NEK9异常表达在乳腺癌发生发展中的作用研究

发布时间：2018-11-28 15:55

【摘要】：目的:乳腺癌是女性常见恶性肿瘤,近年来发病率逐年上升,已跃居女性恶性肿瘤的第一位。手术切除辅以术后化疗和内分泌治疗是乳腺癌治疗的主要方法。近年来,乳腺癌的分子机制研究和治疗取得了巨大进展,但乳腺癌仍然严重危害女性的生命及生活质量,其中肿瘤局部扩散、淋巴结转移和远处脏器转移是影响患者预后和限制生存率的主要原因。深入研究乳腺癌的分子发病机制,探索浸润、转移的机制,寻找新的抗癌策略,可以为乳腺癌患者尤其是缺乏有效治疗的患者提供新的靶向治疗,有效提高病人预后及生存率。Nek9是Nek家族成员之一,Nek9在中心粒复制、分离和纺锤体的形成等细胞周期过程中起重要作用,Nek9的异常表达也可以引起胞质的异常分裂及同源染色体的错误分离,目前为止,关于Nek9的相关研究,尤其是其在肿瘤中作用的报道甚少。本研究拟检测Nek9在乳腺癌及常见良性乳腺疾病中的表达情况,研究其在乳腺癌发生发展中的作用,并初步探讨其作用机制。方法:收集乳腺癌及常见良性乳腺病变标本,并完善相关临床病理资料。采用免疫组化方法检测浸润性乳腺癌及正常乳腺组织、乳腺增生、纤维腺瘤、导管内癌中NEK9蛋白的表达情况,比较同一病例浸润性癌与导管上皮增生、导管内癌、浸润癌以及淋巴结内转移癌之间的表达差异,并分析NEK9表达水平与临床病理指标的之间的关系。分别将siRNA和含Nek9的质粒瞬时转染进激素受体阳性乳腺癌MCF-7细胞系,将含Nek9的质粒转染进三阴性乳腺癌细胞系MDA-MB-468,Western blot检测Nek9的干扰效率和过表达效率。EdU实验检测转染前后肿瘤细胞增殖能力的变化;迁移与浸润实验观察Nek9对肿瘤细胞迁移浸润能力的影响;通过流式细胞技术检测Nek9对细胞周期和细胞凋亡的影响。结果:在316例浸润性导管癌中,NEK9的表达与组织学级别(p0.0001)、肿瘤大小(p=0.0087)、HER-2扩增(p=0.0016)、p53 突变(p0.000l)、ki67 高表达(p=0.0020)显著相关;与ER、PR表达呈负相关,结果有统计学意义(p0.0001);三阴性乳腺癌中NEK9表达降低或缺失明显高于其他分子亚型(p0.0001)。我们发现激素受体阳性乳腺癌中,导管内癌、浸润性癌、腋窝淋巴结转移癌中NEK9表达依次降低(p0.0001),但导管内癌与导乳腺增生之间NEK9表达没有差异。体外实验中证实沉默Nek9可以促进细胞增殖、迁移和浸润,抑制凋亡,同时导致处于G2和M期的肿瘤细胞增多,同样的,将Nek9的质粒瞬时转染进乳腺癌细胞系中,细胞的迁移浸润能力受到抑制,细胞增殖能力下降,促进细胞凋亡,同时处于G2和M期的细胞减少,影响细胞周期过程。结论:1.在乳腺浸润性导管癌中,NEK9的表达与肿瘤大小、组织学级别、HER-2扩增、p53功能失常、高增殖指数呈负相关,与ER、PR的表达水平呈正相关。2.NEK9表达降低或缺失多见于三阴性乳腺癌,细胞实验亦显示封闭Nek9促进三阴性乳腺癌肿瘤细胞生长,Nek9或参与了三阴性乳腺癌的发病。3.同一病例中,从导管内癌到浸润性导管癌到腋窝淋巴结转移,NEK9的表达逐步下降,体外实验也证实干扰Nek9可促进乳腺癌细胞迁移和浸润,提示Nek9在乳腺癌的侵袭转移中发挥重要作用。4.NEK9表达异常可能通过抑制细胞凋亡、细胞周期G2/M检测点失控,从而促进细胞增殖,参与三阴性乳腺癌的发生,并在乳腺癌的浸润、转移过程中发挥作用。5.NEK9有望成为新的生物标记物,帮助发现新的乳腺癌分子亚型,为乳腺癌的诊断、治疗、预后提供新的研究依据。
[Abstract]:Objective: Breast cancer is a common malignant tumor of women. In recent years, the incidence of breast cancer has increased year by year. Surgical removal and postoperative chemotherapy and endocrine therapy are the main methods of the treatment of breast cancer. In recent years, the molecular mechanism of breast cancer has made great progress in the research and treatment, but the breast cancer still seriously harms the life and quality of life of the female, in which the local diffusion of the tumor, the metastasis of the lymph node and the metastasis of distant organs are the main causes of the prognosis and the limited survival rate of the patients. To study the molecular pathogenesis of breast cancer, to explore the mechanism of invasion and metastasis, to find a new anti-cancer strategy, to provide new targeted therapy for patients with breast cancer, especially those who lack effective treatment, and to effectively improve the prognosis and survival rate of the patients. Nek9 is one of the Nek family members, and Nek9 plays an important role in the process of cell cycle such as the replication, separation and spindle formation of the central granule. The abnormal expression of Nek9 can also cause the abnormal division of the cytoplasm and the error separation of the homologous chromosomes. So far, the related research on Nek9, in particular its role in the tumor. Objective To study the expression of Nek9 in breast cancer and common benign breast diseases, and to study the role of Nek9 in the development of breast cancer. Methods: The specimens of breast cancer and common benign breast lesions were collected and the relevant clinical and