CN110551197B - Micro-peptide and cancer treatment drug - Google Patents

Abstract

The invention relates to application of a micro-peptide CORO1C-47aa in treating cancer, and researches the influence of the peptide on angiogenesis in the initial stage of endometrial cancer from the perspective of the peptide coded by circRNA at a translation level. And through the treatment of targeting micro peptide CORO1C-47aa, the micro peptide CORO1C-47aa competitively binds with ARNT PAS-B structure domain, and blocks the binding of TACC3 and ligand PAS-B thereof, so that HIF1 is enriched to the vicinity of HRE promoter of VEGF to inhibit the gene expression thereof. In vitro and in vivo experiments prove that the micro-peptide CORO1C-47aa can obviously inhibit the generation of new vessels in the initial stage of endometrial cancer and has clinical application value on the endometrial cancer.

Description

Micro-peptide and cancer treatment drug

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of a micro-peptide CORO1C-47aa in a medicine for treating cancer.

Background

Endometrial Cancer (EC) is the fifth most common malignancy among women worldwide, accounting for 1-2% of all cancer deaths. Risk factors for EC development include age, socioeconomic status, and factors associated with estrogen overexposure, diabetes or hypertension, but genetic risk factors for this disease are poorly understood.

Non-coding rna (ncrna) represents the majority of transcripts in a cell and is often involved in the development of EC. Circular rna (circrna) is a new class of extensive ncrnas that often regulate gene expression in mammals. circRNA is a closed RNA transcript, produced by reverse splicing of a single pre-mRNA, the expression of which is generally highly conserved across species. In recent years, most of the circrnas reported have been indicated as miRNA sponges to regulate gene expression in different cell types, or to bind and sequester other RNA-binding proteins. However, the biological function of most circrnas has not been determined.

Recent studies have shown that circRNA can be translated to produce proteins due to its cytoplasmic localization. For example, circZNF609 and circMBI may be translated. The Circ-FBXW7, driven by the internal ribosome entry site, encodes a novel 21kDa protein. circPINTexon2 was produced from LncRNAs containing srorf (LINC-PINT), encoding tumor suppressor peptides in human cells. In addition, one study also showed that circRNA can be translated by N6-methyladenosine.

Disclosure of Invention

The invention aims to provide a circ-0000437 coded functional micro-peptide CORO1C-47aa, which acts as a tumor suppressor gene in EC and can effectively suppress endometrial cancer vascular endothelial cells.

The invention is realized by the following technical scheme:

a micro peptide is CORO1C-47aa, and the amino acid sequence is shown in SEQ ID NO. 1.

Further, the CORO1C-47aa is coded by a circ-0000437 gene.

Further, the sequence of the circ-0000437 gene is shown in SEQ ID NO. 2.

A medicament for treating cancer, the application of the above-mentioned micro peptide or its pharmaceutically acceptable salt in the medicament for treating cancer.

Further, the cancer is endometrial cancer.

Further, the drug for cancer treatment includes a pharmaceutical composition of the micro-peptide or a pharmaceutically acceptable salt thereof.

Further, the drug is the active ingredient of the micro peptide or the pharmaceutically acceptable salt thereof alone, or the drug is the active ingredient of the micro peptide or the pharmaceutically acceptable salt thereof in combination with an additional pharmaceutically active compound.

Furthermore, the pharmaceutical composition also contains pharmaceutically acceptable auxiliary materials.

Further, the auxiliary material is selected from one or more of polylactic acid, a copolymer of polyglycolic acid and glycolic acid, a copolymer of p-carboxyphenylpropane and sebacic acid or an ethylene-vinyl acetate copolymer, xylitol, oligosaccharide, chitin, potassium salt, sodium salt, hyaluronic acid, collagen, gelatin or albumin.

Further, the dosage form of the medicament for treating the cancer is a pharmaceutically acceptable dosage form.

