NOVEL MICROPEPTIDE HMMW AND APPLICATION THEREOF

Abstract

A micropeptide HMMW of a new structure and an application thereof, and relates to the field of biomedical research and development is prpvided. The micropeptide HMMW is obtained by encoding human lncRNA, and a recombinant vector is constructed so that objective cells perform high expression on the micropeptide HMMW, which can inhibit proliferation and migration of multiple solid tumors including the head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, stomach cancer, breast cancer, kidney cancer, skin cancer and the like, and growth of tumors in the body. The micropeptide HMMW has potential value for new drug development, important tumor detection and treatment value.

Claims

1. An application of a novel micropeptide HMMW in the preparation of tumor detection reagents or tumor treatment drugs, wherein the amino acid sequences of the micropeptide HMMW are amino acid sequences having at least 85% homology to the amino acid sequence shown in SEQ ID NO: 1.

2. The application of a novel micropeptide HMMW in the preparation of tumor detection reagents or tumor treatment drugs according to claim 1, wherein the amino acid sequences of the micropeptide HMMW contain the amino acid sequence shown in SEQ ID NO: 1.

3. The application of a novel micropeptide HMMW in the preparation of tumor detection reagents or tumor treatment drugs according to claim 1, wherein the novel micropeptide HMMW is any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9.

4. A nucleotide, wherein the nucleotide is any of (a), (b) and (c): (a) a nucleotide sequence encoding micropeptide HMMW containing the amino acid sequence shown in SEQ ID NO: 2; (b) a nucleotide encoding micropeptide HMMW containing the amino acid sequence shown in SEQ ID NO: 1; and (c) nucleotide sequences encoding the nucleotide sequences shown in SEQ ID NO: 10 to SEQ ID NO: 18, with N being any of A/T/G/C.

5. A recombinant vector, wherein the recombinant vector contains any of the nucleotides described in claim 4.

6. An application of the nucleotides in claim 4 or a recombinant vector containing any of the nucleotides described in claim 4 in the preparation of tumor detection reagents or tumor treatment drugs.

7. A tumor detection kit, wherein the kit contains a specific primer pair designed for the nucleotide sequences described in claim 4.

8. The tumor detection kit according to claim 7, wherein the specific primer pair is shown in SEQ ID NO: 19 and SEQ ID NO: 20.

9. The tumor detection kit according to claim 7, wherein the tumors include head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, stomach cancer, breast cancer, kidney cancer and skin cancer of human.

10. A pharmaceutical coposition for treating tumors, wherein the pharmaceutical composition at least includes micropeptide HMMW containing SEQ ID NO: 1 amino acid sequence; or contains the nucleotide described in claim 4; or a recombinant vector containing any of the nucleotides described in claim 4.

Description

BRIEF DESCRIPTION

[0038] Some of the embodiments will be described in detail, with references to the following Figures, wherein like designations denote like members, wherein:

[0039] FIG. 1 is a pcDNA3.1 plasmid profile of overexpression micropeptide HMMW in accordance with an embodiment;

[0040] FIG. 2 shows expression of a target band detected by Western blot in accordance with an embodiment;

[0041] FIG. 3 shows inhibitory effect of micropeptide HMMW on the proliferation of SCC4 cells of human head and neck cancer in accordance with an embodiment;

[0042] FIG. 4 shows the inhibitory effect of micropeptide HMMW on the proliferation of SW579 cells of human thyroid cancer in accordance with an embodiment;

[0043] FIG. 5 shows the inhibitory effect of micropeptide HMMW on the proliferation of A549 cells of human lung cancer in accordance with an embodiment;

[0044] FIG. 6 shows the inhibitory effect of micropeptide HMMW on the proliferation of TE13 cells of esophageal squamous cell carcinoma of human in accordance with an embodiment;

[0045] FIG. 7 shows the inhibitory effect of micropeptide HMMW on the proliferation of MGC803 cells of human stomach cancer in accordance with an embodiment;

[0046] FIG. 8 shows the inhibitory effect of micropeptide HMMW on the proliferation of MDA-MB-231 cells of human breast cancer in accordance with an embodiment;

[0047] FIG. 9 shows the inhibitory effect of micropeptide HMMW on the proliferation of UOK262 cells of human kidney cancer in accordance with an embodiment;

[0048] FIG. 10 shows the inhibitory effect of micropeptide HMMW on the proliferation of A431 cells of human skin cancer in accordance with an embodiment;

[0049] FIG. 11 shows the inhibitory effect of micropeptide HMMW on the tumor growth of SCC4 cells of human head and neck cancer in vivo in accordance with an embodiment;

[0050] FIG. 12 shows the inhibitory effect of micropeptide HMMW on the tumor growth of SW579 cells of human thyroid cancer in vivo in accordance with an embodiment;

[0051] FIG. 13 shows the inhibitory effect of micropeptide HMMW on the tumor growth of A549 cells of human lung cancer in vivo in accordance with an embodiment;

[0052] FIG. 14 shows the inhibitory effect of micropeptide HMMW on the tumor growth of TE13 cells of esophageal squamous cell carcinoma of human in vivo in accordance with an embodiment;

[0053] FIG. 15 shows the inhibitory effect of micropeptide HMMW on the tumor growth of MGC803 cells of human stomach cancer in vivo in accordance with an embodiment;

[0054] FIG. 16 shows the inhibitory effect of micropeptide HMMW on the tumor growth of MDA-MB-231 cells of human breast cancer in vivo in accordance with an embodiment;

[0055] FIG. 17 shows the inhibitory effect of micropeptide HMMW on the tumor growth of UOK262 cells of human kidney cancer in vivo in accordance with an embodiment;

[0056] FIG. 18 shows the inhibitory effect of micropeptide HMMW on the tumor growth of A431 cells of human skin cancer in vivo in accordance with an embodiment; and

[0057] FIG. 19 shows the expression level of micropeptide HMMW in cancer tissue/normal tissue detected by the qPCR method in accordance with an embodiment.

DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with specific embodiments.

Embodiment 1

[0059] In this embodiment, the encoding ability of HMMW micropeptide was detected.

[0060] Construct in vitro an overexpression vector pcDNA-HMMW with a Flag tag and containing cDNA (its nucleotide sequence is shown in SEQ ID NO: 10), wherein the profile of the empty plasmid pcDNA3.1 is shown in FIG. 1. Introduce the above plasmid into 293T cells by lipo3000 lipofection reagent, collect cells 48 h after transfection, centrifuge, discard the supernatant and collect precipitated cells, rinse the precipitated cells with PBS twice, centrifuge, discard the supernatant and collect the precipitated cells. Add RIPA lysis buffer to the collected precipitated cells, lyse on ice for 20 min, and then centrifuging at 12,000 g for 10 min and collect the supernatant. Then add 1×SDS loading buffer, mix well by pipetting, then boil up and degenerate for 5 min. Separate total protein by 10% SDS-PAGE gel, then transfer it onto a PVDF membrane, block with 5% nonfat dry milk at room temperature for 2 h, incubate with Flag primary antibody (abeam) overnight at 4 DEG C, and wash with TBST 3 times. Incubate with the secondary antibody at room temperature for 1 h and wash with TBST three times. Develop with an ECL supersensitive chemiluminescence solution and use the Tannon imaging system to form images and detect whether there is a target band.

[0061] The results are shown in FIG. 2. It was found through Western blot that a target band of micropeptide HMMW appeared after transfection of the recombinant plasmid. It further indicates that the micropeptide HMMW protein has an encoding ability.

Embodiment 2

[0062] In this embodiment, micropeptides HMMW I to IX were obtained by the solid-phase synthesis method and tested.

[0063] The micropeptides HMMW Ito IX (their amino acid sequences are shown in SEQ ID NO: 1 to 9) were synthesized by the peptide solid-phase synthesis method, the synthesized micropeptides HMMW were separated and purified by preparative HPLC, and the purity of the micropeptides HMMW was determined by analytical RP-HPLC. In the solid-phase peptide synthesis method, Fmoc-wang-resin or Fmoc-CTC-resin was used as a starting material and then protected amino acids were used to sequentially ligate dipeptide˜unpentacontapeptide. After peptide ligation and full wash, peptide cutting and post-treatment were conducted to obtain a crude angiogenesis inhibitor. The crude product was dissolved, purified by preparative HPLC twice, concentrated and freeze-dried to obtain a pure product. Finally, after the tertiary purification, a refined micropeptide product was obtained. This method can not only ensure the efficiency of synthesis, but also raise product purity. The details are as follows:

1. Peptide Ligation (Including Ligation of Dipeptide˜Unpentacontapeptide)

[0064] Weigh an appropriate amount of Fmoc-wang-resin or Fmoc-CTC-resin, pour it into a glass sand core reaction column, and add an appropriate amount of CH.sub.2Cl.sub.2 to fully expand the resin.

[0065] a. Removal of protecting group: Add a protecting group removing liquid of hexahydropyridine/N,N-dimethylformamide (DMF), react for a period of time, drain the protecting group removing liquid, wash with DMF once, and add an appropriate amount of the protecting group removing liquid again for reaction to remove Fmoc protecting group;

[0066] b. Wash: Drain the protecting group removing liquid, and wash the resin with DMF to thoroughly remove by-products;

[0067] c. Condensation: Dissolve the protecting amino acid and activator used for peptide ligation in DMF and condensing agent, shake up, control the temperature at about 34 DEG C, and fully react in the reactor;

[0068] d. Wash: Drain the reaction solution and fully wash the resin with DMF to thoroughly remove by-products

2. Peptide Cutting

[0069] Put the drained resin into a round-bottom flask, add a cutting fluid to fully lyse hexatriconta-peptide intermediate, and separate the resin from the peptide by a sand core funnel. The components of the cutting fluid and the volume composition of the components are: trifluoroacetic acid:phenol:water:thioanisole:EDT=90:3:3:2:2.

3. Post-Treatment

[0070] First add anhydrous ether to the cutting fluid to separate out the peptide, then centrifuge, discard the supernatant, then wash the peptide with anhydrous ether, and drain to obtain a peptide crude product.

4. Purification

[0071] a. Dissolution: Weigh the crude product to prepare a 5-20 g/L solution, and filter it with a mixed filter membrane with a pore size of 0.45 μm.

[0072] Preparation: Conduct primary purification, secondary purification and tertiary purification by semi-preparative HPLC to obtain a qualified refined peptide product. Mobile phase: A is acetonitrile, B is 0.1% TFA aqueous solution.

[0073] Primary purification: Equilibrate the preparative column by rinsing with 10%-90% acetonitrile and 20%-80% buffer solution at a flow rate of 50 mL/min-100 mL/min for 10 min. Dissolve the filtered crude product and load it with an infusion pump.

TABLE-US-00001 TABLE 1 Elution gradient for primary purification Time Flow rate Wavelength (min) (mL/min) A % B % nm 0 60 10 90 220 40 60 20 80 220

[0074] Collect solutions with an absorption value of greater than 200 mV at UV wavelength 220 nm, combine the solutions with a detected purity of greater than 95% as the peak top, and keep it for secondary separation and purification.

