A single-cell genomic strategy for alternative transcript start sites identification.

Alternative transcription start sites (TSSs) usage plays a critical role in gene transcription regulation in mammals. However, precisely identifying alternative TSSs remains challenging at the genome-wide level. We report a single-cell genomic technology for alternative TSSs annotation and cell heterogeneity detection. In the method, we utilize Fluidigm C1 system to capture individual cells of interest, SMARTer cDNA synthesis kit to recover full-length cDNAs, then dual priming oligonucleotide system to specifically enrich TSSs for genomic analysis. We apply this method to a genome-wide study of alternative TSSs identification in two different IFN-ß stimulated mouse embryonic fibroblasts (MEFs). The data clearly discriminate two IFN-ß stimulated MEFs. Moreover, our results indicate 81% expressed genes in these two cell types containing multiple TSSs, which is much higher than previous predictions based on Cap-Analysis Gene Expression (CAGE) (58%) or empirical determination (54%) in various cell types. This indicates that alternative TSSs are more pervasive than expected and implies our strategy could position them at an unprecedented sensitivity. It would be helpful for elucidating their biological insights in future.

Exploring the Role of Enhancer-Mediated Transcriptional Regulation in Precision Biology.

The emergence of precision biology has been driven by the development of advanced technologies and techniques in high-resolution biological research systems. Enhancer-mediated transcriptional regulation, a complex network of gene expression and regulation in eukaryotes, has attracted significant attention as a promising avenue for investigating the underlying mechanisms of biological processes and diseases. To address biological problems with precision, large amounts of data, functional information, and research on the mechanisms of action of biological molecules is required to address biological problems with precision. Enhancers, including typical enhancers and super enhancers, play a crucial role in gene expression and regulation within this network. The identification and targeting of disease-associated enhancers hold the potential to advance precision medicine. In this review, we present the concepts, progress, importance, and challenges in precision biology, transcription regulation, and enhancers. Furthermore, we propose a model of transcriptional regulation for multi-enhancers and provide examples of their mechanisms in mammalian cells, thereby enhancing our understanding of how enhancers achieve precise regulation of gene expression in life processes. Precision biology holds promise in providing new tools and platforms for discovering insights into gene expression and disease occurrence, ultimately benefiting individuals and society as a whole.

Research Note: Combined analysis of BSA-seq based mapping and RNA-seq reveals candidate genes associated with sub-Columbian plumage in H line chickens.

Columbian coloration patterns in plumage are widespread phenomena in several standard breeds of poultry, such as the Columbian Plymouth Rock chicken. H line chicken plumage is generally a pure white except in the hackle, wing, and tail plumage, which coloration is very similar to the Columbian plumage pattern, but with the barring substituting for the black vertical striping. Thus, we refer to this plumage coloration as "sub-Columbian" pattern. However, the genetic basis of this phenotype remains unknown. Here, a F3 cross population between yellow plumage roosters and sub-Columbian plumage hens was constructed, for verifying sub-Columbian plumage was sex-linked dominant inheritance. To identify the candidate regions, F2 generation sub-Columbian plumage hens and yellow plumage hens with their parental lines were used for BSA-seq, and sub-Columbian plumage genes were mapped to a 10.46 Mb interval on chromosome Z. Remarkably, by transcriptome analysis of the neck and wing tip follicle tissues of the 2 plumage colors, we demonstrated that within the interval, only 1 gene, SLC45A2 expressed significant differently (P < 0.05). Through KASP, we identified L347M and A10331272T in solute carrier family 45 member 2 (SLC45A2), and B2 haplotype of cyclin-dependent kinase inhibitor 2A (CDKN2A), showed near complete association with the phenotype. Eventually, we designed a hybridization experiment for verifying the locus of sub-Columbian plumage, which is inherited through Z-linked dominant inheritance and is controlled by SLC45A2 and CDKN2A.

An SPRI beads-based DNA purification strategy for flexibility and cost-effectiveness.