The invention has the beneficial effects that:

the effect of the peptides on angiogenesis in the initiation stage of endometrial cancer was investigated in terms of the peptides encoded by the circRNA at the translation level. And through the treatment of targeting peptide CORO1C-47aa, the micro peptide CORO1C-47aa competitively binds with ARNT PAS-B structure domain, blocks the binding of TACC3 and ligand PAS-B thereof, leads HIF1 to be enriched near HRE promoter of VEGF so as to inhibit the gene expression thereof. In vitro and in vivo experiments prove that the micro-peptide CORO1C-47aa can obviously inhibit the generation of new vessels in the initial stage of endometrial cancer and has clinical medicinal value on the endometrial cancer.

Drawings

Fig. 1A to fig. 1D are experimental data graphs and schematic diagrams illustrating the effect of ectopic expression of the micro-peptide cor 1C-47aa on EC cell proliferation in the first embodiment of the present invention, specifically:

figure 1A is an analysis of cell proliferation at different time points for circ-0000437OE, circ-0000437IRES mutation and control EC cells (mean ± SD, n ═ 5);

figure 1B shows tumor growth in xenograft mice subcutaneously implanted with EC cells transfected with the indicated constructs (mean ± SD, n-5,. p < 0.05);

FIG. 1C shows staining of CD31 on tumor tissue sections from xenografted mice from circ-0000437OE, circ-0000437IRES mutant and control groups. ImageJ was used to quantify CD31 staining. Each value is the mean ± SEM of three independent experiments;

figure 1D is an endothelial tube formation assay using HUVECs from HEC-1-B and Ishikawa cells and conditioned media, stably expressing the circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n-3, p < 0.05).

FIGS. 2A to 2J are experimental data and schematic diagrams illustrating the effect of the micro-peptide CORO1C-47aa on the proliferation, migration and differentiation of HUVEC cells in the second embodiment of the present invention, specifically:

FIG. 2A shows CORO1C-47aa inhibits the proliferation of HUVEC cells;

figure 2B is a wound healing assay using HUVEC and conditioned media from HEC-1-B and Ishikawa cells, stably expressing the circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n-3, p < 0.05);

figure 2C is an endothelial migration assay using HUVEC and conditioned media from HEC-1-B and Ishikawa cells stably expressing the circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n-3, p < 0.05);

FIG. 2D is the Phalloidine-FITC staining of microfilaments and immunofluorescence staining of HUVEC cells and conditioned media stably expressing circ-0000437OE, circ-0000437IRES mutations from HEC-1-B and Ishikawa cells. Nuclei were counterstained with DAPI;

FIG. 2E is a representative photograph of an electron microscope under the experimental conditions shown;

FIG. 2F is a representative micrograph of spheroids incubated with CORO1C-47 aa;

FIG. 2G shows the matrix plugs harvested and processed for qRT-PCR. mRNA expression levels of murine CD31 were normalized to the level of human GAPDH and expressed as the murine CD 31/human GAPDH mRNA ratio;

FIG. 2H is a representative photograph of HE staining under the experimental conditions shown;

FIG. 2I is a representative photograph of Matrigel inserted under the specified experimental conditions;

FIG. 2J is a photograph of HUVEC spheroids embedded in fibrin gel incubated with the indicated medium, and then the radially growing cell buds counted;

FIG. 3 is a graph and schematic representation of experimental data showing significant differences in the expression of circ-0000437 between endometrial cancer and paracarcinoma in example III of the present invention; specifically, the method comprises the following steps:

circ-0000437 expresses EC tissues at a low level compared to the matched adjacent normal tissues. The expression level of circ-0000437 was analyzed by qRT-PCR and normalized to GAPDH, and the data are presented as mean ± SEM from three independent experiments.

Detailed Description

In order to clearly understand the technical means of the present invention and to implement the technical means according to the content of the specification, the following detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings is only exemplary and can be used to explain and illustrate the technical solution of the present invention, but not to be construed as limiting the technical solution of the present invention.

