or treatment of tumors is achieved by a pharmaceutical composition containing a micropeptide and a pharmaceutically acceptable carrier thereof. When the pharmaceutical composition contains a micropeptide, the micropeptide may exist in the form of a free peptide, an overexpression vector containing a micropeptide gene, etc.

[0019] The sixth object of the present invention is to provide a tumor detection kit, which contains specific primer pairs and / or antibodies designed for the above-mentioned nucleotide sequence.

[0020] As a preferred embodiment, the nucleotide sequences of the specific primer pair are shown as SEQ ID NO.3 and SEQ ID NO.4.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) This study first discovered that the long noncoding RNA C5ORF66-AS1 has the ability to encode and translate a 36-amino acid micropeptide (named MP36);

[0023] (2) The present invention, through expression profile chip data analysis, found that the expression of MP36 in breast cancer and lung cancer was significantly higher than that in normal tissues, and the expression level in colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer and sarcoma tissues was significantly lower than that in normal tissues, indicating that MP36 can be used as a new tumor marker for auxiliary diagnosis of early tumors;

[0024] (3) The present invention designed specific primers encoding the nucleotide sequence of MP36 and used the fluorescence quantitative PCR method to detect the RNA expression level of MP36 in head and neck cancer or breast cancer samples, as well as the corresponding adjacent cancer tissues. It was found that MP36 was significantly underexpressed in head and neck cancer tissues and significantly overexpressed in breast cancer tissues, suggesting that specific primers designed for the MP36 sequence can be used for tumor diagnosis;

[0025] (4) The present invention prepared specific antibodies against MP36 and used immunohistochemistry to detect MP36 levels in breast cancer and adjacent tissue samples. It was found that MP36 was significantly overexpressed in breast cancer tissue, suggesting that antibodies against MP36 can be used for tumor diagnosis.

[0026] (5) Through extensive experiments, the present invention has found that endogenously overexpressed micropeptide MP36 can significantly inhibit the growth and / or metastasis of head and neck cancer CAL27 cells;

[0027] (6) The present invention designed specific siRNA targeting the nucleotide sequence encoding the micropeptide MP36 and found that it can significantly inhibit the growth and / or metastasis of breast cancer MDA-MB-231 and MCF7 cells, and inhibit the growth of lung cancer A549 cells.

[0028] (7) The present invention conjugated TAT transmembrane peptide with MP36 micropeptide sequence and found that it could significantly inhibit the growth and / or metastasis of head and neck cancer CAL27 and / or KB cells, colon cancer HT29 and / or SW480 cells, brain glioma U87-MG cells, bladder cancer T24 and / or EJ cells, gastric cancer MGC-803 and / or BGC-823 cells, and sarcoma MG63 and / or HT-1080 cells.

[0029] (8) The experiments designed in the present invention are scientific, reasonable, feasible and effective. Based on the above findings, the expression level of MP36 can be used as a new biomarker to help diagnose malignant tumors including lung cancer, breast cancer, colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer and sarcoma. The micropeptide MP36 can be directly used as a therapeutic drug for malignant tumors or as a target for anti-tumor drugs, which greatly expands the therapeutic spectrum of the micropeptide and provides new ideas and prospects for future drug development. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 shows the expression of the target band of MP36 detected by western blot.

[0031] Figure 2 shows the comparison of C5ORF66-AS1 expression levels in human tumor tissues and normal tissues. The data are from the TCGA database.

[0032] Figure 3 shows the expression of micropeptide MP36 in human head and neck cancer tissues and adjacent tissues detected by fluorescence quantitative PCR.

[0033] Figure 4 shows the expression of micropeptide MP36 in human breast cancer tissues and adjacent tissues detected by immunohistochemistry.

[0034] Figure 5 shows the inhibitory effect of micropeptide MP36 on the proliferation of human head and neck cancer CAL27 cells.

[0035] Figure 6 shows the inhibitory effect of micropeptide MP36 on the migration of human head and neck cancer CAL27 cells.

[0036] Figure 7 shows the inhibitory effect of siRNA of micropeptide MP36 on the proliferation of human breast cancer MDA-MB-231 cells.