BACKGROUND: Current solid-phase reversible immobilization (SPRI) beads technology is widely used in molecular biology due to its convenience for DNA manipulation. However, the high performance commercial SPRI beads have no price advantage over our method. Furthermore, the use of commercially available SPRI beads standards does not provide the flexibility required for a number of specific nucleic acid handling scenarios. RESULTS: We report an efficient DNA purification strategy by combining home-made beads-suspension buffer with SPRI beads. The method tests the critical concentrations of polyethylene glycol (PEG) 8000 and beads to maximise recovery. And the composition of the SPRI beads DNA purification system (SDPS) was determined at 20% PEG 8000, 2 M NaCl and 16.3 mM MgCl2, and 1.25 mg/ml beads (1/8th original concentration). Then, we tested the DNA recovery of the SDPS, and the result showed that it was comparable to the control (AMPure XP beads). In the study, we have also developed an adjustment SPRI beads DNA purification system (ASDPS), the volume of ASDPS per reaction is 0.6× reaction volume (beads/samples). The performance of ASDPS is similar to SDPS and the control. But the cost of our methods is only about 1/24th of the control. To further assess its performance, we prepare the DNA-seq libraries to evaluate the yield, library quality, capture efficiency and consistency. We have compared all these results with the performance of the control and confirmed its efficiency. CONCLUSION: We have proposed an alternative DNA purification approach with great flexibility, allowing researchers to manipulate DNA in different conditions. And ultimately, its application will benefit molecular biology research in the future.

Bidirectional Association between Hypertension and NAFLD: A Systematic Review and Meta-Analysis of Observational Studies.

An increasing body of evidence connects non-alcoholic fatty liver disease (NAFLD) to hypertension. The objective of this systematic review and meta-analysis was to estimate the nature and magnitude of the association between NAFLD and hypertension. We systematically searched PubMed, Embase, Cochrane Library, and Web of Science for observational studies published up to May 1, 2021. Cohort studies that reported data on the association between NAFLD and incident hypertension or between hypertension and incident NAFLD were included. We used random-effects models to conduct meta-analysis on the measures of association from individual studies. A total of 11 studies were eligible for inclusion, among which 4 studies including 25,260 participants reported the association between hypertension and new-onset NAFLD. The presence of hypertension was significantly associated with an increased risk of incident NAFLD (HR 1.63, 95% CI: 1.41-1.88; I 2 = 37.6%). On the other hand, 9 studies with data on 46,487 participants analyzed the effects of NAFLD on incident hypertension. Pooled analysis showed that the presence of NAFLD was significantly associated with an increased incidence of hypertension (HR 1.55, 95% CI: 1.29-1.87; I 2 = 80.5%). There was significant heterogeneity among the studies in this analysis (p < 0.01). Sensitivity analyses showed that the magnitude of the association was significantly different in subgroups stratified by a mean age of participants and geographical location, which explains part of the heterogeneity. In conclusion, this meta-analysis indicates the existence of a bidirectional relationship between NAFLD and hypertension independent of traditional cardiometabolic risk factors.

Association of a new 99-bp indel of the CEL gene promoter region with phenotypic traits in chickens.

Carboxyl ester lipase (CEL) encodes a cholesterol ester hydrolase that is secreted into the duodenum as a component of pancreatic juice. The objective of this study was to characterize the CEL gene, investigate the association between the CEL promoter variants and chicken phenotypic traits, and explore the CEL gene regulatory mechanism. An insertion/deletion (indel) caused by a 99-bp insertion fragment was shown for the first time in the chicken CEL promoter, and large differences in allelic frequency were found among commercial breeds, indigenous and feral birds. Association analysis demonstrated that this indel site had significant effects on shank length, shank girth, chest breadth at 8 weeks (p < 0.01), evisceration weight, sebum weight, breast muscle weight, and leg weight (p < 0.05). Tissue expression profiles showed extremely high levels of the CEL gene in pancreatic tissue. Moreover, the expression levels of the genes APOB, MTTP, APOV1 and SREBF1, which are involved in lipid transport, were significantly reduced by adding a 4% oxidized soybean oil diet treatment at the individual level and transfecting the embryonic primary hepatocytes with a CEL-overexpression vector. Interestingly, the results showed that the expression level of the II homozygous genotype was significantly higher than that of the ID and DD genotypes, while individuals with DD genotypes had higher phenotypic values. Therefore, these data suggested that the CEL gene might affect body growth by participating in hepatic lipoprotein metabolism and that the 99-bp indel polymorphism could be a potentially useful genetic marker for improving the economically important traits of chickens.

Combined transcriptomics and proteomics forecast analysis for potential genes regulating the Columbian plumage color in chickens.

