Nianhan Ma

A shRNA Functional Screen Reveals Nme6 and Nme7 Are Crucial for Embryonic Stem Cell Renewal

In contrast to the somatic cells, embryonic stem cells (ESCs) are characterized by its immortalization ability, pluripotency, and oncogenicity. Revealing the underlying mechanism of ESC characteristics is important for the application of ESCs in clinical medicine. We performed systematic functional screen in mouse ESCs with 4,801 shRNAs that target 929 kinases and phosphatases. One hundred and thirty‐two candidate genes that regulate both ESC expansion and stem cell marker expression were identified. Twenty‐seven out of the 132 genes were regarded as most important since knockdown of each gene induces morphological changes from undifferentiated to differentiated state. Among the 27 genes, we chose nonmetastatic cell 6 (Nme6, also named as Nm23‐H6) and nonmetastatic cell 7 (Nme7, also designated as Nm23‐H7) to study first. Nme6 and Nme7 both belong to the members of nucleoside diphosphate kinase family. We demonstrate that Nme6 and Nme7 are important for the regulation of Oct4, Nanog, Klf4, c‐Myc, telomerase, Dnmt3B, Sox2, and ERas expression. Either knockdown of Nme6 or Nme7 reduces the formation of embryoid body (EB) and teratoma. The overexpression of either Nme6 or Nme7 can rescue the stem cell marker expression and the EB formation in the absence of leukemia inhibiting factor. This implies the importance of Nme6 and Nme7 in ESC renewal. This finding not only pinpoints Nme6 or Nme7 can regulate several critical regulators in ESC renewal but also increases our understanding of the ESC renewal and oncogenesis. STEM Cells2012;30:2199–2211

Functional Module Connectivity Map (FMCM): A Framework for Searching Repurposed Drug Compounds for Systems Treatment of Cancer and an Application to Colorectal Adenocarcinoma

Drug repurposing has become an increasingly attractive approach to drug development owing to the ever-growing cost of new drug discovery and frequent withdrawal of successful drugs caused by side effect issues. Here, we devised Functional Module Connectivity Map (FMCM) for the discovery of repurposed drug compounds for systems treatment of complex diseases, and applied it to colorectal adenocarcinoma. FMCM used multiple functional gene modules to query the Connectivity Map (CMap). The functional modules were built around hub genes identified, through a gene selection by trend-of-disease-progression (GSToP) procedure, from condition-specific gene-gene interaction networks constructed from sets of cohort gene expression microarrays. The candidate drug compounds were restricted to drugs exhibiting predicted minimal intracellular harmful side effects. We tested FMCM against the common practice of selecting drugs using a genomic signature represented by a single set of individual genes to query CMap (IGCM), and found FMCM to have higher robustness, accuracy, specificity, and reproducibility in identifying known anti-cancer agents. Among the 46 drug candidates selected by FMCM for colorectal adenocarcinoma treatment, 65% had literature support for association with anti-cancer activities, and 60% of the drugs predicted to have harmful effects on cancer had been reported to be associated with carcinogens/immune suppressors. Compounds were formed from the selected drug candidates where in each compound the component drugs collectively were beneficial to all the functional modules while no single component drug was harmful to any of the modules. In cell viability tests, we identified four candidate drugs: GW-8510, etacrynic acid, ginkgolide A, and 6-azathymine, as having high inhibitory activities against cancer cells. Through microarray experiments we confirmed the novel functional links predicted for three candidate drugs: phenoxybenzamine (broad effects), GW-8510 (cell cycle), and imipenem (immune system). We believe FMCM can be usefully applied to repurposed drug discovery for systems treatment of other types of cancer and other complex diseases.

MicroRNA regulation of DNA repair gene expression in 4-aminobiphenyl-treated HepG2 cells

