【作者】 杨帆；

【导师】 宋东；

【作者基本信息】 吉林大学 ， 临床医学硕士（专业学位）， 2023， 硕士

【摘要】 研究背景:乳腺癌是女性最常见的恶性肿瘤,是造成全球癌症死亡的第五大原因,严重影响患者的健康,给个人、家庭及社会造成了沉重的心理和经济负担。乳腺癌是一种高度异质性疾病,其中,三阴性乳腺癌由于其特殊的分子表型,对内分泌治疗及靶向治疗并不敏感,与其他亚型乳腺癌相比,临床预后更差,发掘更多潜在的治疗靶点对于三阴性乳腺癌的精准治疗至关重要。MYCN基因是MYC癌基因家族中的成员,近来有研究报道,大部分原发性三阴性乳腺癌表达MYCN。目前并没有直接靶向MYCN的药物用于MYCN高表达的治疗,例如BETi是通过抑制MYCN上游BRD家族成员间接抑制MYCN。筛选新的靶向MYCN的药物对于MYCN高表达的三阴性乳腺癌的精准治疗具有重要意义。槐耳作为我国的传统中药,具有良好的抗肿瘤作用,而且,槐耳颗粒在多种肿瘤包括三阴性乳腺癌中具有广泛的临床应用。而槐耳对三阴性乳腺癌中MYCN高表达的治疗效果目前尚不清楚,因此,研究槐耳对MYCN高表达的三阴性乳腺癌细胞的作用,有助于探讨槐耳对三阴性乳腺癌中MYCN高表达患者的治疗效果,建立新的靶向MYCN的三阴性乳腺癌的治疗新策略。研究目的:通过构建MYCN高表达的两种三阴性乳腺癌细胞系MDA-MB-231和BT549,研究MYCN对三阴性乳腺癌细胞的影响及槐耳清膏对所构建的细胞系的作用和机制。研究方法:1、免疫印迹(Western blot)检测三阴性乳腺癌细胞系及临床样本组织中MYCN蛋白的表达;检测所构建的MYCN高表达的三阴性乳腺癌细胞中MYCN蛋白的表达;检测不同浓度槐耳作用后,MYCN高表达的三阴性乳腺癌细胞中凋亡相关蛋白的表达。2、MYCN基因表达载体构建及慢病毒包装,构建MYCN高表达三阴性乳腺癌细胞系MDA-MB-231和BT549。3、q PCR检测所构建的MYCN高表达的三阴性乳腺癌细胞中MYCN m RNA的表达。4、CCK-8法检测MYCN高表达三阴性乳腺癌细胞的存活率;检测不同浓度槐耳清膏作用后,MYCN高表达的三阴性乳腺癌细胞的存活率。5、克隆形成实验检测MYCN高表达三阴性乳腺癌细胞的克隆形成能力。6、划痕实验检测MYCN高表达三阴性乳腺癌细胞的迁移能力;检测不同浓度槐耳清膏作用后,MYCN高表达三阴性乳腺癌细胞的迁移能力。研究结果:1、部分三阴性乳腺癌患者的肿瘤组织中存在MYCN蛋白高表达,三阴性乳腺癌细胞MDA-MB-231和BT549中未见检测到MYCN蛋白表达。2、构建了MYCN高表达的三阴性乳腺癌细胞系MDA-MB-231和BT549,q PCR和Western blot分别从m RNA表达水平和蛋白表达水平上验证所构建的这两种细胞系均能稳定表达MYCN。3、CCK-8检测结果显示,MYCN基因促进三阴性乳腺癌细胞的增殖。4、细胞克隆形成实验结果显示,MYCN基因提高了三阴性乳腺癌细胞的克隆形成能力。5、细胞划痕实验结果显示,MYCN基因增强三阴性乳腺癌细胞的迁移能力。6、CCK-8检测结果显示,与对照组相比,槐耳清膏对MYCN高表达的BT549细胞的存活率有更强的抑制作用。7、细胞划痕实验结果显示,与对照组相比,槐耳清膏对MYCN高表达的BT549细胞的迁移能力有更强的抑制作用。8、Western blot结果显示,不同浓度槐耳清膏处理MYCN高表达的BT549细胞后,Bax蛋白表达增加,Bcl-2蛋白及Survivin蛋白表达降低。结论:1、部分三阴性乳腺癌患者的肿瘤组织中存在MYCN蛋白高表达。2、MYCN基因促进三阴性乳腺癌细胞的增殖、克隆及迁移。3、槐耳清膏通过诱导凋亡抑制MYCN高表达的BT549细胞的存活率。更多还原