Aiming at the current limited treatment means of the endometrial cancer, the therapeutic value of the micro-peptide CORO1C-47aa (human full-length recombinant protein-47 aa) on the endometrial cancer is researched and explored from the perspective of translating the peptide encoded by the circRNA. The technical scheme firstly identifies that the encoding micro-peptide CORO1C-47aa is circular RNA circ-0000437, and the encoding frame is sORF. Next, it was verified that the expression of the micro-peptide CORO1C-47aa was down-regulated in EC. The micro-peptide CORO1C-47aa competitively binds with ARNT PAS-B domain, blocking the binding of TACC3 with its ligand PAS-B, resulting in HIF1 enriched near the HRE promoter of VEGF to inhibit its gene expression. In conclusion, experiments show that in endometrial cancer, the micro-peptide CORO1C-47aa achieves the treatment effect by inhibiting the generation of new blood vessels in the initial stage of cancer.

In some embodiments, a pharmaceutical composition comprising the micro-peptide CORO1C-47aa, or a pharmaceutically acceptable salt thereof, may be employed, which may optionally further comprise one or more additional pharmaceutically active compounds.

The medicine containing the micro-peptide CORO1C-47aa or pharmaceutically acceptable salt has at least one of the following functions:

1) reducing the incidence of chemical-induced endometrial cancer;

2) slowing or stopping the growth of established endometrial cancer foci;

3) slowing or stopping metastasis of established endometrial cancer foci;

4) inducing CTL cells that produce endometrial cancer specificity and have killing on endometrial cancer cells.

The pharmaceutic adjuvant of the technical scheme can be hydrolyzed or degraded by enzyme, acid and alkali or tissue fluid. The medicinal adjuvant is selected from any one or more of biocompatible high molecular polymer, mixture or copolymer of high molecular polymer. Specifically, the pharmaceutical excipients are selected from any one or more of polylactic acid, polyglycolic acid and glycolic acid copolymer, p-carboxyphenylpropane and sebacic acid copolymer or ethylene-vinyl acetate copolymer, for example:

a) polylactic acid with molecular weight of 5000-15000, 10000-20000, 20000-35000 or 30000-50000.

b) A copolymer of polylactic acid and glycolic acid with a molecular weight of 5000-15000, 10000-20000, 25000-35000 or 30000-50000.

c) Ethylene-vinyl acetate copolymer.

d) Copolymer of p-carboxyphenylpropane and sebacic acid, wherein the mass ratio of the p-carboxyphenylpropane to the sebacic acid is 10: 90, 20: 80, 30: 70, 40: 60, 50: 50 or 60: 40.

e) One or more of xylitol, oligosaccharide, chitin, potassium salt, sodium salt, hyaluronic acid, collagen, gelatin or albumin.

The pharmaceutical composition of the technical scheme can be used for preparing various antitumor drugs for treating human and animals, in particular to endometrial cancer resistant drugs.

The medicine of the technical scheme can be prepared into various pharmaceutically acceptable medicinal preparations, such as, but not limited to, turbid suspension, ointment, capsules, pills, tablets, injection and the like; in various shapes such as, but not limited to, granular, flake, spherical, block, needle, rod, and film. The dosage forms and shapes described above are suitable for compositions with or without additives, and the pharmaceutical formulations are prepared using methods conventional in the art, such as, but not limited to, (i) mixing a carrier support powder with the drug and then compressing into an implant, a so-called compounding method; (ii) melting the carrier support, mixing with the drug to be packaged, and then cooling the solid, the so-called melt process; (iii) dissolving the carrier support in a solvent, dissolving or dispersing the drug to be packaged in a polymer solution, followed by evaporation of the solvent and drying, a so-called dissolution method; (iv) spray drying; and (v) freeze-drying method. The dissolving method can be used for preparing microsphere, and the anticancer pharmaceutical composition can also be packaged in liposome.

The medicament of the technical scheme can be administrated by various routes, such as channels, arteries, subcutaneous, muscle, intradermal, intracavity, intratumoral, peritumoral and the like. The route of administration depends on a variety of factors, and in order to achieve an effective concentration at the site of the tumor, the drug may be administered by a variety of other routes, such as selective arterial, intraluminal, intraperitoneal, or intrathoracic, and intravertebral administration.

The administration dosage of the antitumor drug can be properly changed according to specific administration objects, administration routes or different preparation forms of the drug, but on the premise of ensuring that the pharmaceutical composition can achieve effective blood concentration in a mammal body.

