CN110551197A - 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

Translated fromChinese

一种微肽及癌症治疗的药物A kind of micropeptide and medicine for cancer treatment

技术领域technical field

本发明属于生物医药技术领域，特别是指一种微肽CORO1C-47aa在癌症治疗中的药物的应用。The invention belongs to the technical field of biomedicine, in particular to the application of a micropeptide CORO1C-47aa in the treatment of cancer.

背景技术Background technique

子宫内膜癌(EC)是全球女性中第五位最常见的恶性肿瘤，占所有癌症死亡人数的1-2％。发生EC的风险因素包括年龄，社会经济地位，以及与雌激素暴露过多，糖尿病或高血压相关的因素，但对这种疾病的遗传风险因素知之甚少。Endometrial cancer (EC) is the fifth most common malignancy in women worldwide, accounting for 1-2% of all cancer deaths. Risk factors for developing EC include age, socioeconomic status, and factors associated with excess estrogen exposure, diabetes, or hypertension, but little is known about genetic risk factors for this disease.

非编码RNA(ncRNA)代表细胞中的大多数转录物并且经常参与EC的发展。环状RNA(circRNA)是一类新的广泛的ncRNA，常在哺乳动物体内调节基因的表达。circRNA是封闭的RNA转录物，通过单个前mRNA的反向剪接产生，circRNA的表达通常在物种间高度保守。近年来，报道的大多数circRNA被指出可作为miRNA海绵来调节不同细胞类型的基因表达，或结合和隔离其他RNA结合蛋白。然而，大多数circRNA的生物学功能仍未确定。Noncoding RNAs (ncRNAs) represent the majority of transcripts in cells and are often involved in the development of EC. Circular RNAs (circRNAs) are a new class of ubiquitous ncRNAs that often regulate gene expression in mammals. CircRNAs are closed RNA transcripts generated by back-splicing of a single pre-mRNA, and the expression of circRNAs is often highly conserved across species. In recent years, most of the reported circRNAs have been pointed out to act as miRNA sponges to regulate gene expression in different cell types, or to bind and sequester other RNA-binding proteins. However, the biological functions of most circRNAs remain undetermined.

最近研究表明，由于其细胞质定位，circRNA可被翻译产生蛋白质。例如，circZNF609和circMBI可被翻译。由内部核糖体进入位点驱动的Circ-FBXW7编码一种新的21kDa蛋白质。circPINTexon2由含有sORF的LncRNAs(LINC-PINT)产生，编码人类细胞中的肿瘤抑制肽。此外，一项研究还表明，circRNA可以由N6-甲基腺苷驱动翻译。Recent studies have shown that circRNAs can be translated to produce proteins due to their cytoplasmic localization. For example, circZNF609 and circMBI can be translated. Circ-FBXW7, driven by an internal ribosome entry site, encodes a novel 21 kDa protein circPINTexon2 is generated from sORF-containing LncRNAs (LINC-PINT) and encodes a tumor suppressor peptide in human cells. In addition, a study also showed that the translation of circRNAs can be driven by N6-methyladenosine.

发明内容Contents of the invention

本发明的目的是提供一种circ-0000437编码功能性微肽CORO1C-47aa，其在EC中充当肿瘤抑制基因，能有效抑制子宫内膜癌血管内皮细胞。The purpose of the present invention is to provide a functional micropeptide CORO1C-47aa encoded by circ-0000437, which acts as a tumor suppressor gene in EC and can effectively inhibit endometrial cancer vascular endothelial cells.

本发明是通过以下技术方案实现的：The present invention is achieved through the following technical solutions:

一种微肽，为CORO1C-47aa，其氨基酸序列如SEQ ID NO.1所示。A micropeptide is CORO1C-47aa, the amino acid sequence of which is shown in SEQ ID NO.1.

进一步的，所述CORO1C-47aa是由circ-0000437基因所编码。Further, the CORO1C-47aa is encoded by the circ-0000437 gene.

进一步的，所述circ-0000437基因的序列如SEQ ID NO.2所示。Further, the sequence of the circ-0000437 gene is shown in SEQ ID NO.2.

一种癌症治疗的药物，上述任一项微肽或其药学上可接受的盐在癌症治疗的药物方面的应用。A medicine for cancer treatment, the application of any one of the micropeptides or a pharmaceutically acceptable salt thereof in the medicine for cancer treatment.

进一步的，所述癌症为子宫内膜癌。Further, the cancer is endometrial cancer.

进一步的，癌症治疗的药物包括微肽或其药学上可接受的盐的药物组合物。Further, the drug for cancer treatment includes a pharmaceutical composition of micropeptide or a pharmaceutically acceptable salt thereof.