[0075] Secondary purification: After removing by rotary evaporation the organic solvent from the peak top received in the primary purification, load the sample by infusion pump in form of 5-95% acetonitrile and 15-85% buffer at a flow rate of 50-100 mL/min.

TABLE-US-00002 TABLE 2 Elution gradient for secondary purification Time Flow rate Wavelength (min) (mL/min) A % B % nm 0 60 5 95 220 40 60 15 85 220

[0076] The solutions with absorption greater than 200 mV at UV wavelength 220 nm were collected. The solutions were considered qualified if their purity was greater than 98%.

[0077] b. Concentration, filtration and freeze-drying: Concentrate the qualified solution in a rotary evaporator under reduced pressure at 37 DEG C to remove residual solvent and water for injection. Finally, filter it with a 0.22 μm filter membrane, put the filtrate in a freeze-drying tray and freeze-dry it with a freeze dryer to obtain a pure product.

[0078] Tertiary purification: Conduct tertiary purification of the qualified sample with a purification of greater than 98% obtained in the secondary purification, and use 5-95% acetonitrile and 10-90% buffer at a flow rate of 50-100 mL/min to prepare a refined peptide product.

TABLE-US-00003 TABLE 3 Elution gradient for tertiary purification Time Flow rate Wavelength (min) (mL/min) A % B % nm 0 60 5 95 220 40 60 10 90 220

[0079] Collect solutions with an absorption value of greater than 200 mV at UV wavelength 220 nm, combine the samples with a detected purity of greater than 95% as the qualified refined product.

5. Purity Detection

[0080] Collect freeze-dried purified product and detect peptide purity by analytical RP-HPLC. Analysis conditions: Mobile phase: ACN (+0.1% TFA) and H.sub.2O (+0.1% TFA); linear gradient of acetonitrile: 10%-100%; flow rate: 1 mL/min; operation time: 20 min; loading volume: 20 μL; detection wavelength: 220 nm.

TABLE-US-00004 TABLE 4 Purities of micropeptides HMMW I to IX detected by RP-HPLC Name Peak area Purity (%) HMMW-I 1349502 98.87% HMMW-II 5510739 95.56 HMMW-III 2578258 95.68 HMMW-IV 8335415 98.32 HMMW-V 2695695 95.42 HMMW-VI 8429617 95.19 HMMW-VII 5562335 95.74 HMMW-VIII 3662672 95.66 HMMW-IX 3293152 95.56

[0081] The purities of synthesized micropeptides were determined by RP-HPLC. The results showed that the purities of the nine prepared micropeptides HMMW were all greater than 95%, which met the design requirements.

[0082] In this experiment, micropeptides HMMW I to IX were successfully synthesized by solid-phase synthesis method. This method has high repeatability, strong operability and less pollution; two types of resin, i.e.: wang resin and CTC resin, can be used in the experiment to synthesize peptides; in the experiment, when wang resin was used, it was more stable and had fewer side reactions, a better peak pattern of the process crude product and a higher purification yield compared with other resins, so the cost was lower; in the experiment, when CTC resin was used, the reaction was less affected by temperature and the reaction rate was higher; RP-HPLC was used to purify peptide and gradient elution was used with a better effect compared with isocratic elution. In the separation process, the retention time was appropriate and the production efficiency and the purity were high.

Embodiment 3

[0083] In this embodiment, the effects of micropeptides HMMW I to IX on the proliferation ability of human tumor cells were detected.

[0084] After SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer and A431 cells of skin cancer of human were cultured in a 37 DEG C 5% CO.sub.2 incubator until the density was 90%, they were digested and collected by trypsin, the cells were re-suspended in a culture solution and counted under a microscope, the concentration of the cells was adjusted to 3.0×10.sup.4 cells/mL, and the cell suspension was inoculated to 96-well plates, 100 μL per well, and cultured in a 37 DEG C 5% CO.sub.2 incubator overnight. After the cells were completely adherent, different doses of micropeptides HMMW I to IX were added as drug groups, taxol was used as a positive control group, and the culture solution without any drug was used as a blank control group. They were diluted with the culture solution till predetermined concentrations. The diluted solutions were added to 96-well plates, respectively, 100 μL per well, and incubated in a 37 DEG C 5% CO.sub.2 incubator for 48h. 20 μL of 5 mg/mL MTT was added to each well of the 96-well plates, and the culture was continued for 4 h. The culture medium was sucked away, and 100 μL of DMSO was added to each well for dissolution. Absorbance was detected by ELIASA at detection wavelength 570 nm and reference wavelength 630 nm, and proliferation inhibition (PI) was calculated according to Formula PI (%)=1−drug group/negative group. The test was independently repeated three times. The results obtained from the test were expressed with mean±SD and statistical t test was done. *P<0.05 means significant difference, and **P<0.01 means very significant difference.