The following examples, unless otherwise specified, all experimental procedures used are routine in the art; the reagents or materials, if not specifically mentioned, are commercially available. 6-8 week-old female nude mice were purchased from Shanghai laboratory animal center of Chinese academy of sciences (Shanghai, China). All cell lines were purchased from excel life science co. These cell lines were all subjected to DNA fingerprinting and were passaged for less than 6 months. DMEM, MEM and Fetal Bovine Serum (FBS) were purchased from Invitgen. HEC-1-B cells were grown in MEM medium containing 10% fetal bovine serum, and Ishikawa cells were grown in DMEM medium containing 10% fetal bovine serum. All cell lines were grown in penicillin/streptomycin-containing medium at 37 ℃ with air humidity of 5% CO₂。

Example one

Referring to FIGS. 1A-1D, the effect of ectopic expression of the micro-peptide CORO1C-47aa on EC cell proliferation.

Construction of circ-0000437OE vector and IRES mutant vector cloned EC cells: the inventor clones the full-length or mutated circ-0000437IRES sequence between the Rluc and Luc reporter genes to form two groups of Luc/Rluc high-low activity contrast vectors. The circ-0000437OE vector and IRES mutant vector were then transfected into EC cells (HEC-1-B, Ishikawa) to form EC cells that overexpress the circ-0000437 vector and IRES mutation. The inventors have constructed an ORF mutation expression vector (in which the initiation codon ATGGTG has been mutated to ATTGTT) and a FLAG tag to the C-terminus of CORO1C-47aa in order to determine whether the in-frame ATG codon of CORO1C-47aa can promote initiation of translation. CORO1C-47aa-FLAG was detectable in cells transfected with the circ-0000437OE vector and not in cells transfected with the IRES mutant vector using anti-FLAG Western blotting. It was demonstrated that overexpression of the circ-0000437OE vector could increase CORO1C-47aa expression, whereas the circ-0000437-IRES mutant vector could not. The inventor artificially detects the cell location of CORO1C-47aa, and finds that CORO1C-47aa is concentrated in the cell nucleus of EC cells through immunofluorescence assay.

As shown in FIG. 1A, the inventors performed CCK-8 assays on CORO1C-47aa stably upregulated and IRES mutant vector-transfected HEC-1-B and Ishikawa cells, respectively, however, the results indicated that neither the CORO1C-47aa OE group nor the IRES mutant group proliferated EC cells significantly compared to control cells. To explore the biological significance of CORO1C in tumor growth, 5 mice per group were injected subcutaneously with EC cells overexpressing CORO1C-47aa and mutated IRES, respectively. As shown in FIG. 1B, the xenografts of the CORO1C-47aa OE group were significantly inhibited from growing as compared to the control xenografts. Whereas the xenografts of IRES mutant group did not differ significantly from the control group. To investigate whether the inhibitory effect of CORO1C-47aa could be attributed to angiogenic disruption, immunohistological analysis of tumor tissues was performed to detect the expression of CD 31. As shown in FIG. 1C, CD31 was significantly inhibited in the CORO1C-47aa OE group compared to the IRES mutant group and the control group. As shown in fig. 1D, the inventors also performed an endothelial tube formation assay to explore how cor 1C-47aa affects endothelial network formation on matrigel. The results showed that CORO1C-47aa significantly disrupted the ability of HUVEC cells to form capillaries compared to the control.