[0037] Figure 8 shows the inhibitory effect of siRNA of micropeptide MP36 on the proliferation of human breast cancer MCF7 cells.

[0038] FIG9 shows the inhibitory effect of siRNA of micropeptide MP36 on the proliferation of human lung cancer A549 cells.

[0039] FIG10 shows the inhibitory effect of siRNA of micropeptide MP36 on the migration of human breast cancer MDA-MB-231 cells.

[0040] FIG11 shows the inhibitory effect of siRNA of micropeptide MP36 on the migration of human breast cancer MCF7 cells.

[0041] FIG12 shows the effect of siRNA of micropeptide MP36 on the migration of human lung cancer A549 cells.

[0042] FIG13 shows the effects of micropeptide MP36 and its homologous mutants on the proliferation of human breast cancer MDA-MB-231 cells.

[0043] FIG14 shows the effects of micropeptide MP36 and its homologous mutants on the migration of human breast cancer MDA-MB-231 cells. DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to specific embodiments.

[0045] Example 1

[0046] Confirmation of the in vivo coding capacity of C5ORF66-AS1.

[0047] Using CRISPR / Cas9 gene editing technology, a Flag tag was site-specifically inserted before the stop codon of the sORF contained in C5ORF66-AS1. After sequencing confirmed the correct insertion of the target cells, the cells were amplified, harvested, centrifuged, and the supernatant discarded. The cells were rinsed twice with PBS and the supernatant discarded. RIPA lysis buffer was added and lysed on ice for 20 minutes. The cells were centrifuged at 12,000 g for 10 minutes, and the supernatant was collected. 1X SDS loading buffer was added, pipetted to mix, and then denatured by boiling for 5 minutes. Total proteins were separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The membrane was blocked with 5% BSA at room temperature for 2 hours, incubated with a Flag antibody (abcam) overnight at 4°C, and washed three times with TBST. Secondary antibody was incubated for 1 hour at room temperature, and the membrane was washed three times with TBST. The membrane was developed with ECL ultrasensitive chemiluminescence solution, and the presence of the target band was detected using a Tannon imaging system.

[0048] The results are shown in Figure 1 . The Flag band was detected by Western blot, indicating that C5ORF66-AS1 does have coding ability.

[0049] Example 2

[0050] Analysis of MP36 expression in human tumor tissues and normal tissues.

[0051] The TCGA standard method was used to download RNA-seq sequencing files and clinical information of cancerous and normal tissues of 32 tumor types, including head and neck cancer, brain glioma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, liver cancer, gastric cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, colorectal cancer, pancreatic cancer, osteosarcoma, and skin cancer. Statistical analysis was performed using R language (version 3.1.1) software. The packages (heatmap, venndiagram, hist, etc.) needed to be installed and loaded. Then, the DESeq and edgeR packages were used to analyze the expression level of C5ORF66-AS1 and identify tumor types with differential expression (as shown in Table 1). The criteria for differential expression were: (1) |Expression level of cancer / adjacent tumor|>2, (2) P<0.05.

[0052] Table 1 Analysis of MP36 expression in human tumor tissues and normal tissues (cancer / paracancer)

[0053]

[0054] As shown in Table 1 and Figure 2, the expression of MP36 in cancerous tissues and adjacent tissues was analyzed compared with normal tissues. It was found that compared with adjacent tissues, MP36 was significantly overexpressed in breast cancer and lung cancer, and its expression level was significantly reduced in colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer, and sarcoma tissues.

[0055] Example 3

[0056] Fluorescence quantitative PCR was used to detect the expression of MP36 in head and neck cancer patients and normal adjacent tissues.

[0057] (1) Specimen collection

[0058] With the patients' informed consent, head and neck cancer and adjacent tissue specimens were collected during surgery, washed with saline, and stored in liquid nitrogen or a -80°C freezer for later use.

