Sheridan Julie M, Ritchie Matthew E, Best Sarah A, Jiang Kun, Beck Tamara J, Vaillant François, Liu Kevin, Dickins Ross A, Smyth Gordon K, Lindeman Geoffrey J, Visvader Jane E
ACRF Stem Cells and Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.
Molecular Genetics of Cancer Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC, 3052, Australia.
BMC Cancer. 2015 Apr 3;15:221. doi: 10.1186/s12885-015-1187-z.
The molecular regulators that orchestrate stem cell renewal, proliferation and differentiation along the mammary epithelial hierarchy remain poorly understood. Here we have performed a large-scale pooled RNAi screen in primary mouse mammary stem cell (MaSC)-enriched basal cells using 1295 shRNAs against genes principally involved in transcriptional regulation.
MaSC-enriched basal cells transduced with lentivirus pools carrying shRNAs were maintained as non-adherent mammospheres, a system known to support stem and progenitor cells. Integrated shRNAs that altered culture kinetics were identified by next generation sequencing as relative frequency changes over time. RNA-seq-based expression profiling coupled with in vitro progenitor and in vivo transplantation assays was used to confirm a role for candidate genes in mammary stem and/or progenitor cells.
Utilizing a mammosphere-based assay, the screen identified several candidate regulators. Although some genes had been previously implicated in mammary gland development, the vast majority of genes uncovered have no known function within the mammary gland. RNA-seq analysis of freshly purified primary mammary epithelial populations and short-term cultured mammospheres was used to confirm the expression of candidate regulators. Two genes, Asap1 and Prox1, respectively implicated in breast cancer metastasis and progenitor cell function in other systems, were selected for further analysis as their roles in the normal mammary gland were unknown. Both Prox1 and Asap1 were shown to act as negative regulators of progenitor activity in vitro, and Asap1 knock-down led to a marked increase in repopulating activity in vivo, implying a role in stem cell activity.
This study has revealed a number of novel genes that influence the activity or survival of mammary stem and/or progenitor cells. Amongst these, we demonstrate that Prox1 and Asap1 behave as negative regulators of mammary stem/progenitor function. Both of these genes have also been implicated in oncogenesis. Our findings provide proof of principle for the use of short-term cultured primary MaSC/basal cells in functional RNAi screens.
协调乳腺上皮层级中干细胞更新、增殖和分化的分子调节因子仍知之甚少。在此，我们使用针对主要参与转录调控的基因的1295个短发夹RNA（shRNA），在富含原代小鼠乳腺干细胞（MaSC）的基底细胞中进行了大规模的汇集RNA干扰筛选。
用携带shRNA的慢病毒库转导富含MaSC的基底细胞，并将其维持为非贴壁乳腺球，这是一种已知能支持干细胞和祖细胞的系统。通过下一代测序，将改变培养动力学的整合shRNA鉴定为随时间的相对频率变化。基于RNA测序的表达谱分析，结合体外祖细胞和体内移植试验，以确认候选基因在乳腺干细胞和/或祖细胞中的作用。
利用基于乳腺球的试验，该筛选鉴定出了几个候选调节因子。虽然一些基因先前已被认为与乳腺发育有关，但绝大多数发现的基因在乳腺中尚无已知功能。对新鲜纯化的原代乳腺上皮细胞群体和短期培养的乳腺球进行RNA测序分析，以确认候选调节因子的表达。选择分别与乳腺癌转移和其他系统中祖细胞功能有关的两个基因Asap1和Prox1进行进一步分析，因为它们在正常乳腺中的作用尚不清楚。结果表明，Prox1和Asap1在体外均作为祖细胞活性的负调节因子，敲低Asap1导致体内再增殖活性显著增加，这意味着其在干细胞活性中发挥作用。
本研究揭示了许多影响乳腺干细胞和/或祖细胞活性或存活的新基因。其中，我们证明Prox1和Asap1作为乳腺干/祖细胞功能的负调节因子。这两个基因也都与肿瘤发生有关。我们的研究结果为在功能性RNA干扰筛选中使用短期培养的原代MaSC/基底细胞提供了原理证明。
