摘要
目的:筛选新的乳腺癌转移相关基因并进行功能研究,为乳腺癌诊断提供分子标志,为乳腺癌治疗提供靶点,为阐明乳腺癌发生和转移机制提供线索。
方法:利用mRNA差异显示技术筛选乳腺原发癌与配对淋巴结转移癌组织的差异表达基因片段;利用同位素标记的反向斑点杂交和荧光标记的基因芯片杂交方法验证候选差异基因。采用实时定量逆转录-聚合酶链反应方法(qRT-PCR)检测乳腺癌原发癌组织及癌旁正常组织中kinesin-like 4(KNSL4)基因mRNA表达,统计学分析与乳腺癌发生、临床病理因素和患者预后的关系;采用免疫组织化学方法检测乳腺癌组织中KNSL4基因编码的驱动蛋白样DNA结合蛋白Kid的表达,分析Kid的组织细胞定位及与细胞侵袭和转移的关系。克隆KNSL4 siRNA真核表达质粒载体pSilencerTM3.1-KNSL4 siRNA,转染高表达Kid的人乳腺癌细胞系MDA-MB-435S,并建立稳定沉默KNSL4表达的亚克隆细胞,以观察KNSL4及其编码蛋白Kid对细胞体内外生物学行为的影响。采用染色质免疫沉淀联合启动子芯片(ChIP-on-chip)方法筛选Kid下游调控基因,ChIP-qPCR方法验证筛选基因与Kid的结合,qRT-PCR方法分析下调乳腺癌细胞Kid表达对候选基因的转录调节作用。
结果:筛选得到4个未知的基因序列(Expressed Sequence Tag,EST)登录在GenBank,序列号为BG518428、BG518429、BM005520、BM005521;验证证实KNSL4基因在乳腺转移癌中表达下调。KNSL4 mRNA在癌组织中表达,而在正常组织中不表达(P=0.000);KNSL4 mRNA低水平患者5年无病生存期低于高水平患者(P=0.016),KNSL4 mRNA水平是临床I~II期和Her2阳性乳腺癌患者预后预测分子标志物(P=0.023,P=0.041)。Kid蛋白在增殖的肿瘤细胞中高表达,而在侵袭和迁移的细胞中表达下调。KNSL4基因转录沉默的亚克隆细胞增殖能力降低,细胞周期G2期比率增加,细胞有丝分裂阻滞,而细胞的克隆形成率没有影响,体内和体外的侵袭和迁移能力也没有改变。CDC25C和ATF7IP是Kid作为转录因子的下游调节基因,且Kid对二者起负调控作用。
结论:KNSL4基因及其编码蛋白Kid是乳腺癌细胞转移状态的指示者,而非转移生物学过程的调控者。Kid在细胞有丝分裂中起关键作用,与CDC25C形成的负反馈调节环异常可以使细胞有丝分裂异常,进而使细胞恶性转化和恶性增殖。KNSL4基因表达是潜在的乳腺癌预后预测分子标志物和抗肿瘤治疗的分子靶标。
Objective: To identify the genes related to breast cancer metastasis in order to find the new molecular diagnostic markers and therapeutic molecular targets, and to provide clues for understanding the mechanism of carcinogenisis and metastasis of breast cancer.
Methods: The differentially expressed genes between primary breast cancer and paired lymph node metastasis tissues were identified using mRNA differential display, and validated by using reverse dot blot hybridization and cDNA microarray methods. Kinesin-like 4 (KNSL4) mRNA levels in 30 normal breast tissues and in 142 primary breast cancer tissues from the patients with more than five years follow-up were quantified with real time RT-PCR respectively, and the correlations of KNSL4 mRNA level to clinical clinicopathologic factors and prognosis were analyzed. The cellular localization and expression level of Kid protein coded by KNSL4 in breast cancer tissues were detected by immunohistochemistry staining. Furthermore, the pSilencerTM3.1-KNSL4 siRNA recombinant plasmid was constructed and transfected into the breast cancer cell line MDA-MB-435S to establishe the stable sub-cell line of silenced KNSL4 expression, in order to observe the influence of cell biological behavior when KNSL4 expression was down-regulated. Kid regulated target genes were profiled based on the chromatin immunoprecipitation in combination with promoter microarray (ChIP-on-chip) method. The candidate target genes were confirmed by ChIP-qPCR. The mRNA expression of candidate Kid’s target genes in MDA-MB-435S/KNSL4 siRNA cells compare with their parental cells were assayed by real time RT-PCR.
Result: Four unknown gene fragments were cloned and accepted by the Genbank, with the accession numbers as BG518428, BG518429, BM005520, and BM005521, respectively. The kinesin-like 4 (KNSL4) gene expression was down-regulated in lymph node metastasis in comparison to the paired primary tissues. KNSL4 mRNA was highly expressed in breast cancers, but unexpressed in normal breast tissues (P=0.000). However, lower expression of KNSL4 mRNA in primary cancers correlated to a reduced five years disease-free survival (DFS) of breast cancer patients (P=0.016). KNSL4 mRNA level is prognostic predictor for breast cancer patients in stage I~II or with Her2 positive (P=0.023, P=0.041). Consistent with the KNSL4 mRNA expression, Kid protein expression was higher in cancer cells in situ than in normal tissues, but down-regulated in invasive carcinoma cells. In the KNSL4 transcription silenced subclone cell MDA-MB-435S/KNSL4 siRNA, cell proliferation capacity decreased, proportion of G2/M phase of the cell cycle increaced, and the mitotic block was observed. However, changes of capacity of clone formation, invasion and migration change in vitro and in vivo were not observed. Cell division cycle 25 homolog C (CDC25C) and activating transcription factor 7 interacting protein (ATF7IP) were validated as target genes negatively regulated by Kid.
Conclusions: KNSL4 expression is an indicator of metastasis of breast cancer cells but not a regulator in metastasis progress. Kid plays a key role and may form a negative feedback loop with CDC25C in cell mitosis, and its abnormality may cause the cell malignant transformation and proliferation. KNSL4 expression may serves as molecular marker of the prognostic prediction and antitumor molecular target for breast cancer patients.
引文
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