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.

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.

TFAP2C increases cell proliferation by downregulating GADD45B and PMAIP1 in non-small cell lung cancer cells

BACKGROUND: Non-small cell lung cancer (NSCLC) is one of the leading causes of death in the world. NSCLC diagnosed at an early stage can be highly curable with a positive prognosis, but biomarker limitations make it difficult to diagnose lung cancer at an early stage. To identify biomarkers for lung cancer development, we previously focused on the oncogenic roles of transcription factor TFAP2C in lung cancers and revealed the molecular mechanism of several oncogenes in lung tumorigenesis based on TFAP2C-related microarray analysis. RESULTS: In this study, we analyzed microarray data to identify tumor suppressor genes and nine genes downregulated by TFAP2C were screened. Among the nine genes, we focused on growth arrest and DNA-damage-inducible beta (GADD45B) and phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1) as representative TFAP2C-regulated tumor suppressor genes. It was observed that overexpressed TFAP2C resulted in inhibition of GADD45B and PMAIP1 expressions at both the mRNA and protein levels in NSCLC cells. In addition, downregulation of GADD45B and PMAIP1 by TFAP2C promoted cell proliferation and cell motility, which are closely associated with NSCLC tumorigenesis. CONCLUSION: This study indicates that GADD45B and PMAIP1 could be promising tumor suppressors for NSCLC and might be useful as prognostic markers for use in NSCLC therapy.

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.

Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines / Caracterização do HIPSTR destaca o padrão de expressão heterogênea de IncRNAs em embriões humanos e linhagens estáveis de células

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns

Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos

Construction of a luciferase reporter vector containing response element of activator protein 2α and its application in study of osteogenetic differentiation / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a luciferase reporter vector containing the response element of transcription protein AP2α for screening the effect of bone morphogenetic proteins (BMPs) on the transcriptional activity of AP2α.</p><p><b>METHODS</b>Four tandem-linked response elements of AP2α were cloned to the pBGLuc luciferase reporter gene plasmid, which was digested with Bam HI and Mlu I to construct pBGLuc-AP2α-RE vector. The recombinant adenovirus Ad-AP2α and its dominant negative mutant Ad-dnAP2α were used to infect mouse mesenchymal stem cells C3H10; the changes in cellular AP2α mRNA and protein expressions were detected by real-time PCR and Western blotting, and electrophoretic mobility shift assay (EMSA) was carried out to assess the DNA-binding ability of AP2α. C3H10 cells were transfected with pBGLuc-AP2α-RE vector, and AP2α transcriptional activity was measured using luciferase reporter gene assay. In pBGLuc-AP2α-RE vector-transfected C3H10 cells infected with Ad-BMPs, luciferase reporter gene assay was performed to screen the effect of BMPs on AP2α transcriptional activity.</p><p><b>RESULTS</b>The results of PCR, enzyme digestion and sequencing all confirmed correct cloning of AP2α-RE into pBGLuc-AP2α-RE luciferase reporter vector, and Ad-AP2α infection significantly increased AP2α expression and its DNA binding ability. The dominant negative mutants expressed the corresponding mutants, and EMSA results showed that Ad-dnAP2α-δbHLH significantly lowered while Ad-dnAP2α-δTAD enhanced the DNA-binding ability of AP2α. AP2α over-expression promoted AP2α transcriptional activity, which was suppressed by the two dominant negative mutants. AP2α transcriptional activity increased in the cells infected with the recombinant adenovirus BMPs, especially in cells with BMP9 infection.</p><p><b>CONCLUSIONS</b>The luciferase reporter vector containing the response element of AP2α we constructed allows detection of AP2α transcriptional activity. BMP9 can significantly enhance AP2α transcriptional activity.</p>

Mutations of TFAP2B in congenital heart disease patients in Mysore, South India.