进一步的，所述药物为所述微肽或其药学上可接受的盐单独作为活性成分，或者，所述药物为所述微肽或其药学上可接受的盐与额外的药物活性化合物联合应用。Further, the drug is the micropeptide or its pharmaceutically acceptable salt alone as an active ingredient, or the drug is the combination of the micropeptide or its pharmaceutically acceptable salt and an additional pharmaceutically active compound .

进一步的，所述药物组合物中还含有药学上可接的辅料。Further, the pharmaceutical composition also contains pharmaceutically acceptable excipients.

进一步的，所述辅料选自聚乳酸、聚乙醇酸和羟基乙酸的共聚物、对羧苯基丙烷与葵二酸共聚物或乙烯-乙酸乙烯酯共聚物、木糖醇、低聚糖、甲壳素、钾盐、钠盐、透明质酸、胶原蛋白、明胶或白蛋白中的任一种或多种。Further, the auxiliary material is selected from polylactic acid, polyglycolic acid and glycolic acid copolymer, p-carboxyphenylpropane and sebacic acid copolymer or ethylene-vinyl acetate copolymer, xylitol, oligosaccharides, chitosan Any one or more of vitamins, potassium salts, sodium salts, hyaluronic acid, collagen, gelatin or albumin.

进一步的，所述癌症治疗的药物的剂型为药学上可以接受的剂型。Further, the dosage form of the drug for cancer treatment is a pharmaceutically acceptable dosage form.

本发明的有益效果是：The beneficial effects of the present invention are:

从翻译水平circRNA编码的肽角度，研究肽对子宫内膜癌起始阶段血管生成的影响。并通过靶向肽CORO1C-47aa的治疗，使微肽CORO1C-47aa与ARNT PAS-B结构域竞争性结合，阻断TACC3与其配体PAS-B的结合，导致HIF1富集至VEGF的HRE启动子附近从而抑制其基因表达。通过体外和体内实验证明，微肽CORO1C-47aa能明显抑制子宫内膜癌起始阶段新生血管的生成，对子宫内膜癌具有临床药用价值。From the perspective of peptides encoded by circRNAs at the translational level, the effects of peptides on angiogenesis at the initiation stage of endometrial cancer were studied. And by targeting the peptide CORO1C-47aa, the micropeptide CORO1C-47aa competes with the ARNT PAS-B domain, blocking the binding of TACC3 to its ligand PAS-B, resulting in the enrichment of HIF1 to the HRE promoter of VEGF nearby to inhibit gene expression. In vitro and in vivo experiments proved that the micropeptide CORO1C-47aa can significantly inhibit the formation of new blood vessels at the initial stage of endometrial cancer, and has clinical medicinal value for endometrial cancer.

附图说明Description of drawings

图1A至图1D为本发明实施例一中微肽CORO1C-47aa异位表达对EC细胞增殖的影响的实验数据图及示意图，具体的：Figure 1A to Figure 1D are the experimental data diagrams and schematic diagrams of the effect of ectopic expression of the micropeptide CORO1C-47aa on the proliferation of EC cells in Example 1 of the present invention, specifically:

图1A为circ-0000437OE，circ-0000437IRES突变和对照EC细胞在不同时间点的细胞增殖分析(平均值±SD，n＝5)；Figure 1A is the cell proliferation analysis of circ-0000437OE, circ-0000437IRES mutant and control EC cells at different time points (mean ± SD, n = 5);

图1B为异种移植小鼠中的肿瘤生长皮下植入用指定构建体转染的EC细胞(平均值±SD，n＝5，*p<0.05)；Figure 1B. Tumor growth in xenograft mice implanted subcutaneously with EC cells transfected with the indicated constructs (mean ± SD, n = 5, *p < 0.05);

图1C为CD31染色在来自circ-0000437OE，circ-0000437IRES突变和对照组的异种移植小鼠肿瘤组织切片上进行。使用ImageJ定量CD31染色。每个值是三次独立实验的平均值±SEM；Figure 1C shows CD31 staining on tumor tissue sections from circ-0000437OE, circ-0000437IRES mutant and control xenografted mice. CD31 staining was quantified using ImageJ. Each value is the mean ± SEM of three independent experiments;

图1D为使用来自HEC-1-B和Ishikawa细胞的HUVEC和条件培养基进行内皮管形成测定，其稳定表达circ-0000437OE，circ-0000437IRES突变(平均值±SD，n＝3，*p<0.05)。Figure 1D. Endothelial tube formation assay using HUVEC and conditioned media from HEC-1-B and Ishikawa cells stably expressing circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n = 3, *p < 0.05 ).

