【作者】 刘磊；

【导师】 许乃寒；

【作者基本信息】 清华大学 ， 生物学， 2017， 硕士

【摘要】 本文系统性的研究了miR-20a通过抑制细胞自噬从而促进乳腺癌发生发展的分子机制。分析癌症基因组图谱(TCGA)中乳腺癌的基因表达数据发现在乳腺癌,特别是三阴性乳腺癌中miR-20a的表达上调。基因集富集分析(GSEA)表明miR-20a的表达与自噬/溶酶体途径负相关。乳腺癌细胞过表达miR-20a后,其基础水平与代谢压力诱导的自噬流活性被抑制,导致细胞内ROS水平升高,DNA损伤增加。当用LNA-20a抑制内源性miR-20a时,细胞自噬水平与溶酶体活性升高。研究发现miR-20a可以靶向多个关键自噬调节基因,包括BECN1、ATG16L1与SQSTM1。实时荧光定量PCR(qRT-PCR)与蛋白质免疫印迹实验(Western Blot)发现miR-20a下调BECN1、ATG16L1、SQSTM1的mRNA与蛋白表达水平。荧光素酶报告实验确证miR-20a结合BECN1、ATG16L1与SQSTM1的mRNA的3’-UTR从而抑制基因的表达。沉默内源BECN1、ATG16L1与SQSTM1具有与miR-20a同样的效果,即细胞内DNA损伤增加,ROS水平升高。引入外源性的BECN1、ATG16L1与SQSTM1解除了miR-20a对自噬的抑制作用,减少了DNA损伤。分析乳腺肿瘤组织样本发现,miR-20a与其靶基因的表达呈现负相关。而且,与其他亚型相比,低水平的BECN1、ATG16L1与SQSTM1在三阴性乳腺癌中更常见。具有较高miR-20a水平的乳腺癌患者中也检测到大量的基因组拷贝数的改变与DNA突变。最后,我们用三阴性乳腺癌细胞构建了异种移植小鼠模型,结果表明miR-20a较明显的促进肿瘤起始和生长。综上所述的研究结果证明miR-20a介导的细胞自噬的下调可能是乳腺肿瘤发生过程中miR-20a致癌作用的新机制。这一研究结果加深了我们对乳腺癌发病机制的理解,并有可能有为乳腺癌的诊断与治疗提供一定的指导意义。更多还原

【Abstract】 In this study,we systematically investigated the molecular mechanism of miR-20 a in regulating autophagy and mammary tumorigenesis.Gene expression data from studies of breast cancer in The Cancer Genome Atlas(TCGA)revealed miR-20 a is upregulated in human breast cancer,especially in triple negative subtype.Gene Set Enrichment Analysis(GSEA)suggested that miR-20 a expression negatively correlates with autophagy/lysosome pathway.In this study,we show that overexpression of miR-20 a suppresses the basal level and metabolic stress induced autophagy and lysosomal proteolytic activity,increases intracellular ROS levels and DNA damage in breast cancer cells.On the contrary,LNA-modified miR-20 a inhibitor enhances autophagy and lysosomal proteolytic activity.Using miRNA targets prediction software,qRT-PCR,Western Blot and luciferase assay we demonstrate that miR-20 a directly targets several key regulators of autophagy,including BECN1,ATG16L1 and SQSTM1.Reintroduction of exogenous BECN1,ATG16L1 or SQSTM1 restores the inhibitory effect of miR-20 a on autophagy and decreases DNA damage.The expression of miR-20 a inversely correlates with its target genes in human breast cancer tissues.Low expression of BECN1,ATG16L1 and SQSTM1 is more common in triple negative cancers compared with other subtypes.A great fraction of copy number altered genome and DNA mutations are also detected in cancer patients with higher miR-20 a expression.Subsequent studies in xenograft mouse model further show that miR-20 a promotes tumor initiation and tumor growth in vivo.Collectively,these findings indicate that miR-20 a mediated loss of autophagy may serve as a new mechanism underling the oncogenic function of miR-20 a during breast tumorigenesis.This study has deepened our understanding and may provide some guidance for the diagnosis and treatment of breast cancer.更多还原