[0059] (2) Primer design

[0060] Specific primers were designed based on the sequence information of MP36, and the sequences are as follows:

[0061] Upstream primer (SEQ ID NO. 3): aggcccctccacggaggt

[0062] Downstream primer (SEQ ID NO.4): ctccctcggggcgtaggct

[0063] (3) Real-time quantitative PCR was used to detect the expression of MP36 in head and neck cancer patients and normal adjacent tissues.

[0064] Total RNA from the collected samples was extracted using Trizol according to Tiangen Bio's instructions. The purity and concentration of the extracted RNA were quantified using a NanoDrop ND-1000 nucleic acid quantifier. Agarose gel quality control was performed to ensure the integrity of the extracted RNA. The extracted total RNA was reverse transcribed into cDNA using the TaKaRa PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time). qPCR reactions were performed using the TaKaRa SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) kit. The reaction system is shown in Table 2:

[0065] Table 2 PCR reaction system

[0066]

[0067] After mixing the above components evenly, the following procedure was followed: pre-denaturation at 95°C for 30 s, 40 cycles; 95°C for 5 s, 60°C for 30 s.

[0068] The specificity of the reaction was determined based on the melting curve, and the formula 2 -ΔΔCtThe relative expression of MP36 was calculated. The results are shown in Figure 3. In approximately 75% of head and neck cancer samples, MP36 expression levels were significantly lower than those in normal adjacent tissues. The expression trend was similar to that analyzed in the TCGA database, indicating that MP36 could be a potential biomarker for the diagnosis of head and neck cancer.

[0069] Example 4

[0070] Immunohistochemistry was used to detect the expression of MP36 in breast cancer patients and normal adjacent tissues.

[0071] Paraffin microarrays of 40 breast cancer clinical tissue samples and 10 adjacent adjacent tissue samples were collected and immunohistochemistry was performed using the monoclonal antibody against MP36. The specific procedures were as follows:

[0072] (1) After obtaining the tissue wax block, slice it and bake it at 60℃ for 3 hours.

[0073] (2) Dewaxing: Soak in xylene, xylene, and 50% ethanol for 10 min each.

[0074] (3) Rehydration: Soak in 100% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol for 10 minutes in this order, and finally wash in 50% ethanol in running water for 5 minutes.

[0075] (4) Wash with PBS three times, 10 min each time.

[0076] (5) Soak the sample in 3% hydrogen peroxide at room temperature for 10 minutes to inactivate endogenous HPR.

[0077] (6) Wash with PBS three times, 10 min each time.

[0078] (7) Use a microwave to heat the sample for antigen repair and then cool it to room temperature in a water bath.

[0079] (8) Wash with PBS three times, 10 min each time, circle the tissue with an immunohistochemistry pen, and dry for 15 s.

[0080] (9) Add 5% sheep serum blocking solution and block for 70 minutes.

[0081] (10) Incubate with MP36 primary antibody at 4°C overnight and wash three times with PBST, each time for 10 min.

[0082] (11) Incubate with secondary antibody at room temperature for 1 h, then wash with PBST three times, 10 min each time.

[0083] (12) Develop with DAB for 10 min, then rinse with PBST to terminate staining.

[0084] (13) Restain with hematoxylin for 7 min and rinse for 5 min.

[0085] (14) Dehydrate, seal the slides, and observe under a microscope.

[0086] The results are shown in Figure 4. Similar to the analysis of the TCGA database, MP36 was significantly overexpressed in breast cancer compared with adjacent adjacent tissue samples, indicating that MP36 can be used as a potential new indicator for breast cancer diagnosis.

[0087] Example 5

[0088] Effect of overexpression of MP36 on the proliferation of human head and neck cancer cells

[0089] MP36 was overexpressed in head and neck cancer cell CAL27 cells (named MP36-OE) using lentiviral transfection technology. The control CAL27 cells were named Ctrl. The cells in both groups were cultured in a 37°C, 5% CO2 incubator until the density exceeded 90%. The cells were then digested and harvested using trypsin. The cells were resuspended in culture medium and counted under a microscope. The cell concentration was adjusted to 3.0×10 4 Cell suspensions were plated into 96-well plates at a concentration of 100 μL per well and incubated in a 37°C, 5% CO2 incubator for 48 h. In a dark-protected environment, 10 μL of CCK8 reagent was added to each well and incubated in a 37°C, dark-protected water bath for 4 h. A microplate reader was preheated 30 min in advance. After the incubation, the absorbance of each well was measured at 450 nm. Data were analyzed. The experiment was repeated three times. Results are expressed as mean ± SD. T-tests were performed. *P < 0.05 indicated significant differences, and **P < 0.01 indicated extremely significant differences.

