Yuxiang Zhou

Genomic Analysis of Anti-Hepatitis B Virus (HBV) Activity by Small Interfering RNA and Lamivudine in Stable HBV-Producing Cells

ABSTRACT Hepatitis B virus (HBV) causes acute and chronic hepatitis and hepatocellular carcinoma. Small interfering RNA (siRNA) and lamivudine have been shown to have anti-HBV effects through different mechanisms. However, assessment of the genome-wide effects of siRNA and lamivudine on HBV-producing cell lines has not been reported, which may provide a clue to interrogate the HBV-cell interaction and to evaluate the siRNAs side effect as a potential drug. In the present study, we designed seven siRNAs based on the conserved HBV sequences and tested their effects on the expression of HBV genes following sorting of siRNA-positive cells. Among these seven siRNAs, siRNA-1 and siRNA-7 were found to effectively suppress HBV gene expression. We further addressed the global gene expression changes in stable HBV-producing cells induced by siRNA-1 and siRNA-7 by use of human genome-wide oligonucleotide microarrays. Data from the gene expression profiling indicated that siRNA-1 and siRNA-7 altered the expression of 54 and 499 genes, respectively, in HepG2.2.15 cells, which revealed that different siRNAs had various patterns of gene expression profiles and suggested a complicated influence of siRNAs on host cells. We further observed that 18 of these genes were suppressed by both siRNA-1 and siRNA-7. Interestingly, seven of these genes were originally activated by HBV, which suggested that these seven genes might be involved in the HBV-host cell interaction. Finally, we have compared the effects of siRNA and lamivudine on HBV and host cells, which revealed that siRNA is more effective at inhibiting HBV expression at the mRNA and protein level in vitro, and the gene expression profile of HepG2.2.15 cells treated by lamivudine is totally different from that seen with siRNA.

Identification of the gene transcription and apoptosis mediated by TGF-β-Smad2/3-Smad4 signaling

Transforming growth factor‐β (TGF‐β) signaling is known to depend on the formation of Smad2/3‐Smad4 transcription regulatory complexes. However, the signaling functions of Smad2/3‐Smad4 during TGF‐β‐induced responses are obscure as TGF‐β also initiates a number of other signaling pathways. In this study, we systematically assessed the contribution of TGF‐β‐Smad2/3‐Smad4 signaling to both target gene transcription and apoptosis. Individual Smads were selectively knocked down in Hep3B cells by stable RNA interference (RNAi). We identified TGF‐β‐responsive genes using genome‐wide oligonucleotide microarrays and confirmed their dependency on Smad2, Smad3, or Smad4 by the combination of RNAi and microarray assay. The major finding from our microarray analysis was that of the 2,039 target genes seen to be regulated via TGF‐β induction, 190 were differentially transcriptionally controlled by Smad2‐Smad4 and Smad3‐Smad4 signaling and the latter control mechanism appeared to be functionally more important. We also found indirect evidence of competition between Smad2 and Smad3 for their activation when controlling the transcription of target genes. Functional analysis revealed that Smad3 and Smad4 were the predominant mediators of TGF‐β‐induced apoptosis in Hep3B cells. We provide evidence that up‐regulation of Bcl‐2‐interacting mediator of cell death (Bim), under the transcriptional control of Smad3‐Smad4 signaling, is crucial to TGF‐β‐induced apoptosis in Hep3B cells. J. Cell. Physiol. 215: 422–433, 2008.

Identification of activity-dependent gene expression profiles reveals specific subsets of genes induced by different routes of Ca2+ entry in cultured rat cortical neurons

Neuronal activity‐dependent gene transcription is a key feature of long‐lasting synaptic strengthening associated with learning and memory, as well as activity‐dependent neuroprotection. To comprehensively determine the molecular alterations, we carried out genome‐wide microarray analysis in cultured rat cortical neurons treated with specific pharmacological agents, a model with alterations in neuronal activity, which were monitored by multi‐site electrophysiological recordings. Of the approximately 27,000 genes, the expression of 248 genes was strongly changed in response to enhanced activity. These genes encompass a large number of members of distinct families, including synaptic vesicle proteins, ion channels, signal transduction molecules, synaptic growth regulators, and others. Two subsets of these genes were further confirmed to be specifically induced by Ca2+ influx through N‐methyl‐D‐aspartate (NMDA) receptors and L‐type voltage‐gated Ca2+ channels (VGCCs). In addition, those genes dynamically regulated by the enhanced activity were also elucidated, as well as those candidate genes associated with synaptic plasticity and neuroprotection. Our findings therefore would help define the molecular mechanisms that occur in response to neuronal activity and identify specific clusters of genes that contribute to activity‐dependent and Ca2+‐inducible modulation of brain development and function. J. Cell. Physiol. 212: 126–136, 2007.