BACKGROUND: Coloration is one of the most recognizable characteristics in chickens, and clarifying the coloration mechanisms will help us understand feather color formation. "Yufen I" is a commercial egg-laying chicken breed in China that was developed by a three-line cross using lines H, N and D. Columbian plumage is a typical feather character of the "Yufen I" H line. To elucidate the molecular mechanism underlying the pigmentation of Columbian plumage, this study utilizes high-throughput sequencing technology to compare the transcriptome and proteome differences in the follicular tissue of different feathers, including the dorsal neck with black and white striped feather follicles (Group A) and the ventral neck with white feather follicles (Group B) in the "Yufen I" H line. RESULTS: In this study, we identified a total of 21,306 genes and 5,203 proteins in chicken feather follicles. Among these, 209 genes and 382 proteins were differentially expressed in two locations, Group A and Group B, respectively. A total of 8 differentially expressed genes (DEGs) and 9 differentially expressed proteins (DEPs) were found to be involved in the melanogenesis pathway. Additionally, a specifically expressed MED23 gene and a differentially expressed GNAQ protein were involved in melanin synthesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis mapped 190 DEGs and 322 DEPs to 175 and 242 pathways, respectively, and there were 166 pathways correlated with both DEGs and DEPs. 49 DEPs/DEGs overlapped and were enriched for 12 pathways. Transcriptomic and proteomic analyses revealed that the following pathways were activated: melanogenesis, cardiomyocyte adrenergic, calcium and cGMP-PKG. The expression of DEGs was validated by real-time quantitative polymerase chain reaction (qRT-PCR) that produced results similar to those from RNA-seq. In addition, we found that the expression of the MED23, FZD10, WNT7B and WNT11 genes peaked at approximately 8 weeks in the "Yufen I" H line, which is consistent with the molting cycle. As both groups showed significant differences in terms of the expression of the studied genes, this work opens up avenues for research in the future to assess their exact function in determining plumage color. CONCLUSION: Common DEGs and DEPs were enriched in the melanogenesis pathway. MED23 and GNAQ were also reported to play a crucial role in melanin synthesis. In addition, this study is the first to reveal gene and protein variations in in the "Yufen I" H line during Columbian feather color development and to discover principal genes and proteins that will aid in functional genomics studies in the future. The results of the present study provide a significant conceptual basis for the future breeding schemes with the "Yufen I" H line and provide a basis for research on the mechanisms of feather pigmentation.

[Adenanthin Induces Differentiation of Acute Promyelocytic Leukemia Cells by Targeting Peroxiredoxin III].

OBJECTIVE: To investigate the differentiation of acute promyelocytic leukemia (APL) cells induced by adenosine targeting Prx III. METHODS: HL-60 cells were divided into four groups: control group, all-trans retinoic acid (ATRA) group, adenanthin group and ATRA+adenanthin group. Cell morphologic changes were observed under optical microscope. The influence of adenanthin on the differentiation of HL-60 was observed by nitro blue tetrazolium chloride (NBT) test. Cell surface differentiation antigens CD11b expression was measured by flow cytometry. The protein expression of Prx III was detected by immunohistochemical assay. RESULTS: Adenanthin could induce the differentiation of HL-60 cells; the NBT reduction positive rate in ATRA+adenanthin group was significantly higher than that in ATRA group and adenanthin group (P＜0.05). The percentage of CD11b positive cells in ATRA+adenanthin group (43.62％±1.38％) was higher than that in adenanthin group (28.15％±1.78％), ATRA group (36.72％±1.33％) and control group (7.99％±1.78％) (P＜0. 05). The content of Prx Ⅲ protein in adenanthin group was significantly higher than that in control group and ATRA group (P＜0.05). CONCLUSION: Adenanthin and ATRA have a synergistic effect on the differentiation and maturation of HL-60 cells, and its mechanism may be related with regulation of Prx III expression.

A multiallelic indel in the promoter region of the Cyclin-dependent kinase inhibitor 3 gene is significantly associated with body weight and carcass traits in chickens.

