Dual-luciferase Reporter Assay System Research Articles

MiR-23b controls TGF-β1 induced airway smooth muscle cell proliferation via direct targeting of Smad3

BackgroundMicroRNAs are small yet versatile gene tuners that regulate a variety of cellular processes, including cell growth and proliferation. Here we report that miR-23b inhibited airway smooth muscle cells (ASMCs) proliferation through directly targeting of Smad3. MethodsWe obtained ASMCs by laser capture microdissection of normal and asthmatic mice lung tissues. Mice ASMCs were cultured and induced by TGF-β1. The implication between TGF-β1 and miR-23b in ASMCs were detected by RT-PCR. The effects of miR-23b on ASMCs proliferation and apoptosis were assessed by transient transfection of miR-23b mimics and inhibitor. The expression of Smad3 in ASMCs were detected by RT-PCR and Western blotting analysis. Dual-Luciferase Reporter Assay System will be applied to identify whether Smad3 is a target gene of miR-23b. ResultsTGF-β1 and miR-23b mRNA expression of in-situ bronchial ASMCs collected by laser capture microdissection were increased in asthmatic mice compared to non-asthma controls. This is accompanied by an increase in miR-23b mRNA expression in TGF-β1 induced ASMCs. miR-23b up-regulation significantly inhibited TGF-β1-induced ASMCs proliferation and promoted apoptosis. MiR-23b negatively regulates the expression of Smad3 in ASMCs. Dual-Luciferase Reporter Assay System demonstrated that Smad3 was a direct target of miR-23b. ConclusionsMiR-23b may function as an inhibitor of asthma airway remodeling by suppressing ASMCs proliferation via direct targeting of Smad3.

Cell Signaling Pathway in 12-O-Tetradecanoylphorbol-13-acetate-Induced LCN2 Gene Transcription in Esophageal Squamous Cell Carcinoma.

LCN2 is involved in various cellular functions, including transport of small hydrophobic molecules, protection of MMP9 from proteolytic degradation, and regulating innate immunity. LCN2 is elevated in multiple human cancers, frequently being associated with tumor size, stage, and invasiveness. Our previous studies have shown that LCN2 expression could be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in esophageal squamous cell carcinoma (ESCC) by the binding of five nucleoproteins (MISP, KLF10, KLF15, PPP1R18, and RXRβ) at a novel TPA-responsive element (TRE), at −152~−60 bp of the 5′ flanking region of the LCN2 promoter. However, much is unknown about whether these proteins can respond to TPA stimulation to regulate LCN2 transactivation and which cell signaling pathways mediate this process. In this study, expression plasmids encoding these five nucleoproteins were stably transfected into EC109 cells. Then, stable transfectant was characterized by a Dual-Luciferase Reporter Assay System. RT-PCR, real-time PCR, western blotting, specific kinase inhibitor treatment, and bioinformatics analyses were applied in this study. We found that MISP, KLF10, KLF15, PPP1R18, and RXRβ proteins could strongly respond to TPA stimulation and activate LCN2 transcriptional expression. MEK, ERK, JNK, and P38 kinases were involved in the LCN2 transactivation. Furthermore, the MEK-ERK signal pathway plays a major role in this biological process but does not involve PKCα signaling.

