WO2024050861A1 - Runx2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用 - Google Patents

Runx2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用 Download PDF

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WO2024050861A1
WO2024050861A1 PCT/CN2022/118895 CN2022118895W WO2024050861A1 WO 2024050861 A1 WO2024050861 A1 WO 2024050861A1 CN 2022118895 W CN2022118895 W CN 2022118895W WO 2024050861 A1 WO2024050861 A1 WO 2024050861A1
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runx2
breast
cells
malignant
tumors
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聂燕
宋尔卫
雷蓉
黄红颜
贺诗施
艾力菲热***
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中山大学孙逸仙纪念医院
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  • the invention relates to the field of medical biotechnology, and specifically relates to the application of RUNX2 inhibitors in the preparation of drugs against malignant phyllodes tumors of the breast.
  • Phyllodes tumor is a rare fibroepithelial tumor, accounting for 0.3% to 1% of all breast tumors. According to existing literature research, borderline and malignant phyllodes tumors not only grow rapidly, but are also prone to local recurrence and distant metastasis. Blood metastasis is the most common way of metastasis, and lung and bone are the most common metastasis sites. The recurrence rate of malignant phyllodes tumors of the breast is as high as 53.1%, and the metastasis rate is as high as 43.1%. Once recurrence and metastasis occur, the patient will die within a short period of time. The mortality rate of malignant phyllodes tumors is approximately 16.3%.
  • breast phyllodes tumors The treatment of breast phyllodes tumors is mainly surgical. Malignant phyllodes tumors have poor clinical prognosis due to their high recurrence rate and high metastasis rate. Unlike breast cancer, adjuvant treatments such as chemotherapy and radiotherapy are not effective after surgery for malignant phyllodes tumors of the breast. There are currently no studies reporting clinical benefit from targeted therapy and immunotherapy. Moreover, because phyllodes tumors of the breast are relatively rare, there is a lack of large-scale clinical data and basic research, especially research on specific molecular markers and treatment targets, and no effective progress has been made, further resulting in the lack of research or screening. Progress in effective treatments has stalled.
  • RUNX2 is an important member of the RUNX family of transcription factors and is named because it contains a runt domain.
  • the biological role of RUNX2 is mainly as a specific transcription factor for osteogenic differentiation, regulating the transcription of genes such as type I collagen, osteomodulin, osteocalcin, col1a1, col1a2, bone sialoprotein (BSP) and fibronectin. It plays an important role in the formation and differentiation of osteoblasts, differentiation and maturation of chondrocytes, the formation and resorption of osteoclasts, and the synthesis of bone matrix proteins.
  • BSP bone sialoprotein
  • RUNX2 is related to the biological behavior of malignant phyllodes tumors of the breast.
  • the present invention discloses the technical application of RUNX2 as a biological behavioral marker and drug target of malignant phyllodes tumors of the breast.
  • RUNX2 is highly expressed in malignant breast phyllodes tumor cells and promotes the progression of malignant breast phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of malignant phyllodes tumors of the breast.
  • RUNX2 can be used as a marker and drug target for malignant phyllodes tumors of the breast, and has high application value in the auxiliary diagnosis, screening and preparation of targeted therapeutic drugs for breast phyllodes tumors. The following applications are thus exposed:
  • RUNX2 as a marker in screening or preparing drugs against malignant breast phyllodes tumors.
  • detection of RUNX2 expression in test tumor cells can be used as a means to evaluate drug efficacy.
  • RUNX2 as a drug target in screening or preparing drugs against malignant phyllodes tumors of the breast.
  • Drugs targeting RUNX2 as a therapeutic target can be prioritized for screening as potential drugs against breast malignant phyllodes tumors.
  • the drug CADD522 inhibits the binding of RUNX2 to DNA.
  • the invention further discloses the application of the RUNX2 inhibitor CADD522 in the preparation of drugs against malignant phyllodes tumors of the breast.
