Identification and Expression Analysis of AP2/ERF Family in Stress Responses of Pinellia ternata Based on Transcriptome Data / 中国实验方剂学杂志

ObjectiveTo identify the functions of the AP2/ERF family members in Pinellia ternata and promote the genetic improvement of P. ternata varieties. MethodWe identified and conducted a systematic bioinformatics analysis of the AP2/ERF family member genes in P. ternata based on the three generations of transcriptome data. Real-time polymerase Chain reaction　（Real-time） PCR was employed to determine the expression pattern of AP2/ERF genes in different tissues and under different stress conditions. ResultA total of eight full-length AP2/ERF family members were identified from the transcriptome data， which were classified into three sub-gene families： AP2， ERF， and DREB. The deduced AP2/ERF proteins in P. ternata had the length of 251-512 aa， the theoretical pI of 5.29-11.72， the instability index of 45.90-82.41， subcellular localization in the nucleus， and conserved domains and motifs. AP2/ERF genes were expressed in different tissues of P. ternata， with high expression levels in the leaf. The stress response experiments showed that PtERF1 mainly responded to NaCl stress. The expression of PtERF2 and PtERF4 was significantly up-regulated under low temperature and polyethylene glycol （PEG）-simulated stress. PtERF3 responded to both low temperature and NaCl stress. The expression of PtERF5 was induced by high temperature， low temperature， NaCl and PEG stress. The expression of PtERF7 was up-regulated under high temperature， while that of PtERF8 under low temperature. ConclusionThe AP2/ERF genes in P. ternata can respond to stress and have the potential functions of regulating photosynthesis and improving root stress resistance.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.@*METHODS@#To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.@*RESULTS@#One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.@*CONCLUSION@#This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Identification and analysis of AP2/ERF gene family of Panax notoginseng and function of PnDREB84 gene / 药学学报

The AP2/ERF gene family is one of the largest transcription factor families in the plant kingdom, and plays an important role in response to biological and abiotic stresses, plant hormone responses, and plant growth and development. In this study, the AP2/ERF family of Panax notoginseng was identified by bioinformatics methods, and the physicochemical properties, structure, phylogenetic relationship, expression pattern and function of PnDREB4 gene of the family were analyzed. The results showed that 140 AP2/ERF family members were identified in P. notoginseng, which were divided into DREB, ERF, AP2, RAV and Sololit subgroups. The physicochemical properties and motifs of proteins were similar among the subgroups. There were 34 differentially expressed genes in the AP2/ERF family of Fusarium oxysporum infected P. notoginseng plants, and 19 genes were up-regulated. The expression level of PnDREB84 was up-regulated with the extension of Fusarium oxysporum infection time in the range of 0-96 h. The content of ABA and SA in P. notoginseng plants overexpressing PnDREB84 gene increased after 4 ℃ stress. The results showed that PnDREB84 gene plays a dual regulatory role in the process of biological stress and abiotic stress. PnDREB84 gene can be used as a potential molecular marker for the breeding of new varieties of P. notoginseng. The identification of AP2/ERF transcription factor and function analysis of PnDREB84 gene of P. notoginseng provided data support for the analysis of stress resistance mechanism of P. notoginseng and the breeding of new varieties.

VIPR1 promoter methylation promotes transcription factor AP-2α binding to inhibit VIPR1 expression and promote hepatocellular carcinoma cell growth in vitro / 南方医科大学学报