Prevalence and Spectrum of NKX2-5 Mutations Associated With Sporadic Adult-Onset Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM), the most common form of primary myocardial disease, is a leading cause of congestive heart failure and the most common indication for heart transplantation. Recently, NKX2-5 mutations have been involved in the pathogenesis of familial DCM. However, the prevalence and spectrum of NKX2-5 mutations associated with sporadic DCM remain to be evaluated. In this study, the coding regions and flanking introns of the NKX2-5 gene, which encodes a cardiac transcription factor pivotal for cardiac development and structural remodeling, were sequenced in 210 unrelated patients with sporadic adult-onset DCM. A total of 300 unrelated healthy individuals used as controls were also genotyped for NKX2-5. The functional effect of the mutant NKX2-5 was investigated using a dual-luciferase reporter assay system. As a result, two novel heterozygous NKX2-5 mutations, p.R139W and p.E167X, were identified in 2 unrelated patients with sporadic adult-onset DCM, with a mutational prevalence of approximately 0.95%. The mutations were absent in 600 referential chromosomes and the altered amino acids were completely conserved evolutionarily across species. Functional assays revealed that the NKX2-5 mutants were associated with significantly reduced transcriptional activity. Furthermore, the mutations abrogated the synergistic activation between NKX2-5 and GATA4 as well as TBX20, two other cardiac key transcription factors that have been causally linked to adult-onset DCM. This study is the first to associate NKX2-5 loss-of-function mutations with enhanced susceptibility to sporadic DCM, which provides novel insight into the molecular etiology underpinning DCM, and suggests the potential implications for the genetic counseling and personalized treatment of the DCM patients.

MEF2C loss-of-function mutation contributes to congenital heart defects.

Congenital heart disease (CHD) is the most common type of developmental abnormality in humans, and is a leading cause for substantially increased morbidity and mortality in affected individuals. Increasing studies demonstrates a pivotal role of genetic defects in the pathogenesis of CHD, and presently mutations in more than 60 genes have been associated with CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic basis underpinning CHD in a large proportion of patients remains unclear. In the present study, the whole coding exons and splicing donors/acceptors of the MEF2C gene, which codes for a transcription factor essential for normal cardiovascular development, were sequenced in 200 unrelated patients affected with CHD, and a novel heterozygous missense mutation, p.L38P, was identified in an index patient with patent ductus arteriosus (PDA) and ventricular septal defect (VSD). Genetic scan of the mutation carrier's family members available showed that the mutation was present in all affected family members but absent in unaffected family members. Analysis of the proband's pedigree revealed that the mutation co-segregated with PDA, which was transmitted as an autosomal dominant trait with complete penetrance. The mutation changed the amino acid that was completely conserved evolutionarily, and did not exist in 300 unrelated, ethnically-matched healthy individuals used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant MEF2C protein had a significantly reduced transcriptional activity. Furthermore, the mutation significantly diminished the synergistic activation between MEF2C and GATA4, another cardiac core transcription factor that has been causally linked to CHD. In conclusion, this is the first report on the association of a MEF2C loss-of-function mutation with an increased vulnerability to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD, implying potential implications for early diagnosis and timely prophylaxis of CHD.

TBX20 loss-of-function mutation responsible for familial tetralogy of Fallot or sporadic persistent truncus arteriosus.

Congenital heart disease (CHD), the most common form of developmental abnormality in humans, remains a leading cause of morbidity and mortality in neonates. Genetic defects have been recognized as the predominant causes of CHD. Nevertheless, CHD is of substantial genetic heterogeneity and the genetic defects underlying CHD in most cases remain unclear. In the current study, the coding regions and splicing junction sites of the TBX20 gene, which encodes a T-box transcription factor key to cardiovascular morphogenesis, were sequenced in 175 unrelated patients with CHD, and a novel heterozygous TBX20 mutation, p.K274X, was identified in an index patient with tetralogy of Fallot (TOF). Genetic analysis of the proband's available family members showed that his father, elder brother and son had also TOF. In addition, his father and elder brother had also atrial septal defect, and his niece had persistent truncus arteriosus and ventricular septal defect. Analysis of the pedigree revealed that the mutation co-segregated with CHD transmitted in an autosomal dominant fashion, with complete penetrance. The nonsense mutation, which was absent in the 800 control chromosomes, was predicted to produce a truncated protein with only the amino terminus and partial T-box domain left. Functional analyses by using a dual-luciferase reporter assay system showed that the mutant TBX20 lost the ability to transactivate the target gene ANF. Furthermore, the mutation reduced the synergistic activation between TBX20 and NKX2.5 as well as GATA4, two other transcriptional factors previously associated with various CHD, encompassing TOF. This study firstly links TBX20 loss-of-function mutation to familial TOF or sporadic persistent truncus arteriosus, providing novel insight into the molecular pathogenesis of CHD.

