BMAL1 facilitates trophoblast migration and invasion via SP1-DNMT1/DAB2IP pathway in recurrent spontaneous abortion

Abstract

The underlying mechanism about rhythms and epigenetics leading to aberrant trophoblast migration and invasion in recurrent spontaneous abortion (RSA) remains unknown. Brain and muscle ARNT-like protein 1 (BMAL1) is considered as a crucial role in fertility, and polymorphism of BMAL1 gene has been reported to be associated with risk of miscarriage. However, the functional role of BMAL1 in RSA is not fully understood. Previous study shows the descended expression of DNA 5'-cytosine-methyltransferases 1 (DNMT1) in the villous of early pregnancy loss. Thus, understanding of the regulation of DNMT1 expression may be of significance for the elucidation of the process of RSA. Using HTR-8/SVneo and JEG-3 cell lines, we certified the induction of specificity protein 1 (SP1) to DNMT1 and DAB2 interaction protein (DAB2IP), respectively, both of which further activated matrix metallo-proteinase 2/9 (MMP2/9), bringing out changes in trophoblast migration and invasion. Notably, BMAL1 functioned as a positive upstream factor of SP1 only in HTR-8/SVneo cells but not in JEG-3 cells, inducing SP1-DNMT1/DAB2IP pathway and facilitating migration and invasion of trophoblasts. In addition, progesterone might restore the down-regulation of BMAL1 and downstream pathway in a dose-dependent manner. Last but not least, the decreased abundance of BMAL1 was correlated positively with that of SP1, DNMT1, DAB2IP, MMP2 and MMP9 in human villous specimens of RSA. Our results demonstrate that the induction of BMAL1 to SP1 contributes to the expression of DNMT1 and DAB2IP, respectively, activating trophoblast migration and invasion. The deregulation of the BMAL1-mediated pathway in RSA can be rescued by progesterone.

Keywords: BMAL1; Pathology Section; migration and invasion; progesterone; recurrent spontaneous abortion; trophoblast.

Figure 1. BMAL1, SP1, DNMT1 and DAB2IP induced migration and invasion of HTR-8/SVneo cells

A. Scratch-wound assay after BMAL1 knock-down (magnification: 50 ×). The left panel shows the representative images of Scratch-wound assay and the right panel clarifies the statistic results of the wound recovery of Scratch-wound assay. B. Scratch-wound assay after SP1 knock-down (magnification: 50 ×). C. Scratch-wound assay after DNMT1 knock-down (magnification: 50 ×). D. Scratch-wound assay after DAB2IP knock-down (magnification: 50 ×). E. Transwell assay and MTT after BMAL1 knock-down (magnification: 100 ×). The top panel shows the representative images of transwell assay. The bottom panel from left to right presents the statistic result of invaded cells, the statistic result of MTT and the cell count. F. Transwell assay and MTT after SP1 knock-down (magnification: 100 ×). G. Transwell assay and MTT after DNMT1 or DAB2IP knock-down (magnification: 100 ×). Images are representative, and data are means ± SEM from three experiments. *P < 0.05, **P < 0.01 against si-NC cells.

Figure 2. BMAL1 promoted SP1-induced DNMT1 and DAB2IP, and further regulated MMP2 and MMP9 in HTR-8/SVneo cells

A. The mRNA and protein abundance of DNMT1, MMP2 and MMP9 after DNMT1 knock-down in HTR-8/SVneo cells. The panel from left to right is the representative images of western blot assays, the immunoreactive bands densitometrically quantified and mRNA abundance. B. The mRNA and protein abundance of DAB2IP, MMP2 and MMP9 after DAB2IP knock-down in HTR-8/SVneo cells. C. The mRNA and protein abundance of SP1, DNMT1, DAB2IP, MMP2 and MMP9 after SP1 knock-down in HTR-8/SVneo cells. D. The mRNA and protein abundance of SP1, DNMT1, DAB2IP, MMP2 and MMP9 after SP1 over-expression in HTR-8/SVneo cells. E. The mRNA and protein abundance of BMAL1, SP1, DNMT1, DAB2IP, MMP2 and MMP9 after BMAL1 knock-down in HTR-8/SVneo cells. F. The mRNA and protein abundance of BMAL1, SP1, DNMT1, DAB2IP, MMP2 and MMP9 after BMAL1 over-expression in HTR-8/SVneo cells. β-Actin was used as a loading control. Blots are representative, and data are means ± SEM from four to five experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 against si-NC cells or against control-vec cells.