TABLE-US-00005 TABLE 5 Inhibitory effects of micropeptides HMMW I to IX on proliferation of human tumor cells Dose Tumor type (inhibition rate/%) Group (μM) SCC4 SW579 A549 TE13 MGC803 MDA-MB-231 UOK262 A431 HMMW-I 1 20.18 18.98 14.71 13.09 15.93 18.90 16.92 17.04 2 32.67 24.87 19.37 17.27 19.91 23.17 24.15 20.98 4 44.89 36.23 21.87 23.98 24.09 36.99 38.19 31.09 8 58.19 42.17 37.09 36.09 38.18 45.06 43.54 41.21 16 64.29 59.02 43.92 43.53 45.10 56.19 59.21 48.02 32 79.18 68.01 58.90 61.02 68.09 69.18 72.10 65.09 HMMW-II 1 19.58 17.90 14.09 12.45 16.23 17.38 16.23 16.44 2 31.62 26.81 18.21 16.89 18.57 22.45 23.32 22.18 4 42.34 33.19 22.45 22.68 23.18 35.57 34.29 30.29 8 55.41 41.57 35.05 35.16 39.16 46.48 42.34 40.41 16 60.15 54.15 42.54 44.37 44.15 55.24 52.51 45.32 32 72.14 63.25 56.87 60.13 63.24 61.35 70.70 62.19 HMMW-III 1 17.18 17.91 15.73 14.04 16.90 16.94 17.90 18.34 2 30.67 23.80 18.34 18.21 18.95 22.12 23.15 23.58 4 41.89 35.21 23.85 20.90 25.01 35.91 35.16 34.03 8 53.19 41.12 35.01 34.03 39.13 44.05 41.53 42.11 16 60.29 55.00 41.90 44.50 47.16 52.14 57.20 49.06 32 73.18 64.00 54.84 60.09 65.05 67.13 70.16 67.19 HMMW-IV 1 17.54 18.94 15.06 14.43 15.20 16.34 17.23 15.43 2 30.32 27.21 19.23 17.59 19.37 23.25 24.12 23.28 4 41.31 35.39 24.41 23.62 24.15 36.53 36.25 34.25 8 53.21 40.51 36.35 32.46 37.36 47.28 43.24 42.31 16 62.13 52.05 41.50 43.31 45.12 53.21 54.31 48.30 32 70.34 61.21 54.27 58.33 61.54 60.25 69.75 60.29 HMMW-V 1 16.13 16.51 16.76 15.07 15.93 17.92 18.95 17.35 2 32.27 22.86 19.14 19.11 19.92 21.32 21.35 22.38 4 40.84 34.11 22.80 22.94 24.05 33.95 33.11 31.05 8 52.09 40.10 36.31 32.53 38.11 42.35 40.43 40.31 16 61.24 54.40 42.95 41.57 46.36 50.12 55.24 47.05 32 70.28 63.05 55.54 59.39 63.00 64.33 67.26 65.39 HMMW-VI 1 15.41 17.58 16.01 15.83 16.10 15.54 18.53 14.43 2 32.18 25.20 18.13 18.49 18.27 22.22 25.15 22.21 4 40.21 33.35 23.40 22.32 23.13 34.33 33.15 33.35 8 52.20 41.50 34.30 31.44 36.26 45.24 41.20 40.30 16 60.03 50.45 40.54 42.51 44.10 51.31 53.11 46.20 32 68.14 60.20 52.23 54.30 60.34 62.27 64.73 58.24 HMMW-VII 1 15.33 15.53 15.86 16.17 15.93 18.52 19.45 16.25 2 31.21 21.56 18.13 18.18 19.92 20.12 20.31 21.35 4 41.54 33.14 22.60 21.54 24.05 32.45 32.21 30.15 8 51.02 41.00 35.30 33.50 38.11 41.15 41.40 39.30 16 63.14 52.45 43.75 40.47 46.36 52.02 52.14 44.25 32 71.20 61.15 53.51 57.33 63.00 63.23 64.25 64.32 HMMW-VIII 1 16.18 16.91 13.73 15.04 15.90 17.94 18.90 16.34 2 31.67 22.80 17.34 19.21 19.95 21.12 22.15 22.58 4 40.89 32.21 21.85 21.90 23.01 34.91 33.16 33.03 8 51.19 40.12 32.01 33.03 35.13 43.05 40.53 41.11 16 61.29 52.00 40.90 42.50 46.16 50.14 54.20 48.06 32 70.18 60.00 53.84 61.09 63.05 64.13 71.16 66.19 HMMW-IX 1 16.33 16.53 16.86 17.17 18.93 19.52 18.45 18.25 2 33.21 22.56 19.13 19.18 21.92 23.12 22.31 23.35 4 42.54 31.14 23.60 22.54 25.05 34.45 33.21 31.15 8 50.02 45.00 36.30 35.50 39.11 42.15 43.40 39.30 16 64.14 54.45 45.75 43.47 47.36 53.02 54.14 43.25 32 72.20 63.15 54.51 58.33 64.00 62.23 65.25 61.32 Taxol 10 (μg/ml) 87.02 82.83 84.29 79.21 81.28 83.92 83.92 85.19

[0085] The results are shown in FIG. 3 to FIG. 10. Compared with the negative control group, micropeptide HMMW-I at a dose of 1 to 32 μM could significantly inhibit the proliferation of SCC4 cells of head and neck cancer (FIG. 3), SW579 cells of thyroid cancer (FIG. 4), A549 cells of lung cancer (FIG. 5), TE13 cells of esophageal squamous cell carcinoma (FIG. 6), MGC803 cells of stomach cancer (FIG. 7), MDA-MB-231 cells of breast cancer (FIG. 8), UOK262 cells of kidney cancer (FIG. 9), A431 cells of skin cancer (FIG. 10) of human to various extent, and showed a dose-dependent relation. The results are shown in Table 5. Compared with the negative control, micropeptides HMMW I to IX at a dose of 1 to 32 μM all could significantly inhibit the proliferation of SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, A431 cells of skin cancer of human, and showed a dose-dependent relation. It indicates that the peptides with more than 85% homology to the original sequence HMMW I all have an inhibitory effect on the proliferation of tumor cells, and it can be considered to use micropeptides HMMW I to IX as candidate anti-tumor drugs.

