Qinghua Cui

PP2A regulates the pro-apoptotic activity of FOXO1.

FOXO1, a member of the evolutionarily conserved forkhead family of transcription factors, regulates expression of a number of genes that play critical roles in cell cycle and apoptosis. A pivotal regulatory mechanism of FOXO is reversible phosphorylation, catalyzed by kinases and phosphatases. Phosphorylation of FOXO1 is associated with 14-3-3 binding and cytosolic localization, whereas dephosphorylated FOXO1 translocates to the nucleus and is transcriptionally active. Experiments were performed to identify the serine/threonine phosphatase that dephosphorylates FOXO1. PP2A inhibitors, okadaic acid and fostriecin, increased FOXO1 phosphorylation in vitro and in cells. Microcystin-agarose pull-downs suggested that a phosphatase binds to FOXO1, and PP2A catalytic subunit was identified in endogenous FOXO1 immunocomplexes, indicating that PP2A is a FOXO1 phosphatase. Purified PP2A interacted directly with FOXO1 and dephosphorylated FOXO1 in vitro. Silencing of PP2A protected FOXO1 from dephosphorylation and delayed FOXO1 nuclear translocation, confirming the physiologic role of PP2A in the regulation of FOXO1 function. Furthermore, inhibition of PP2A phosphatases rescued FOXO1-mediated cell death by regulating the level of the pro-apoptotic protein BIM. We conclude that PP2A is a physiologic phosphatase of FOXO1.

G0 function of BCL2 and BCL-xL requires BAX, BAK, and p27 phosphorylation by Mirk, revealing a novel role of BAX and BAK in quiescence regulation.

BCL2 and BCL-xL facilitate G0 quiescence by decreasing RNA content and cell size and up-regulating p27 protein, but the precise mechanism is not understood. We investigated the relationship between cell cycle regulation and the anti-apoptosis function of BCL2 and BCL-xL. Neither caspase inhibition nor abrogation of mitochondria-dependent apoptosis by BAX and BAK deletion fully recapitulated the G0 effects of BCL2 or BCL-xL, suggesting that mechanisms in addition to anti-apoptosis are involved in the cell cycle arrest function of BCL2 or BCL-xL. We found that BCL2 and BCL-xL expression in bax-/- bak-/- cells did not confer cell cycle effects, consistent with the G0 function of BCL2 and BCL-xL being mediated through BAX or BAK. Stabilization of p27 in G0 in BCL2 or BCL-xL cells was due to phosphorylation of p27 at Ser10 by the kinase Mirk. In bax-/- bak-/- cells, total p27 and p27 phosphorylated at Ser10 were elevated. Re-expression of BAX in bax-/- bak-/- cells and silencing of BAX and BAK in wild type cells confirmed that endogenous BAX and BAK modulated p27. These data revealed a novel role for BAX and BAK in the regulation of G0 quiescence.

Transcriptome-Wide Identification and Characterization of MicroRNAs from Castor Bean (Ricinus communis L.)

Background MicroRNAs (miRNAs) are endogenously encoded small RNAs that post-transcriptionally regulate gene expression and play essential roles in numerous developmental and physiological processes. Currently, little information on the transcriptome and tissue-specific expression of miRNAs is available in the model non-edible oilseed crop castor bean (Ricinus communis L.), one of the most important non-edible oilseed crops cultivated worldwide. Recent advances in sequencing technologies have allowed the identification of conserved and novel miRNAs in many plant species. Here, we used high-throughput sequencing technologies to identify and characterize the miRNAs in castor bean. Results Five small RNA libraries were constructed for deep sequencing from root tips, leaves, developing seeds (at the initial stage, seed1; and at the fast oil accumulation stage, seed2) and endosperms in castor bean. High-throughput sequencing generated a large number of sequence reads of small RNAs in this study. In total, 86 conserved miRNAs were identified, including 63 known and 23 newly identified. Sixteen miRNA isoform variants in length were found from the conserved miRNAs of castor bean. MiRNAs displayed diverse organ-specific expression levels among five libraries. Combined with criteria for miRNA annotation and a RT-PCR approach, 72 novel miRNAs and their potential precursors were annotated and 20 miRNAs newly identified were validated. In addition, new target candidates for miRNAs newly identified in this study were proposed. Conclusions The current study presents the first high-throughput small RNA sequencing study performed in castor bean to identify its miRNA population. It characterizes and increases the number of miRNAs and their isoforms identified in castor bean. The miRNA expression analysis provides a foundation for understanding castor bean miRNA organ-specific expression patterns. The present study offers an expanded picture of miRNAs for castor bean and other members in the family Euphorbiaceae.

microRNA-375 inhibits colorectal cancer cells proliferation by downregulating JAK2/STAT3 and MAP3K8/ERK signaling pathways