Many studies have reported that cyclin-dependent kinase inhibitor 3 (CDKN3) is involved in the cell cycle. However, the function of CDKN3 has not been well elucidated in organisms. In this study, a multiallelic indel caused by a 19-bp fragment and a 2 × 19 bp fragment was shown for the first time to be inserted into the promoter of the CDKN3 gene in 1994 chickens from 9 different breeds. In addition, 6 genotypes (C5C5, C4C4, C3C3, C4C5, C3C4, and C3C5) were observed (C3C3, C4C4, C5C5 have 3 × 19 bp, 4 × 19 bp, and 5 × 19 bp, respectively). Among these genotypes, the C4C4 genotype was the most dominant genotype in 9 breeds. The results of χ2 analysis of CDKN3 gene in different breeds showed that there were significant differences in the distribution of genotypes among different cultivars (P < 0.01). In addition, association study with F2 chicken resource population which produced by Anka and Gushi chickens showed that the C3C4 genotypes had the greatest semi-evisceration weight (SEW, 1163.94 ± 46.84), evisceration weight (EW, 964.15 ± 41.16), head weight (HW, 45.55 ± 1.43), claw weight (CW, 63.42±2.86), wing weight (WW, 129.15±5.48), liver weight (LW, 29.96±1.27), carcass weight (cW, 1286.96±49.53), weight at 10 (1190.68±45.68) and 12 (1430.65±54.45) wk, followed by C3C3, C4C4, C5C5, C4C5, whereas C3C5 genotypes having the lowest SEW (989.21±47.71), EW (841.38±40.55), HW (41.03±1.46), CW (54.36±2.81), WW (116.31±5.39), LW (27.31±1.25), cW (1093.29±49.99), weight at 10 (1036.10±44.99) and 12 (1246.28±53.59) wk. Expression levels of CDKN3 in breast muscle of chickens with C4C4 (0.72±0.02), C3C3 (0.95±0.41), and C4C5 (0.74±0.13) genotypes were significantly lower than those with C5C5 (1.80±0.01) and C3C5 (2.14±0.17) genotypes (P < 0.05). In conclusion, we investigated the effect of a multiallelic indel in the CDKN3 gene on the economic traits of chickens, and this indel was significantly associated with growth and carcass traits in chickens. Collectively, our findings provide useful information about the repeat sequence indel in the promoter region of the CDKN3 gene as a potential molecular marker for chicken breeding.

Features and Associated Factors of the Behavioral Development of 24-month-old Children in Rural China: Follow-up Evaluation of a Randomized Controlled Trial.

The aim of this study was to assess the risk factors associated with the behavioral development among 24-month-old children in rural northwestern China. A total of 657 children whose mothers had participated in a double-blinded, randomized, controlled trial of antenatal micronutrient supplementation in western China were followed until 24 months of age. Their mental, psychomotor, and behavioral development were assessed by the Bayley Scales of Infant Development. Multivariate logistic regression models were used to examine the factors associated with infant behavioral development. Six behavioral factors of infants were presented: activity, social adaptability, reactivity, endurance, concentration, and motor coordination. Further analysis demonstrated that maternal malnutrition, exposure to risk factors during pregnancy, and adverse birth outcomes negatively affected the behavioral development of children at 24 months, which is a common co-occurrence with cognitive and emotional problems. These results suggest that strategies to improve infant behavioral development should consider the maternal pregnancy status.

The extent and nature of television food advertising to children in Xi'an, China.

BACKGROUND: To explore the extent and nature of television food advertising especially unhealthy food advertising to primary school children in Xi'an, China. METHODS: Television data were recorded for 2 weekdays and 2 weekend days between 6:00 and 22:00 during May and June in 2012 from a total of five television channels most popular with children in Xi'an. Pearson χ (2) tests and logistic regression were applied to determine differences in the proportion of healthy food, unhealthy food and miscellaneous food advertisements for different channels, programs, dates, viewing periods and the use of persuasive marketing tactics. RESULTS: Of the 5527 advertisements transcribed, 25.5 % were for food, among which 48.1 % were considered to be unhealthy. The frequency of food advertisements was 6 per hour per channel, including 3 unhealthy food advertisements. Compared with healthy and miscellaneous food advertisements, more unhealthy food advertisements were shown during afternoon, weekends and children's non-peak viewing times as well as on children's television channels, central television channels and non-children's programmes. Unhealthy foods contributed the highest proportion of all food advertisements containing promotional characters (51.7 %) and premium offers (59.1 %). Both promotional characters and premium offers appeared more on non-children's television channels. CONCLUSIONS: The majority of food advertisements were for unhealthy food. More unhealthy food ads were shown in children's non-peak time and afternoon as well as non-children's channels. More children-oriented persuasive marketing tactics were used in unhealthy food ads especially in non-children's channels. Therefore, intervening in the entrance of unhealthy foods into the market and establishing regulations related to food advertising especially unhealthy food advertisements are important strategies to prevent children's exposure to unhealthy food and childhood obesity.

