OBJECTIVES: To study the effect of CDC20 knockdown on proliferation, migration and invasion of cervical cancer cells and its underlying mechanism. METHODS: CDC20 expression in cervical cancer tissues was analyzed using the TCGA database, and the protein expressions of CDC20 and β-Catenin in clinical specimens of cervical cancer and adjacent tissues were detected using immunohistochemistry. A dual target sgRNA2&7 sequence for CDC20 gene was designed for CDC20 gene knockdown in cervical cancer C33A cells using CRISPR/Cas9 technology, and CDC20 mRNA and protein expression levels in the transfected cells were detected using qRT-PCR and Western blotting. The changes in proliferation, cell cycle, apoptosis, migration and invasiveness of the transfected cells were evaluated using colony-forming assay, fluorescence activated cell sorting (FACS) and Transwell assay. In the animal experiment, naïve C33A cells and the cells with CDC20 knockdown were injected subcutaneously into the left and right axillae of nude mice (n=5) to observe tumor growth. The expressions of CDC20 and β-Catenin proteins in transfected cells and the xenograft were analyzed using Western blotting, and their interaction was confirmed by co-immunoprecipitation (CoIP) and immunofluorescence co-localization assays. RESULTS: Cervical cancer tissues expressed significantly higher CDC20 and β‑Catenin levels than the adjacent tissues. C33A cells with CDC20 knockdown showed reduced proliferation, increased apoptosis, and lowered migration and invasion abilities. CDC20 knockdown significantly suppressed the growth of C33A cell xenograft in nude mice, and the tumor-bearing mice did not exhibit obvious body mass changes. CDC20 and β-Catenin levels were both significantly lowered in C33A cells with CDC20 knockdown. Co-immunoprecipitation and co-localization assays confirmed the interaction between CDC20 and β‑Catenin. CONCLUSIONS CDC20 is highly expressed in cervical cancer tissues, and CDC20 knockdown can suppress proliferation, invasion, and metastasis while enhancing apoptosis of C33A cells, which is closely related with the regulation of the Wnt/β-Catenin signaling pathway.
Humans ; Uterine Cervical Neoplasms/metabolism* ; Female ; Cdc20 Proteins/genetics* ; Cell Proliferation ; Animals ; Cell Movement ; Neoplasm Invasiveness ; Apoptosis ; Mice, Nude ; beta Catenin/metabolism* ; CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis

BACKGROUND: Present therapeutic tool cannot supplement infarct myocardium. Studies have shown that stem cell transplantation can promote regeneration of myocardium and vessels and improve heart function and prognosis.OBJECTIVE: To observe changes in morphology and hemodynamics in myocardium following embryonic stem cell transplantation in and surrounding the acute myocardial infarct site.DESIGN, TIME AND SETTING: The randomized, controlled, animal study was performed at the Laboratory of Neurobiology,Department of Human Anatomy and Neurobiology, Xiangya Medical College, Central South University from March 2007 to October 2008.MATERIALS: A total of 40 SPF grade Wistar rats were equally randomized into 4 groups, normal control, infarct model,central transplantation and peripheral transplantation groups. Embryonic stem cells-D3 (ES-D3) and Buffalo rat hepatocytas were supplied by Shanghai Cell Institute, Chinese Academy of Sciences.METHODS: Following resuscitation, ES-D3 cells at (2.0-5.0)×107/L were incubated in a flask, and induced to in vitro differentiate in conditioned medium containing Buffalo rat hepatocytes. Except normal control group, rat models of acute myocardial infarction were established by ligating left anterior descending coronary artery in the infarct model, central transplantation and peripheral transplantation groups. At 1 week following model induction, ES-D3 cells were labeled by BrdU for 1 day, and implanted at 1×109/L. Three sites were selected in the infarct site in the central transplantation group. 10 μ L cell suspension (104 cells) was implanted in the ventricular wall through each site. In the peripheral transplantation group, an equal volume of cell suspension was separately implanted in three peripheral infarct sites by the same method.MAIN OUTCOME MEASURES: Results of immunohistochemistry and hemodynamics were measured.RESULTS: ES-D3 cells in buffalo rat hepatocyte conditioned medium presented regular colony-shaped. At 8 days following differentiation, some embryo proper had spontaneous rhythmic contraction, showed positive reaction of cardiac troponin T after immunostaining. Under the electron microscope, myotube and muscle fiber appeared, which verified the differentiation of cardiomyocytes. Cells were positive for BrdU in the peripheral transplantation group, but negative in the central transplantation group. Cells were also positive for cardiac troponin T. 4 weeks following transplantation, left ventricular systolic pressure,minimum/maximum rate of ventricular pressure (±dp/dtmax) were significantly reduced (P ＜ 0.01), but left ventricular end diastolic pressure was significantly increased (P ＜ 0.01), left ventricular mass and left ventricular mass index were significantly increased (P ＜ 0.01 ) in the infarct model group compared with the normal control group. Compared with the infarct model group, no significant changes in hemodynamics indices were found in the central transplantation group (P ＞ 0.05); left ventricular systolic pressure, ±dp/dtmax were significantly increased (P ＜ 0.01), left ventricular end diastolic pressure was significantly decreased (P ＜ 0.01 ), left ventricular mass, left ventricular mass index and infarct area were significantly reduced(P ＜ 0.01) in the peripheral transplantation group.