MicroRNA-375 is involved in many types of alimentary system cancers. Our previous studies showed that microRNA-375 was significantly down-regulated in carcinoma tissues compared with para-carcinoma tissues, which strongly indicates that microRNA-375 might suppress the occurrence and development of colorectal cancer. However, the mechanism underlying the microRNA-375 regulation in colorectal cancer remains unclear. In this study, we first sorted out jak2, map3k8 and atg7 as microRNA-375 targeted genes from multiple databases, and found that jak2, map3k8 and their downstream genes stat3 and erk were up-regulated in carcinoma tissues. Secondly, we over-expressed microRNA-375 in colorectal cancer cell lines (HCT116, Caco2 and HT29). Our results showed that in microRNA-375 over-expressing cells, JAK2/STAT3 and MAP3K8/ERK proteins were down-regulated, cell proliferation was inhibited, cell migration rate did not change. There was no significant difference on ATG7 expression between the control group and microRNA-375 over-expressing HT29/Caco2 cells, whereas microRNA-375 down-regulated ATG7 specifically in HCT116 cells. Finally, we demonstrated that expressing microRNA-375 suppressed tumor formation in nude mice. In conclusion, microRNA-375 might function as a tumor-repressive gene to inhibit cell proliferation, mainly through targeting both JAK2/STAT3 and MAP3K8/ERK signaling pathways in colorectal cancer. These findings suggest miR-375 as a promising diagnostic marker and a therapeutic drug for colorectal cancer.

Nutrient deprivation-related OXPHOS/glycolysis interconversion via HIF-1α/C-MYC pathway in U251 cells.

Although the Warburg effect is a dominant metabolic phenotype observed in cancers, the metabolic changes and adaptation occurring in tumors have been demonstrated to extend beyond the Warburg effect and thus considered a secondary effect to the transformation process of carcinogenesis, including nutritional deficiencies. However, the role of nutritional deficiencies in this metabolic reprogramming (e. g., oxidative phosphorylation (OXPHOS)/glycolysis interconversion) is not completely known yet. Here, we showed that under regular culture condition, the proliferation of U251 cells, but not other tumor cell lines, preferentially performed the Warburg effect and was remarkably inhibited by oxamic acid which can inhibit the activity of lactate dehydrogenase (LDH); whereas under serum starvation, glycolysis was depressed, tricarboxylic acid cycle (TCA) was enhanced, and the activity of OXPHOS was reinforced to maintain cellular ATP content in a high level, but interestingly, we observed a decreased expression of reactive oxygen species (ROS). Moreover, the upregulated activity of mitochondrial complex I was confirmed by Western blots and showed that the mitochondrial-related protein, NDUFA9, NDUFB8, ND1, and VDAC1 were remarkably increased after serum starved. Mechanistically, nutritional deficiencies could reduce hypoxia-inducible factor α (HIF-1α) protein expression to increase C-MYC protein level, which in turn increased nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM) transcription to enhance the activity of OXPHOS, suggesting that metabolic reprogramming by the changes of microenvironment during the carcinogenesis can provide some novel therapeutic clues to traditional cancer treatments.

Genome-Wide Identification, Evolutionary Analysis, and Stress Responses of the GRAS Gene Family in Castor Beans

Plant-specific GRAS transcription factors play important roles in regulating growth, development, and stress responses. Castor beans (Ricinus communis) are important non-edible oilseed plants, cultivated worldwide for its seed oils and its adaptability to growth conditions. In this study, we identified and characterized a total of 48 GRAS genes based on the castor bean genome. Combined with phylogenetic analysis, the castor bean GRAS members were divided into 13 distinct groups. Functional divergence analysis revealed the presence of mostly Type-I functional divergence. The gene structures and conserved motifs, both within and outside the GRAS domain, were characterized. Gene expression analysis, performed in various tissues and under a range of abiotic stress conditions, uncovered the potential functions of GRAS members in regulating plant growth development and stress responses. The results obtained from this study provide valuable information toward understanding the potential molecular mechanisms of GRAS proteins in castor beans. These findings also serve as a resource for identifying the genes that allow castor beans to grow in stressful conditions and to enable further breeding and genetic improvements in agriculture.

Bax-/- bak-/- cells exhibit p27 Thr198 phosphorylation and autophagy.

Cell cycle arrest in G0 and autophagy share features in common. The Bcl-2 family exerts cell cycle effects in addition to regulating apoptosis. Bcl-2 and Bcl-xL upregulate p27 and promote G0 arrest. Recently, we asked whether autophagy was involved in Bcl-2 and Bcl-xL-mediated cell cycle arrest, and found that autophagy was activated, but not required, for G0 arrest. We also discovered that the cell cycle function of Bcl-2 and Bcl-xL was dependent on Bax and Bak, and in bax-/- bak-/- double knockout cells, features of G0 quiesecence were already present and p27 was constitutively elevated. Here, we queried the presence of autophagy in bax-/- bak-/- double knockout cells, and report the phosphorylation of p27 at Thr198, which is known to occur in autophagy, as well as constitutive Atg5 induction and LC3-I to -II conversion. These findings in bax-/- bak-/- cells suggest that a physiological role of Bax and Bak may be the suppression of autophagy.

CXC chemokine CXCL12 and its receptor CXCR4 in tree shrews (Tupaia belangeri): structure, expression and function.

