Earticle

The cryopreservation of unfertilized oocytes is valuable for both livestock and human. However, freezing procedure to oocyte induce many kinds damages such as cytoskeleton disorganization, and chromosome and DNA abnormalities. Especially, during mammalian oocyte meiotic maturation, a process called oocyte polarization occurs, which leads to a unique asymmetric division. Actin play key roles during oocyte polarization experiences several steps including spindle migration, spindle anchoring and cortical reorganization. However, how chilling stress affect to actin nucleation in oocytes is poorly studied. In this study, we investigated expressions and feature of Arp2/3 (actin activators), WAVE2 and JMY (nucleation promoting factors) in MІІ stage oocytes after different freezing procedure in oocytes. Results indicated that survival, IVF, developmental rates of vitrified-thawed oocytes were significantly higher than those of slow freezed oocytes. Especially, expressions of Arp2/3, WAVE2 and JMY in vitrified-thawed oocytes were similar with normal oocytes. However, expression of actin nucleation related genes in slow freezed group was significantly higher or lower than normal oocytes. Our results may suggested that slow freezing procedure to oocyte induced abnormal actin neucleation results in low survival and developmental rates.

RNF2, also known as Ring1B/Ring2, is a component of the polycomb repression complex 1. RNF2 is highly expressed in many tumors, suggesting that it might have an oncogenic function, but the mechanism is unknown. Here, we show that knockdown of RNF2 significantly inhibits both cell proliferation and colony formation in soft agar, and inducesapoptosis in cancer cells. Knockdown of RNF2 in HCT116 p53(+/+) cells resulted in significantly more apoptosis than was observed in RNF2 knockdown HCT116 p53(—/—) cells, indicating that RNF2 knockdown-induced apoptosis is partially dependent on p53. Various p53-targeted genes were increased in RNF2 knockdown cells. Further studies revealed that in RNF2 knockdown cells, the p53 protein level was increased, the half-life of p53 was prolonged and p53 ubiquitination was decreased. In contrast, cells overexpressing RNF2 showed a decreased p53 protein level, a shorter p53 half-life and increased p53 ubiquitination. Importantly, we found that RNF2 directly binds with both p53 and MDM2 and promotes MDM2-mediated p53 ubiquitination. RNF2 overexpression could also increase the half-life of MDM2 and inhibit its ubiquitination. The regulation on p53 and MDM2 stability by RNF2 was also observed during the etoposide-induced DNA damage response. These results provide a possible mechanism explaining the oncogenic function of RNF2, and because RNF2 is important for cancer cell survival and proliferation, it might be an ideal target for cancer therapy or prevention.

Autophagy is a critical cellular process required for maintaining cellular homeostasis in health and disease states, but the molecular mechanisms and impact of autophagy on cancer is not fully understood. Here, we found that Sox2, a key transcription factor in the regulation of the "stemness" of embryonic stem cells and induced-pluripotent stem cells, strongly induced autophagic phenomena, includingintracellular vacuole formation and lysosomal activation in colon cancer cells. The activation occurred through Sox2-mediated ATG10 gene expression and resulted in the inhibition of cell proliferation and anchorage-independent colony growth ex vivo and tumor growth in vivo. Further, we found that Sox2-induced-autophagy enhanced cellular senescence by up-regulating tumor suppressors or senescence factors, including p16(INK4a), p21 and phosphorylated p53 (Ser15). Notably, knockdown of ATG10 in Sox2-expressing colon cancer cells restored cancer cell properties. Taken together, our results demonstrated that regulation of autophagy mediated by Sox2 is a mechanism-driven novel strategy to treat human colon cancers.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription- 3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

JMY (junction-mediating and regulatory protein) is a transcriptional co-factor of p53, little information is available on porcine oocyte. Herein we demonstrated that JMY robustly expressed in oocytes, where it was localized predominantly in cytoplasm, and JMY mRNA expressed largely from germinal vesicle to metaphase I stage, and gradually decreased during metaphase II (MII) stages. Depletion of JMY by double-stranded RNA (dsRNA) influenced actin and Arp2/3 expression, further blocking maturation of porcine oocyte in vitro. The p53 transcription JMY responsed in DNA damage conditions, undergoing nuclear accumulation and mediated p53 expression. On the basis of our results, we proposed that p53 co-factor JMY regulated actin and Arp2/3 linked in vitro meiotic maturation in porcine oocyte and related to DNA damage with the p53 response.

LINE-1 is an autonomous non-LTR retrotransposon in mammalian genomes and encodes ORF1P and ORF2P. ORF2P has been clearly identified as the enzyme supplier needed in LINE-1 retrotransposition. However, the role of ORF1P is not well explored and requires further elucidation. In this study, we depleted ORF1P to investigate its role in mouse oocyte meiotic maturation. The results showed that depletion of ORF1P caused oocyte arrest at the germinal vesicle (GV) stage as well as down-regulation of Cdc2 and Cyclin B1, components of the maturation promoting factor (MPF). Further analysis demonstrated a decreased expression of the P21 upstream factors Smad4 and Dcp1a after ORF1P depletion. However, SMAD4 and DCP1A became accumulated in the nucleus. This translocation would up-regulate the expression of P21. Furthermore, ORF1P knockdown also increased the expression of microRNA-494, which could decrease the expression of Cyclin B1 and Cdc2. Propidium Iodide (PI) staining after ORF1P depletion revealed abnormal chromatin configuration in the GV of mouse oocytes. Taken together, these results indicate that ORF1P is involved in the TGF-β pathway to modulate the GV breakdown (GVBD) during mouse oocyte meiotic maturation.

Actin-related protein 2/3 (Arp2/3) complex plays a central role in the de novo nucleation of filamentous actin as branches on existing filaments. Small molecule inhibitor CK-666 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we shed light on the functional aspects of Arp2/3 by CK-666 inhibitor on porcine early embryo development. Our data indicated that CK-666 destabilizes the complex, blocking movement of the Arp2 and Arp3 subunits, causing actin degradation and failure of cell division which results in failure of early embryo development. Our results also shows there is decrease in mRNA expression of ICM and trophectoderm related genes such as Sox2, Pou5f1, Nanog. These results provided key insights into the relationship between early embryo dvelopement and critical role of Arp2/3 complex.

We have analysed the expression and function of cell cycle and cell death regulator Aven in porcine oocytes. Aven is expressed in a wide variety of adult tissue and cell lines, and there is increased evidence of that its role in cell cycle regulation. The mechanism by which anti apoptotic activity of Aven is poorly understood. Although Aven ortholog have been reported in other mammalian species, no Aven gene has been reported in porcine oocytes. Here we shed light on this issue for the first time in porcine oocytes demonstrating that Aven can be potential oocyte quality marker and a novel inhibitor of caspase activation. Our results revealed there is gradual increase in Aven m-RNA expression upto 4-cell stage and then decreased drastically.This result shows Aven is a maternal gene and is regulated dynamically. In addition. Aven protein localisation,caspase-3 activation and mitochondrial distribution were studied by immunofloroscence. Induction of apoptosis by etoposide drug treatment resulted in changes in Aven localisation within COC, corresponding to caspase-3 activation and altered mitochondrial distribution. These results could be an excellent example for studying the function of Aven and identifying cellular factors that influence its activity,revealing information that may be relevant to human disease.