HAND1 Loss-of-Function Mutation Causes Tetralogy of Fallot.

As the most prevalent form of birth defect in humans worldwide, congenital heart disease (CHD) is responsible for substantial morbidity and is still the leading cause of birth defect-related demises. Increasing evidence demonstrates that genetic defects play an important role in the pathogenesis of CHD, and mutations in multiple genes, especially in those coding for cardiac core transcription factors, have been causally linked to various CHDs. Nevertheless, CHD is a genetically heterogeneous disease and the genetic determinants underpinning CHD in an overwhelming majority of patients remain elusive. In the current study, genomic DNA was extracted from venous blood samples of 165 unrelated patients with CHD, and the coding exons and splicing junction sites of the HAND1 gene, which encodes a basic helix-loop-helix transcription factor essential for cardiovascular development, were sequenced. As a result, a novel heterozygous mutation, p.R118C, was identified in a patient with tetralogy of Fallot (TOF). The missense mutation, which was absent in 600 referential chromosomes, altered the amino acid that was completely conserved evolutionarily. Biological assays with a dual-luciferase reporter assay system revealed that the R118C-mutant HAND1 protein had significantly reduced transcriptional activity when compared with its wild-type counterpart. Furthermore, the mutation significantly decreased the synergistic activation of a downstream target gene between HAND1 and GATA4, another cardiac core transcription factor associated with TOF. To our knowledge, this is the first report on the association of a HAND1 loss-of-function mutation with enhanced susceptibility to TOF in humans. The findings provide novel insight into the molecular etiology underlying TOF, suggesting potential implications for the improved prophylactic and therapeutic strategies for TOF.

Deregulation of microRNA-188-5p in prostate carcinoma and effects on proliferation and metastasis of prostate carcinoma cell lines

Objective To measure the expression level of microRNA (miRNA, miR)-188-5p in prostate carcinoma (PCa) and investigate its biological functions on prostate carcinoma cell lines. Methods In 30 PCa and 40 benign prostatic hyperplasia (BPH) samples, quantificational real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was used to detect the expression of miR-188-5p in prostate tissues. In different prostate cell lines, miR-188-5p expression was also detected using RT-qPCR. The miR-188-5p mimic was re-introduced into PC-3 and LNCaP cells by Lipfectamine 2000. The function of miR-188-5p on PCa cells was explored by colony formation assays, Transwell migration and invasion assays. The candidate targets for miR-188-5p were investigated using prediction algorithm provided by miRanda. Luciferase activity was measured using the dual luciferase reporter assay system. Results The expression level of miR-188-5p was significantly lower in PCa tissues than in pericarcinous and BPH tissues (0.996±0.024, 0.990±0.094, 0.385±0.031; P 0.05). It was also found that miR-188-5p was significantly down-regulated in PCa cell lines as compared with that in RWPE-1 (0.409±0.034, 0.358±0.024, 0.255±0.018, 1.022±0.012; P<0.01). The colony formation assays showed that ectopic miR-188-5p expression significantly inhibited colony formation compared to control PC-3 and LNCaP cells (21±5, 47±3; 25±4, 51±5; P<0.01). We next found ectopic miR-188-5p expression significantly reduced the cell invasion and migration compared to control PC-3 and LNCaP cells (P<0.05). Through computational analysis, the binding site for miR-188-5p at 3’-untranslated region (3’-UTR) of lysosome-associated protein transmembrane 4 beta (LAPTM4B) was depicted. The luciferase-based assay validated LAPTM4B was regulated by miR-188-5p. Conclusion The miR-188-5p was significantly down-regulated in PCa. Restoration of miR-188-5p in PCa cells significantly suppresses proliferation, migration and invasion in vitro. The miR-188-5p can negatively regulate LAPTM4B expression by directly binding to its 3’-UTR. Key words: MicroRNA-188-5p; Lysosome-associated protein transmembrane 4 beta; Prostate carcinoma

