The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

Cell Biochemistry & Function

2020

Preeclampsia (PE) is a serious disease that occurs after 20 weeks during pregnancy.There are some aberrant microRNAs (miRNAs) that are associated with the etiology of PE. As discovered by scholars, there was an increased level of miR-215-5p in plasma of PE patients compared with the control group; nonetheless, there is still no knowledge of the mechanism of miR-215-5p in PE. We carried out the comparison of the expression levels of miR-215-5p, and the supposed target gene cell division cycle 6 (CDC6) in 30 placentas from PE patients as well as 30 placentas from normal pregnant women. The verification of the impacts of miR-215-5p and CDC6 was carried out by functional assays in HTR-8/SVneo cells transfected with the miR-215-5p mimic or siR-CDC6. As indicated by findings, miR-215-5p showed an apparent increase; conversely, CDC6 was inhibited in the experiment group. The upregulation of miR-215-5p inhibited both the migration and invasive potential of trophoblasts, besides decreasing the G1-S transition and downregulating CDC6 in HTR-8/SVneo cells; nonetheless, it did not significantly impact the cell proliferation. Furthermore, siR-CDC6 replicated the functions of the miR-215-5p mimic. Also, the miR-215-5p mimic and siR-CDC6 both decreased the epithelial-mesenchymal transition (EMT) with additional E-cadherin level and decreased the expressions of N-cadherin as well as vimentin in trophoblast cells. To conclude, miR-215-5p decreased not only the migration but also the invasion of trophoblasts through regulating CDC6, which indicated that miR-215-5p might be associated with the etiology of PE.Significance of the study: More and more attention has been paid on the roles of miRNAs in the pathogenesis of PE. However, there is no study of miR-215-5p in the etiology of PE. We first investigated the mechanism of miR-215-5p in placental tissues and HTR-8/SVneo cells. It was suggested that miR-215-5p decreased the abilities of migration and invasion of trophoblasts through regulating CDC6 in PE. miR-215-5p might be used as an target for the early diagnosis and treatment of PE in the future. K E Y W O R D S preeclampsia, miR-215-5p, CDC6, EMT, placenta Abbreviations: CCK-8, Cell Counting Kit-8; CDC6, cell division cycle 6; cDNA, complementary DNA; DMEM, Dulbecco's modified Eagle's medium; EMT, epithelial-mesenchymal transition.; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; miRNA, microRNA; PBS, phosphate-buffered saline solution; PE, preeclampsia; PI, propidium iodide; PVDF, polyvinylidene fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Section: Discussionsupporting

confidence: 72%

miR‐215‐5p decreases migration and invasion of trophoblast cells through regulating CDC6 in preeclampsia

Yang

Cell Biochemistry & Function

2020

“…γ-tubulin is a major component of MTOCs and it is important for nucleating microtubules (Sobel & Snyder, 1995). A recent study showed that CDC6 localizes to centrosomes during the S-and G2-phases of the cell cycle of human cells, and it inhibits the microtubule-organizing activity of the centrosome in mitosis (Lee et al, 2017). In the present study, we found that CDC6 Depletion of CDC6 resulted in displacement of γ-tubulin from spindle poles, implying that CDC6 may contribute to spindle organization by partially impairing the function of MTOCs.…”

Section: Discussionmentioning

confidence: 99%

“…The mouse CDC6 binds chromatin and associates with the spindle during mitosis. A recent study suggests a novel function for CDC6 in coordinating centrosome assembly and function (Lee et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

CDC6 regulates both G2/M transition and metaphase‐to‐anaphase transition during the first meiosis of mouse oocytes

Journal Cellular Physiology

et al. 2020

Cell division cycle protein, CDC6, is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule‐organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from pro‐metaphase I (MI) to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (germinal vesicle breakdown [GVBD]) through regulation of Cdh1 and cyclin B1 expression and CDK1 (CDC2) phosphorylation in a GVBD‐inhibiting culture system containing milrinone. Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6‐depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ‐tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation, and spindle assembly checkpoint (SAC) activation, leading to significant pro‐MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time‐lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase‐to‐anaphase transition in meiotic oocytes.

“…A recent study showed that CDC6 inhibits the microtubule-organizing activity of the centrosome in mitosis (Lee et al, 2017). In Hela cells, phosphorylation of CDC6 by Plk1 is required for CDK1 and cyclin B inhibition, which is necessary for separase activation and exit from mitosis (Hyungshin and Erikson, 2010).…”

Section: Discussionmentioning

confidence: 99%

“…The mouse CDC6 binds chromatin and associates with the spindle during mitosis. A recent study suggests a novel function for Cdc6 in coordinating centrosome assembly and function (Lee et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

CDC6 regulates both G2/M transition and metaphase-to-anaphase transition during the first meiosis of mouse oocytes

et al. 2019

Preprint

Cell division cycle protein CDC6 is essential for the initiation of DNA replication. CDC6 was recently shown to inhibit the microtubule-organizing activity of the centrosome. Here, we show that CDC6 is localized to the spindle from Pro-MI to MII stages of oocytes, and it plays important roles at two critical steps of oocyte meiotic maturation. CDC6 depletion facilitated the G2/M transition (GV breakdown, GVBD) through regulation of Cdh1 and cyclin B1 expression and CDK1 phosphorylation in a GVBD-inhibiting culture system containing milrinone.Furthermore, GVBD was significantly decreased after knockdown of cyclin B1 in CDC6-depleted oocytes, indicating that the effect of CDC6 loss on GVBD stimulation was mediated, at least in part, by raising cyclin B1. Knockdown of CDC6 also caused abnormal localization of γ -tubulin, resulting in defective spindles, misaligned chromosomes, cyclin B1 accumulation and spindle assembly checkpoint (SAC) activation, leading to significant Pro-MI/MI arrest and PB1 extrusion failure. These phenotypes were also confirmed by time-lapse live cell imaging analysis. The results indicate that CDC6 is indispensable for maintaining G2 arrest of meiosis and functions in G2/M checkpoint regulation in mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes.Summary statement：We show that CDC6 is indispensable for maintaining G2 arrest of mouse oocytes. Moreover, CDC6 is also a key player regulating meiotic spindle assembly and metaphase-to-anaphase transition in meiotic oocytes. resumption of meiosis in mouse oocytes. Hum Mol Genet. 21:2476. Althoff, F., Karess, R.E. and Lehner, C.F.( 2012). Spindle checkpoint-independent inhibition of mitotic chromosome segregation by Drosophila Mps1. Mol Biol Cell. 23:2275. Anger, M., Stein, P. and Schultz, R.M. (2005). CDC6 requirement for spindle formation during maturation of mouse oocytes. Biol Reprod. 72:188-194.