图2A至图2J为本发明实施例二中微肽CORO1C-47aa对HUVEC细胞增殖、迁移和分化的影响的实验数据图及示意图，具体的：Figure 2A to Figure 2J are the experimental data diagrams and schematic diagrams of the effect of the micropeptide CORO1C-47aa on the proliferation, migration and differentiation of HUVEC cells in Example 2 of the present invention, specifically:

图2A为CORO1C-47aa抑制HUVEC细胞的增殖；Figure 2A shows that CORO1C-47aa inhibits the proliferation of HUVEC cells;

图2B为使用HUVEC和来自HEC-1-B和Ishikawa细胞的条件培养基进行伤口愈合测定，其稳定表达circ-0000437OE，circ-0000437IRES突变(平均值±SD，n＝3，*p<0.05)；Figure 2B Wound healing assay using HUVEC and conditioned medium from HEC-1-B and Ishikawa cells stably expressing circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n = 3, *p < 0.05) ;

图2C为使用HUVEC和来自HEC-1-B和Ishikawa细胞的条件培养基进行内皮迁移测定，其稳定表达circ-0000437OE，circ-0000437IRES突变(平均值±SD，n＝3，*p<0.05)；Figure 2C. Endothelial migration assay using HUVEC and conditioned medium from HEC-1-B and Ishikawa cells stably expressing circ-0000437OE, circ-0000437IRES mutation (mean ± SD, n = 3, *p < 0.05) ;

图2D为微丝的Phalloidine-FITC染色和HUVEC细胞的免疫荧光染色和来自HEC-1-B和Ishikawa细胞的稳定表达circ-0000437OE，circ-0000437IRES突变的条件培养基。细胞核用DAPI复染；Figure 2D is Phalloidine-FITC staining of microfilaments and immunofluorescence staining of HUVEC cells and conditioned medium from HEC-1-B and Ishikawa cells stably expressing circ-0000437OE, circ-0000437IRES mutations. Nuclei were counterstained with DAPI;

图2E为在所示实验条件下的电子显微镜的代表性照片；Figure 2E is a representative photograph of an electron microscope under the indicated experimental conditions;

图2F为用CORO1C-47aa孵育的球状体的代表性显微照片；Figure 2F is a representative photomicrograph of spheroids incubated with CORO1C-47aa;

图2G为收获基质胶塞并加工用于qRT-PCR。将鼠CD31的mRNA表达水平标准化为人GAPDH的水平，并表达为鼠CD31/人GAPDH mRNA比率；Figure 2G. Matrigel plugs were harvested and processed for qRT-PCR. The mRNA expression level of murine CD31 was normalized to the level of human GAPDH and expressed as murine CD31/human GAPDH mRNA ratio;

图2H为在所示实验条件下HE染色的代表性照片；Figure 2H is a representative photograph of HE staining under the indicated experimental conditions;

图2I为Matrigel的代表性照片在指定的实验条件下插入；Figure 2I is a representative photograph of Matrigel inserted under the indicated experimental conditions;

图2J为将包埋在纤维蛋白凝胶中的HUVEC球状体与指定的培养基一起温育，然后，计数径向生长的细胞芽；Figure 2J is the incubation of HUVEC spheroids embedded in fibrin gel with the indicated medium, and then counting the radially growing cell buds;

图3为本发明实施例三中circ-0000437在子宫内膜癌与癌旁表达有显着差异的实验数据图及示意图；具体的：Fig. 3 is the experimental data diagram and schematic diagram of the significant difference between the expression of circ-0000437 in endometrial cancer and adjacent cancer in Example 3 of the present invention; specifically:

为与匹配的相邻正常组织相比，circ-0000437以低水平表达EC组织。通过qRT-PCR分析circ-0000437的表达水平并标准化为GAPDH，数据表示为来自三个独立实验的平均值±SEM。circ-0000437 expressed EC tissue at low levels compared to matched adjacent normal tissue. The expression level of circ-0000437 was analyzed by qRT-PCR and normalized to GAPDH, and the data were expressed as mean±SEM from three independent experiments.

具体实施方式Detailed ways

为了能够更清楚了解本发明的技术手段，并可依照说明书的内容予以实施，以下以本发明的较佳实施例并配合附图详细说明，以下通过实施仅是示例性的，仅能用来解释和说明本发明的技术方案，而不能解释为是对本发明技术方案的限制。In order to be able to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the following is a detailed description of the preferred embodiments of the present invention with the accompanying drawings. The following implementation is only exemplary and can only be used for explanation and explain the technical solution of the present invention, but should not be construed as limiting the technical solution of the present invention.

