UNRAVELING THE IMPACT OF BMAL1-MEDIATED CIRCADIAN RHYTHM DISRUPTION ON TROPHOBLAST INVASION AND MIGRATION THROUGH WNT/Β-CATENIN AND INTEGRINMEDIATED SIGNALING PATHWAYS

Abstract

Objective

Circadian rhythm (CR) orchestrates various physiological processes on a 24-hour basis, governed by molecular clock genes (MCGs); BMAL1, CLOCK, PERs, and CRYs. Clinical data indicate that disruption of maternal CR during pregnancy is associated with intrauterine growth restriction (IUGR), a condition linked to defects in trophoblast migration and invasion, yet the underlying molecular mechanisms need to be explored. This study aims to elucidate rhythmic expression pattern of BMAL1 in human villous trophoblast (HVT) and to investigate the specific pathways governed by BMAL1 related to trophoblast migration and invasion.

Materials and methods

We utilized a pLenti-BMAL1-DLuc lentivirus for in vitro circadian rhythm monitoring of BMAL1 gene. BMAL1 expression was analyzed using JTK-Cycle, robust non-parametric algorithm for detecting rhythmic elements in genome-scale data. Non-target, and BMAL1-silenced groups were established using HVT cells. BMAL1-silenced group was created using shRNA-silencing strategy. Following transduction and subsequent selection, cells were harvested every six hours over 48-hour period, corresponding to Zeitgeber Time (ZT) points 0- 48, following dexamethasone treatment used for cell-cycle synchronization. CLOCK, PERs, CRYs, RORα, and NR1D1 expression were quantified every 6 hours via qPCR. Trophoblast migration/invasion, influenced by BMAL1, were assessed through transwell assay, and related protein expressions (β-catenin, integrin-β1, integrin-ɑ5) were evaluated using western blotting. Statistical significance was determined at p<0.05 using two-way ANOVA and Tukey's post hoc test.

Results

The outcomes from JTK-Cycle analysis, revealed the inherent rhythmic expression pattern of BMAL1 in HVT cells. This natural expression displayed an average period of 23.3 hours and an average amplitude of 429,7 abs. BMAL1 silencing was confirmed at the protein level. Rhythmic expression of other MCGs; CLOCK, PER1/2/3, CRY1/2, RORα, and NR1D1 genes were altered significantly in BMAL1-silenced group(p<0.05). Following BMAL1 silencing, a notable reduction in the migration and invasion capabilities of HVT cells was observed(p<0.05). Additionally, BMAL1 depletion correlated with a significant decrease in β-catenin expression from ZT30 onwards, and a decrease in integrin-β1 expression from ZT36 onwards(p<0.05). Conversely, there was a significant increase in integrin-ɑ5 protein expression beginning at ZT24(p<0.05).

Conclusions

BMAL1 is rhythmically expressed in HVT cells, and its silencing disrupts rhythmic expression of key MCGs and leads to alterations in trophoblast migration and invasion-related pathways. Specifically, BMAL1 depletion dysregulates β-catenin, integrin-β1, and integrin-ɑ5 protein expressions, revealing its pivotal role in orchestrating trophoblast function.