Expression data of Brti1 shRNA knockdown and control shRNA in MCF-10A cells. Homo sapiens

Homologous recombination-mediated DNA repair deficiency (HRD) predisposes to cancer development, but also provides therapeutic opportunities Here, we identified an HRD gene signature that robustly predicted HRD status Unexpectedly, concurrent loss of PTEN in BRCA1-deficient cells might extensively rewire the HR repair network and confer resistance to PARP inhibitor, partially through over-expression of TTK We used the HRD gene signature as a drug discovery tool and found several PARP-inhibitor-synergizing agents through the connectivity map Thus gene expression profiling can be used to define the functional status of the HR repair network providing prognostic and therapeutic information Overall design: Various shRNAs that target genes involved in homologous recombination (HR) were transfected in MCF-10A non-transformed breast cells lines Stable HR gene knockdown MCF-10A cells were seeded 200000 at 10 cm plate Cells were harvested after 48 hours culturing and used for gene expression profiling The shRNA that target Brit1 genes was transfected in MCF-10A non-transformed breast cell line by lentiviral particles and selected stable Brit1 knockdown MCF-10A cells. Scrambled control shRNA-transfected MCF-10A cells were applying as control. Both stable Brit1 knockdown and control MCF-10A cells were seeded with 2 x 10^5 cells at 10 cm culture plate. Cells were cultured in MCF-10A medium and harvested after 48 hours culturing. mRNA was extracted from collected cells and performing gene expression profiling. Three or four biological replicates were applied.

同源重组介导的DNA修复缺陷（Homologous recombination-mediated DNA repair deficiency, HRD）可增加癌症发生风险，同时亦为癌症治疗带来潜在机遇。本研究鉴定出可稳健预测HRD状态的基因特征。令人意外的是，BRCA1缺陷细胞中同时发生PTEN缺失，可能会广泛重塑同源重组修复网络，并通过上调TTK的表达部分介导对PARP抑制剂（PARP inhibitor）的耐药性。我们将该HRD基因特征作为药物发现工具，通过连通性图谱（Connectivity Map）筛选出数种可与PARP抑制剂产生协同作用的化合物。综上，基因表达谱分析可用于界定同源重组修复网络的功能状态，为临床预后判断与治疗决策提供参考依据。
整体实验设计：将靶向同源重组（Homologous Recombination, HR）相关基因的多种短发夹RNA（short hairpin RNA, shRNA）转染至MCF-10A非转化乳腺细胞系。将稳定敲低HR基因的MCF-10A细胞以2×10^5个细胞接种于10 cm培养皿中，培养48小时后收集细胞，用于基因表达谱分析。
此外，通过慢病毒颗粒将靶向Brit1基因的shRNA转染至MCF-10A非转化乳腺细胞系，并筛选获得稳定敲低Brit1的MCF-10A细胞系；以转染乱序对照shRNA的MCF-10A细胞作为对照。将稳定敲低Brit1的细胞与对照组细胞均以2×10^5个细胞接种于10 cm培养皿中，使用MCF-10A培养基培养，培养48小时后收集细胞。从收集的细胞中提取mRNA并进行基因表达谱分析。本实验设置了3至4次生物学重复。