针对目前对子宫内膜癌治疗手段有限的情况，从翻译水平circRNA所编码的肽的角度，研究探索微肽CORO1C-47aa(人源全长重组蛋白-47aa)对子宫内膜癌的治疗价值。本技术方案首先鉴定了编码微肽CORO1C-47aa为环状RNA circ-0000437，其编码框为sORF。接着验证了微肽CORO1C-47aa在EC中表达下调。微肽CORO1C-47aa与ARNT PAS-B结构域竞争性结合，阻断TACC3与其配体PAS-B的结合，导致HIF1富集至VEGF的HRE启动子附近从而抑制其基因表达。总之，实验发现在子宫内膜癌中，微肽CORO1C-47aa通过抑制癌症起始阶段新生血管的生成来达到治疗的效果。In view of the current limited treatment options for endometrial cancer, from the perspective of the peptide encoded by the translational circRNA, we explored the therapeutic value of the micropeptide CORO1C-47aa (human full-length recombinant protein-47aa) on endometrial cancer. In this technical solution, the coding micropeptide CORO1C-47aa is firstly identified as circular RNA circ-0000437, and its coding frame is sORF. Next, it was verified that the micropeptide CORO1C-47aa was down-regulated in EC. The micropeptide CORO1C-47aa competitively binds to the ARNT PAS-B domain, blocks the binding of TACC3 to its ligand PAS-B, and leads to the enrichment of HIF1 near the HRE promoter of VEGF, thereby inhibiting its gene expression. In conclusion, the experiment found that in endometrial cancer, micropeptide CORO1C-47aa achieves the therapeutic effect by inhibiting the formation of angiogenesis in the initial stage of cancer.

在一些实施例中，可采用含有微肽CORO1C-47aa或其药学上可接受的盐的药物组合物，所述药物组合物可任选地还包含一种或多种额外的药物活性化合物。In some embodiments, a pharmaceutical composition comprising the micropeptide CORO1C-47aa or a pharmaceutically acceptable salt thereof, optionally further comprising one or more additional pharmaceutically active compounds, may be employed.

含有微肽CORO1C-47aa或药学上可接受的盐的药物具有以下至少一种功能：The medicine containing micropeptide CORO1C-47aa or a pharmaceutically acceptable salt has at least one of the following functions:

1)降低化学物诱导的子宫内膜癌的发生率；1) Reduce the incidence of chemical-induced endometrial cancer;

2)减缓或停止已建立的子宫内膜癌肿瘤灶的生长；2) Slow down or stop the growth of established endometrial cancer tumor foci;

3)减缓或停止已建立的子宫内膜癌肿瘤灶的转移；3) Slow down or stop the metastasis of established endometrial cancer tumor foci;

4)诱导产生子宫内膜癌特异性并对子宫内膜癌细胞具有杀伤的CTL细胞。4) Inducing and producing endometrial cancer-specific CTL cells capable of killing endometrial cancer cells.

本技术方案的药用辅料可经酶、酸碱或组织液水解或降解。药用辅料选自生物可容性的高分子多聚物、高分子多聚物的混合物或共聚物中的任一种或多种。具体地，药用辅料选自聚乳酸、聚乙醇酸和羟基乙酸的共聚物、对羧苯基丙烷与葵二酸共聚物或乙烯-乙酸乙烯酯共聚物中的任一种或多种，例如：The pharmaceutical adjuvant of the technical solution can be hydrolyzed or degraded by enzyme, acid-base or tissue fluid. The pharmaceutical excipient is selected from any one or more of biocompatible polymers, mixtures or copolymers of polymers. Specifically, the pharmaceutical excipient is selected from any 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, for example :

a)分子量为5000-15000、10000-20000、20000-35000或30000-50000的聚乳酸。a) Polylactic acid with a molecular weight of 5000-15000, 10000-20000, 20000-35000 or 30000-50000.

b)分子量为5000-15000、10000-20000、25000-35000或30000-50000的聚乳酸和羟基乙酸的共聚物。b) A copolymer of polylactic acid and glycolic acid with a molecular weight of 5000-15000, 10000-20000, 25000-35000 or 30000-50000.

c)乙烯-乙酸乙烯酯共聚物。c) Ethylene-vinyl acetate copolymers.

d)对羧苯基丙烷与癸二酸的共聚物，其中，对羧苯基丙烷∶癸二酸质量比为10∶90、20∶80、30∶70、40∶60、50∶50或60∶40。d) A copolymer of p-carboxyphenylpropane and sebacic acid, wherein the mass ratio of p-carboxyphenylpropane: sebacic acid is 10:90, 20:80, 30:70, 40:60, 50:50 or 60 : 40.

e)木糖醇、低聚糖、甲壳素、钾盐、钠盐、透明质酸、胶原蛋白、明胶或白蛋白中的一种或多种。e) One or more of xylitol, oligosaccharides, chitin, potassium salt, sodium salt, hyaluronic acid, collagen, gelatin or albumin.

本技术方案的药物组合物可以用于制备治疗人及动物的各种抗肿瘤药物，具体为抗子宫内膜癌药物。The pharmaceutical composition of the technical solution can be used to prepare various antitumor drugs for treating humans and animals, specifically anti-endometrial cancer drugs.