OBJECTIVE@#To explore the transcriptional regulation mechanism and biological function of low expression of vasoactive intestinal peptide receptor 1 (VIPR1) in hepatocellular carcinoma (HCC).@*METHODS@#We constructed plasmids carrying wild-type VIPR1 promoter or two mutant VIPR1 promoter sequences for transfection of the HCC cell lines Hep3B and Huh7, and examined the effect of AP-2α expression on VIPR1 promoter activity using dual-luciferase reporter assay. Pyrosequencing was performed to detect the changes in VIPR1 promoter methylation level in HCC cells treated with a DNA methyltransferase inhibitor (DAC). Chromatin immunoprecipitation was used to evaluate the binding ability of AP-2α to VIPR1 promoter. Western blotting was used to assess the effect of AP-2α knockdown on VIPR1 expression and examine the differential expression of VIPR1 in the two cell lines. The effects of VIPR1 overexpression and knockdown on the proliferation, cell cycle and apoptosis of HCC cells were analyzed using CCK8 assay and flow cytometry. We also observed the growth of HCC xenograft with lentivirus-mediated over-expression of VIPR1 in nude mice.@*RESULTS@#Compared with the wild-type VIPR1 promoter group, co-transfection with the vector carrying two promoter mutations and the AP-2α-over-expressing plasmid obviously restored the luciferase activity in HCC cells (P < 0.05). DAC treatment of the cells significantly decreased the methylation level of VIPR1 promoter and inhibited the binding of AP-2α to VIPR1 promoter (P < 0.01). The HCC cells with AP-2α knockdown showed increased VIPR1 expression, which was lower in Huh7 cells than in Hep3B cells. VIPR1 overexpression in HCC cells caused significant cell cycle arrest in G2/M phase (P < 0.01), promoted cell apoptosis (P < 0.001), and inhibited cell proliferation (P < 0.001), while VIPR1 knockdown produced the opposite effects. In the tumor-bearing nude mice, VIPR1 overexpression in the HCC cells significantly suppressed the increase of tumor volume (P < 0.001) and weight (P < 0.05).@*CONCLUSION@#VIPR1 promoter methylation in HCC promotes the binding of AP-2α and inhibits VIPR1 expression, while VIPR1 overexpression causes cell cycle arrest, promotes cell apoptosis, and inhibits cell proliferation and tumor growth.

Gold nanorods-mediated efficient synergistic immunotherapy for detection and inhibition of postoperative tumor recurrence

Tumor recurrence after surgery is the main cause of treatment failure. However, the initial stage of recurrence is not easy to detect, and it is difficult to cure in the late stage. In order to improve the life quality of postoperative patients, an efficient synergistic immunotherapy was developed to achieve early diagnosis and treatment of post-surgical tumor recurrence, simultaneously. In this paper, two kinds of theranostic agents based on gold nanorods (AuNRs) platform were prepared. AuNRs and quantum dots (QDs) in one agent was used for the detection of carcinoembryonic antigen (CEA), using fluorescence resonance energy transfer (FRET) technology to indicate the occurrence of

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata / 中国天然药物

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Research progress on effect of AP2/ERF transcription factors in regulating secondary metabolite biosynthesis / 中国中药杂志

AP2/ERF transcription factor is a kind of transcription factors widely existing in plants, and contains at least a conserved AP2/ERF domain composed of about 60-70 amino acids. AP2/ERF transcription factors are widely involved in a variety of physiological processes in plants, including plant development, fruit ripening, flower development and other plant development processes, as well as such stress response processes as damage, pathogen defense, high-salt condition and drought. In recent years, secondary metabolic engineering that takes transcription factors as genetic manipulation targets has developed rapidly in improving the content of active ingredients and the quality of medicinal plants. This paper reviews the recent progress in the regulation of secondary metabolites biosynthesis with AP2/ERF transcription factors, and provides theoretical basis for the exploration of efficient regulatory targets, the regulation of secondary metabolites in medicinal plants, the targeted improvement of the content of active ingredients in traditional Chinese medicine, and the sustainable supply of high-quality traditional Chinese medicines.

Regulation of flowering time via miR172-mediated APETALA2-like expression in ornamental gloxinia (Sinningia speciosa) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.