  • the present invention has the following beneficial effects:
  • the present invention discloses for the first time that the overexpression of RUNX2 in breast malignant phyllodes tumor specimens is significantly related to the biological behavior of breast malignant phyllodes tumors. Knocking down the expression of RUNX2 can inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumors. It is believed that RUNX2 can be used as a marker and therapeutic target for screening drugs against malignant phyllodes tumors of the breast, and has high application value in the screening and preparation of drugs targeting malignant phyllodes tumors of the breast.
  • CADD522 can significantly inhibit the proliferation, migration, and invasion of breast malignant phyllodes tumor cells in vitro, and can significantly inhibit the growth of tumors in vivo.
  • CADD522 has great potential clinical application value in the development of targeted therapeutic drugs for breast phyllodes tumors.
  • Figure 1 A is a histogram of RUNX2 mRNA expression levels in benign and malignant breast phyllodes tumor tissues; B is a protein immunoblot gel imaging image of RUNX2 protein expression test in benign and malignant breast phyllodes tumor tissues; C is benign , RUNX2 expression levels in paraffin sections of malignant phyllodes tumors and RUNX2 expression levels in paraffin sections of multiple recurrences of tumor tissue from the same patient.
  • Figure 2 A is a graphical representation of the test results of knocking down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01 (i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A) and changing the cell proliferation ability.
  • SYSH-MPT-01 i.e., the cell line HJP-0320, disclosed in the patent application with publication number CN111019898A
  • B knocks down the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of the test on changes in cell colony formation ability
  • C shows the knockdown of the RUNX2 gene of the breast malignant phyllodes tumor cell line SYSH-MPT-01, Graphical representation of cell cycle change test results
  • D is a graphic representation of the knockdown of the RUNX2 gene in the breast malignant phyllodes tumor cell line SYSH-MPT-01, and the results of a cell migration and invasion ability change test
  • E is a graphic representation of the knockdown of the breast malignant phyllodes tumor cell line Illustration of the test results of the RUNX2 gene of SYSH-MPT-01 and the changes in cell collagen contraction ability.
  • FIG. 3 A is a graphical representation of the test results of changes in cell proliferation ability overexpressing RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 (i.e., the cell line GLK-1010, disclosed in the patent application with publication number CN111019897A) ; B is a graphical representation of the test results of the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in the clonogenic ability of the cells; C is the overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01 RUNX2, a graphical representation of the test results of cell cycle changes; D is a graphical representation of the test results of overexpression of RUNX2 in the benign breast phyllodes tumor cell line SYSH-BPT-01, and the changes in cell migration and invasion capabilities; E is a graphic representation of the test results of the benign breast phyllodes tumor RUNX2 is overex
  • Figure 4 is a graphical representation of the results of a trial on the impact of RUNX2 on the prognosis of patients with breast phyllodes tumors.
  • Figure 5 is a graphical representation of the results of a test to knock down RUNX2 in malignant phyllodes tumor cells on subcutaneous tumor formation ability and tumor growth; A is the change in tumor volume at the corresponding time point, and B is a picture of the tumors in each group.
  • FIG. 6 is a diagram showing the effect of CADD522 on the malignant phyllodes tumor cell lines SYSH-BPT-01 and SYSH-MPT-02 (i.e., the cell line LJ-0429, disclosed in the patent application with publication number CN111019899A);
  • A is CADD522 The survival rate curve acting on the malignant phyllodes tumor cell line, illustrating the determination of the half inhibitory concentration (IC50) of CADD522 on the malignant phyllodes tumor cell line;
  • B is the growth of the malignant phyllodes tumor cell line SYSH-MPT-01 inhibited by CADD522 , showing a dose- and time-dependent diagram;
  • C is a diagram showing the effect of CADD522 on the migration and invasion ability of breast malignant phyllodes tumor cells.
  • Figure 7 is a diagram showing the effect of CADD522 on malignant phyllodes tumors in vivo.
  • A is the inhibition of tumor growth by intraperitoneal injection of CADD522 in the PDX model of malignant phyllodes tumors, and the changes in tumor volume at corresponding time points.
  • B is the pictures of tumors in each group.