[0090] The results are shown in Figure 5. Compared with the control group, overexpression of MP36 can significantly inhibit the proliferation of head and neck cancer CAL27 cells.

[0091] This indicates that the new micropeptide MP36 can exert anti-tumor effects by inhibiting the proliferation of head and neck cancer cells.

[0092] Example 6

[0093] Effect of overexpression of MP36 on the migration ability of human head and neck cancer cells

[0094] MP36-overexpressing head and neck cancer cells and control cells, constructed using lentiviral transfection technology, were seeded into transwell chambers, with 100 μL per well. 0.6 mL of complete culture medium containing 10% FBS was then added to the lower chamber of the transwell to stimulate cell migration. Cells were incubated at 37°C in a 5% CO2 atmosphere for 48 h. The culture medium was discarded, and the cells were fixed with methanol for 30 min at room temperature. The cells were then stained with 0.1% crystal violet for 10 min at room temperature and rinsed with water. Unmigrated cells in the upper layer were removed with a cotton swab, and the cells were observed under a microscope, with four fields of view selected for imaging and counting. The experiment was repeated three times. Results are expressed as mean ± SD, and statistical t-tests were performed. *P < 0.05 indicates a significant difference, and **P < 0.01 indicates a highly significant difference.

[0095] The results are shown in Figure 6. Compared with the control group, overexpression of MP36 can significantly inhibit the migration of head and neck cancer CAL27 cells.

[0096] This indicates that the new micropeptide MP36 can exert anti-tumor effects by inhibiting the migration of head and neck cancer cells.

[0097] Example 7

[0098] Effects of MP36 Interference on the Proliferation of Human Breast Cancer and Lung Cancer Cells

[0099] A specific siRNA was designed based on the nucleotide sequence of MP36, and the specific sequence is shown in SEQ ID NO:5.

[0100] Breast cancer cells MDA-MB-231, MCF7 and lung cancer cells A549 were cultured in a 37°C, 5% CO2 incubator. The cells were digested 24 h in advance and plated in 12-well plates at a density of 8 × 10 4 / mL, and cell transfection was performed when the cell confluence reached 60%. Add 50 μL of DMEM medium to a 1.5 mL sterile EP tube, add 8 μL of transfection reagent, pipette to mix, and let it stand at room temperature for 5 minutes. Add 50 μL of DMEM medium to two other EP tubes, add 6 μL of siRNA (si-MP36 and si-NC) respectively, pipette to mix, and let it stand for 5 minutes. Add the transfection reagent mixture obtained in the first step dropwise to the second EP tube, pipette to mix, and incubate at room temperature for 20 minutes. While incubating, aspirate the original medium in the 6-well plate and replace it with fresh complete medium. After the incubation is completed, add the transfection mixture dropwise to the 6-well plate, shake to mix, and place it in the incubator for culture. Change the medium after 6 hours, and digest and carry out functional experiments after 48 hours. The cell proliferation experiment was performed as described in Example 4, except that the groups were si-NC and si-MP36. The experiment was repeated three times. The results were expressed as mean ± SD and subjected to statistical T test. *P < 0.05 indicated a significant difference, and **P < 0.01 indicated a very significant difference.

[0101] The results are shown in Figures 7-9. Compared with the control group, knockdown of MP36 can significantly inhibit the proliferation of breast cancer cells MDA-MB-231, MCF7 and lung cancer cells A549, indicating that targeted inhibition of the new micropeptide MP36 exerts an anti-tumor effect by inhibiting the proliferation of breast cancer and lung cancer cells.

