Direct somatic embryogenesis and related gene expression networks in leaf explants of Hippeastrum ‘Bangkok Rose’

Hippeastrum, a highly diverse genus in the Amaryllidaceae family, is a valuable ornamental bulbous flowering plant. Somatic embryogenesis(SE) is an efficient method for mass production of Hippeastrum plantlets. Previous studies have been devoted to the in vitro propagation of Hippeastrum, but the SE and its regulatory networks are rarely reported. In this study, we established a direct SE method of Hippeastrum Bangkok Rose' using leaf bases as explants. MS supplemented with 1.00 mg·L^(-1)NAA +1.00 mg·L^(-1)KT + 0.25 mg·L^(-1)TDZ was the optimal medium for SE. Histological observations showed that the bipolar somatic embryo originated from the epidermal cell layer and underwent initiation,globular, scutellar and coleoptile stages. During SE, endogenous hormones of IAA, CTK, ABA, and SA were highly accumulated. Transcriptomic analysis revealed the genes encoding auxin biosynthesis/metabolic enzymes and efflux carriers were induced, while the auxin receptor of TIR1 and ARF transcriptional repressor of Aux/IAA were down-regulated and up-regulated, respectively, leading to suppression of auxin signaling. In contrast, cytokine signaling was promoted at the early stage of SE, as biosynthesis, transport, and signaling components were up-regulated.Various stress-related genes were up-regulated at the early or late stages of SE. Chromatin remodeling could also be dynamically regulated via distinct expression enzymes that control histone methylation and acetylation during SE. Moreover, key SE regulators, including WOXs and SERKs were highly expressed along with SE. Overall, the present study provides insights into the SE regulatory mechanisms of the Hippeastrum.

Regulatory genes controlling neural stem cells differentiation into neurons

The recent progress in neural stem cells （NSCs） research has shed lights on possibility of repair and restoration of neuronal function in neurodegenerative diseases using stem cells. Induction of stem cells differentiate into mature neurons is critical to achieve the clinical applications of NSCs. At present, molecular mechanisms modulating NSC differentiation are not fully understood. Differentiation of stem cells into neuronal and glial cells involves an array of changes in expression of transcription factors. Transcription factors then trigger the expression of a variety of central nervous system （CNS） genes that lead NSCs to differentiate towards different cell types. In this paper, we summarized the recent findings on the gene regulation of NSCs differentiation into neuronal cells.

Gene Expression and Regulation of Blastocyst Formation

Blastocyst formation is a crucial stage of early embryo development.Cell junction proteins and cell adhesion associated proteins are involved in the establishment of cell junction,and subsequently induce cell compaction,blastocyst formation,differentiation of trophectoderm and maintenance of blastocyst expansion.Genes regulating development and differentiation participate in embryo development and differentiation of inner cell mass and trophectoderm,which controls the transition from the undifferentiation to differentiation state.Furthermore,cytokine and growth factor have influence on the proliferation of cells of inner cell mass.In a word,many proteins and factors are involved in the gene expression and regulation of blastocyst formation.

Regulation of cytokine signaling pathways by PIAS proteins

Cytokines activate multiple signal transduction pathways to regulate gene expression. STATs and NF-kB are two important families of transcription factors activated by cytokines. Abnormal regulation of STAT and NF-kB activities has been associated with human diseases. The protein inhibitor of activated STAT （PIAS） protein family has been proposed to interact with over 60 proteins, many of which are transcription factors involved in the immune system. PIAS proteins regulate transcription through several mechanisms, including blocking the DNA-binding activity of transcription factors, recruiting transcriptional co-repressors and promoting protein sumoylation. This article is to review the role of PIAS proteins in the regulation of STAT and NF-kB signaling pathways.

Expression and regulation of IL-22 in the IL-17.producing CD4+T lymphocytes

IL-22 is a novel cytokine in the IL-10 family that functions to promote innate immunity of tissues against infection. Although CD4＋ helper T lymphocytes （TH） were found as a source of IL-22, the regulation of this cytokine has been poorly understood. Here, we show that IL-22 is expressed at both mRNA and protein levels by a novel subset of TH cells that also makes IL-17. IL-22 and IL-17 were found to be coordinately regulated by TGFI3 and IL-6 during TH differentiation by real-time PCR as well as ELISA analysis. However, IL-22 does not regulate TH differentiation; exogenous IL-22 or an IL-22 antagonist had no effect on TH differentiation. These data demonstrate a novel cytokine expressed by IL-17-producing T cells, and suggest interaction and synergy of IL-22 and IL-l 7 signaling pathways in tissue inflammation and autoimmune diseases.