MicroRNA let - 7c inhibits proliferation of hepatocellular carcinoma cells by targeting cyclin dependent kinase 6

Objective To explore let-7c’s influence on the proliferation of human hepatocellular carcinoma cells and to clarify its target gene. Methods The let-7c expression in human hepatocellular carcinoma cells (MHCC-97L, HepG2, HCCLM3, SMMC-7721) and human hepatic cell L02 were detected by real-time quantitative polymerase chain reaction (Real-time PCR). MicroRNAs (miRNAs) were transfected into HCCLM3 cells via Lipofectamine™2000. The cells were divided into three groups: let-7c group transfected with let-7c, negative control group transfected with negative control miRNA, blank control group transfected with no miRNAs. The proliferation of cells was assessed via cell counting kit-8 (CCK-8), cell cycle of each group was detected by flow cytometry, Western blotting was used to evaluate the protein expression of cyclin dependent kinase 6 (CDK6) after transfection. The 3’untranslated region (3’-UTR) of CDK6 containing the putative binding sites of let-7c was inserted into a firefly luciferase reporter vector named pMIR-REPORT. MiRNAs, pMIR-REPORT and renilla luciferase control plasmid were transfected into 293T cells together, the Dual-Luciferase Reporter Assay System was used to assay the luciferase activity. Results The expression level of let-7c in MHCC-97L, HepG2, HCCLM3, SMMC-7721 were (3.22±0.08)×10-4, (2.34±0.11)×10-4, (1.85±0.03)×10-4 and (3.03±0.11)×10-4 respectively, lower than that in L02 [(4.37±0.09)×10-4,P 0.05). The percentage of G1 stage in let-7c group was (56.95±2.40)% at 72 h after transfection, higher than that of the other two groups (P 0.05). The protein level of CDK6 in let-7c group was lower than that of the other two groups at 48 h after transfection (P 0.05). The Dual-Luciferase Reporter Assay System revealed that let-7c could suppress the luciferase’s activity compared with negative control miRNA (P<0.05). Conclusion The expression level of let-7c is lower in the hepatocellular carcinoma cells compared with normal hepatic cell. let-7c can inhibit the proliferation of hepatocellular carcinoma cells and cause a delay in G1-S transition, CDK6 is its target gene. Key words: Carcinoma, hepatocellular; MicroRNAs; Cyclin-dependent kinase 6; Let-7c

Abstract 13426: Suppression of Epsin Expression Rescues Diabetes Triggered Impaired Lymphatic Function by Limiting VEGFR3 Degradation

Rationale: Impaired lymphatic circulation causes lymphedema and contributes to pathological conditions, such as diabetes. VEGF-C/VEGFR3 signaling is a crucial regulator of lymphatic development; however, whether manipulation in signaling improves lymphatic circulation under pathological conditions is unclear. Objective: We recently uncovered that loss of epsins in lymphatic endothelial cells (LECs) caused heightened VEGFR3 signaling and delayed lymphatic formation during development. Herein, we investigated whether elevated VEGFR3 signaling caused by epsin loss is critical for pathological lymphatic circulation in adult mice. Methods and Results: Using innovative near-infrared lymphatic imaging, and tail lymphedema models, we demonstrated that epsin loss accelerated recovery from diabetic secondary lymphedema. High-glucose-induced oxidative stress results in reduced VEGFR3 signaling, impaired lymphangiogenesis and delayed recovery from diabetic secondary lymphedema. The dual luciferase reporter assay system and ChIP assay showed in STZ-induced diabetic mice drove Src- and AP-1- mediated upregulation of epsin expression. Epsin deletion in primary LECs caused heightened VEGFR3 signaling and promoted in vitro lymphangiogenesis by preventing VEGFR3 downregulation. Mechanistically, we showed that by protecting cell surface VEGFR3 from degradation, lack of epsin rescued high-glucose-induced VEGFR3 loss, defective lymphangiogenesis in vitro , and ameliorated STZ-induced diabetic secondary lymphedema in vivo . Conclusions: Epsins essentially regulate lymphatic circulation through the modulation of VEGFR3 signaling. In STZ- induced diabetic mice, Src induces epsin expression via transcription factor AP-1. Epsin depletion ameliorated diabetic secondary lymphedema under hyperglycemic condition by protecting VEGFR3 from oxidative stress-induced degradation. Our findings highlight the therapeutic potential for targeted inhibition of epsins within the lymphatic endothelium as a treatment for secondary lymphedema caused by uncontrolled diabetes. Key Words: Epsin, VEGFR3, Lymphatics, Secondary Lymphedema, Diabetes