  • Example 1 Analysis of RUNX 2 expression profile of breast phyllodes tumors
  • RUNX2-specific primers to perform fluorescence quantitative PCR to detect the expression levels of RUNX2 mRNA in different types of breast phyllodes tumor tissues.
  • Reverse transcription to synthesize cDNA Reverse transcription to synthesize cDNA: Add 1 ⁇ g of template RNA and 4 ⁇ l of reverse transcriptase SuperScript II mix (containing Buffer, dNTP, HiScript II reverse transcriptase, RNase, Random primers/Oligo dT) in the PCR tube.
  • Real-time quantitative PCR amplification Dilute the template cDNA 3 times and mix well for later use. Each experimental group was set up with 3 parallel tubes. Reaction system: 1 ⁇ l cDNA, 5 ⁇ l SYBR green dye, 0.3 ⁇ l forward primer, 0.3 ⁇ l reverse primer, 3.4 ⁇ l ddH 2 O, centrifuge and mix. Reaction conditions: 95°C for 5min, 95°C for 30 seconds, 55°C-60°C (depending on the annealing temperature) for 30s, a total of 40 cycles. GAPDH was used as an internal reference to analyze the relative transcription levels of genes. The primer sequences are as follows:
  • Example 2 Effects of knocking down RUNX2 on the proliferation, migration, invasion, cell cycle and collagen contraction ability of malignant phyllodes tumor cells
  • siRNA The sequence of siRNA is as follows:
  • Example 6 Effect of RUNX2 small molecule inhibitor CADD522 on IC50, proliferation, migration and invasion of malignant phyllodes tumor cells
  • the RUNX2 small molecule inhibitor CADD522 inhibits the migration and invasion of malignant phyllodes tumor cells
  • the RUNX2 small molecule inhibitor CADD522 inhibits PDX tumor growth

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Abstract

RUNX2作为标志物和治疗靶点在筛选和制备抗乳腺恶性叶状肿瘤药物中的应用，以及RUNX2 抑制剂 CADD522 在制备抗乳腺恶性叶状肿瘤药物中的应用。公开了RUNX2 的过表达与乳腺恶性叶状肿瘤的生物学行为相关，敲低 RUNX2表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。并经试验证明，RUNX2 特异性小分子抑制剂 CADD522 在体外可以抑制乳腺恶性叶状肿瘤细胞的增殖、迁移、侵袭，在体内可以抑制肿瘤的生长。

Description

RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用 技术领域
本发明涉及医药生物技术领域，具体涉及RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用
背景技术
乳腺叶状肿瘤(Phyllodes tumor,PT)是一种罕见的纤维上皮性肿瘤，占所有乳腺肿瘤的0.3％至1％。根据现有的文献研究，交界性、恶性叶状肿瘤不仅生长快，而且极易局部复发和远处转移，血液转移是最常见的转移方式，肺、骨是最常见的转移部位。乳腺恶性叶状肿瘤的复发率高达53.1％，转移率高达43.1％。一旦出现复发、转移，患者会在短期内死亡，恶性叶状肿瘤的死亡率约为16.3％。乳腺叶状肿瘤的治疗主要是手术治疗，恶性叶状肿瘤由于其具有高复发率、高转移率等特点，其临床预后差。不同于乳腺癌，乳腺恶性叶状肿瘤术后的辅助治疗手段如化疗、放疗等治疗效果均不佳，靶向治疗及免疫治疗目前也无研究报道具有临床获益。且由于乳腺叶状肿瘤较为罕见，缺乏大规模的临床数据与基础研究，特别在特异性分子标志物和治疗靶点方面的研究更为匮乏，一直没有取得有效的进展，进一步造成在寻求或筛选有效治疗药物方面停滞不前。
RUNX2是转录因子RUNX家族的重要成员，其因含有runt结构域而被命名。RUNX2的生物学作用主要是作为成骨分化的特异性转录因子，调控一型胶原、骨调素、骨钙蛋白、col1a1、col1a2、骨涎蛋白(BSP)和纤维连接蛋白等基因的转录，在成骨细胞形成和分化、软骨细胞分化和成熟、破骨细胞的形成和吸收及骨基质蛋白的合成中起到重要作用。但未见有RUNX2与乳腺恶性叶状肿瘤生物学行为相关的报道。
发明内容
为克服以上技术问题，本发明公开了RUNX2作为乳腺恶性叶状肿瘤生物学行为标志物及药物靶点相关的技术应用。
发明人在临床样本和体外细胞实验中中发现，RUNX2在乳腺恶性叶状肿瘤细胞中高表达，促进乳腺恶性叶状肿瘤的进展。敲低RUNX2的表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。RUNX2可作为乳腺恶性叶状肿瘤标志物及药物 靶点，在乳腺叶状肿瘤辅助诊断，筛选和制备靶向治疗药物开发中具有较高的应用价值。由此公开了以下应用：
1.RUNX2作为标志物在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。在筛选乳腺恶性叶状肿瘤药物中，可通过检测试验肿瘤细胞的RUNX2表达作为其中的评价药效手段。
2.RUNX2作为药物靶点在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。可优先筛选针对RUNX2作为治疗靶点的药物作为潜在的抗乳腺恶性叶状肿瘤药物。
例如RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用。
药物CADD522，具有抑制RUNX2与DNA结合的作用。本发明进一步公开RUNX2抑制剂CADD522在制备抗乳腺恶性叶状肿瘤药物中的应用。
与现有技术相比，本发明具有如下的有益效果：
1.本发明首次公开乳腺恶性叶状肿瘤标本RUNX2的过表达与乳腺恶性叶状肿瘤的生物学行为显著相关，敲低RUNX2的表达可以抑制乳腺恶性叶状肿瘤的增殖、迁移、侵袭。认为RUNX2可作为筛选抗乳腺恶性叶状肿瘤药物的标志物和治疗靶点，在筛选和制备靶向抗乳腺恶性叶状肿瘤药物方面均具有较高应用价值。
2.经试验证明，RUNX2特异性小分子抑制剂CADD522在体外可以显著抑制乳腺恶性叶状肿瘤细胞的增殖、迁移、侵袭，在体内可以显著抑制肿瘤的生长。CADD522在乳腺叶状肿瘤靶向治疗药物开发中具有很好潜在临床应用价值。
附图说明
图1:A是良性和恶性乳腺叶状肿瘤组织中RUNX2的mRNA表达水平的柱状图；B是良性和恶性乳腺叶状肿瘤组织中RUNX2蛋白表达试验的蛋白免疫印迹凝胶成像图；C是良、恶性叶状肿瘤石蜡切片中的RUNX2表达水平及同一患者多次复发的肿瘤组织石蜡切片中RUNX2表达水平情况图示。
图2：A是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01(即细胞系HJP-0320,公开于公布号为CN111019898A的专利申请中)的RUNX2基因，细胞增殖能力改变试验结果图示；B敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞克隆形成能力改变试验结果图示；C是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞周期改变试验结果图示；D是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞迁移和侵袭能力改变试验结果图 示；E是敲降乳腺恶性叶状肿瘤细胞系SYSH-MPT-01的RUNX2基因，细胞胶原收缩能力改变试验结果图示。