pathological data were improved. The expression of NEK9 protein in invasive breast cancer and normal breast tissue, breast hyperplasia, fibroadenoma and intraductal carcinoma was detected by immunohistochemistry. The relationship between the expression level of NEK9 and the clinical pathological index was analyzed. The siRNA and the plasmid containing Nek9 were transiently transfected into the human breast cancer MCF-7 cell line of the hormone receptor, and the plasmid containing Nek9 was transfected into the three-negative breast cancer cell line MDA-MB-468, and the interference efficiency and the overexpression efficiency of the Nek9 were detected by Western blot. The effects of Nek9 on cell cycle and cell apoptosis were measured by flow cytometry. Results: In 316 cases of invasive ductal carcinoma, the expression of NEK9 was significantly related to the histological grade (p0.01), tumor size (p = 0.0087), HER-2 amplification (p = 0. 0016), p53 mutation (p0. 000l), ki67 high expression (p = 0.0020), negative correlation with ER and PR expression and statistical significance (p0.01); The decrease or deletion of NEK9 in three-negative breast cancer was significantly higher than that of other molecular subtypes (p0.01). In the case of hormone receptor-positive breast cancer, the expression of NE9 in the ductal carcinoma, invasive carcinoma and axillary lymph node metastasis was decreased in turn (p0.01), but there was no difference in the expression of NEK9 between the intraductal carcinoma and the mammary gland hyperplasia. In vitro experiments, it is confirmed that the silence Nek9 can promote cell proliferation, migration and infiltration, inhibit the apoptosis, and at the same time lead to the increase of the tumor cells in the G2 and M phase, and the same, the plasmid of the Nek9 is transiently transfected into the breast cancer cell line, the migration and infiltration capacity of the cells is inhibited, The cell proliferation ability is decreased, the cell apoptosis is promoted, and the cells in the G2 and M phase are reduced and the cell cycle process is affected. Conclusion: 1. In the breast invasive ductal carcinoma, the expression of NEK9 was negatively correlated with the tumor size, the histological grade, HER-2 amplification, the abnormal p53 function and the high proliferation index, which was positively correlated with the expression level of ER and PR. The cell experiment also revealed that the closure of Nek9 promoted the growth of three-negative breast cancer cells, Nek9, or the onset of three-negative breast cancer. In the same case, the expression of NEK9 was gradually decreased from the intraductal carcinoma to the invasive ductal carcinoma to the axillary lymph node metastasis, and the in vitro experiment also confirmed that the interference with Nek9 could promote the migration and infiltration of breast cancer cells, It is suggested that Nek9 plays an important role in the invasion and metastasis of breast cancer. 5. NEK9 is expected to be a new biomarker to help discover new molecular subtypes of breast cancer and provide a new basis for the diagnosis, treatment and prognosis of breast cancer.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.9

【相似文献】