MiR-449介导KDM4C-Notch通路在三阴性乳腺癌增殖转移中的调控研究

Triple-negative breast cancer (TNBC), defined by a lack of expression of both estrogen and progesterone receptor (ER,PR) as well as human epidermal growth factor receptor 29(HER-2). TNBC represents an important clinical challenge characterized by high invasiveness and poor clinical prognosis, because these cancers do not respond to endocrine therapy or other available targeted agents. In our previous studies, KDM4C gene is amplified and overexpressed in TNBC, which is positively correlated with the clinical stage and distant metastasis status. Activation of “KDM4C-Notch” pathway may related to the malignant behavior of TNBC. To investigate the microRNAs which may regulate KDM4C, TargetScan software was used and predicted that miR-449 may be the one and specific miRNA that regulate KDM4C. Our preliminary experiment also showed that the expression of miR-449 in clinical TNBC specimen is significantly lower than normal breast tissue. Therefore, we speculate that miR-449 may directly regulate its target gene-KDM4C,by doing so, influence the proliferation and metastasis of TNBC. In this study, the expression and correlation of miR-449 and KDM4C will be validated in TNBC samples and breast cancer cell lines by real-time quantitative PCR and Western blot. Further, to clarify the regulatory mechanism of miR-449 for KDM4C, miR-449 will be transfected into low-expression breast cancer cell lines while knocked down in high-expression cell lines with lentiviral expression system.Then, phenotype changes such as potential of proliferation, invasion and migration will be observed in vitro. At last, the tumorigenicity and distant metastasis capability will be studied in SCID mice xenograft model. Through the above experiments, our studies aim to explore the regulation mechanism between miR-449 and KDM4C in proliferation and metastasis of TNBC, and may provide novel insight for the molecular mechanisms, experimental and theoretical basis for TNBC.

三阴性乳腺癌（ER、PR和HER-2均阴性，TNBC）侵袭性强、预后差。与ER阳性或HER-2阳性乳腺癌相比，缺乏较有效的治疗选择与靶向药物。我们发现：TNBC中多有KDM4C的扩增与高表达，"DM4C-Notch"通路的异常激活与其恶性生物学行为有关。TargetScan软件预测可能调控KDM4C的miRNA,仅发现miR-449是特异性候选miRNA。预实验miR-449在临床TNBC样本中的表达明显低于癌旁正常乳腺组织。因此推测miR-449可能通过调控其靶基因KDM4C从而影响TNBC增殖与转移。本研究拟检测TNBC组织与乳癌细胞系miR-449、KDM4C的表达及相关性；并分别对miR-449高表达和低表达乳腺癌细胞系抑制和转染miR-449，观察增殖、侵袭和迁移、SCID小鼠检测成瘤和转移变化。阐明miR-449调控KDM4C在TNBC增殖转移中的机制提供实验基础和理论依据。

三阴性乳腺癌侵袭性强、预后差，缺乏较有效的治疗选择与靶向药物。本课题主要研究miR-449在三阴性乳腺癌发生演化中的作用，主要包括miR-449调控KDM4C的分子机理和生物学功能研究，并在动物模型中加以验证。. 本课题发现：（1）miR-449的靶点位于 KDM4C基因的3’UTR 区域（300-307）；（2）miR-449在乳腺癌组织中的表达明显低于自身正常乳腺组织，KDM4C 表达与miR-449表达负相关；（3）miR-449能够显著抑制乳腺癌细胞系的增殖、迁移和侵袭能力；（4）miR-449有效降低乳腺癌细胞株的成瘤能力和肺转移能力。. 总之miR-449能够有效抑制三阴性乳腺癌的进展，本项目为新型分子靶点的确认与药物研发提供理论依据，具有重大的现实意义和临床应用前景。