图3：A是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01(即细胞系GLK-1010,公开于公布号为CN111019897A的专利申请中)中过表达RUNX2，细胞增殖能力改变试验结果图示；B是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞的克隆形成能力改变试验结果图示；C是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞周期的改变试验结果图示；D是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞迁移及侵袭能力改变试验结果图示；E是在乳腺良性叶状肿瘤细胞系SYSH-BPT-01中过表达RUNX2，细胞胶原收缩能力改变试验结果图示。
图4是RUNX2对乳腺叶状肿瘤患者预后影响试验结果图示。
图5是是敲降恶性叶状肿瘤细胞的RUNX2，对皮下成瘤能力及肿瘤生长影响试验结果图示；A是相应时间点肿瘤体积的变化情况,B是各组肿瘤图片。
图6是CADD522对恶性叶状肿瘤细胞系SYSH-BPT-01，SYSH-MPT-02(即细胞系LJ-0429,公开于公布号为CN111019899A的专利申请中)作用试验效果图示；A是CADD522作用于恶性叶状肿瘤细胞系的存活率曲线，确定CADD522对恶性叶状肿瘤细胞系的半数抑制浓度(IC50)的图示；B是CADD522抑制恶性叶状肿瘤细胞系SYSH-MPT-01的生长，呈剂量和时间依赖性图示；C是CADD522对乳腺恶性叶状肿瘤细胞的迁移、侵袭能力影响图示。
图7是CADD522对体内恶性叶状肿瘤试验效果图示。A是在恶性叶状肿瘤PDX模型中，腹腔注射CADD522对肿瘤生长的抑制，相应时间点肿瘤体积的变化情况图示,B是各组肿瘤图片。
具体实施方式
下面结合实施例对本发明作进一步的说明。
实施例1：乳腺叶状肿瘤RUNX 2表达谱分析
1.采用RUNX2特异性引物，进行荧光定量PCR，检测不同类型乳腺叶状肿瘤组织中RUNX2mRNA表达水平。
(1)分别选取3例良性叶状肿瘤组织标本、10例恶性叶状肿瘤组织标本。按以下步骤进行实验：将组织用冷冻研磨机研碎，加入Trizol裂解液，用移液器 将裂解物转移至1.5mlEP管中，反复吹打或振荡以裂解细胞。室温静置5min，按每1ml Trizol裂解液加入0.2ml氯仿，用力振荡15s，室温静置2-3min后，12000x g 4℃离心15min，取上层水相至新EP管中，加入等体积异丙醇沉淀RNA；12000x g 4℃离心10min；加入75％乙醇清洗，7500x g 4℃离心5min，弃上清；室温下超静台内晾干RNA沉淀，溶解于适量Rnase-free水中，测定RNA浓度及纯度。反转录合成cDNA：反转录合成cDNA：在PCR管中加入模板RNA 1μg，4μl反转录酶SuperScript II mix(含Buffer、dNTP、HiScript II逆转录酶、RNA酶、Random primers/Oligo dT)，补充RNase-free水至20μl，65℃ 5min，冰上放置5min后加入5×buffer 8μl及0.1M DTT 2μl，混匀后补加ddH2O至40μl，反应条件：50℃ 15min，85℃ 15s，4℃保存。
实时定量PCR扩增：将模板cDNA稀释3倍并混匀备用。每个实验组设置3个平行管。反应体系：cDNA 1μl，SYBR green染料5μl，正向引物0.3μl，反向引物0.3μl，ddH 2O 3.4μl，离心混匀。反应条件：95℃ 5min，95℃ 30秒，55℃-60℃(视退火温度而定)30s，共40个循环。以GAPDH作为内参分析检测基因的相对转录水平。引物序列如下：
RUNX2 Forward：5’-CGCCTCACAAACAACCACAG-3’(SEQ ID NO.1)
RUNX2 Reverse：5’-TCACTGTGCTGAAGAGGCTG-3’(SEQ ID NO.2)
定量分析mRNA相对表达水平。统计数据根据三次重复实验结果计算平均值，显著性差异由T-检验来判断，以P<0.05定义为有统计学差异。