本技术方案的药物可制成多种药学可以接受的药物制剂，如，但不限于，浑悬液、软膏、胶囊、丸剂、片剂或注射剂等；呈各种形状，如，但不限于，颗粒样、片状、球形、块状、针状、棒状及膜状。上述剂型和形状适用于含或不含添加剂的组合物，且所述药物制剂采用本领域常规的制备方法进行制备，如，但不限于，(i)把载体支持物粉末与药物混合然后压制成植入剂，即所谓的混合法；(ii)把载体支持物熔化，与待包装的药物相混合，然后固体冷却，即所谓的熔融法；(iii)把载体支持物溶解于溶剂中，把待包装的药物溶解或分散于聚合物溶液中，然后蒸发溶剂，乾燥，即所谓的溶解法；(iv)喷雾干燥法；及(v)冷冻干燥法等。其中溶解法可用于微球的制造，抗癌药物组合物也可包装脂质体中。The medicine of this technical solution can be made into a variety of pharmaceutically acceptable pharmaceutical preparations, such as, but not limited to, suspensions, ointments, capsules, pills, tablets or injections, etc.; in various shapes, such as, but not limited to, Granular, flaky, spherical, blocky, needle-like, rod-like and film-like. The above-mentioned dosage forms and shapes are suitable for compositions with or without additives, and the pharmaceutical preparations are prepared by conventional preparation methods in the art, such as, but not limited to, (i) mixing the carrier support powder with the drug and then compressing it into Implants, the so-called mixing method; (ii) melting the carrier support, mixing with the drug to be packaged, and then cooling the solid, the so-called melting method; (iii) dissolving the carrier support in a solvent, putting The drug to be packaged is dissolved or dispersed in the polymer solution, and then the solvent is evaporated and dried, which is the so-called dissolution method; (iv) spray drying method; and (v) freeze drying method, etc. The dissolving method can be used in the manufacture of microspheres, and the anticancer drug composition can also be packaged in liposomes.

本技术方案的药物可经各种途径给药，如经脉、动脉、皮下、肌肉、皮内、腔内、瘤内、瘤周等。给药途径取决于多种因素，为于肿瘤所在部位获有效浓度，药物可经其它多种途径给予，如选择性地动脉灌注，腔内灌注(intracavitary)，腹腔(intraperitoneal)或胸腔(intrapleural)及椎管内给药。The medicine of the technical solution can be administered through various routes, such as meridian, artery, subcutaneous, muscle, intradermal, intracavitary, intratumoral, peritumoral, etc. The route of administration depends on many factors. In order to obtain an effective concentration at the tumor site, the drug can be administered through other routes, such as selective arterial infusion, intracavitary, intraperitoneal or intrapleural and intraspinal administration.

所述抗肿瘤药物的给药剂量可根据具体给药对象、给药途径或药物的制剂形式不同进行适当的变化，但以保证该药物组合物在哺乳动物体内能够达到有效的血药浓度为前提。The dosage of the antitumor drug can be appropriately changed according to the specific administration object, the route of administration or the preparation form of the drug, but it is based on the premise that the pharmaceutical composition can reach an effective blood concentration in the mammalian body. .

以下实施例，如未特别说明，所采用的实验方法均为本领域的常规技术；所述试剂或材料，如未特别说明，均来源于商业渠道。6-8周龄雌性裸鼠购自中国科学院上海实验动物中心(中国上海)。所有细胞系均购自Procell生命科技有限公司。这些细胞系均经DNA指纹图谱分析，传代不到6个月。DMEM、MEM和胎牛血清(FBS)购自Invitgen公司。HEC-1-B细胞在含10％胎牛血清的MEM培养基中生长，Ishikawa细胞在含10％胎牛血清的DMEM培养基中生长。所有细胞株均在含青霉素/链霉素的培养基中生长，温度37℃，空气湿度为5％CO₂。In the following examples, unless otherwise specified, the experimental methods used are conventional techniques in the art; the reagents or materials, unless otherwise specified, are sourced from commercial sources. 6-8-week-old female nude mice were purchased from Shanghai Experimental Animal Center, Chinese Academy of Sciences (Shanghai, China). All cell lines were purchased from Procell Life Sciences Co., Ltd. These cell lines were analyzed by DNA fingerprinting and 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 a temperature of 37°C and an air humidity of 5% CO₂ .

实施例一Embodiment one

请参见图1A至图1D，微肽CORO1C-47aa异位表达对EC细胞增殖的影响。Please refer to Figure 1A to Figure 1D, the effect of ectopic expression of micropeptide CORO1C-47aa on the proliferation of EC cells.