Example two

Referring to FIGS. 2A to 2J, this example investigated the effect of the micro-peptide CORO1C-47aa on HUVEC cell proliferation, migration and differentiation. The inventors cultured HUVEC cells in conditioned medium of HEC-1-B and Ishikawa cells transfected with CORO1C-47aa over-expression and IRES mutation. As shown in FIG. 2A, the results of HUVEC cell proliferation assay showed that the HUVEC cell proliferation of the CORO1C-47aa OE group was significantly reduced. To explore the effect of overexpression of CORO1C-47aa on migration of HUVEC cells, the inventors performed wound healing assays and Transwell using HUVEC cells cultured in conditioned medium. As shown in FIGS. 2B and 2C, both the wound healing assay and Transwell demonstrated that the migration ability of HUVEC cells was significantly inhibited by overexpression of CORO1C-47aa, whereas the IRES mutant group was not significantly different from the empty vector transfected group. The inventors further investigated the effect of CORO1C-47aa on endothelial cytoskeleton, and as shown in FIG. 2D, F-actin in the group CORO1C-47aa OE could not rearrange into cytoskeleton, which is detrimental to cell migration. As shown in FIG. 2E, the inventors also examined the intercellular junctions of HUVEC cells by electron microscopy, and as a result, they showed that the intercellular junctions of the control group and the IRES mutant group were tight, whereas the intercellular junctions of HUVEC cells were significantly induced and the intercellular spaces were narrowed in the CORO1C-47aa OE group. To further demonstrate the possibility that CORO1C-47aa can inhibit angiogenesis, the inventors performed a Matigel plug assay in mice. As shown in FIGS. 2F, 2G and 2H, in the CORO1C-47aa OE group, angiogenesis was significantly inhibited in mice injected subcutaneously with Matigel. Finally, the inventors evaluated the role of CORO1C-47aa in a three-dimensional model of angiogenesis in vitro, HUVEC spheroids invading the three-dimensional fibrin matrix, producing endothelial buds due to the co-action of local breakdown of the extracellular matrix with HUVEC migration and growth. As shown in FIGS. 2I and 2J, CORO1C-47aa resulted in reduced HUVEC sprouting. These results indicate that CORO1C-47aa is capable of inhibiting angiogenesis in the initial stage by inhibiting endothelial cell proliferation, migration and differentiation.

EXAMPLE III

Referring to FIG. 3, circ-0000437 showed significant differences in endometrial and paracarcinoma expression, and the inventors used RT-qPCR assays to determine the level of circ-0000437 in a total of 198 paired EC samples and matched non-cancerous tissues in the eastern (Suzhou) and central northern China (Beijing). Circ-0000437 is expressed at higher levels in paracancerous tissues compared to matched cancerous tissues.

In summary, the following steps: the invention researches the influence of the peptide on the neovasculature of the endometrial cancer at the initial stage of the endometrial cancer from the perspective of the peptide encoded by the circRNA at the translation level, and proves the important function of the peptide in the endometrial cancer. Due to the competitive binding of the micro-peptide CORO1C-47aa and ARNT PAS-B domain, the blocking of the binding of TACC3 and the ligand PAS-B thereof leads to the enrichment of HIF1 near the HRE promoter of VEGF so as to inhibit the gene expression thereof, thereby achieving the effect of treating endometrial cancer. Moreover, the targeted therapy through the micro-peptide CORO1C-47aa can up-regulate the expression of the micro-peptide CORO1C-47aa, and has clinical medicinal value on endometrial cancer.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

<110> Suzhou university

<120> a micro-peptide and a drug for cancer treatment

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Claims (7)

1. A medicament for treating endometrial cancer, which is characterized by comprising a oligopeptide with an amino acid sequence shown as SEQ ID NO.1 or a pharmaceutically acceptable salt thereof.

2. The medicament of claim 1, wherein the mini-peptide is encoded by the circ-0000437 gene;

the sequence of the circ-0000437 gene is shown in SEQ ID NO. 2.

3. The medicament of claim 1, wherein the medicament comprises a pharmaceutical composition of a mini-peptide or a pharmaceutically acceptable salt thereof.

4. The agent for the treatment of endometrial cancer according to claim 1, wherein said agent is said oligopeptide or a pharmaceutically acceptable salt thereof alone as an active ingredient or in combination with an additional pharmaceutically active compound.

5. The medicament for treating endometrial cancer according to claim 1, 3 or 4, wherein a pharmaceutically acceptable excipient is further contained in the medicament.

6. The drug for treatment of endometrial cancer according to claim 5, wherein said adjuvant is selected from one or more of polylactic acid, polyglycolic acid and glycolic acid copolymer, p-carboxyphenylpropane and sebacic acid copolymer or ethylene-vinyl acetate copolymer, xylitol, oligosaccharide, chitin, potassium salt, sodium salt, hyaluronic acid, collagen, gelatin or albumin.

7. The medicament for the treatment of endometrial cancer of claim 1, wherein said medicament is in a pharmaceutically acceptable dosage form.

Images