Bioelectrical impedance assay to monitor changes in aspirin-treated human colon cancer HT-29 cell shape during apoptosis

Abstract Physiological cell death occurs primarily through an evolutionarily conserved form of cell suicide termed apoptosis, which may be triggered by cytokines, depletion of growth factors, or certain chemicals. It is morphologically characterized by severe alterations in cell shape like cell shrinkage and disintegration of cell–cell contacts. Aspirin and other nonsteroidal anti‐inflammatory drugs (NSAIDs) inhibit proliferation of human colon cancer cells in vitro. We applied a noninvasive bioelectrical impedance assay referred to as electric cell–substrate impedance sensing (ECIS) in order to monitor the apoptosis‐induced changes in human colon cancer HT‐29 cell shape in an integral and quantitative fashion with a time resolution on the order of minutes. In whole‐cell biosensors the cells are grown directly on the surface of small gold‐film electrodes. From readings of the electrical impedance of the cell‐covered electrode, we deduced alterations of aspirin on HT‐29 cells in cell–cell and cell–substrate contacts. And the apoptosis was verified by transmission electron microscope.

Genomewide oligonucleotide microarray analysis on placentae of pre-eclamptic pregnancies

Objective: Human placentae from normal and pre-eclamptic pregnancies were evaluated for possible changes in gene expression by microarray analysis to uncover new clues for the research of the etiology of pre-eclampsia. Methods: Placentae from five normal pregnancies and five pregnancies complicated by pre-eclampsia were collected. mRNA levels of five pre-eclamptic placentae were examined using genome-wide 70-mer oligonucleotide microarrays (CapitalBio, Beijing, China) in comparison with the pooled control consisting of total RNA from five normotensive placentae. Results: Ninety-six genes were found consistently down- or up-regulated in at least four pre-eclamptic samples. Most of them were related to an imbalance of reactive oxygen metabolites in placenta, abnormal trophoblast invasion, disorders of lipoprotein metabolism and signal transduction, or some that have been reported to have close correlation to the pathology of pre-eclampsia. The microarray data were also confirmed by the measurement of real-time PCR. Conclusion: DNA microarray is a high throughput and time-saving method to monitor altered gene expression. The results could provide interesting clues to the etiology of pre-eclampsia and lead to further studies in a more targeted fashion.

Microarray and biochemical analysis of bufalin-induced apoptosis of HL-60 Cells

Bufalin is a natural toxin with anti-leukemic properties. It induces cell differentiation and apoptosis, as well as increasing the sensitivity of leukemia cells to other chemotherapeutic agents. We investigated the biological effects and molecular mechanisms of bufalin triggered apoptosis in HL-60 cells by gene expression profiling. The broad transcriptional response to bufalin was consistent with bufalin’s action of regulating HL-60 cell proliferation and apoptosis, as well as its synergistic effect with other drugs. Further transcription factor ELISA experiments suggested that the transcription factors NFκB and AP-1 were activated to promote bufalin-induced HL-60 cell apoptosis. Our study provides new insights into the molecular mechanisms of bufalin, might prove to be beneficial in leukemia therapy.