(2)实验结果：如图1(A)所示，RUNX2在良性叶状肿瘤组织中低表达，在恶性叶状肿瘤组织高表达，恶性中RUNX2mRNA的表达水平约为良性叶状肿瘤组织的25倍。
2.采用蛋白免疫印迹(Western blot)实验检测不同类型叶状肿瘤组织中RUNX2蛋白表达水平。
(1)实验方法：SDS-PAGE电泳、转膜、封闭之后，采用RUNX2特异性抗体作为一抗孵育，进一步采用辣根过氧化物酶标二抗孵育之后进行凝胶成像分析。
(2)实验结果:如图(1)B显示，10例恶性叶状肿瘤组织中RUNX2蛋白表达量较高，而在3例良性叶状肿瘤组织中RUNX2蛋白不表达或低表达。
3.使用RUNX2的特异性抗体，进行免疫组织化学染色，评估良、乳腺恶性叶 状肿瘤组织中RUNX2蛋白水平。
(1)实验方法：实验所采用病理切片均来自于临床确诊病例。按以下步骤进行石蜡包埋组织免疫组化染色：60℃烘烤处理，入预热二甲苯脱蜡两次，每次5min；脱蜡后的切片经100％-95％-80％-70％-50％乙醇梯度及蒸馏水水化，每个梯度放置5min；0.01M(pH6.0)柠檬酸缓冲液高压热修复10min，自然冷却后PBS洗三次，每次5min；入0.3％过氧化氢溶液处理30min，消除内源性过氧化物酶活性；PBS洗三次，每次5min；10％羊血清37℃封闭1h；滴加羊血清稀释一抗工作液(AA4)，4℃孵育过夜，PBS清洗；加入生物素标记二抗工作液，37℃孵育20min，PBS清洗三次；加入辣根过氧化物酶标记链霉亲和素，37℃孵育20min，PBS清洗三次；滴加DAB显色液，室温避光显色2min，PBS洗去多余显色液；苏木素复染，蒸馏水清洗；50％-70％-80％-90％-100％-100％乙醇梯度逐级脱水，每个梯度5min；中性树脂封片；显微成像***拍片。
(2)实验结果:如图1(C)所示：RUNX2在乳腺恶性叶状肿瘤细胞中高表达，在良性中低表达(C)。随着肿瘤复发次数的增加，RUNX2在组织中的表达量也同步增加(C)。
实施例2：敲低RUNX2对恶性叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及胶原收缩能力的影响
1.敲低RUNX2对恶性叶状肿瘤细胞增殖的影响
(1)实验方法：接种恶性叶状肿瘤细胞于96孔板和6孔板中，采用1ipo3000瞬时转染siRNA，至乳腺恶性叶状肿瘤细胞中，敲低RUNX2的表达接种敲低RUNX2表达及未进行基因敲低的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种敲低RUNX2表达及未进行基因敲低的对照组细胞至60mm中皿的完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。
siRNA的序列如下：
sense:GGACGAGGCAAGAGTTTCA,(SEQ ID NO.3)
antisense:CCAAATTTGCCTAACCAGA(SEQ ID NO.4)
(2)实验结果:如图2(A-B)所示。
2.敲低RUNX2对恶性叶状肿瘤细胞周期的影响
(1)实验方法：接种敲低表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。
(2)实验结果：如图2(C)敲低RUNX2后的细胞阻滞在G1期。
3.敲低RUNX2对恶性叶状肿瘤细胞迁移、侵袭能力的影响
(1)实验方法：将敲低RUNX2表达及对照组细胞以2x10 4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，用4％多聚甲醛固定15min，小心擦去膜上层细胞，利用0.5％结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(2)实验结果；如图2(D)显示，RUNX2敲低表达组细胞迁移及侵袭能力显著下降。
4.敲低RUNX2对恶性叶状肿瘤胶原收缩能力的影响
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。
(2)实验结果如图2(E)所示。
实施例3.过表达RUNX2对叶状肿瘤细胞增殖、迁移、侵袭、细胞周期及胶原收缩能力的影响
1.过表达RUNX2对良性乳腺叶状肿瘤细胞增殖的影响
(1)实验方法:接种原代良性叶状肿瘤细胞至96孔板和6孔板中，使用转染试剂lipo3000和p3000转染RUNX2过表达质粒(4μg/孔)，转染4-6小时后更 换正常完全培养基，收集细胞提取RNA样品，检测RUNX2过表达水平。接种过表达RUNX2及未进行处理的对照组细胞，采用CCK8法，测试不同时间点(1-4天)细胞存活率，绘制细胞增殖曲线。接种过表达RUNX2及未进行处理的对照组细胞至60mm中皿的完全培养基中，每皿200个细胞，培养14天后，计数克隆形成数量。
