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. 2025 Sep 28;7(4):54.
doi: 10.3390/clockssleep7040054.

Reporter-Mediated Evaluation of the Circadian Oscillations of SNAIL Across In Vitro Models

Kaitlyn Chhe  1 , Bhavna Kalyanaraman  1 , Sophie A Spielberger  2 , Hui-Hsien Lin  1 , Stephanie R Taylor  3 , Michelle E Farkas  1
Affiliations

Reporter-Mediated Evaluation of the Circadian Oscillations of SNAIL Across In Vitro Models

Kaitlyn Chhe et al. Clocks Sleep. .

Abstract

The protein SNAIL has been widely studied for its roles in promoting cancer invasion and resistance to apoptosis. There are multiple contributors to its expression, including self- and circadian regulation, and it has been posited that SNAIL oscillates in a circadian manner. Given the multiple factors involved, we sought to determine whether this is indeed the case. We developed a luciferase reporter that was used to demonstrate SNAIL's rhythmic nature (SNAIL:luc) in the circadian model cell line, U2OS. Considering SNAIL's relevance in breast cancer, we also assessed its oscillations in cellular models representing different levels of aggression. We incorporated the SNAIL:luc reporter in MCF10A breast epithelial cells, and MCF7 and MDA-MB-231 breast cancer cell lines, which are less and more aggressive, respectively. We found that SNAIL oscillations were present but weak in MCF7 and arrhythmic in MDA-MB-231 cells, correlating with those of core clock genes (BMAL1 and PER2) in these models. Surprisingly, MCF10A cells, whose core clock genes possess robust circadian expression patterns, did not have rhythmic oscillations of SNAIL. Our findings suggest that SNAIL is under circadian control, but this is cell line/tissue dependent, setting the stage for additional studies to better understand the impacts of various factors contributing to its expression.

Keywords: MCF10A cells; MCF7 cells; MDA-MB-231 cells; SNAIL; U2OS cells; breast cancer; cancer biology; circadian rhythms; epithelial-to-mesenchymal transition (EMT); luciferase reporters; rhythmicity.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Bioluminescence time-series for Per2:luc (A,B), Bmal1:luc (C,D), and SNAIL:luc (E,F). Excluding a 12 h transient, shown are raw time-series (A,C,E) and time-series after de-trending (B,D,F) by removing the average of a 24 h moving window. The mean (raw or de-trended) time-series is plotted as a solid line, with the standard error of the mean as a semi-transparent envelope around it. (N = 3 for Per2:luc, N = 3 for Bmal1:luc, and N = 15 for SNAIL:luc).
Figure 2
Figure 2
Shown are the period (above) and phase-offset (below) values estimated by fitting a damped cosine curve to de-trended Per2:luc, Bmal1:luc, and SNAIL:luc time-series. (N = 3 for Per2:luc, N = 3 for Bmal1:luc, and N = 15 for SNAIL:luc).
Figure 3
Figure 3
Bioluminescence time-series for SNAIL:luc in MCF10A (A,B), MCF7 (C–F), and MDA-MB-231 (G,H) cell lines. Excluding a 12 h transient, shown are raw time-series (A,C,E,G) and time-series after de-trending (B,D,F,H) by removing the average of a 24 h moving window. The mean (raw or de-trended) time-series is plotted as a solid line, with the standard error of the mean as a semi-transparent envelope around it. Seven MCF7 time-series were deemed circadian (C,D; rhythmic with a period in the range of 16 to 32 h). The remaining ten MCF7 (E,F) and all MCF10A (A,B) and MDA-MB-231 (G,H) time-series were non-circadian. (N = 15 for MCF10A-SNAIL:luc, N = 7 for MCF7-SNAIL:luc (circadian), N = 10 for MCF7-SNAIL:luc (non-circadian), N = 6 for MDA-MB-231-SNAIL:luc).
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