【Abstract】 Background:Breast cancer is the most common malignant tumor among women and the fifth leading cause of cancer death worldwide.It seriously affects the health of patients and causes heavy psychological and economic burden to individuals,families and society.Breast cancer is a highly heterogeneous disease.Among them,triple negative breast cancer is not sensitive to endocrine therapy and targeted therapy due to its special molecular phenotype.Compared with other subtypes of breast cancer,its clinical prognosis is worse.Today,in the pursuit of precise and personalized treatment,more potential therapeutic targets are worth exploring.MYCN gene is a member of MYC oncogene family.Recently,there have been reports that most primary triple negative breast cancer express MYCN,and bromine domain and external motif inhibitors(BETi)combined with mitogen activated protein kinase inhibitors(MEKi)can effectively inhibit triple negative breast cancer expressing MYCN.It provides a new therapeutic target for the treatment of triple negative breast cancer.As a traditional Chinese medicine in China,huaier has a good anti-tumor effect,and huaier granules have a wide range of clinical applications in a variety of tumors,including triple negative breast cancer.It is unclear whether huaier has better therapeutic effect on triple negative breast cancer patients with high MYCN expression.Therefore,studying the effect of huaier on triple negative breast cancer cells with high MYCN expression will help to explore the therapeutic effect of hauier on triple negative breast cancer patients with high MYCN expression,in order to provide theoretical basis for MYCN as a therapeutic target for triple negative breast cancer.Objective:By constructing two triple negative breast cancer cell lines MDA-MB-231 and BT549 with high expression of MYCN,the effects of MYCN on triple negative breast cancer cells and the effects and mechanisms of huaier aqueous extract on the two constructed cell lines were studied.Methods:1.Western blot was used to detect the expression of MYCN in triple negative breast cancer cell lines and clinical samples,the expression of MYCN protein in the constructed triple negative breast cancer cells with overexpressed MYCN,the expression of apoptosis related proteins in the triple negative breast cancer cells with overexpressed MYCN after the treatment of hauier aqueous extract with different concentrations.2.MYCN gene expression vector construction and lentivirus packaging were used to construct triple negative breast cancer cell lines MDA-MB-231 and BT549 with overexpressed MYCN.3.The expression of MYCN m RNA in the constructed triple negative breast cancer cells with overexpressed MYCN was detected by q PCR.4.CCK-8 method was used to detect the cell viability of triple negative breast cancer cells with overexpressed MYCN,to detect the survival rate of triple negative breast cancer cells with overexpressed MYCN after the treatment of different concentrations of huaier aqueous extract.5.The clonal formation ability of triple negative breast cancer cells with overexpressed MYCN was tested by clonal formation assay.6.The migration ability of triple negative breast cancer cells with overexpressed MYCN and the migration ability of triple negative breast cancer cells with overexpressed MYCN after the treatment of huaier aqueous extract were detected by wound-healing assay.Results:1.Some triple negative breast cancer patients had high expression of MYCN protein in tumor tissue,and no MYCN protein expression was detected in triple negative breast cancer cells MDA-MB-231 and BT549.2.Triple negative breast cancer cell lines MDA-MB-231 and BT549 with overexpressed MYCN were constructed.q PCR and western blot verified that the two cell lines constructed could stably express MYCN from m RNA expression level the and protein expression level respectively.3.CCK-8 detection results showed that overexpression of MYCN gene promoted the proliferation of triple negative breast cancer cells.4.The results of cell cloning experiment showed that MYCN gene enhanced the cloning ability of triple negative breast cancer cells.5.The results of wound-healing assay showed that MYCN gene enhanced the migration ability of triple negative breast cancer cells.6.CCK-8 detection results showed that compared with the control group,huaier aqueous extract had a stronger inhibitory effect on the survival rate of BT549 cells with overexpressed MYCN.7.The results of wound-healing assay showed that huaier aqueous extract had a stronger inhibitory effect on the migration ability of BT549 cells with overexpressed MYCN.8.Western blot results showed that the expression of Bax protein increased and the expression of Bcl-2 protein and Survivin protein decreased after treatment with different concentrations of huaier aqueous extract on BT549 cells with overexpressed MYCN.Conclusion:1.Some triple negative breast cancer patients had high expression of MYCN protein in tumor tissue.2.MYCN gene promoted the proliferation,cloning and migration of triple negative breast cancer cells.3.Huaier aqueous extract inhibited the survival rate of BT549 cells with overexpressed MYCN by inducing apoptosis.更多还原