(2)实验结果如图3(A-B)所示。
2.过表达RUNX2对良性叶状肿瘤细胞周期的影响
(1)实验方法：接种过表达及对照细胞，培养24h后，收集细胞，用预冷的PBS洗细胞2-3次，离心(1500rpm，4min)弃掉上清，在沉淀中加入少量PBS，重悬细胞，再将重悬细胞加入到4℃预冷的70％冰乙醇中固定，封口膜封口，4℃过夜。PBS洗两次离心去上清液(2000rpm 4min)。加入100ul 100ug/ml RNase A和0.2％Triton X-100重悬细胞。加入400ul 50ug/ml PI，涡旋振荡混匀，室温避光孵育30min。流式细胞仪检测细胞周期，一般计数10万个细胞，在激发波长488nm波长处检测红色荧光，而后用FlowJo软件分析细胞周期时相分布，分析时，使用FL2-w和FL2-A显示，去除粘连在一起的细胞。
(2)实验结果：如图3(C)过RUNX2后的细胞活跃在S期。
3.过表达RUNX2对乳腺良性叶状肿瘤细胞迁移和侵袭能力的影响
(1)实验方法：将过表达RUNX2及未进行处理的对照组细胞以1*10 4/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，小心擦去膜上层细胞，利用结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
(2)实验结果如图3(D)所示。
4.过表达RUNX2对恶性叶状肿瘤胶原收缩能力的影响
(1)实验方法：用含乙酸和NaOH的DMEM培养基稀释Ⅰ型鼠尾胶原，叶状肿瘤细胞悬液与稀释后的鼠尾胶原1:1混合后，接种到24孔板中，加入含血清的培养基4小时后，换上无血清DMEM培养基孵育过夜，使胶与孔壁分离，8小时后观察、测量收缩后的直径。
(2)实验结果如图3(E)所示。
实施例4.RUNX2对乳腺叶状肿瘤患者预后的影响
1.RUNX2对乳腺叶状肿瘤患者预后的影响
(1)实验方法：取237例乳腺叶状肿瘤患者的新鲜冰冻切片，进行免疫组化染色，由两位病理科医生分别阅读切片，根据染色强度和染色阳性比例，分为RUNX2高表达组和RUNX2低表达组，评估RUNX2表达与总生存期OS的影响，无进展生存期DFS的影响。
(2)实验结果：如图4(A-B)所示，RUNX2高表达组的OS和DFS显著低于RUNX2低表达组。
实施例5.敲低RUNX2基因影响小鼠体内肿瘤的生长
1.敲低RUNX2基因抑制小鼠皮下移植瘤成瘤及生长的影响
(1)实验方法：构建乳腺恶性叶状肿瘤皮下移植瘤模型：取恶性叶状肿瘤细胞、稳定敲降RUNX2的叶状肿瘤细胞，稀释至5x10 7/ml，取100μl混匀的细胞悬液，于对照组小鼠、实验组小鼠分别于左前肢腋窝部皮下***脂肪垫处接种，每4天测量一次肿瘤长径和短径，根据公式V＝1/2(L×W 2)计算移植瘤平均体积。待对照组肿瘤体积达到直径1.5cm时处死小鼠，并绘制肿瘤生长变化曲线。
(2)实验结果：如图5(A-B)所示，与对照组相比，接种稳定敲降RUNX2叶状肿瘤细胞小鼠的肿瘤成瘤及生长明显被抑制。
实施例6.RUNX2小分子抑制剂CADD522对恶性叶状肿瘤细胞的IC50、增殖、迁移、侵袭的影响
1.RUNX2小分子抑制剂CADD522在恶性叶状肿瘤细胞中的IC50
(1)实验方法：接种乳腺恶性叶状肿瘤细胞至96孔板，接种密度为5000个/孔，24h后加入RUNX2小分子抑制剂CADD522，设置浓度梯度为200μM、100μM、50μM、25μM、12.5μM、6.125μM、3.0625μM，培养72小时，采用CCK8试剂检测细胞活力，绘制细胞IC50曲线。
(2)实验结果如图6(A)所示。
2.RUNX2小分子抑制剂CADD522对恶性叶状肿瘤细胞增殖的影响
(1)实验方法：接种乳腺恶性叶状肿瘤细胞至96孔板，接种密度为5000个/孔，24h后加入RUNX2小分子抑制剂CADD522，接种密度为3000个/孔，分别培养24、48、72、96小时，采用CCK8试剂检测细胞活力，绘制细胞增殖曲线。
(2)实验结果如图6(B)所示。
2.RUNX2小分子抑制剂CADD522抑制恶性叶状肿瘤细胞迁移和侵袭
(1)实验方法：将恶性肿瘤细胞以1*104/孔的密度接种于Transwell板(Costar)上层小室无血清培养基中或预铺Matrigel薄层上层培养室中，下层为含16％胎牛血清的完全培养基，接种细胞的同时加入RUNX2小分子抑制剂CADD522共培养，每组设置三个重复孔；37℃培养箱分别培养8小时或24小时后，小心擦去膜上层细胞，利用结晶紫对下室细胞进行染色，显微镜下计数发生迁移或侵袭的细胞数目。