Circular RNAs in colorectal cancer:Possible roles in regulation of cancer cells

Colorectal cancer(CRC) is the third most commonly diagnosed cancer in the world and the fourth principal cause of cancer deaths worldwide. Currently, there is a lack of low cost and noninvasive screening tests for CRC, becoming a serious health problem. In this context, a potential biomarker for the early detection of CRC has recently gained attention. Circular RNAs(circ RNA), a re-discovered, abundant RNA specie, is a type of noncoding covalent closed RNAs formed from both exonic and intronic sequences. These circular molecules are widely expressed in cells, exceeding the abundance of the traditional linear m RNA transcript. They can regulate gene expression, acting as real sponges for mi RNAs and also regulate alternative splicing or act as transcriptional factors and inclusive encoding for proteins. However, little is known about circ RNA and its relationship with CRC. In this review, we focus on the biogenesis, function and role of these circ RNAs in relation to CRC, including their potential as a new biomarker.

Regulation of epithelium-specific Ets-like factors ESE-1 and ESE-3 in airway epithelial cells： potential roles in airway inflammation

Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1β （IL-1β） and tumor necrosis factor-α （TNF-α） in bronchial epithelial cell lines. Treatment of these cells with IL-1β and TNF-α resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-κB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-κB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in E/f3 （homologous to human ESE-1） knockout mice, the expression of the inflammatory cytokine interleukin-6 （IL-6） is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

Differentially expressed genes in hepatocellular carcinoma induced by woodchuck hepatitis B virus in mice

INTRODUCTIONHepatocellular carcinoma(HCC)is one of the major causes of death in the word.The mechanism of carcinogenesis is unknown,although it is widely accepted that HBV and HCV are clsely related to liver cancer[1-5[1-5].Previously,a variety of studies have described the differences in gene expression which distinguished tumor from nontumor[6-11].Cloning of the genes,especially the genes associated with HBV and HCV,is still very important to account for the development of liver cancer.

Identification of differentially expressed genes in normal mucosa,adenoma and adenocarcinoma of colon by SSH

AIM: To construct subtracted cDNA libraries and further identify differentially expressed genes that are related to the development of colorectal carcinoma(CRC). METHODS: Suppression subtractive hybridization(SSH) was done on cDNAs of normal mucosa, adenoma and adenocarcinoma tissues from the same patient. Three subtracted cDNA libraries were constructed and then hybridized with forward and backward subtracted probes for differential screening. Positive clones from each subtracted cDNA library were selected for sequencing and BLAST analysis. Finally, virtual Northern Blot confirmed such differential expression. RESULTS: By this way, there were about 3-4 X 10(2) clones identified in each subtracted cDNA library, in which about 85% positive clones were differentially screened. Sequencing and BLAST homology search revealed some clones containing sequences of known gene fragments and several possibly novel genes showing few or no sequence homologies with any known sequences in the database. CONCLUSION: All results confirmed the effectiveness and sensitivity of SSH. The differentially expressed genes during the development of CRC can be used to shed light on the pathogenesis of CRC and be useful genetic markers for early diagnosis and therapy.