[0102] Example 8

[0103] Effects of MP36 Interference on Migration Ability of Human Breast Cancer and Lung Cancer Cells

[0104] . Similarly, breast cancer cells MDA-MB-231, MCF7 and lung cancer cells A549 transfected with si-NC and si-MP36 respectively

[0105] 100 μL of culture medium was seeded into transwell chambers. Cell migration was stimulated by adding 0.6 mL of complete culture medium containing 10% FBS to the lower chamber of the transwell. The cells were incubated at 37°C in a 5% CO2 atmosphere for 48 h. The culture medium was discarded, and the cells were fixed with methanol for 30 min at room temperature. The cells were then stained with 0.1% crystal violet for 10 min at room temperature. The cells were rinsed with water, and the upper layer of unmigrated cells was removed with a cotton swab. The cells were observed under a microscope, and four fields of view were photographed and counted. The experiment was repeated three times. The results are expressed as mean ± SD, and statistical t-tests were performed. *P < 0.05 indicates a significant difference, and **P < 0.01 indicates a highly significant difference.

[0106] The results are shown in Figures 10-12. Compared with the control group, knockdown of MP36 can significantly inhibit the migration of breast cancer cells MDA-MB-231 and MCF7, but has no effect on the migration of lung cancer cells A549, indicating that targeted inhibition of the new micropeptide MP36 exerts an anti-tumor effect by inhibiting the migration of breast cancer.

[0107] Example 9

[0108] Using homologous mutations, we determined that MP36 acts as a micropeptide rather than a lncRNA.

[0109] To prove that it was the micropeptide MP36 rather than the lncRNA C5ORF66-AS1 that played a functional role, we used synonymous mutations to construct a lentiviral vector with the same amino acid sequence but altered RNA sequence and secondary structure, thereby overexpressing the micropeptide MP36 without changing the lncRNA level. The cell line with the MP36 synonymous mutation obtained by transfection was named 231-66TY cell line. At the same time, the start codon ATG of the MP36 open reading frame was mutated to ATT by lentiviral transfection, making this sequence unable to translate the micropeptide MP36, and the cell line was named 231-66Lnc cell line.

[0110] The results are shown in Figures 13-14. Compared with the control group, the 231-66TY cell line can promote cell proliferation and migration, while the 231-66Lnc cell line has no significant effect on cell proliferation and migration, proving that it is the micropeptide MP36 rather than the lncRNA that plays a functional role.

Claims

1. A micropeptide, characterized in that: The amino acid sequence of the micropeptide is any of the following: (a) the amino acid sequence shown in SEQ ID NO.1; (b) an amino acid sequence having more than 85% homology with the amino acid sequence shown in SEQ ID NO.1; (c) An amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID NO.

1.

2. A nucleotide sequence encoding the micropeptide according to claim 1.

3. A recombinant vector containing the nucleotide sequence of claim 2.

4. Use of the micropeptide according to any one of claims 1 to 3 in the preparation of reagents or drugs for detecting, preventing or treating tumors.

5. The use according to claim 4, characterized in that The tumor is one or more of human lung cancer, breast cancer, colon cancer, glioma, bladder cancer, gastric cancer, head and neck cancer and sarcoma.

6. The use according to claim 4, characterized in that: The tumor treatment is achieved by overexpressing the nucleotide sequence shown in SEQ ID NO.2 in the tumor; or using a specific small interfering RNA of the nucleotide sequence shown in SEQ ID NO.2; or coupling the micropeptide with a membrane-penetrating peptide.

7. The use according to claim 4, characterized in that: The nucleotide sequence of the specific small interfering RNA is shown in SEQ ID NO:

5.

8. The use according to claim 4, characterized in that: The prevention or treatment of tumors includes inhibiting tumor cell growth and / or metastasis.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the micropeptide according to claim 1 or a specific small interfering RNA of the micropeptide or a molecule that promotes the degradation of the micropeptide and a pharmaceutically acceptable carrier thereof.

10. A tumor detection kit, characterized in that: The kit contains a specific primer pair and / or an antibody designed for the nucleotide sequence of claim 2; preferably, the nucleotide sequence of the specific primer pair is as shown in SEQ ID NO.3 and SEQ ID NO.4.

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