构建circ-0000437OE载体和IRES突变载体克隆的EC细胞：发明人将全长或突变的circ-0000437IRES序列克隆在Rluc和Luc报道基因之间，形成两组Luc/Rluc高低活性对比的载体。接着将circ-0000437OE载体和IRES突变载体转染到EC细胞(HEC-1-B，Ishikawa)中，形成可过表达circ-0000437载体与IRES突变的EC细胞。发明人为确定CORO1C-47aa的框内ATG密码子是否可以促进翻译的启动，构建了ORF突变表达载体(其中起始密码子ATGGTG已突变为ATTGTT)和FLAG标记至CORO1C-47aa的C末端。使用抗FLAG蛋白质印迹在转染circ-0000437OE载体的细胞中可检测到CORO1C-47aa-FLAG，而在转染IRES突变载体的细胞中则无法检测。证明circ-0000437OE载体过表达可以提高CORO1C-47aa表达，而circ-0000437-IRES突变载体不能。发明人为检测CORO1C-47aa的细胞定位，通过免疫荧光测定，发现CORO1C-47aa集中在EC细胞的细胞核中。Construction of EC cells cloned by circ-0000437OE vector and IRES mutant vector: The inventors cloned the full-length or mutated circ-0000437IRES sequence between the Rluc and Luc reporter genes to form two sets of vectors for comparing the high and low activity of Luc/Rluc. Then, the circ-0000437OE vector and the IRES mutation vector were transfected into EC cells (HEC-1-B, Ishikawa) to form EC cells that could overexpress the circ-0000437 vector and the IRES mutation. In order to determine whether the in-frame ATG codon of CORO1C-47aa can promote the initiation of translation, the inventors constructed an ORF mutation expression vector (in which the initiation codon ATGGTG has been mutated to ATTGTT) and FLAG tagged to the C-terminus of CORO1C-47aa. CORO1C-47aa-FLAG was detectable in cells transfected with the circ-0000437OE vector but not in cells transfected with the IRES mutant vector using anti-FLAG western blotting. It was proved that the overexpression of circ-0000437OE vector can increase the expression of CORO1C-47aa, while the circ-0000437-IRES mutant vector could not. In order to detect the cellular localization of CORO1C-47aa, the inventors found that CORO1C-47aa was concentrated in the nucleus of EC cells by immunofluorescence assay.

如图1A所示，发明人分别对CORO1C-47aa稳定上调以及转染IRES突变载体的HEC-1-B和Ishikawa细胞进行CCK-8测定，然而结果表明，与对照细胞相比，无论是CORO1C-47aaOE组或IRES突变组的EC细胞增殖均不显着。发明人为探究CORO1C在肿瘤生长中的生物学意义，将每组5只小鼠分别进行皮下注射CORO1C-47aa过表达和IRES突变的EC细胞。如图1B所示，与对照异种移植物相比，CORO1C-47aa OE组的异种移植物的生长受到显着抑制。而IRES突变组与对照组的异种移植物没有显着差异。为探究CORO1C-47aa的抑制作用是否可归因于血管生成破坏，进行肿瘤组织的免疫组织化分析以检测CD31的表达。如图1C所示，与IRES突变组和对照组相比，在CORO1C-47aa OE组中CD31被显着抑制。如图1D所示，发明人还进行了内皮管形成测定以探究CORO1C-47aa如何影响基质胶上的内皮网络形成。结果显示，与对照组相比，CORO1C-47aa有显着破坏HUVEC细胞形成毛细管的能力。As shown in Figure 1A, the inventors performed CCK-8 assays on HEC-1-B and Ishikawa cells that were stably upregulated by CORO1C-47aa and transfected with an IRES mutant vector, however, the results showed that neither CORO1C-47aa nor EC cell proliferation was not significant in either the 47aaOE group or the IRES mutation group. In order to explore the biological significance of CORO1C in tumor growth, the inventors subcutaneously injected EC cells with CORO1C-47aa overexpression and IRES mutation into 5 mice in each group. As shown in Figure 1B, the growth of xenografts in the CORO1C-47aa OE group was significantly inhibited compared with control xenografts. However, xenografts from the IRES mutant group were not significantly different from the control group. To explore whether the inhibitory effect of CORO1C-47aa could be attributed to disrupted angiogenesis, immunohistochemical analysis of tumor tissues was performed to detect the expression of CD31. As shown in Figure 1C, CD31 was significantly suppressed in the CORO1C-47aa OE group compared with the IRES mutation group and the control group. As shown in Figure ID, the inventors also performed an endothelial tube formation assay to explore how CORO1C-47aa affects endothelial network formation on Matrigel. The results showed that CORO1C-47aa significantly disrupted the ability of HUVEC cells to form capillaries compared with the control group.