Molecular aspects of carcinogenesis in pancreatic cancer

BACKGROUND:Pancreatic cancer(PCa)is one of the most aggressive human solid tumors,with rapid growth and metastatic spread as well as resistance to chemotherapeutic drugs,leading rapidly to virtually incurable disease.Over the last 20 years,however,significant advances have been made in our understanding of the molecular biology of PCa,with a focus on the cytogenetic abnormalities in PCa cell growth and differentiation. DATA SOURCES:A MEDLINE search and manual cross- referencing were utilized to identify published data for PCa molecular biology studies between 1986 and 2008, with emphasis on genetic alterations and developmental oncology. RESULTS:Activation of oncogenes,deregulation of tumor suppressor and genome maintenance genes,upregulation of growth factors/growth factor receptor signaling cascade systems,and alterations in cytokine expression,have been reported to play important roles in the process of pancreatic carcinogenesis.Alterations in the K-ras proto- oncogene and the p16INK4a,p53,FHIT,and DPC4 tumor suppressor genes occur in a high percentage of tumors. Furthermore,a variety of growth factors are expressed at increased levels.In addition,PCa often exhibits alterations in growth inhibitory pathways and evades apoptosis through p53 mutations and aberrant expression of apoptosis-regulating genes,such as members of the Bcl family.Additional pathways in the development of an aggressive phenotype,local infiltration and metastasis are still under ongoing genetic research.The present paper reviews recent studies on the pathogenesis of PCa,and includes a brief reference to alterations reported for other types of pancreatic tumor. CONCLUSIONS:Advances in molecular genetics and biology have improved our perception of the pathogenesis of PCa.However,further studies are needed to better understand the fundamental changes that occur in PCa,thus leading to better diagnostic and therapeutic management.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Cloning of differentially expressed genes in human hepatocellular carcinoma and nontumor liver

INTRODUCTIONThe mechanism of hepatocellular carcinoma(HCC)is still unclear,although some genes have been found to play a role in the transformation of liver cells,and a variety of studies have described differences in gene expression which distinguished tumor from nontumor[1-6].The new genes,especially the functional genes directly related with tumor are still worth being found.The purpose of our study is to find the different genes between human liver tumor and normal tissues using suppression subtractive hybridization.

Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp (Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Thansgenic peanut plants obtained by particle bombardment via somatic embryogenesis regeneration system

After pre-culture and treatment of osmosis, cotyledons of immature peanut (Arachis hypogaea L.) zygotic embryos were transformed via particle bombardment with a plasmid containing a chimeric hph gene conferring resistance to hygromycin and a chimeric intron-gus gene. Selection for hygromycin resistant calluses and somatic embryos was initiated at 10th d post-bombardment on medium containing 10-25 mg/L hygromycin. Under continuous selection, hygromycin resistant plantlets were regenerated from somatic embryos and were recovered from nearly 1.6% of the bombarded cotyledons. The presence and integration of foreign DNA in regenerated hygromycin resistant plants was confirmed by PCR (polymerase chain reaction) for the intron-gus gene and by Southern hybridization of the hph gene. GUS enzyme activity was detected in leaflets from transgenic plants but not from control, non-transformed plants. The production of transgenic plants are mainly based on a newly improved somatic embryogenesis regeneration system developed by us.

Transcriptional regulation of adult neural stem/progenitor cells: tales from the subventricular zone

In rodents,well characterized neurogenic niches of the adult brain,such as the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus,support the maintenance of neural/stem progenitor cells(NSPCs)and the production of new neurons throughout the lifespan.The adult neurogenic process is dependent on the intrinsic gene expression signatures of NSPCs that make them competent for self-renewal and neuronal differentiation.At the same time,it is receptive to regulation by various extracellular signals that allow the modulation of neuronal production and integration into brain circuitries by various physiological stimuli.A drawback of this plasticity is the sensitivity of adult neurogenesis to alterations of the niche environment that can occur due to aging,injury or disease.At the core of the molecular mechanisms regulating neurogenesis,several transcription factors have been identified that maintain NSPC identity and mediate NSPC response to extrinsic cues.Here,we focus on REST,Egr1 and Dbx2 and their roles in adult neurogenesis,especially in the subventricular zone.We review recent work from our and other laboratories implicating these transcription factors in the control of NSPC proliferation and differentiation and in the response of NSPCs to extrinsic influences from the niche.We also discuss how their altered regulation may affect the neurogenic process in the aged and in the diseased brain.Finally,we highlight key open questions that need to be addressed to foster our understanding of the transcriptional mechanisms controlling adult neurogenesis.