Exosomes from GATA-4-overexpressing bone marrow mesenchymal stem cells suppress the apoptosis of cardiomyocytes by inhibiting the expression of Ap2m1 via miR-330-3p / 医学研究生学报

Objective Exosomes secreted from mouse bone marrow mesenchymal stem cells (BMSC) overexpressing the cardiomyocyte transcription factor GATA-4 (BMSCGATA-4-exosome) may play a key role in repairing myocardial injury. This study aimed to investigate the molecular regulatory network of BMSCGATA-4-exosome for inhibiting the apoptosis of cardiomyocytes. Methods Exosomes extracted from GATA-4-overexpressing BMSCs of the mouse cultured with miR-330-3p-mimic were cultured with myocardial cells under hypoxic and serum-free conditions for 24 hours (the experimental group), the overexpressed GATA-4, empty vector and BMSCs were taken as the confounding factor control (CFC), the myocardial cells cultured under hypoxic and serum-free conditions for 24 hours were used as the positive control, and those cultured under the normal condition for 24 hours as the negative control. The apoptosis rates of myocardial cells in different groups were measured by flow cytometry, the expression levels of miR-330-3p in the myocardial cells determined by RT-PCR, and those of the corresponding miR-330-3p target gene Ap2m1 and transcriptional protein Cnot4 detected by Western blot. Results CD29 was expressed in 99.71% of the mouse BMSCs, CD44 in 97.28%, SCA-1 in 99.40%, and CD11b overexpressed in only 0.1%. The early apoptosis rate of myocardial cells was significantly higher in the experimental than in the negative control group ([7.90 ± 0.34]% vs [2.30 ± 0.09]%, P < 0.05) but lower than in the positive control ([51.48 ± 0.40]%), BMSC ([18.32 ± 3.03]%), empty vector ([16.99 ± 2.93]%) and overexpressed GATA-4 groups ([10.22 ± 0.35]%) (P < 0.05). The expression of miR-330-3p in the myocardial cells was markedly higher in the experimental ([396.10 ± 1.02]%) than in the negative control ([1.37 ± 0.33]%), positive control ([0.26±0.32]%), BMSC ([1.40 ± 0.42]%), empty vector ([1.41 ± 0.27]%) and overexpressed GATA-4 groups ([3.80 ± 0.62]%) (P < 0.05). The expressions of Ap2m1 and Cont4 in the myocardial cells were remarkably decreased in the experimental group compared with those in the other five groups (P < 0.05). Conclusion Overexpressed BMSCGATA-4-exosomes suppress the apoptosis of myocardial cells by inhibiting the expression of the Ap2m1 protein via miR-330-3p.

Regulation of flowering time via miR172-mediated APETALA2-like expression in ornamental gloxinia (Sinningia speciosa) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

We investigated the microRNA172 (miR172)-mediated regulatory network for the perception of changes in external and endogenous signals to identify a universally applicable floral regulation system in ornamental plants, manipulation of which could be economically beneficial. Transgenic gloxinia plants, in which miR172 was either overexpressed or suppressed, were generated using Agrobacterium-mediated transformation. They were used to study the effect of altering the expression of this miRNA on time of flowering and to identify its mRNA target. Early or late flowering was observed in transgenic plants in which miR172 was overexpressed or suppressed, respectively. A full-length complementary DNA (cDNA) of gloxinia (Sinningia speciosa) APETALA2-like (SsAP2-like) was identified as a target of miR172. The altered expression levels of miR172 caused up- or down-regulation of SsAP2-like during flower development, which affected the time of flowering. Quantitative real-time reverse transcription PCR analysis of different gloxinia tissues revealed that the accumulation of SsAP2-like was negatively correlated with the expression of miR172a, whereas the expression pattern of miR172a was negatively correlated with that of miR156a. Our results suggest that transgenic manipulation of miR172 could be used as a universal strategy for regulating time of flowering in ornamental plants.