Embodiment 4

Effects of Micropeptides HMMW I to IX on the Migration Ability of Human Tumor Cells

[0086] Inoculate SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, and A431 cells of skin cancer of human to transwell cells, 100 μL per well, and meanwhile add micropeptides HMMW I to IX at different doses to every cell. Add 0.6 mL of complete medium containing 10% FBS to the transwell cells to stimulate cell migration, and culture in 5% CO.sub.2 at 37 DEG C for 24 h. Discard the medium in the wells, fix with 90% alcohol at room temperature for 30 min, stain with 0.1% crystal violet at room temperature for 10 min, rinse with clear water, gently wipe off the non-migrated cells in the upper layer with a cotton swab, observe under a microscope and select four fields of view to take pictures and count. Calculate the migration inhibition rate (MIR) of the cells according to the following formula:

[00001]$\mathrm{MIR}\left(\%\right)=1-\frac{N_{\mathrm{test}}}{N_{\mathrm{control}}}\times 100\%$

[0087] where N.sub.test is the number of migrated cells in the test groups (the groups at a dose of 1, 4 or 14 μM in the table), N.sub.control is the number of migrated cells in the blank control groups (the groups at a dose of 0μM in the table).

[0088] The test was repeated independently three times. Mean±SD was calculated based on the test results. Statistical t test was done. Here, “the test was repeated independently three times” means that every dose of any type of cells was tested repeatedly three times, and then the above formula was used to calculate the number of migrated cells (Mean±SD). Value P was used to express statistical difference. The statistical significance of the results is a method for estimating how true the results are (the totality can be represented), *P<0.05 means significant difference, and **P<0.01 means very significant difference.

TABLE-US-00006 TABLE 6 Inhibitory effects of micropeptides HMMW I to IX on the migration ability of human tumor cells Dose Tumor type (inhibition rate) Group (μM) SCC4 SW579 A549 TE13 MGC803 MDA-MB-231 UOK262 A431 HMMW-I 1 17.85% 19.81% 35.00% 23.76% 27.61% 28.06% 15.39% 33.19% 4 42.97% 51.03% 53.95% 42.29% 53.45% 50.34% 42.38% 55.17% 16 77.00% 70.98% 73.46% 68.32% 76.80% 75.38% 71.47% 73.22% HMMW-II 1 18.81% 18.80% 34.01% 22.74% 25.64% 27.07% 16.29% 31.14% 4 43.67% 50.13% 52.91% 40.23% 52.41% 51.24% 41.28% 53.12% 16 75.04% 71.92% 72.36% 65.31% 73.82% 73.18% 70.43% 71.12% HMMW-III 1 16.81% 18.83% 33.01% 22.76% 25.61% 27.06% 16.39% 32.13% 4 43.94% 50.04% 54.93% 41.29% 52.45% 51.34% 41.38% 54.27% 16 74.01% 71.92% 72.42% 65.32% 74.80% 74.38% 70.47% 72.21% HMMW-IV 1 16.84% 18.81% 33.00% 22.76% 25.60% 29.06% 16.32% 32.19% 4 43.91% 50.03% 52.91% 41.29% 52.45% 51.34% 41.38% 54.17% 16 75.04% 71.91% 71.46% 65.32% 74.80% 73.38% 70.47% 71.24% HMMW-V 1 18.36% 17.45% 35.01% 23.74% 27.64% 28.07% 18.29% 30.14% 4 44.67% 51.13% 53.91% 42.23% 54.41% 50.24% 42.28% 54.12% 16 71.04% 71.93% 71.36% 64.31% 75.82% 74.18% 72.43% 70.12% HMMW-VI 1 16.69% 19.81% 32.00% 21.76% 26.64% 27.06% 17.32% 33.19% 4 42.81% 52.03% 51.90% 40.21% 51.45% 50.34% 40.38% 55.17% 16 71.04% 70.91% 70.46% 66.32% 72.80% 74.38% 72.47% 70.23% HMMW-VII 1 17.42% 20.80% 35.01% 23.64% 26.60% 28.27% 19.24% 32.10% 4 44.61% 53.13% 53.91% 41.21% 51.31% 50.14% 40.21% 52.22% 16 72.34% 70.92% 71.36% 63.30% 70.62% 72.15% 72.40% 70.11% HMMW-VIII 1 17.80% 18.81% 32.06% 23.73% 24.60% 26.14% 18.29% 33.10% 4 42.92% 52.05% 51.90% 42.23% 53.43% 50.24% 43.48% 53.37% 16 72.35% 70.90% 73.40% 63.31% 73.81% 73.32% 71.45% 71.20% HMMW-IX 1 18.81% 17.81% 36.00% 21.76% 25.61% 26.06% 17.39% 33.19% 4 43.97% 50.03% 52.95% 40.29% 51.45% 52.34% 43.38% 55.17% 16 74.00% 71.98% 71.46% 65.32% 74.80% 73.38% 70.47% 73.22% Avastin 10 68.64% 59.81% 66.21% 58.19% 71.25% 62.19% 61.39% 64.63%

[0089] The results are shown in Table 6. Compared with the negative control group, micropeptides HMMW I to IX at a dose of 1 to 16 μM all could significantly inhibit the migration of SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, A431 cells of skin cancer of human to various extent, and showed a dose-dependent relation. It indicates that the peptides with more than 85% homology to the original sequence HMMW I all have an inhibitory effect on the migration of tumor cells and can be used as treatment drugs to inhibit the migration ability of malignant tumor cells.