Comparison of Different Methods for Preparing Single Stranded DNA for Oligonucleotide Microarray

Abstract Single stranded DNAs (ssDNA) were crucial reagents for nucleic acid analysis on oligonucleotide microarrays. Methods for preparing ssDNAs have been reported, but the systematic study about them was still lacking now, especially for oligonucleotide microarrays. Using oligonucleotide microarrays, some of the most popular methods were compared in this report, including asymmetric polymerase chain reaction (PCR), magnetic beads based method, denaturation of double stranded DNA (dsDNA) by heating or by alkaline treatment. All the ssDNA targets prepared by the four methods were applied to oligonucleotide microarrays. The results indicate that the ssDNA targets generated by asymmetric PCR and magnetic beads based method have shown high sensitivity and specificity in hybridization, but those generated by denaturation of dsDNA via heating or alkaline treatment produced serious false negative signals under the same hybridization conditions. Considering the simplicity and the cost of the method, asymmetric PCR is a more favorable approach for oligonucleotide microarrays. In view of the fact that most of the present asymmetric PCR procedures were in two steps, and a purification process was necessary before the second step amplification was carried out, a one-step asymmetric PCR was attempted in this work. The results demonstrate that it was a preferred approach for ssDNA preparation for oligonucleotide microarrays use.

Hepatitis B Virus Encoded X Protein Suppresses Apoptosis by Inhibition of the Caspase-Independent Pathway

Hepatitis B virus (HBV) encoded X protein (HBx) has been implicated in apoptotic and related pathogenic events during hepatocellular carcinoma. However, the underlying molecular mechanism through which HBx acts is largely unclear. We used tandem affinity purification under mild conditions to gain insight into the HBx interactome in HBV-producing HepG2.2.15 cells and identified 49 proteins by mass spectrometry that are potentially associated with HBx. Two of the key proteins of the caspase-independent apoptosis pathway were newly identified, apoptosis-inducing factor (AIF) and the homologous AMID (AIF-homologue mitochondrion-associated inducer of death). We confirmed the interactions of HBx with AIF and with AMID by reciprocal coimmunoprecipitation experiments, respectively. We observed the expression of HBx-reduced AIF-mediated apoptosis and HBx colocalization with AIF and AMID, principally in the cytoplasm. Furthermore, the elevated cytoplasmic levels of HBx could inhibit mitochondrion-to-nucleus translocation of AIF. Here, we present the first detailed molecular evidence that HBx can repress apoptosis via inhibition of the caspase-independent apoptosis pathway. This inhibition of apoptosis involves the repression of the mitochondrion-to-nucleus translocation of AIF, although tests with AMID were not conclusive. These findings provide important insights into the new mechanism of the apoptosis inhibition by HBV.

Microarray profiling of monocytic differentiation reveals miRNA‐mRNA intrinsic correlation

MiRNAs (microRNAs) are small non‐coding RNAs involved in mammalian gene expression of cellular processes including differentiation, apoptosis and cancer development. Both specific miRNAs and mRNAs have been identified during monocytic differentiation, but their interactions have not been fully characterized. Here we report that by genome‐wide microarray analysis for U937 monocytic differentiation induced by TPA, a large number of miRNAs and mRNAs were differentially expressed, and by bioinformatics analysis could demonstrate that their functional pathway patterns overlap strongly. While expected negative correlation between the expression levels of miRNAs and their target mRNAs was seen, several positive correlations between miRNAs and host mRNAs were also observed, such as C13orf25/miR17, MCM7/miR93, and MGC14376/miR22. These microarray data were verified by quantitative RT‐PCR, and the TPA‐induced differentiation of U937 cells was confirmed by flow cytometric analysis. Our study suggests an intrinsic correlation between miRNAs and mRNAs underlying their interactions which would provide new insights for defining the mechanisms occurring during monocytic differentiation. J. Cell. Biochem. 112: 2443–2453, 2011.

One-Tube Nested RT-PCR Enabled by Using a Plastic Film and its Application for the Rapid Detection of SARS-Virus

A general strategy based on the use of a plastic film that allows reverse transcription and nested-PCR in a single closed-tube has been developed. The reaction mixture for the second PCR amplification is quarantined in the cap of the reaction tube during the first round amplification by a piece of plastic film, and later introduced into the PCR amplicons from the first round reaction by centrifugation without opening the reaction tube. The main advantages of our method are its high sensitivity, specificity, simplicity, cost effectiveness, low risk of contamination and the ease in establishment of conditions for nested-PCR. The method has been successfully applied to the detection of the genomic RNA from SARS-CoV, the detection limit of this method is comparable to that of the conventional two-tube nested PCR system. This method could be easily adapted to the detection of other targets.