Chemokines are small secreted proteins functionally involved in the immune systems regulation of lymphocyte migration across numerous mammalian species. Given its growing popularity in immunological models, we investigated the structure and function of chemokine CXCL12 protein in tree shrews. We found that CXCL12 and its receptor CXCR4 in tree shrew had structural similarities to their homologous human proteins. Phylogenetic analysis supports the view that tree shrew is evolutionarily-close to the primates. Our results also showed that the human recombinant CXCL12 protein directly enhanced the migration of tree shrews lymphocytes in vitro, while AMD3100 enhanced the mobilization of hematopoietic progenitor cells (HPCs) from bone marrow into peripheral blood in tree shrew in vivo. Collectively, these findings suggested that chemokines in tree shrews may play the same or similar roles as those in humans, and that the tree shrew is a viable animal model for studying human immunological diseases.

PGC-1α regulates the cell cycle through ATP and ROS in CH1 cells

Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) is a transcriptional co-activator involved in mitochondrial biogenesis, respiratory capacity, and oxidative phosphorylation (OXPHOS). PGC-1α plays an important role in cellular metabolism and is associated with tumorigenesis, suggesting an involvement in cell cycle progression. However, the underlying mechanisms mediating its involvement in these processes remain unclear. To elucidate the signaling pathways involved in PGC-1α function, we established a cell line, CH1 PGC-1α, which stably overexpresses PGC-1α. Using this cell line, we found that over-expression of PGC-1α stimulated extra adenosine triphosphate (ATP) and reduced reactive oxygen species (ROS) production. These effects were accompanied by up-regulation of the cell cycle checkpoint regulators CyclinD1 and CyclinB1. We hypothesized that ATP and ROS function as cellular signals to regulate cyclins and control cell cycle progression. Indeed, we found that reduction of ATP levels down-regulated CyclinD1 but not CyclinB1, whereas elevation of ROS levels down-regulated CyclinB1 but not CyclinD1. Furthermore, both low ATP levels and elevated ROS levels inhibited cell growth, but PGC-1α was maintained at a constant level. Together, these results demonstrate that PGC-1α regulates cell cycle progression through modulation of CyclinD1 and CyclinB1 by ATP and ROS. These findings suggest that PGC-1α potentially coordinates energy metabolism together with the cell cycle.中文概要目 的探讨在CH1 细胞中过氧化物酶体增殖物受体γ 共激活因子1α(PGC-1α)调控细胞周期时三磷 酸腺苷(ATP)和活性氧(ROS)的作用机制。创新点构建了稳定表达PGC-1α 的CH1 细胞株, 并系统 地研究了PGC-1α 调控细胞周期是通过ATP 和 ROS 调节CyclinD1 和CyclinB1 的行使功能。方 法以慢病毒质粒pBABE 为载体构建了PGC-1α 稳 定表达的CH1 PGC-1α 细胞株(PGC-1α), 同时 转染空质粒pBABE 作为对照(PB), 结合RNA 干扰CH1 PGC-1α 中PGC-1α 的过表达(Si), 测定了ATP 和ROS 水平。用流式细胞术检测了 细胞周期和免疫印迹检测了CyclinB1/D1 的表达, 并进一步分别用寡霉素抑制PGC-1α 细胞中的 ATP 生成, 用H2O2 处理细胞以增加外源ROS 水 平。然后检测ATP 和ROS 改变后, 对CyclinB1/D1 表达及细胞周期的影响, 以明确ATP 和ROS 是 否参与PGC-1α 对细胞周期的调控作用。结 论本实验成功构建了稳定表达PGC-1α 的细胞株 (图1 和图2a), 与PB 对照和RNA 干扰PGC-1α 比较, 过表达PGC-1α 具有升高ATP、降低ROS 和促进细胞周期的作用(图3 和图4)。进一步 用寡霉素抑制ATP 合成后发现CyclinD1 明显下 调(图5), 而加入H2O2 增加外源ROS 后发现 CyclinB1 显著上调(图6)。通过本实验我们提 出PGC-1α调控细胞周期是通过升高ATP水平抑 制CyclinD1 表达和降低ROS 水平促进CyclinB1 表达来实现。

Digital gene expression profiling analysis of DNA repair pathways in colon cancer stem population of HT29 cells

Cancer stem cells (CSCs) contribute to the relapse and development of new neoplasm lesions. While most available clinical approaches, such as chemical and radiation therapies, will kill the majority of cancer cells, they do not kill them all. Some resisting cells, like CSCs, are able to survive due to their excellent self-maintaining capabilities, even in challenging environments. In the present study, we investigated the mRNA level of DNA repair genes of colon CSCs from the HT29 cell line in response to single-strand damage and double-strand breaks, as well as the evident upregulation of key genes in base excision repair, mismatch repair, non-homologous end-joining, and homologous recombination pathways in these cells. Digital gene expression analysis identified upregulated genes in CD44+ HT29 cells that may play important roles in DNA repair. Our results reveal that colon CSCs bear efficient DNA repair abilities, which might explain the survival of colon CSCs after repeated chemical and radiation therapy.