Embodiment 5

Effects of Micropeptides HMMW I to IX on the Invasion Ability of Human Tumor Cells

[0090] Dilute 10 mg/mL Matrigel with culture medium at 1:3, spread it on membranes of transwell cells and dry it in the air at room temperature. Use trypsin to digest and collect SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, and A431 cells of skin cancer of human, which were cultured to the logarithmic phase, wash with PBS twice and re-suspend with a blank culture medium. Adjust cell concentration to 1×10.sup.5 cells/mL. Inoculate the cells to transwell cells, 100 μL per well, and meanwhile add micropeptides HMMW I to IX at different doses to every cell. Add 0.6 mL of complete medium containing 10% FBS to the transwell cells to stimulate cell invasion, and culture in 5% CO.sub.2 at 37 DEG C for 24 h. Discard the medium in the wells, fix with 90% alcohol at room temperature for 30 min, stain with 0.1% crystal violet at room temperature for 10 min, rinse with clear water, gently wipe off the non-invaded cells in the upper layer with a cotton swab, observe under a microscope and select four fields of view to take pictures and count. Calculate the invasion inhibition rate (IIR) of the cells according to the following formula:

[00002]$\mathrm{IIR}\left(\%\right)=1-\frac{N_{\mathrm{test}}}{N_{\mathrm{control}}}\times 100\%$

[0091] where N.sub.test is the number of invaded cells in the test groups (the groups at a dose of 1, 4 or 16 μM in the table), N.sub.control is the number of invaded cells in the blank control groups (the groups at a dose of 0 μM in the table). The test was repeated independently three times. Mean±SD was calculated based on the test results. Statistical t test was done. Value P was used to express statistical difference. The statistical significance of the results is a method for estimating how true the results are (the totality can be represented), *P<0.05 means significant difference, and **P<0.01 means very significant difference.

TABLE-US-00007 TABLE 7 Inhibitory effects of micropeptides HMMW I to IX on the invasion ability of human tumor cells Tumor type (inhibition rate) Group Dose SCC4 SW579 A549 TE13 MGC803 MDA-MB-231 UOK262 A431 HMMW-I 1 20.75% 19.63% 31.23% 20.43% 26.12% 27.97% 33.80% 32.74% 4 46.24% 44.76% 51.32% 42.86% 50.08% 48.95% 53.12% 51.22% 16 74.84% 64.89% 64.08% 66.51% 68.62% 70.49% 66.67% 69.37% HMMW-II 1 21.72% 22.13% 33.25% 21.41% 27.52% 25.93% 34.70% 31.71% 4 45.14% 45.78% 55.12% 43.46% 51.04% 47.75% 54.16% 53.41% 16 73.80% 66.73% 65.09% 64.56% 66.51% 71.52% 65.64% 68.25% HMMW-III 1 22.75% 18.63% 32.13% 21.23% 25.11% 28.91% 34.37% 33.70% 4 48.24% 42.76% 52.30% 41.81% 53.38% 49.82% 55.15% 53.21% 16 75.84% 63.89% 66.09% 63.31% 67.61% 68.36% 67.23% 67.33% HMMW-IV 1 22.79% 23.10% 34.22% 22.40% 23.50% 24.91% 38.40% 36.25% 4 47.34% 46.71% 56.11% 42.42% 54.21% 46.73% 58.36% 57.19% 16 74.40% 67.72% 65.24% 67.51% 63.49% 71.64% 67.62% 71.36% HMMW-V 1 20.34% 19.09% 31.19% 20.29% 26.53% 27.25% 33.43% 32.19% 4 46.26% 44.23% 51.27% 42.74% 50.24% 48.37% 53.27% 51.35% 16 74.71% 64.14% 64.25% 66.69% 68.38% 70.50% 66.14% 69.29% HMMW-VI 1 22.83% 23.29% 34.15% 22.25% 23.42% 24.83% 38.15% 36.83% 4 47.82% 46.63% 56.32% 42.39% 54.19% 46.57% 58.28% 57.35% 16 74.16% 67.58% 65.35% 67.47% 63.35% 71.43% 67.51% 71.16% HMMW-VII 1 19.70% 20.11% 32.22% 25.11% 29.50% 27.91% 36.74% 34.70% 4 43.11% 43.72% 52.42% 45.36% 54.34% 49.55% 56.26% 55.51% 16 70.84% 63.71% 64.02% 67.52% 69.57% 74.32% 67.62% 69.24% HMMW-VIII 1 23.71% 19.61% 34.03% 24.21% 26.01% 27.90% 35.27% 34.50% 4 49.14% 43.56% 55.20% 42.31% 57.18% 45.81% 54.14% 55.11% 16 76.64% 64.79% 68.03% 61.51% 70.60% 64.26% 68.21% 69.43% HMMW-IX 1 24.83% 25.21% 30.15% 21.25% 26.42% 27.83% 39.15% 34.83% 4 48.82% 48.60% 60.32% 40.39% 57.19% 49.57% 59.28% 54.35% 16 75.16% 62.48% 70.35% 65.47% 68.35% 75.43% 69.51% 74.16% Avastin 10 68.41% 67.40% 61.35% 69.31% 63.48% 70.03% 72.16% 70.39%

[0092] The results are shown in Table 7. Micropeptides HMMW I to IX all can significantly inhibit the migration of SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, A431 cells of skin cancer of human to various extent, and showed a dose-dependent relation. It indicates that the peptides with more than 85% homology to the original sequence HMMW I all have an inhibitory effect on the invasion of tumor cells and can be used as treatment drugs to inhibit the invasion ability of malignant tumor cells.