Cloning of ERF1 transcription factor from Carthamus tinctorius and construction of plant expression vector / 中草药

Objective: To clone a coding region sequence of AP2/ERF transcription factor family from Carthamus tinctorius, and construct a plant expression vector. Methods: A gene (CtERF1) of AP2/ERF family transcription factor was cloned by RT-PCR based on the sequence of C. tinctorius transcription sequencing, the phylogenetic tree was constructed by ClustalW 1.83 software, Spe I and Xba I restriction sites were introduced to construct over-expression vector pBASTA-CtERF1 containing 35S promoter. Results: CtERF1 gene had a functional domain of a typical AP2/ERF gene encoding 297 amino acids, and contained an AP2 region speculated to be located in cytoplasm and nucleus, which was ERF subprotein. Systematic evolution analysis showed that CtERF1 gene had some homology with other plant species, among which the relationship with Populus deltoides and Panax japonicus were the closest. The pBASTA-CtERF1 plant expression vector was constructed successfully by molecular biology. Conclusion: A CtERF1 gene of C. tinctorius AP2/ERF transcription factor family was cloned and the plant expression vector pBASTA-CtERF1 was constructed successfully.

The role of combined analysis of E2F3a and CASP8AP2 expression in prognosis evaluation in pediatric acute lymphoblastic leukemia / 中华实用儿科临床杂志

Objective To investigate the role of combined analysis of E2F3a and CASP8AP2 expression in prognosis evaluation in pediatric acute lymphoblastic leukemia (ALL). Methods The study included 141 newly diag-nosed pediatric ALL patients enrolled at the Hematology Center,Beijing Children′s Hospital,Capital Medical Universi-ty between March 2008 and July 2010,including 97 boys and 44 girls(aged 1. 2 - 15. 5 years,median 5. 2 years). E2F3a and CASP8AP2 expressions were quantified in 141 children with ALL by adopting real - time quantitative poly-merase chain reaction (qPCR). Receiver operating characteristic (ROC)curve was used to find the best cut - off point to divide the patients into E2F3a or CASP8AP2 low - and high - expression groups,and the treatment outcome between the groups was compared. Cox regression was used to analyze the prognostic significance of the combined expression of E2F3a and CASP8AP2. Results The estimated 5 - year relapse free survival(RFS)rate,event free survival(EFS) rate and overall survival (OS)rate of patients with low - E2F3a and low - CASP8AP2 expression were (58. 9 ± 10. 0)％,(56. 0 ± 9. 9)％ and (72. 0 ± 9. 0)％,respectively. They were significantly lower than those of patients with high - E2F3a and high - CASP8AP2 expression,whose RFS,EFS and OS were (94. 9 ± 2. 5)％,(93. 7 ± 2. 7)％ and (96. 2 ± 2. 2)％,and the differences were all statistically significant(all P < 0. 05),respectively. Compared with other patients,the one with low expression of both E2F3a and CASP8AP2 had a poorer prognosis. In addition to MLL rear-rangements and minimal residual disease level at the end of remission induction,low expression of both E2F3a and CASP8AP2 remained as independent prognostic factors. Conclusion Low expressions of E2F3a and CASP8AP2 pre-dict poor prognosis in pediatric ALL. Combined assessment of E2F3a and CASP8AP2 expression could predict poor prognosis and relapse more accurately.

Porcine transcription factor AP-2δ promotes porcine circovirus type 2 replication through enhancing the activity of the rep gene promoter / 生物工程学报

Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.