Background & objectives: Cardiac malformations in the young constitute a major portion of clinically significant birth defects. Congenital heart disease (CHD) is a common congenital cardiac birth defect, affecting nearly 1 per cent of all live births. Patent ductus arteriosus (PDA) is clinically significant foetal circulation anomaly, second most common form of CHD which constitutes approximately 10 per cent of total CHDs. The study aimed to screen for TFAP2B mutations in CHD patients of Mysore. Methods: With informed consent, 100 clinically diagnosed CHD patients and 50 healthy controls in Mysore, south India, were recruited for the analysis of screening of mutations. MassARRAY analysis of 5 prominent mutations of TFAP2B was performed. Results: The analysis did not show any of the five mutations of TFAP2B screened by massARRAY in patients and controls, indicating that these mutations were not involved in the manifestation of CHD in the patients at Mysore, south India. Interpretation & Conclusions: The findings suggest the lack of involvement of known mutations of TFAP2B with syndromic or nonsyndromic CHDs in Mysore patients.

Cloning and bioinformatic analysis of PqERF1 gene in Panax quinquefolius / 药学学报

ERF family transcription factor (TF) represented ethylene-responsive protein which harbored a conserved AP2 domain. After searching the plant transcription factor database, a total of 75 unigenes was found which contained AP2 domain from the transcriptome dataset of Panax quinquefolius L. One unique sequence of ERF transcript, named as PqERF1, was cloned with entire open reading frame of 933 base pairs (bp). Protein prediction result indicated that the gene was localized in nucleus and had a conserved AP2 domain. PqERF1 gene could be induced by methyl jasmonate (MeJA) which was consistent to the inducing profile of triterpene ginsenosides. InterproScan prediction indicated that PqERF1 was probably a pathogenesis-related gene. Sequence alignment and phylogenetic analysis demonstrated PqERF1 was with high identity and had relative close relationship to the NtERF4 (Nicotiana tabacum), PhERF12 (Petunia x hybrida) and DcERF1 (Daucus carota) which was related to plant defense, regulation of secondary metabolism and the flower senescence respectively. Therefore, the gene was likely involved in regulation of secondary metabolism, plant defense and physical processes which would provide gene resource for further study on secondary metabolite synthesis and molecular breeding of P. quinquefolius.

Mutation of TFAP-2B gene in patients with patent ductus arteriosus / 中华儿科杂志

<p><b>OBJECTIVE</b>To identify novel genetic mutations in Chinese patients with congenital patent ductus arteriosus (PDA).</p><p><b>METHOD</b>Clinical data and peripheral blood specimens from a kindred spanning 3 generations in which 5 of 16 individuals had PDA and a cohort of 95 unrelated subjects with PDA were collected, and 100 unrelated healthy individuals were included as controls. The coding exons and flanking introns of TFAP-2B gene were amplified by polymerase chain reaction (PCR) with specific primers. We aligned the acquired sequences with which publicized in GenBank by the aid of program BLAST. Reverse transcription-polymerase chain reaction (RT-PCR) was used to amplify the parts of TFAP-2B and sequencing was performed on PCR products forward and reversely directly.</p><p><b>RESULT</b>Sequencing of TFAP-2B identified that there was a splice-junction in intron 3 [intron 3(+5)G > A] and a 60 bp deletion was found in exon 3 by nested PCR. Additionally, a novel single nucleotide polymorphism (SNP) where a transition of guanine (G) to adenine (A) was identified at 34 bp front of transcription initiation site in TFAP-2B gene. There were significant differences in the prevalence of alleles G and A between controls and PDA patients (Z = -2.513, P = 0.012).</p><p><b>CONCLUSION</b>We identified a novel splice-junction in TFAP-2B gene which might lead to hereditary PDA in a Chinese family. However, the mechanism by which this mutation results in PDA is still to be ascertained.</p>

Dual role of transcription factor AP-2 in carcinogenesis / 浙江大学学报·医学版

Activating protein-2 (AP-2) is a cell type-specific DNA binding transcription factor family with the ability to regulate the expression of specific target genes. Five isoforms of AP-2 have been discovered, they are AP-2alpha, AP-2beta, AP-2gamma, AP-2delta and AP-2epsilon. AP-2s are involved in the regulation of cell proliferation, differentiation and apoptosis as well as embryogenesis of mammary animals. Recently, the function of AP-2 in neoplasm has attracted increasing attention. Researches reveal that the modulation of AP-2 in tumorigenesis may be dual, either inhibitory or promoting, which depends on the specific tissues,stages of cancer progression and difference between five family members. This review summarizes recent research progress on the role of AP-2 in the oncogenesis and their potential applications in clinical practice.