Embodiment 6

Effects of Micropeptide HMMW on In Vivo Growth of Human Tumor Cells

[0093] (1) Massively culture SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, and A431 cells of skin cancer of human, digest with a 0.25% pancreatin solution, centrifuge the cell suspension at 1,000 rpm for 5 min after termination of digestion, re-suspend the cells by serum-free DMEM culture medium, then count the cells and adjust cell concentration to 5×10.sup.7 cells/ml;

[0094] (2) Inoculate each nude mouse (female mice at the age of 4-6 weeks and with a weight of 14-16 g were ordered and adaptively reared for 1 week in an SPF animal breeding room) with 100 μl of the cell suspension of the corresponding group in the left armpit, and the number of cells injected is 5×10.sup.6;

[0095] (3) After inoculation, the tumor growth at the inoculation sites of nude mice was closely observed. On the 7.sup.th day after inoculation, white scabs appeared at the inoculation sites, which could move subcutaneously after being touched. With the growth of tumor tissue, hard tumor masses were gradually formed at the inoculation sites. On about the 14.sup.th day, the average volume of the tumor tissue reached 100 mm.sup.3. BALB/c nude mice were randomly divided into three groups (the normal saline group was a blank control group, the micropeptide HMMW at a dose of 10 mg/kg was a low-dose group, and the micropeptide HMMW at a dose of 15 mg/kg was a high-dose group), 6 mice in each group, and the animals weighed 16-18 g at the beginning of administration;

[0096] (4) The volume of the transplanted tumor was measured and recorded every two days. The calculation formula of tumor volume (TV) is shown below:

TV=0.5×a×b{circumflex over ( )}2

[0097] where, a is the length of the transplanted tumor (mm), and b is the width of the transplanted tumor (mm).

[0098] The results are shown in FIG. 11 to FIG. 18. Compared with the normal saline control group, micropeptide HMMW could significantly inhibit the tumorigenicity in vivo of SCC4 cells of head and neck cancer (FIG. 11), SW579 cells of thyroid cancer (FIG. 12), A549 cells of lung cancer (FIG. 13), TE13 cells of esophageal squamous cell carcinoma (FIG. 14), MGC803 cells of stomach cancer (FIG. 15), MDA-MB-231 cells of breast cancer (FIG. 16), UOK262 cells of kidney cancer (FIG. 17), A431 cells of skin cancer (FIG. 18) of human to various extent and showed a dose-dependent relation, so it can be considered to use micropeptide HMMW as a new type of antitumor peptide.

Embodiment 7

Effects of Micropeptides HMMW I to IX on In Vivo Growth of Human Tumor Cells

[0099] (1) Massively culture SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, and A431 cells of skin cancer of human, digest with a 0.25% pancreatin solution, centrifuge the cell suspension at 1,000 rpm for 5 min after termination of digestion, re-suspend the cells by serum-free DMEM culture medium, then count the cells and adjust cell concentration to 5×10.sup.7 cells/ml;

[0100] (2) Inoculate each nude mouse (female mice at the age of 4-6 weeks and with a weight of 14-16 g were ordered and adaptively reared for 1 week in an SPF animal breeding room) with 100 μl of the cell suspension of the corresponding group in the left armpit, and the number of cells injected is 5×10.sup.6;

[0101] (3) After inoculation, the tumor growth at the inoculation sites of nude mice was closely observed. On the 7.sup.th day after inoculation, white scabs appeared at the inoculation sites, which could move subcutaneously after being touched. With the growth of tumor tissue, hard tumor masses were gradually formed at the inoculation sites. On about the 14.sup.th day, the average volume of the tumor tissue reached 100 mm.sup.3. BALB/c nude mice were randomly divided into three groups (the normal saline group was a blank control group, and each of micropeptides HMMW I to IX formed a group at a dose of 15 mg/kg), 6 mice in each group, and the animals weighed 16-18 g at the beginning of administration;

[0102] (4) The volume of the transplanted tumor was measured and recorded every two days. The calculation formula of tumor volume (TV) is shown below:

TV=0.5×a×b{circumflex over ( )}2

[0103] where, a is the length of the transplanted tumor (mm), and b is the width of the transplanted tumor (mm).

TABLE-US-00008 TABLE 8 Inhibitory effects of micropeptides HMMW I to IX on in vivo tumor growth of human tumor cells Group (n = 6) Tumor type (inhibition rate) SCC4 SW579 A549 TE13 MGC803 MDA-MB-231 UOK262 A431 HMMW-I 72.56% 66.63% 69.23% 65.43% 68.12% 67.97% 63.80% 62.74% HMMW-II 66.24% 64.76% 71.32% 62.86% 70.08% 68.95% 63.12% 71.22% HMMW-III 70.84% 71.89% 73.08% 66.31% 69.62% 69.49% 61.67% 69.54% HMMW-IV 71.72% 72.13% 68.25% 61.41% 67.52% 65.93% 64.70% 71.71% HMMW-V 67.14% 70.78% 71.12% 63.46% 71.04% 67.75% 64.16% 69.41% HMMW-VI 73.54% 66.79% 65.27% 65.56% 66.51% 71.18% 65.19% 68.25% HMMW-VII 72.75% 68.63% 69.13% 71.23% 65.11% 68.91% 64.37% 67.70% HMMW-VIII 68.24% 71.76% 69.30% 68.81% 69.38% 69.82% 65.15% 68.21% HMMW-IX 71.84% 67.89% 69.09% 65.31% 68.61% 69.36% 65.23% 64.33%

[0104] The results are shown in FIG. 8. Compared with the normal saline control group, micropeptides HMMW I to IX could significantly inhibit the tumorigenicity in vivo of SCC4 cells of head and neck cancer, SW579 cells of thyroid cancer, A549 cells of lung cancer, TE13 cells of esophageal squamous cell carcinoma, MGC803 cells of stomach cancer, MDA-MB-231 cells of breast cancer, UOK262 cells of kidney cancer, and A431 cells of skin cancer of human and showed a dose-dependent relation. It indicates that the peptides with more than 85% homology to the original sequence HMMW I all have an inhibitory effect on the in vivo growth of tumor cells, so it can be considered to use micropeptides HMMW I to IX as a new type of antitumor peptides.