实施例二Embodiment two

请参见图2A至图2J，本实施例研究了微肽CORO1C-47aa对HUVEC细胞增殖、迁移和分化的影响。发明人在转染了CORO1C-47aa过表达和IRES突变的HEC-1-B和Ishikawa细胞的条件培养基中培养HUVEC细胞。如图2A所示，HUVEC细胞增殖试验结果表明，CORO1C-47aa OE组的HUVEC细胞增殖显着降低。为探究CORO1C-47aa的过表达对HUVEC细胞迁移的影响，发明人使用条件培养基培养的HUVEC细胞进行伤口愈合测定和Transwell。如图2B和图2C所示，伤口愈合测定和Transwell均证明HUVEC细胞的迁移能力因CORO1C-47aa的过表达而受到显着抑制，而IRES突变组与空载体转染组相比没有显着差异。发明人对CORO1C-47aa对内皮细胞骨架的影响进行进一步探究，如图2D所示，CORO1C-47aa OE组中的F-肌动蛋白无法重排成为细胞骨架，不利于细胞的迁移。如图2E所示，发明人还通过电子显微镜检测HUVEC细胞的胞间连接，结果显示对照组和IRES突变组的细胞间连结紧密，而CORO1C-47aa OE组中，HUVEC细胞的胞间连结被显着诱导，细胞间距缩小。为进一步证明CORO1C-47aa可抑制血管生成的可能性，发明人进行了小鼠的Matigel栓测定。如图2F、2G和2H所示，在CORO1C-47aaOE组中，用Matigel皮下注射的小鼠体内的新生血管生成被显着抑制。最后，发明人在体外血管生成三维模型中评估了CORO1C-47aa的作用，HUVEC球状体侵入三维纤维蛋白基质，由于细胞外基质的局部分解与HUVEC迁移与生长共同作用产生内皮芽。如图2I和2J所示，CORO1C-47aa导致HUVEC发芽减少。这些结果表明CORO1C-47aa能够通过抑制内皮细胞增殖，迁移和分化来抑制起始阶段的血管生成。Please refer to FIG. 2A to FIG. 2J , this example studies the effects of the micropeptide CORO1C-47aa on the proliferation, migration and differentiation of HUVEC cells. The inventors cultured HUVEC cells in the conditioned medium of HEC-1-B and Ishikawa cells transfected with CORO1C-47aa overexpression and IRES mutation. As shown in Figure 2A, the results of the HUVEC cell proliferation assay showed that the HUVEC cell proliferation in the CORO1C-47aa OE group was significantly reduced. To explore the effect of overexpression of CORO1C-47aa on HUVEC cell migration, the inventors used HUVEC cells cultured in conditioned medium for wound healing assay and Transwell. As shown in Figure 2B and Figure 2C, both the wound healing assay and Transwell demonstrated that the migration ability of HUVEC cells was significantly inhibited by the overexpression of CORO1C-47aa, while there was no significant difference between the IRES mutant group and the empty vector transfection group . The inventors further explored the effect of CORO1C-47aa on the endothelial cytoskeleton. As shown in Figure 2D, F-actin in the CORO1C-47aa OE group could not be rearranged into the cytoskeleton, which was not conducive to cell migration. As shown in Figure 2E, the inventors also detected the intercellular junctions of HUVEC cells by electron microscopy, and the results showed that the intercellular junctions of the control group and the IRES mutation group were tight, while in the CORO1C-47aa OE group, the intercellular junctions of HUVEC cells were significantly With induction, the intercellular space shrinks. To further demonstrate the possibility that CORO1C-47aa can inhibit angiogenesis, the inventors performed a Matigel plug assay in mice. As shown in Figures 2F, 2G, and 2H, in the CORO1C-47aaOE group, neovascularization in mice injected subcutaneously with Matigel was significantly inhibited. Finally, the inventors assessed the role of CORO1C-47aa in a 3D model of angiogenesis in vitro, in which HUVEC spheroids invade a 3D fibrin matrix and generate endothelial buds due to local breakdown of the extracellular matrix combined with HUVEC migration and growth. As shown in Figures 2I and 2J, CORO1C-47aa resulted in reduced sprouting of HUVECs. These results suggest that CORO1C-47aa is able to inhibit angiogenesis at the initial stage by inhibiting endothelial cell proliferation, migration and differentiation.

实施例三Embodiment three

请参见图3，circ-0000437在子宫内膜癌与癌旁表达有显着差异，发明人运用RT-qPCR测定来在中国东部(苏州)及中国北部中心(北京)的共198个配对的EC样品和匹配的非癌组织中circ-0000437的水平。与匹配的癌症组织相比，circ-0000437在癌旁组织中以更高水平表达。Please refer to Figure 3. The expression of circ-0000437 is significantly different between endometrial cancer and adjacent tumors. The inventors used RT-qPCR to detect a total of 198 paired ECs in eastern China (Suzhou) and northern China (Beijing). Levels of circ-0000437 in samples and matched noncancerous tissues. circ-0000437 was expressed at higher levels in paracancerous tissues compared with matched cancer tissues.