In Silico Cloning and Bioinformatics Analysis of AP2/ERF Family Gene from Brassica Oleracea / 中国药师

Objective:To obtain AP2/ERF genes from Brassica oleracea by using in silico cloning. Methods: AP2/ERF genes were cloned by retnieving the EST database and using the bioinformatics software with the Arabidopsis thaliana AP2/ERF as a querying probe. Results:Two AP2/ERF family transcriptional regulators (BoAP2/ERF1 and BoAP2/ERF2) were isolated from Brassica olera-cea by the in silico cloning method. Some characters of AP2/ERF protein were analyzed and predicted by the tools of bioinformatics in the following aspects including the composition of amino acid sequence, hydrophilicity and hydrophobicity, subcellular localization, secondary and tertiary structure of protein and function. Conclusion:Bioinformatical analysis shows BoAP2/ERF1 and BoAP2/ERF2 gene encode 40. 85kDa and 39. 44kDa protein with 371 and 352 amino acids. The domain is predicted to locate on nucleus. Sequence analysis indicates the protein may be involved in signaling transducer and stressing response roles in plantbiotic stresses.

Expression analysis of bZIP transcription factor encoding genes in response to water deficit stress in rice.

In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate several developmental processes and activate genes in response to biotic and abiotic stresses. Role of stress responsive bZIP transcription factors was studied in paddy in relation to different stages of development and water deficit stress (WDS) in a drought tolerant cultivar N22 and susceptible IR 64. Further, relative water content (RWC), membrane stability index (MSI) and abscisic acid (ABA) content were measured as indices of WDS at different stages of development and levels of stress. Expression of stress responsive bZIP transcription factors was directly correlated to developmental stage and WDS and indirectly to RWC, MSI and ABA content.

Cloning and characterization of a water deficit stress responsive transcription factor gene from Oryza sativa L.

Understanding the biochemical and molecular basis of drought mechanism in rice is important as drought is one of the major causes affecting rice crop adversely. A 1017 bp gene sequence encoding AP2/ERF family TF was isolated from Oryza sativa sp. Indica cv N22 encoding a protein of 338 amino acid residues, with a molecular weight of 36.58 kDa, and no intron in the ORF. The gene was named as AP2/ERF-N22(2) different from the drought responsive gene AP2/ERF-N22 that we reported earlier. AP2/ERF-N22(2) has entirely different characteristics from that of AP2/ERF-N22. It has a single AP2 domain of 55 amino acid residues and a cluster of acidic amino acid residues at the C-terminal region, which could function as a trans-activation domain. Presence of NLS indicates that it is a nuclear localized transcription factor encoding gene. It falls in group VI L, sharing characteristic similarities. Arabidopsis members of group VI L have been shown to be involved in response to cytokinin under drought stress.

Research progress on AP2/ERF transcription factors regulating biosynthesis of active ingredients in medicinal plants / 中草药

AP2/ERF transcription factors are widely present in plants. Each member of AP2/ERF family contains a much conserved DNA binding domain, AP2/EREBP binding domain, which is composed by about 60 amino acids. The studies about plant AP2/ERF transcription factors mainly focused on the developmental and physiological regulation function. In recent years, the function of regulating secondary metabolites is being concerned. Here the research progress on AP2/ERF regulating the biosynthesis of active ingredient of medicinal plants was reviewed, which could be used for reference in further studies on improving the accumulation of active ingredients in medicinal plants by applying metabolic engineering.

miR-200a inhibits cell proliferation by targeting AP-2γexpression in neuroblastoma cells SK-N-AS / 天津医药

