We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3β, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.

我们之前曾报道过， BMAL1基因的缺失对于其对多阶段癌发生过程中有效途径的贡献具有相反的影响。 BMAL1缺失使几乎正常的乳腺上皮细胞（MCF10A）和侵袭性乳腺癌细胞（MDA-MB-231）对顺铂和阿霉素诱导的细胞凋亡敏感，同时这种缺失也加剧了MDA-MB-231细胞的侵袭潜力。然而， BMAL1缺失对致癌过程看似相反的贡献的机制关系在全基因组水平上尚不清楚。在这项研究中，采用RNA-seq方法揭示了用顺铂和阿霉素处理BMAL1敲除（KO）或野生型（WT）MDA-MB-231细胞以启动细胞凋亡后的差异表达基因（DEG）和通路。 DEG 的基因集富集分析表明，多个基因/途径的富集有助于以BMAL1依赖性方式对顺铂或多柔比星诱导的细胞凋亡敏感。此外，我们的 DEG 分析表明非编码转录 RNA（例如 lncRNA 和加工后的假基因）可能在顺铂或多柔比星诱导的细胞凋亡中发挥作用。从顺铂和阿霉素治疗中的常见 DEG 获得的蛋白质-蛋白质相互作用网络表明，GSK3β、NACC1 和 EGFR 是调节 KO 细胞反应的主要基因。此外，对未处理的BMAL1 KO 和 WT 细胞的 DEG 分析表明，KO 细胞中上皮-间质转化基因上调。 作为阴性对照，我们还分析了用内质网 (ER) 应激诱导剂衣霉素治疗后的 DEG，该药物受BMAL1缺失的影响最小。总的来说，本研究表明BMAL1调节许多基因/途径，其中BMAL1 KO 细胞中的改变可能有助于观察到的多效性表型。