NRF2转录激活CBR3-AS1双靶向调控乳腺癌药物敏感性的分子机制

MEK4/JNK1 pathway plays an important role in regulating drug sensitivity of breast cancer, but the mechanism is still unclear. Our group found that: CBR3-AS1, transcription factor NRF2 and JNK pathway key factor MEK4, JNK1 were high expressed in drug resistant breast cancer cells and miR-25-3p was low expressed; MEK4, JNK1 and CBR3-AS1 share the same MRE sequence predicted to bind to miR-25-3p, the CBR3-AS1 promoter include 3 NRF2 binding sites. Whether CBR3-AS1 is activated by NRF2 and involved in the double-targeted inhibition of the MEK4/JNK1 pathway by miR-25-3p, regulating drug sensitivity in breast cancer? This study try to figure out whether CBR3-AS1 and MEK4/JNK1 could bind to miR-25-3p, intervention expression of CBR3-AS1 and miR-25-3p both or respectively could change breast cancer cells drug sensitivity, and confirm whether or not MEK4/JNK1 were involved. To elucidate the mechanism of NRF2 activate CBR3-AS1 then regulate breast cancer drug sensitivity.

MEK4/JNK1通路在调控乳腺癌药物敏感性中发挥重要作用，但具体调控机制仍不明确。课题组发现：乳腺癌耐药细胞中CBR3-AS1、转录因子NRF2及JNK通路关键因子MEK4、JNK1高表达，miR-25-3p低表达；预测发现MEK4、JNK1和CBR3-AS1具有相同的与miR-25-3p结合的MRE序列，CBR3-AS1启动子区具有NRF2结合位点。那么CBR3-AS1是否被NRF2激活参与miR-25-3p双靶向抑制MEK4/JNK1通路，调控乳腺癌药物敏感性？本课题拟在明确CBR3-AS1及MEK4/JNK1与miR-25-3p特异性结合的基础上，比较分别及相互干预CBR3-AS1与miR-25-3p时乳腺癌细胞药物敏感性的改变与MEK4/JNK1水平变化的相关性，考察NRF2对CBR3-AS1的激活作用。阐明NRF2激活CBR3-AS1双靶向调控乳腺癌药物敏感性的作用与分子机制。

背景：阿霉素（ADR）耐药性是影响乳腺癌患者临床预后的主要因素之一。长链非编码RNA（lncRNAs）可以调控细胞行为，但这些RNA在乳腺癌抗ADR活性中的作用尚不清楚。在这里，我们的目的是研究一种特殊的长非编码RNA--lncRNA CBR3反义RNA 1（CBR3-AS1）的失衡及其在ADR耐药性中的作用。.方法：采用基因芯片技术对乳腺癌耐药细胞CBR3-AS1进行鉴定。采用CCK-8法和集落形成法检测乳腺癌细胞对ADR的敏感性。采用双荧光素酶报告、RNA pulldown、IHC和westernblot等方法验证CBR3-AS1、miRNA的表达与靶基因的关系。在体内实验中，在异种移植瘤模型中观察CBR3-AS1对乳腺癌耐药的影响。通过分析TCGA、CCLE和GDSC数据库的临床乳腺癌标本证实了CBR3-AS1在对乳腺癌ADR敏感性的影响。.结果：CBR3-AS1在乳腺癌组织中的表达明显增高，且与预后不良密切相关。CBR3-AS1过表达促进乳腺癌细胞体外和体内的ADR耐药性。在机制上，我们发现CBR3-AS1作为miR-25-3p一种竞争性的内源性RNA抑制其功能，MAPK途径的MEK4和JNK1被确定为乳腺癌细胞CBR3-AS1/miR-25-3p轴的直接下游蛋白。.结论：我们的研究结果表明CBR3-AS1通过介导miR-25-3p/MEK4/JNK1调控轴在乳腺癌的化疗耐药中发挥关键作用；证实了CBR3-AS1具有作为乳腺癌靶向癌基因和治疗性生物标志物的潜力。