Who Should Be Targeted for the Prevention of Birth Defects? A Latent Class Analysis Based on a Large, Population-Based, Cross-Sectional Study in Shaanxi Province, Western China.

BACKGROUND: The wide range and complex combinations of factors that cause birth defects impede the development of primary prevention strategies targeted at high-risk subpopulations. METHODS: Latent class analysis (LCA) was conducted to identify mutually exclusive profiles of factors associated with birth defects among women between 15 and 49 years of age using data from a large, population-based, cross-sectional study conducted in Shaanxi Province, western China, between August and October, 2013. The odds ratios (ORs) and 95% confidence intervals (CIs) of associated factors and the latent profiles of indicators of birth defects and congenital heart defects were computed using a logistic regression model. RESULTS: Five discrete subpopulations of participants were identified as follows: No folic acid supplementation in the periconceptional period (reference class, 21.37%); low maternal education level + unhealthy lifestyle (class 2, 39.75%); low maternal education level + unhealthy lifestyle + disease (class 3, 23.71%); unhealthy maternal lifestyle + advanced age (class 4, 4.71%); and multi-risk factor exposure (class 5, 10.45%). Compared with the reference subgroup, the other subgroups consistently had a significantly increased risk of birth defects (ORs and 95% CIs: class 2, 1.75 and 1.21-2.54; class 3, 3.13 and 2.17-4.52; class 4, 5.02 and 3.20-7.88; and class 5, 12.25 and 8.61-17.42, respectively). For congenital heart defects, the ORs and 95% CIs were all higher, and the magnitude of OR differences ranged from 1.59 to 16.15. CONCLUSIONS: A comprehensive intervention strategy targeting maternal exposure to multiple risk factors is expected to show the strongest results in preventing birth defects.

A novel mutation in fibrillin-1 gene identified in a Chinese family with marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant inheritary disorder of the connective tissue. We report clinical features of a Chinese family with MFS and identify mutations in fibrillin-1 gene (FBN1). In this study, all three members of this family underwent complete ophthalmologic examinations. Two of the three members were diagnosed with MFS. Molecular genetic analysis was performed on the three members. All coding exons of FBN1 were amplified by polymerase chain reaction (PCR). The amplified products were sequenced and compared with a reference sequence in the database. Possible structural and functional changes of the protein induced by amino acids variance were predicted by bioinformatic analysis. A novel heterozygous mutation c.4504 T>A (p.C1502S) in exon 36 was identified in the two affected members, but not in the unaffected member. To our knowledge, this FBN1 mutation has not been reported beforein MFS or other patients.

Axonal Transport in the Rat Optic Nerve Following Short-Term Reduction in Cerebrospinal Fluid Pressure or Elevation in Intraocular Pressure.

PURPOSE: To examine the influence of short-term reduction in cerebrospinal fluid pressure (CSFP) as compared with short-term elevation in IOP on axonal transport. METHODS: The study included 111 adult Sprague-Dawley rats. For 6 hours, IOP was unilaterally elevated to 40 mm Hg (IOP40-group; n = 27), IOP was unilaterally increased to a value of 25 mm Hg below the mean blood pressure (PP25-group; n = 27), or CSFP was reduced by continuous aspiration of cerebrospinal fluid (Low-CSFP-group; n = 27). A sham control group (with a trocar in cisterna magna without cerebrospinal fluid release) included 24 rats. The left eyes of the IOP40 study group and PP25 study group served as additional contralateral control group. Orthograde axonal transport was examined by intravitreally injected rhodamine-ß-isothiocyanate; retrograde axoplasmic flow was assessed by fluorogold injected into the superior colliculi. RESULTS: At 24 hours after baseline, rhodamine-ß-isothiocyanate (RITC) staining intensity of the optic nerve was lower (P < 0.05) in the IOP40-group, PP25-group, and Low-CSFP-group than in the control groups. At 6 hours after the fluorogold injection, fluorogold fluorescence was significantly lower in the IOP40-group, the PP25-group, and the Low-CSFP-group than in the control groups. At 5 days after baseline, the fluorogold fluorescence no longer differed significantly between the IOP40-group or the Low-CSFP-group and the control groups. At 1 week after baseline, retinal ganglion cell density was markedly reduced only in the PP25-group. CONCLUSIONS: Both short-term lowering of CSFP and short-term rise in IOP were associated with a disturbance of both the orthograde and retrograde axonal transport. The findings support the notion of an association between abnormally low CSFP and optic nerve damage.