实验结果如图6(C)所示。
实施例7.使用RUNX2抑制剂影响小鼠体内肿瘤的生长
2.RUNX2小分子抑制剂CADD522抑制PDX肿瘤生长
(1)实验方法：构建乳腺恶性叶状肿瘤PDX模型：取乳腺恶性叶状肿瘤组织，将其剪成直径约1mm组织块，在距离左前肢腋窝部皮下***脂肪垫约2cm处做3mm切口，套管针通过皮下隧道将3-4个小组织块送至皮下脂肪垫。待肿瘤生长至100m3左右时，对荷瘤小鼠进行随机分组，每组5只，分别腹腔注射RUNX2小分子抑制剂CADD522及对照试剂(DMSO)，注射剂量为10mg/kg和20mg/kg，每周注射两次，连续注射8次。持续监测肿瘤大小，根据公式V＝1/2(L×W2)计算移植瘤平均体积。待对照组肿瘤体积达到直径1.5cm时停止注射，并绘制肿瘤生长变化曲线。
(2)实验结果：如图7(A-B)所示，与对照组相比，RUNX2小分子抑制剂CADD522注射组肿瘤生长明显被抑制。

Claims (4)

  1. RUNX2作为标志物在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。
  2. RUNX2作为药物靶点在筛选或制备抗乳腺恶性叶状肿瘤药物中的应用。
  3. RUNX2抑制剂在制备抗乳腺恶性叶状肿瘤药物中的应用。
  4. CADD522在制备抗乳腺恶性叶状肿瘤药物中的应用。
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KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CEDEÑO YESSENIA CEDEÑO, POLSTER BRIAN M., MARTINEZ RAMON, SHAPIRO PAUL, KESARI SANTOSH, NUR: "Targeting breast cancer metabolism with a novel inhibitor of mitochondrial ATP synthesis", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 11, no. 43, 27 October 2020 (2020-10-27), United States , pages 3863 - 3885, XP093146967, ISSN: 1949-2553, DOI: 10.18632/oncotarget.27743 *
KIM MYOUNG SOOK, GERNAPUDI RAMKISHORE, CHOI EUN YONG, LAPIDUS RENA G., PASSANITI ANTONINO: "Characterization of CADD522, a small molecule that inhibits RUNX2-DNA binding and exhibits antitumor activity", ONCOTARGET, IMPACT JOURNALS LLC, UNITED STATES, vol. 8, no. 41, 19 September 2017 (2017-09-19), United States , pages 70916 - 70940, XP093134573, ISSN: 1949-2553, DOI: 10.18632/oncotarget.20200 *
ZUO ZHONGKUN, YE FEI, LIU ZIRU, HUANG JIANGSHENG, GONG YI: "MicroRNA‑153 inhibits cell proliferation, migration, invasion and epithelial‑mesenchymal transition in breast cancer via direct targeting of RUNX2", EXPERIMENTAL AND THERAPEUTIC MEDICINE, SPANDIDOS PUBLICATIONS, GR, vol. 18, 5 April 2019 (2019-04-05), GR , XP093146968, ISSN: 1792-0981, DOI: 10.3892/etm.2019.7470 *

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