Objective To investigate whether miR-200a suppresses cell proliferation by targeting AP-2γexpression, and reveal molecular mechanism that miR-200a functions as a tumor-suppressor in neuroblastoma cells. Methods Dual-luciferase reporter gene assay was employed to examine the effect of miR-200a on AP-2γpromotor luciferase activity. Neu-roblastoma cells were transfected with miR-200a mimics, and the expressions of AP-2γmRNA and protein were detected by RT-PCR and Western blot assay. The effects of AP-2γdown-regulation on cell proliferation were observed after AP-2γshRNA was transfected into neuroblastoma cells. Neuroblastoma cell proliferation was detected by MTS assay after being co-transfected with miR-200a mimics and AP-2γplasmid. Results Results showed that miR-200a could inhibit proliferation of neuroblastoma cells at cell viability (66.33 ± 5.13) compared with that of control group (100 ± 0), and also miR-200a can bind to the 3'untranslated region of AP-2γpromotor and inhibit its luciferase activity with an inhibit ratio at (0.624±0.051). AP-2γmRNA and protein expressions were significantly down-regulated when miR-200a was over-expressed in neuroblas-toma cells. Furthermore, results showed that shRNA-mediated down-regulation of AP-2γthat suppressed the cell prolifera-tion of neuroblastoma at (62.5±2.4) by comparing with the control group (100±0). Moreover, restoring AP-2γexpression re-versed the effect of miR-200a with a cell viability suppression at (92.4±1.4). Conclusion miR-200a suppresses cell prolif-eration by targeting AP-2γexpression in neuroblastoma cells.

Targeted inhibition of microRNA-200c on expression of AP-2α to enhance the proliferation of colon cancer cells in vitro / 肿瘤研究与临床

Objective To investigate the impact of miR-200c overexpression on colon cancer cell proliferation ability and the related mechanism.Methods MicroRNAs which may combined with the transcription factor AP-2α were screened and forecasted by the bioinformatics database,while its eukaryotic expression plasmids and specific inhibitor were synthesized.Plasmids PEZX-miR-200c,PEZX-NC,pmirGLO-AP-2α3'UTR,pmir-GLO and the specific inhibitors miR-67-inhibtor,miR-200c-inhibitor were transfected in vitro into colon cancer HCT-116 and SW480 cells and the HEK293T cell by Lipofectamine2000.The expression of AP-2α mRNA and protein in colon cancer cells was analyzed by qRT-PCR,Westem blot and immunocytochemical staining.CCK-8 assay and flow cytometry were adopted to observe the effect of miR-200c on colon cancer cells proliferation and apoptosis.Dual-Luciferase assay experiments were performed to observe the relative luciferase activity induced by miR-200c.Results The proliferation activity was significantly decreased in anti-miR-200c/SW480 group,while in PEZX-miR-200c/HCT-116 group,it was higher than that in PEZX-NC/HCT-116 group.The apoptosis ability was significantly increased in anti-miR-200c/SW480 group [(78±0.7) ％ vs (66±1.1) ％,P ＜ 0.05].The expression of AP-2o both in mRNA and protein levels was decreased in PEZX-miR-200c/HCT-116 group,while the protein level was increased in Anti-miR-200c/SW480 group.The relative luciferase activity inhibited by miR-200c was decreased in HEK-293T cells transfected with PEZX-miR-200c and pmir-GLO-AP-2α3' UTR (0.51±0.09 vs 0.98±0.04,P ＜ 0.01).Conclusion MicroRNA-200c could promote cell proliferation ability by targeting transcriptional factor AP-2α in human colorectal cancer cells.

Isolation and molecular characterization of an ethylene response factor NtERF1-1 in Nicotiana tabacum cv. Xanthi.

Apetala2/Ethylene Response Factors (AP2/ERF) play important roles in regulating gene expression under abiotic and biotic stress in the plant kingdom. Here, we isolated a member of the AP2/ERF transcription factors, NtERF1-1, from Nicotiana tabcum cv. Xanthi NN carrying the N gene, which is resistant to Tobacco mosaic virus (TMV). NtERF1-1 encoded a putative protein of 229 amino acids with a predicted molecular mass of 24.58 kDa. Nucleotide sequence analysis showed that NtERF1-1 contained a conserved DNA-binding domain at the N-terminal. Comparison of amino acid sequences revealed that NtERF1-1 possessed high similarities to ERFs from diverse plants. Semi-quantitative and real-time quantitative RT-PCR analyses indicated that NtERF1-1 was up-regulated following TMV infection. In addition, we speculated that NtERF1-1 might participate in the signal transduction pathway of defence response inducted by the interaction between the N gene and TMV.