Effect on Survivin Regulation of Transcription Level by p21^(waf1) Overexpression in HepG2 Hepatocellular Carcinoma Cells

The effect of cyclin-dependent kinase inhibitors Cip1/Wafl （p21） on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin （DOX） was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction （RQ-PCR）. Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction （RT-PCR） was used to measure the levels of E2F-1 and p300. The results showed that： （1） After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; （2） After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; （3） Overexpressed p21 resulted in GI/G0 phase arrest （F=31.59, P〈0.01）, meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls （FE2F-1=I25.28, P〈0.05; Fp300= 46.01, P〈0.01）. It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

MicroRNAs Involved in the Pathogenesis of Phytophthora Root Rot of Soybean (Glycine max)

Phytophthora root rot is one of the most prevalent diseases in the world,which can infect the seedlings and plants,with substantial negative impact on soybean yield and quality.MicroRNAs （miRNAs） are a class of post-transcriptional regulators of gene expression during growth and development of organisms.A soybean disease-resistance variety Suinong 10 was inoculated with Phytophthora sojae race No.1,and the specific miRNA resistant expression profile was acquired by microarray for the first time.Different expressional miRNAs have been found after comparing the results of the treated sample with the control sample.Furthermore,the target genes of different expressional miRNAs were predicted.Two miRNAs,cbr-mir-241 and ath-miR854a,regulated the disease-resistance process directly through their targets,some enzymes.Another two miRNAs,gma-miR169a and ath-miR169h,participated in disease-resistance regulation as transcription factors.Similarly,one miRNA,ptc-miR164f,has been reported to regulate the plant development.All of these studies would be served as the foundation for exploring the resistance mechanism.

Applying a highly specific and reproducible cDNA RDA method to clone garlic up-regulated genes in human gastric cancer cells

AIM: To develop and optimize cDNA representational difference analysis (cDNA RDA) method and to identify and clone garlic up-regulated genes in human gastric cancer (HGC) cells. METHODS: We performed cDNA RDA method by using abundant double-stranded cDNA messages provided by two self-constructed cDNA libraries (Allitridi-treated and paternal HGC cell line BGC823 cells cDNA libraries respectively). Bam H I and Xho I restriction sites harbored in the library vector were used to select representations. Northern and Slot blots analyses were employed to identify the obtained difference products. RESULTS: Fragments released from the cDNA library vector after restriction endonuclease digestion acted as good marker indicating the appropriate digestion degree for library DNA. Two novel expressed sequence tags (ESTs) and a recombinant gene were obtained. Slot blots result showed a 8-fold increase of glia-derived nexin/protease nexin 1 (GDN/PN1) gene expression level and 4-fold increase of hepatitis B virus x-interacting protein (XIP) mRNA level in BGC823 cells after Allitridi treatment for 72h. CONCLUSION: Elevated levels of GDN/PN1 and XIP mRNAs induced by Allitridi provide valuable molecular evidence for elucidating the garlic's efficacies against neurodegenerative and inflammatory diseases. Isolation of a recombinant gene and two novel ESTs further show cDNA RDA based on cDNA libraries to be a powerful method with high specificity and reproducibility in cloning differentially expressed genes.

Transcriptional regulation of human polo-like kinases and early mitotic inhibitors

Human polo-like kinases （PLK1-PLK4） have been implicated in mitotic regulation and carcinogenesis. PLK1 phosphorylates early mitotic inhibitor 1 （Emil） to ensure mitosis entry, whereas Emi2 plays a key role during the meiotic cell cycle. Transcription factor E2F is primarily considered to regulate the G1/S transition of the cell cycle but its involvement in the regulation of mitosis has also been recently suggested. A gap still exists between the molecular basis of E2F and mitotic regulation. The present study was designed to characterize the transcriptional regulation of human PLK and Emi genes. Adenoviral overexpression of E2F1 increased PLK1 and PLK3 mRNA levels in A549 cells. A reporter gene assay revealed that the putative promoter regions of PLK1, PLK3, and PLK4 genes were responsive to activators E2F, E2F1-E2F3. We further characterized the putative promoter regions of Emil and Emi2 genes, and these could be regulated by activators E2F and E2F1-E2F4, respectively. Finally, PLK1-PLK4, Emil, and Emi2 mRNA expression levels in human adult, fetal tissues, and several cell lines indicated that each gene has a unique expression pattern but is uniquely expressed in common tissues and cells such as the testes and thymus. Collectively, these results indicate that E2F can integrate G1/S and G2/M to oscillate the cell cycle by regulating mitotic genes PLK and Emi, leading to determination of the cell fate.

Expression of vascular endothelial growth factor and its role in oncogenesis of human gastric carcinoma

AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.