Abstract
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.
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Funding
This work was supported by the Turkish Scientific and Technical Research Council (TUBITAK) grant 114S446 (to N.O.) and Gebze Technical Research Program grant G.T.Ü. BAP 2018-A-105-39 (to N.O.)
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ESM 1
Supp. Fig. 1 Confirmation of authenticity of MDA-MB-231 WT and KO cells A) The BMAL1 protein level was detected in parental (+), BMAL1 knockout (KO) and mock-infected (WT) MDA-MB-231 cells. B) In agreement with previous findings, BMAL1 deletion sensitized MDA-MB-231 cells to cisplatin (CIS)- and C) doxorubicin (DOX)- induced apoptosis as probed for c-PARP. D) The cells were also treated with tunicamycin (TUN) and spliced (active) form of XBP1 (sXBP1) was detected as the output marker for tunicamycin-induced ER stress response. Since sXPB1 signal and a cross-reacting band were merged, we run the samples for a longer period to confirm that sXBP1 signal was not much different between WT and KO cells in agreement with the transcriptome data that revealed much less DEGs in compared to CIS and DOX treatments. Supp. Fig. 2 Statistical analysis of migration rates of WT and KO cells. A) Migration properties of KO and WT MDA-MB-231 cells were analyzed using wound-healing assay. Representative images show the scratch (wound) at t = 0 h, t = 24 h and t = 48 h. Marks (red lines) were placed to locate the open area on the scratch. Images were taken by using Leica inverted microscope. Magnification 5X. B) The graph shows the percentage of wound healing in WT, and KO cells under starvation condition. The open area was calculated at the indicated time points by ImageJ Software (Free Accessible Image Processing Program, www.imagej.net). All results are representative of four independent experiments. Statistical Analysis was done with 24-h and 48-h data by one-way ANOVA followed by Tukey’s post hoc tests for pairwise comparison. (****P < 0.0001). Supp. Fig. S3 Gene Set Enrichment Analysis after TUN treatment. In agreement with similar sXBP1 formation in both WT and KO cells, no significant GSEA was detected after TUN treatment. TUN, tunicamycin. Supp. Fig. S4 Gene Set Enrichment Analysis and KEGG GO term of DEGs without treatment. In order to reveal the genes enriched in KO cells, DEGs were used for GSEA (KO vs WT). A) In agreement with the observed phenotype, HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION was the most significant one. B) BMAL1 gene deletion affected many genes as KEGG GO term of DEGs was shown. (PDF 701 kb)
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Table S1.
List of proteasomal or polymerase-related genes excluded from network analysis (XLSX 18 kb)
Table S2.
List of common DEGs in CIS and DOX treatments used to construct PPI network (XLSX 33 kb)
Table S3.
List of additional DEGs after 0-FPKM correction (XLSX 57 kb)
Table S4.
List of oligos used for qPCR analysis (XLSX 12 kb)
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Emisoglu-Kulahli, H., Gul, S., Morgil, H. et al. Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects. Funct Integr Genomics 21, 1–16 (2021). https://doi.org/10.1007/s10142-020-00757-6
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DOI: https://doi.org/10.1007/s10142-020-00757-6