Active fragments-guided drug discovery and design of selective tropane alkaloids using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry coupled with virtual calculation and biological evaluation.

Tropane alkaloids (TAs), rich in the plant of Physochlaina infundibularis Kuang, which is named Huashanshen (HSS) in China, showed good effects on types of spasms. However, no data were collected to explore the relationship between the specificity for muscarinic receptor subtypes and the structures of these TAs. To address this issue, an extracted ion chromatogram (EIC) strategy using ultra-high performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q/TOF-MS) based on the fragmentation behavior of the TA standards was established to rapidly capture the varied TAs from HSS. Based on the provided structural information of diagnostic ions or neutral loss, 29 TAs were efficiently profiled, especially some trace ingredients. In additional, via virtual validation combined with molecular dynamic simulation, approximately a dozen alkaloids were found with high selectivity for muscarinic receptors. In additional, N-acetyl convolicine was chosen for selectivity evaluation of M2 or M3 receptors through the use of a dual-luciferase reporter assay system at the cellular level and an ACh-induced constricted strip test in vitro. After summarizing the active fragments and the structure-activity relationship (SAR) information, a new modified TA that takes advantage of both the high affinity and high selectivity for M3 receptors was proposed and evaluated successfully. This study provided an effective approach for the discovery and design of natural products based on highly selective drugs by UPLC-Q/TOF-MS coupled with virtual calculation and biological evaluation. Graphical Abstract Active fragments-guided strategy for selective inhibitors from HSS.

CASZ1 loss-of-function mutation associated with congenital heart disease

As the most common form of birth defect in humans, congenital heart disease (CHD) is associated with substantial morbidity and mortality in both children and adults. Increasing evidence demonstrates that genetic defects play a pivotal role in the pathogenesis of CHD. However, CHD is of great heterogeneity, and in an overwhelming majority of cases, the genetic determinants underpinning CHD remain elusive. In the present investigation, the coding exons and flanking introns of the CASZ1 gene, which codes for a zinc finger transcription factor essential for the cardiovascular morphogenesis, were sequenced in 172 unrelated patients with CHD. As a result, a novel heterozygous CASZ1 mutation, p.L38P, was identified in an index patient with congenital ventricular septal defect (VSD). Genetic scanning of the mutation carrier's available family members revealed that the mutation was present in all affected patients but absent in unaffected individuals. Analysis of the proband's pedigree showed that the mutation co-segregated with VSD, which was transmitted as an autosomal dominant trait with complete penetrance. The missense mutation, which altered the amino acid that was highly conserved evolutionarily, was absent in 200 unrelated, ethnically-matched healthy subjects used as controls. Functional deciphers by using a dual-luciferase reporter assay system unveiled that the mutant CASZ1 had significantly reduced transcriptional activity as compared with its wild-type counterpart. To the best of our knowledge, the current study firstly identifies CASZ1 as a new gene predisposing to CHD in humans, which provides novel insight into the molecular mechanisms underlying CHD and a potential therapeutic target for CASZ1-associated CHD, suggesting potential implications for personalized prophylaxis and therapy of CHD.

MiRNAs Regulate hERG.