综上所述：本发明从翻译水平circRNA编码的肽角度，研究肽对子宫内膜癌起始阶段新生血管的影响，证明了肽在子宫内膜癌中的重要作用。鉴于微肽CORO1C-47aa与ARNTPAS-B结构域竞争性结合，阻断TACC3与其配体PAS-B的结合，导致HIF1富集至VEGF的HRE启动子附近从而抑制其基因表达，以此达到治疗子宫内膜癌的效果。并且，通过微肽CORO1C-47aa进行的靶向治疗，上调微肽CORO1C-47aa的表达，对子宫内膜癌具有临床药用价值。In summary: From the perspective of peptides encoded by circRNA at the translation level, the present invention studies the effect of peptides on neovascularization at the initial stage of endometrial cancer, and proves the important role of peptides in endometrial cancer. In view of the fact that the micropeptide CORO1C-47aa competes with the ARNTPAS-B domain, blocking the binding of TACC3 to its ligand PAS-B, resulting in the enrichment of HIF1 near the HRE promoter of VEGF and inhibiting its gene expression, so as to achieve the treatment of uterine effect on endometrial cancer. Moreover, targeted therapy through the micropeptide CORO1C-47aa up-regulates the expression of the micropeptide CORO1C-47aa, which has clinical medicinal value for endometrial cancer.

以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。The various technical features of the above-mentioned embodiments can be combined arbitrarily. To make the description concise, all possible combinations of the various technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, should be considered as within the scope of this specification.

以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

序列表sequence listing

<110> 苏州大学<110> Soochow University

<120> 一种微肽及癌症治疗的药物<120> A micropeptide and a drug for cancer treatment

<160> 2<160> 2

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 47<211> 47

<212> PRT<212> PRT

<213> 微肽CORO1C-47aa(人工合成)<213> micropeptide CORO1C-47aa (artificial synthesis)

<400> 1<400> 1

Met Asn Pro Arg Thr Ser Thr Thr Ser Thr His Ser Ala Ala Arg SerMet Asn Pro Arg Thr Ser Thr Thr Ser Thr His Ser Ala Ala Arg Ser

1 5 10 151 5 10 15

Leu Arg Glu Gly Trp Val Thr Cys Pro Pro Gly Asp Leu Met Leu ThrLeu Arg Glu Gly Trp Val Thr Cys Pro Gly Asp Leu Met Leu Thr

20 25 30 20 25 30

Asn Val Arg Pro Glu Lys Tyr Ala Gly Thr Asn Cys Ser Ser SerAsn Val Arg Pro Glu Lys Tyr Ala Gly Thr Asn Cys Ser Ser Ser

35 40 45 35 40 45

<210> 2<210> 2

<211> 144<211> 144

<212> RNA<212> RNA

<213> 基因circ-0000437(人工合成)<213> Gene circ-0000437 (artificially synthesized)

<400> 2<400> 2

augaaucccc guacguccac uaccucaaca cauucagcag caaggagccu cagagaggga 60augaaucccc guacguccac uaccucaaca cauuucagcag caaggagccu cagagaggga 60

uggguuacau gcccaagagg ggacuugaug uuaacaaaug ugagauugcc agaaaaauau 120uggguuacau gcccaagagg ggacuugaug uuaacaaaug ugagauugcc agaaaaauau 120

gcaggaacca auugcucuuc auga 144gcaggaacca auugcucuuc auga 144

Claims (10)

1. A micro-peptide is characterized by being CORO1C-47aa, and the amino acid sequence of the micro-peptide is shown as SEQ ID NO. 1.

2. the oligopeptide according to claim 1, wherein the CORO1C-47aa is encoded by the circ-0000437 gene.

3. The oligopeptide according to claim 2, wherein the sequence of the circ-0000437 gene is shown in SEQ ID No. 2.

4. A medicament for the treatment of cancer, characterized in that the oligopeptide according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof is used in the medicament for the treatment of cancer.

5. The medicament for the treatment of cancer according to claim 4, wherein said cancer is endometrial cancer.

6. The medicament for cancer treatment according to claim 4, wherein the medicament comprises a pharmaceutical composition of a micro peptide or a pharmaceutically acceptable salt thereof.

7. the medicament for cancer treatment according to claim 4, wherein the medicament is the micro-peptide or a pharmaceutically acceptable salt thereof alone as an active ingredient, or the medicament is the micro-peptide or a pharmaceutically acceptable salt thereof in combination with an additional pharmaceutically active compound.

8. The medicament for treating cancer according to claim 4, 6 or 7, wherein the medicament further comprises pharmaceutically acceptable excipients.

9. The drug for cancer treatment according to claim 8, wherein the adjuvant is 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, xylitol, oligosaccharide, chitin, potassium salt, sodium salt, hyaluronic acid, collagen, gelatin or albumin.

10. The agent for cancer therapy according to claim 4, wherein the agent for cancer therapy is in a pharmaceutically acceptable dosage form.