We examined the role of miRNAs in DNA damage response in HepG2 cells following exposure to 4-aminobiphenyl (4-ABP). The arylamine 4-ABP is a human carcinogen. Using the Comet assay, we showed that 4-ABP (18.75-300μM) induces DNA damage in HepG2 cells after 24h. DNA damage signaling pathway-based PCR arrays were used to investigate expression changes in genes involved in DNA damage response. Results showed down-regulation of 16 DNA repair-related genes in 4-ABP-treated cells. Among them, the expression of selected six genes (UNG, LIG1, EXO1, XRCC2, PCNA, and FANCG) from different DNA repair pathways was decreased with quantitative real-time PCR (qRT-PCR). In parallel, using the miRNA array, we reported that the expression of 27 miRNAs in 4-ABP-treated cells was at least 3-fold higher than that in the control group. Of these differential 27 miRNAs, the most significant expression of miRNA-513a-5p and miRNA-630 was further validated by qRT-PCR, and was predicted to be implicated in the deregulation of FANCG and RAD18 genes, respectively, via bioinformatic analysis. Both FANCG and RAD18 proteins were found to be down-regulated in 4-ABP-treated cells. In addition, overexpression and knockdown of miRNA-513a-5p and miRNA-630 reduced and increased the expression of FANCG and RAD18 proteins, respectively. Based on the above results, we indicated that miRNA-513a-5p and miRNA-630 could play a role in the suppression of DNA repair genes, and eventually lead to DNA damage.

Akt suppresses DLK for maintaining self-renewal of mouse embryonic stem cells

Mouse embryonic stem cells (ES cells) can proliferate indefinitely. To identify potential signals involved in suppression of self-renewal, we previously screened a kinase/phosphatase expression library in ES cells, and observed that inhibition of Dual Leucine zipper-bearing Kinase (DLK) increased relative cell numbers. DLK protein was detected in both the pluripotent and differentiated states of mouse ES cells while DLK kinase activity increased upon differentiation. Overexpression of DLK in mouse ES cells displayed reductions in relative cell/colony numbers and Nanog expression, suggesting a suppressive role of DLK in self-renewal. By examining protein sequences of DLK, we identified 2 putative Akt phosphorylation sites at S584 and T659. Blocking PI3K/Akt signaling with LY-294002 enhanced DLK kinase activity dramatically. We found that Akt interacts with and phosphorylates DLK. Mutations of DLK amino acid residues at putative Akt phosphorylation sites (S584A, T659A, or S584A and T659A) diminished the level of DLK phosphorylation. While the mutated DLKs (S584A, T659A, or S584A and T659A) were expressed, a further reduction in cell/colony numbers and Nanog expression appeared in mouse ES cells. In addition, these mutant DLKs (S584A, T659A, or S584A and T659A) exhibited more robust kinase activity and cell death compared to wild type DLK or green fluorescence (GFP) controls. In summary, our results show that DLK functions to suppress self-renewal of mouse ES cells and is restrained by Akt phosphorylation.

Global assessment of Antrodia cinnamomea-induced microRNA alterations in hepatocarcinoma cells.

Recent studies have demonstrated a potent anticancer potential of medicinal fungus Antrodia cinnamomea, especially against hepatocarcinoma. These studies, however, were performed with prolonged treatments, and the early anticancer events remain missing. To probe the early anticancer mechanisms of A. cinnamomea, we treated SK-Hep-1 liver cancer cell with A. cinnamomea fruiting body extract for 2 and 4 hours, sequenced RNA samples with next-generation sequencing approach, and profiled the genome-wide miRNA and mRNA transcriptomes. Results unmistakably associated the early anticancer effect of A. cinnamomea fruiting body extract with a global downregulation of miRNAs which occurred solely in the A. cinnamomea fruiting body extract-treated SK-Hep-1 cells. Moreover, the inhibitory effect of A. cinnamomea fruiting body extract upon cancer miRNAs imposed no discrimination against any particular miRNA species, with oncomirs miR-21, miR-191 and major oncogenic clusters miR-17-92 and miR-106b-25 among the most severely downregulated. Western blotting further indicated a decrease in Drosha and Dicer proteins which play a key role in miRNA biogenesis, together with an increase of XRN2 known to participate in miRNA degradation pathway. Transcriptome profiling followed by GO and pathway analyses indicated that A. cinnamomea induced apoptosis, which was tightly associated with a downregulation of PI3K/AKT and MAPK pathways. Phosphorylation assay further suggested that JNK and c-Jun were closely involved in the apoptotic process. Taken together, our data indicated that the anticancer effect of A. cinnamomea can take place within a few hours by targeting multiple proteins and the miRNA system. A. cinnamomea indiscriminately induced a global downregulation of miRNAs by simultaneously inhibiting the key enzymes involved in miRNA maturation and activating XRN2 protein involved in miRNA degradation. Collapsing of the miRNA system together with downregulation of cell growth and survival pathways and activation of JNK signaling unleash the extrinsic and intrinsic apoptosis pathways, leading to the cancer cell death.