The human ether-a-go-go-related gene (hERG) is the major molecular component of the rapidly activating delayed rectifier K+ current (Ikr ). Impairment of hERG function is believed to be a mechanism causing long-QT syndromes (LQTS). Growing evidences have shown that microRNAs (miRNAs) are involved in functional modulation of the hERG pathway. The purpose of this study was to screen and validate miRNAs that regulate the hERG pathway. The miRNAs identified in this study will provide new tools to assess the mechanism of LQTS. Six miRNAs were selected by algorithm predictions based on potential interaction with hERG. The effects of each miRNA on hERG were assessed by use of the Dual-Luciferase Reporter assay system, qRT-PCR, Western blotting, and confocal fluorescence microscopy. Furthermore, whole-cell patch clamp technique was used to validate the effect of miR-103a-1 on the electrophysiological characteristic of the Ikr of the hERG protein channel. miR-134, miR-103a-1, miR-143, and miR-3619 significantly downregulated luciferase activity (P < 0.05) in a reporter test system. These 4 miRNAs significantly suppressed expression of hERG mRNA and protein in U2OS cells (P < 0.05).Corresponding AMOs rescued expression of hERG mRNA and protein. Confocal microscopy showed that all 4 miRNAs reduced the expression of both immature and mature hERG protein. miR-103a-1 decreased the maximum current and tail current amplitudes of hERG channel. Expression and functions of hERG are regulated by specific miRNAs.

Altered expression of interleukin-17A and its targeting microRNAs in oral lichen planus: a pilot study

To investigate the potential role of interleukin (IL)-17A and its targeting microRNAs (miRNAs) in oral lichen planus(OLP). We compared the IL-17A expression levels in biopsies from patients with OLP (n=50) against normal controls (n=19) by using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analyses, identified the targeting miRNAs of IL-17A, and examined the miRNA expression levels in OLP. IL-17A expression was significantly increased in patients with OLP compared with controls. The dual-luciferase reporter assay system demonstrated that miR-635 and miR-578 were the target miRNAs of IL-17A in human embryonic kidney 293cells, which was consistent with predictions from bioinformatics software analyses. In contrast with IL-17A, the expression levels of miR-635 and miR-578 in OLP were significantly decreased compared with controls. This pilot study found that low levels of miR-635 and miR-578 expression were associated with high expression of IL-17A, suggesting that IL-17A and its targeting miRNAs contribute to the pathogenesis of OLP.

MGMT DNA repair gene promoter/enhancer haplotypes alter transcription factor binding and gene expression.

The O6-methylguanine-DNA methyltransferase (MGMT) protein removes O6-alkyl-guanine adducts from DNA. MGMT expression can thus alter the sensitivity of cells and tissues to environmental and chemotherapeutic alkylating agents. Previously, we defined the haplotype structure encompassing single nucleotide polymorphisms (SNPs) in the MGMT promoter/enhancer (P/E) region and found that haplotypes, rather than individual SNPs, alter MGMT promoter activity. The exact mechanism(s) by which these haplotypes exert their effect on MGMT promoter activity is currently unknown, but we noted that many of the SNPs comprising the MGMT P/E haplotypes arelocated within or in close proximity to putative transcription factor binding sites. Thus, these haplotypes could potentiallyaffect transcription factor binding and, subsequently, alter MGMT promoter activity. In this study, we test the hypothesis that MGMT P/E haplotypes affect MGMT promoter activity by altering transcription factor (TF) binding to the P/E region. We used a promoter binding TF profiling array and a reporter assay to evaluate the effect of different P/E haplotypes on TF binding and MGMT expression, respectively. Our data revealed a significant difference in TF binding profiles between the different haplotypes evaluated. We identified TFs that consistently showed significant haplotype-dependent binding alterations (p≤0.01) and revealed their role in regulating MGMT expression using siRNAs and a dual-luciferase reporter assay system. The data generated support our hypothesis that promoter haplotypes alter the binding of TFs to the MGMT P/E and, subsequently, affect their regulatory function on MGMT promoter activity and expression level.

Functional and Activity Analysis of Cattle UCP3 Promoter with MRFs-Related Factors.