Figure 3. BMAL1 didn't function as an upstream factor of SP1-mediated DNMT1, DAB2IP and downstream pathway in JEG-3 cells

A. The mRNA and protein abundance of DNMT1, MMP2 and MMP9 after DNMT1 knock-down in JEG-3 cells. The panel from left to right is the representative images of western blot assays, the immunoreactive bands densitometrically quantified and mRNA abundance. B. The mRNA and protein abundance of DAB2IP, MMP2 and MMP9 after DAB2IP knock-down in JEG-3 cells. C. The mRNA and protein abundance of SP1, DNMT1, DAB2IP, MMP2 and MMP9 after SP1 knock-down in JEG-3 cells. D. The mRNA and protein abundance of BMAL1, SP1, DNMT1, DAB2IP, MMP2 and MMP9 after BMAL1 knock-down in JEG-3 cells. β-Actin was used as a loading control. Blots are representative, and data are means ± SEM from three to four experiments.* P < 0.05, ** P < 0.01,*** P < 0.001 against si-NC cells.

Figure 4. Progesterone promoted BMAL1-mediated pathway in HTR-8/SVneo cells

A. The mRNA and protein abundance of BMAL1, SP1, DNMT1 and DAB2IP after treatment with progesterone for 24 hours at the concentration of 0, 5, 10 and 15 μmmol/L in HTR-8/SVneo cells. The panel from left to right is the representative images of western blot assays, the immunoreactive bands densitometrically quantified and mRNA abundance. B. The mRNA and protein abundance of BMAL1, SP1, DNMT1 and DAB2IP after BMAL1 knock-down and further incubation with 15 μmmol/L progesterone for 24 hours in HTR-8/SVneo cells. C. The mRNA abundance of MMP2 and MMP9 after treatment with progesterone for 24 hours at the concentration of 0, 5, 10 and 15 μmmol/L in HTR-8/SVneo cells. D. The mRNA abundance of MMP2 and MMP9 after BMAL1 knock-down and further incubation with 15 μmmol/L progesterone for 24 hours in HTR-8/SVneo cells. E. The mRNA abundance of MMP2 and MMP9 after BMAL1 over-expression and further incubation with 15 μmmol/L progesterone for 24 hours in HTR-8/SVneo cells. β-Actin was used as a loading control. Blots are representative, and data are means ± SEM from three to four experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 against si-NC cells or against control-vec cells; # P < 0.05, ## P < 0.01, ### P < 0.001 against si-NC+P4 cells or against control-vec +P4 cells.

Figure 5. The decreased expression of BMAL1 in human villous specimens of RSA was consistent with SP1, DNMT1, DAB2IP, MMP2 and MMP9

A. The mRNA abundance of BMAL1, SP1, DNMT1, DAB2IP, MMP2 and MMP9 in human villous specimens. IA: induced abortion (n = 50). SA: sporadic abortion (n = 38). RSA: recurrent spontaneous abortion (n = 11). B. The protein abundance of BMAL1, SP1, DNMT1, DAB2IP, MMP2 and MMP9 in human villous specimens. The top panel shows the representative images of western blot assays. The bottom panel clarifies the proteins levels of detected specimens. IA: induced abortion (n = 25). SA: sporadic abortion (n = 20). RSA: recurrent spontaneous abortion (n = 8). β-Actin was used as a loading control. Blots are representative. * P < 0.05, ** P < 0.01, *** P < 0.001.

Figure 6. A proposed signaling pathways underpinning that BMAL1 induced trophoblast migration and invasion in recurrent spontaneous abortion

As an upstream factor of SP1, BMAL1 facilitated DNMT1 and DAB2IP, respectively, and then promoted migration and invasion of trophoblasts via MMP2 and MMP9. Additionally, the deregulation of the BMAL1-mediated pathway in RSA can be rescued by progesterone.