Cytotoxicity of Postmodified Zeolitic Imidazolate Framework-90 (ZIF-90) Nanocrystals: Correlation between Functionality and Toxicity.

Using a simple method, the aldehyde groups of zeolitic imidazolate framework-90 (ZIF-90) nanocrystals were converted into carboxyl, amino, and thiol groups, without affecting the integrity of the framework. Notably, for the first time, correlations between functionality and cytotoxicity are also demonstrated via in vitro cytotoxicity assays. The positive charged aminated-ZIF-90 presumably results in either perturbation of cell membrane, more efficient cell uptake, or both. Therefore, the half-maximal effective (EC50 ) concentration of aminated-ZIF-90 has a higher cytotoxicity of about 30 μg mL(-1) .

A green and facile approach to obtain 100 nm zeolitic imidazolate framework-90 (ZIF-90) particles via leveraging viscosity effects

For the first time, ∼100 nm zeolitic imidazolate framework-90 (ZIF-90) particles were produced by a water–alcohol-based system, taking advantage of viscosity effects using an optimized H2O/tert-butanol/glycerol/PVP system. Furthermore, an in vitro cytotoxicity assay revealed the half-maximal effective concentration (EC50) of ZIF-90 nanoparticles to be ∼92 μg ml−1.

Novel expression and regulation of TIMP-1 in Epstein Barr virus-infected cells and its impact on cell survival.

Epstein Barr virus (EBV) uses various strategies to manipulate host cytokine production in favor of the survival of infected B-cells. Microarray and cytokine protein array assays revealed that tissue inhibitor of metalloproteinase-1 (TIMP-1) was significantly up-regulated in EBV-infected primary B cells and maintained in abundance in EBV-immortalized lymphoblastoid cell lines (LCLs). TIMP-1 plays critical roles in extracellular matrix homeostasis and regulates signaling pathways. In this study, we demonstrated that the EBV-encoded immediate early lytic protein, Zta, upregulates mainly TIMP-1 expression by binding to the AP-1 site within the TIMP-1 promoter. Moreover, knockdown of TIMP-1 expression promoted cisplastin and cold shock-induced death of LCLs. This study provides a mechanistic link between EBV-induced TIMP-1 expression and its impact on LCL survival.

Genotypes of cancer stem cells characterized by epithelial-to-mesenchymal transition and proliferation related functions.

Cancer stem cells (CSCs), or cancer cells with stem cell-like properties, generally exhibit drug resistance and have highly potent cancer inducing capabilities. Genome-wide expression data collected at public repositories over the last few years provide excellent material for studies that can lead to insights concerning the molecular and functional characteristics of CSCs. Here, we conducted functional genomic studies of CSC based on fourteen PCA-screened high quality public CSC whole genome gene expression datasets and, as control, four high quality non-stem-like cancer cell and non-cancerous stem cell datasets from the Gene Expression Omnibus database. A total of 6,002 molecular signatures were taken from the Molecular Signatures Database and used to characterize the datasets, which, under two-way hierarchical clustering, formed three genotypes. Type 1, consisting of mainly glia CSCs, had significantly enhanced proliferation, and significantly suppressed epithelial-mesenchymal transition (EMT), related functions. Type 2, mainly breast CSCs, had significantly enhanced EMT, but not proliferation, related functions. Type 3, composed of ovarian, prostate, and colon CSCs, had significantly suppressed proliferation related functions and mixed expressions on EMT related functions.

Array‐Based High‐Throughput Screening in Mouse Embryonic Stem Cells with shRNAs

High-throughput short-hairpin RNA (shRNA) lentivirus screening is a powerful tool for identifying multiple functional regulators in embryonic stem cells (ESCs). shRNA libraries can efficiently down-regulate target genes persistently with high efficiency. The concurrent measurement of relative cell number by alamarBlue (AB) assay and undifferentiated ESC markers via an alkaline phosphatase (ALP) activity assay in the same cell culture well provides an efficient and economical way to pinpoint factors crucial for ESC pluripotency and/or expansion. Most of the renewal pathways affect ALP activity. Thus, multiple positive and negative regulators can be identified by this method. In addition, morphological changes and/or the expression levels of specific pluripotency or differentiation markers examined by immunofluorescence can be used as secondary screens for target-gene selection. In summary, we describe an efficient way to identify multiple regulators of ESC renewal using shRNAs. Curr. Protoc.