Ligustilide treatment promotes functional recovery in a rat model of spinal cord injury via preventing ROS production.

Ligustilide from traditional Chinese medicine extract, angelica sinensis is one of the main active components, and has many pharmacological activities related to the effectiveness. This study sought to determine whether neuro-protection of ligustilide promotes functional recovery in a rat model of spinal cord injury (SCI) via preventing ROS production. Male Sprague-Dawley (SD) rats were induced using operation for model SCI. Furthermore, Basso, Beattie, Bresnahan (BBB) scale and footprint analysis of gait was used to assess the neuro-protection of ligustilide on SCI. The intracellular reactive oxygen species (iROS), prostaglandin E(2) (PGE(2)), interleukin-1ß (IL-1ß) and tumor necrosis factor (TNF)-α production levels were measured by monoclonal enzyme immunoassay kit. Inducible nitric oxide synthase (iNOS) gene expression, activator protein-1 (AP-1) and c-Jun N-terminal kinase (JNK) protein expressions were detected using Quantitative real-time reverse transcription polymerase chain reaction (Q-PCR) and western blot analyses, respectively. Interestingly, treatment with ligustilide significantly increased BBB scale and reduced recovery of coordination in SCI rats. After SCI, the iROS, PGE(2), IL-1ß, TNF-α production levels and iNOS gene expression were significantly suppressed in SCI rats. These results suggest that the neuro-protection of ligustilide promotes functional recovery in a rat model of spinal cord injury via preventing ROS production.

Fasudil hydrochloride could promote axonal growth through inhibiting the activity of ROCK.

OBJECTIVE: This study aims to investigate the neuroprotective effect of Rho kinase inhibitor fasudil hydrochloride in ischemia/reperfusion injury N2a neuron. METHODS: In vitro, N2a cells induced by ischemia and ischemia-reperfusion were treated with fasudil hydrochloride, cell damage was analyzed by MTT. On the other hand, the cytoskeleton of N2a cells was scanned through immunofluorescence techniques by Confocal Laser Microscopy which stained with FITC-phalloidin for F-actin visualization. RESULTS: The activation of ROCK-II increased significantly in the damaged local during the following phase of ischemia/reperfusion injury. Ischemia induced a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity of the peripheral filament actin bands and formation of the long and thick stress fibers, but pretreatment of Fasudil hydrochloride could reversed the changes of ultra-structure on the cellular surface. MTT assay showed that Fasudil hydrochloride could prolong the survival time of the N2a cells after mimic ischemia-reperfusion for 24 h. CONCLUSIONS: The activation of ROCK-II has an exceptional hoist after ischemia/reperfusion injury, it is likely to induce the collapse of the growth cone through MLC-P. Fasudil hydrochloride could promote axonal growth on inhibitory of ROCK activity.

miR-200a inhibits tumor proliferation by targeting AP-2γ in neuroblastoma cells.

BACKGROUND: MicroRNA-200a (miR-200a) has been reported to regulate tumour progression in several tumours but little is known about its role in neuroblastoma. Our aim was to investigate the potential role and mechanism of miR-200a in neuroblastomas. MATERIALS AND METHODS: Expression levels of miR-200a in tissues were determined using RT-PCR. The effect of miR-200a and shAP-2γ on cell viability was evaluated using MTS assays, and target protein expression was determined using Western blotting and RT-PCR. Luciferase reporter plasmids were constructed to confirm direct targeting. RESULTS were reported as mean±S.E.M and differences were tested for significance using the 2-tailed Students t-test. RESULTS: We determined that miR-200a expression was significantly lower in neuroblastoma tumors than the adjacent non-cancer tissue. Over-expression of miR-200 are reduced cell viability in neuroblastoma cells and inhibited tumor growth in mouse xenografts. We identified AP-2γ as a novel target for miR-200a in neuroblastoma cells. Thus miR-200a targets the 3'UTR of AP-2γ and inhibits its mRNA and protein expression. Furthermore, our result showed that shRNA knockdown of AP-2γ in neuroblastoma cells results in significant inhibit of cell proliferation and tumor growth in vitro, supporting an oncogenic role of AP-2γ in neuroblastoma. CONCLUSIONS: Our study revealed that miR-200a is a candidate tumor suppressor in neuroblastoma, through direct targeting of AP-2γ. These findings re-enforce the proposal of AP-2γ as a therapeutic target in neuroblastoma.