Embodiment 8

Expression of HMMW in Tumor Patients and Normal Para-Carcinoma Tissue

[0105] Download RNA-seq sequencing files and clinical information of cancer tissues and normal tissues of 16 tumors including head and neck cancer, brain glioma, thyroid cancer, esophageal squamous cell carcinoma, lung cancer, liver cancer, stomach cancer, kidney cancer, breast cancer, ovarian cancer, cervical cancer, bladder cancer, colorectal cancer, pancreatic cancer, osteosarcoma and skin cancer by the TCGA standard method, and analyze the differential expression of micropeptide HMMW (judgment criterion: (1)| Cancer/paracancer expression quantity |>2, (2)P<0.05).

TABLE-US-00009 TABLE 8 Analysis of expression quantity of micropeptide HMMW in human tumor tissue and normal tissue (cancer paracancer) Number of Fold change in Tumor type cases expressiot Value P Head and neck cancer 528 −20.895 6.13E−22 Thyroid cancer 507 −8.285 4.05E−10 Brain glioma 516 1.394 0.0359 Lung cancer 504 −12.345 1.38E−7 Esophageal squamous 185 −11.201 7.17E−5 cell carcinoma Ovarian cancer 608 1.381 0.0683 Cervical cancer 307 0.984 0.0284 Stomach cancer 443 −6.103  0.00018 Breast cancer 1098 −8.193 2.48E−5 Bladder cancer 412 0.895 0.1935 Liver cancer 377 1.237  0.00014 Osteosarcoma 381 1.035  0.05213 Stomach cancer 291 −19.351 4.15E−8 Skin cancer 470 −15.245  0.00219 Colorectal cancer 461 0.818 0.0426 Pancreatic cancer 185 1.781 0.0503

[0106] As shown in Table 9, compared with normal tissues, the expression levels of micropeptide HMMW in eight tumor tissues of head and neck cancer, thyroid cancer, lung cancer, esophageal squamous cell carcinoma, gastric cancer, breast cancer, kidney cancer and skin cancer of human were significantly reduced. It indicates that the expression of micropeptide HMMW is significantly negatively correlated with the development of various tumors.

Embodiment 9

Expression of HMMW in Clinical Patients with Head and Neck Cancer and Normal Paracancer Tissues

(1) Collection of Specimens

[0107] With the informed consent of the patients, head and neck cancer and paracancer tissue specimens were collected during the operation, washed with normal saline, and stored in liquid nitrogen or −80° C. refrigerator for future use.

(2) Primer Design

[0108] Primer Premier5.0 was used to design primers according to the nucleotide sequence corresponding to the micropeptide HMMW, and the sequence is as follows:

[0109] Forward primer (the sequence is shown in SEQ ID NO: 3)

[0110] Reverse primer (the sequence is shown in SEQ ID NO: 4)

(3) Detection of HMMW Expression in Head and Neck Cancer Patients and Normal Paracancer Tissues by Real-Time Quantitative PCR

[0111] Extract and collect the total RNA in the sample according to the Trizol manual of Life Technologies, then quantify the purity and concentration of the extracted RNA by the NanoDrop ND-1000 nucleic acid quantifier, and ensure the integrity of the extracted RNA through quality inspection by agarose. Use TaKaRa kit PrimeScript™ RT kit with gDNA Eraser (Perfect Real Time) to reversely transcribe the extracted total RNA to synthesize cDNA. Use TaKaRa kit SYBR® Premix Ex Taq™ II (TliRNaseH Plus) to conduct qPCR reaction. The reaction system is shown in the table below:

TABLE-US-00010 TABLE 9 PCR reaction system Reagent Dose (μL) SYBR Premix Ex Taq II (TliRNaseH Plus) (2×) 12.5 PCR Forward Primer (10 μM) 1 PCR Reverse Primer (10 μM) 1 DNA template (<100 ng) 2 Sterilized water 8.5 Total 25

[0112] After mixing the above components evenly, follow the procedure below: Initially denature at 95 DEG C for 30 s at 40 cycles; 95 DEG C for 5 s and 60 DEG C for 30 s. Judge the specificity of the reaction according to the melting curve and calculate the relative expression quantity of HMMW by the 2.sup.−ΔΔ.sup.Ct method. The result is shown in FIG. 19. In the head and neck cancer sample of about 75%, the expression level of HMMW was significantly lower than that of normal paracancer tissue.

[0113] Although the present invention has been disclosed in the form of preferred embodiments and variations thereon, it will be understood that numerous additional modifications and variations could be made thereto without departing from the scope of the invention.

[0114] For the sake of clarity, it is to be understood that the use of “a” or “an” throughout this application does not exclude a plurality, and “comprising” does not exclude other steps or elements. The mention of a “unit” or a “module” does not preclude the use of more than one unit or module.