Uncoupling protein 3 (UCP3) is mainly expressed in muscle. It plays an important role in muscle, but less research on the regulation of cattle UCP3 has been performed. In order to elucidate whether cattle UCP3 can be regulated by muscle-related factors, deletion of cattle UCP3 promoter was amplified and cloned into pGL3-basic, pGL3-promoter and PEGFP-N3 vector, respectively, then transfected into C2C12 myoblasts cells and UCP3 promoter activity was measured using the dual-Luciferase reporter assay system. The results showed that there is some negative-regulatory element from −620 to −433 bp, and there is some positive-regulatory element between −433 and −385 bp. The fragment (1.08 kb) of UCP3 promoter was cotransfected with muscle-related transcription factor myogenic regulatory factors (MRFs) and myocyte-specific enhancer factor 2A (MEF2A). We found that UCP3 promoter could be upregulated by Myf5, Myf6 and MyoD and downregulated by MyoG and MEF2A.

A HAND2 Loss-of-Function Mutation Causes Familial Ventricular Septal Defect and Pulmonary Stenosis.

Congenital heart disease (CHD) is the most common developmental abnormality, and is the leading noninfectious cause of mortality in neonates. Increasing evidence demonstrates that genetic defects play an important role in the pathogenesis of CHD. However, CHD exhibits substantial heterogeneity, and the genetic determinants for CHD remain unknown in the overwhelming majority of cases. In the current study, the coding exons and flanking introns of the HAND2 gene, which encodes a basic helix-loop-helix transcription factor essential for normal cardiovascular development, were sequenced in 192 unrelated patients with CHD, and a novel heterozygous mutation, p.S65I, was identified in a patient with congenital ventricular septal defect (VSD). Genetic analysis of the index patient’s pedigree revealed that the mutation was present in all seven affected family members available, but absent in the 13 unaffected family members examined. Besides, in addition to VSD, five of the proband’s close relatives also had pulmonary stenosis (PS), and the proband’s son also had double outlet right ventricle (DORV). The missense mutation, which altered an evolutionarily conserved amino acid, was absent in 300 unrelated, ethnically matched healthy individuals. Biological analyses using a dual-luciferase reporter assay system showed that the mutant HAND2 was associated with significantly diminished transcriptional activity. Furthermore, the mutation abolished the synergistic activation between HAND2 and GATA4, as well as NKX2.5—two other cardiac core transcriptional factors that have been causally linked to CHD. These findings indicate that HAND2 loss-of-function mutation contributes to human CHD, perhaps via its interaction with GATA4 and NKX2.5.

Abstract WMP43: Microrna-210 Improves Long-term Outcome in the Ischemic Mice via La Regulation of BDNF

Objective: MicroRNAs played an important role in the ischemia brain injury and repair. However, the key microRNA and its function in the stroke patients remain unclear. Methods: The blood miRNAs profile was analyzed by quantitative Polymerase Chain Reaction (PCR) methods in ischemic stroke patients in the acute phase (48 hours) and subacute phase (10 days), compared to healthy controls with vascular risk factors. The predicted network of differences in the expression of miRNAs was further explored by Ingenuity Pathway Analysis (IPA). The key miRNA was selected and its function and mechanism were explored in ischemic mice subjected to lentivirus vector mediated overexpression. The predicted target was confirmed by the dual luciferase reporter assay system. Results: After cerebral ischemia, 24 microRNAs were high or low differently expressed in the peripheral blood of stroke patients. The bioinformatics analysis showed a regulated network of stroke cascade, which was consisted of 10 miRNAs including mir-210 and their predicted targets. The lentivirus vector mediated miR-210 overexpression increased microvessel density and the number of neural progenitor cells via regulating brain-derived neurotrophic factor (BDNF) expression in normal and ischemia mice brain. MiR-210 overexpression could ameliorate neurobehavioral outcomes in ischemic mice. The dual luciferase reporter assay system identified that BDNF was the direct target of miR-210. Conclusions: MiR-210 is a key stroke related miRNA. Overexpression of miR-210 promotes focal angiogenesis and neurogenesis via directely regulating BDNF expression. Furthermore, miR-210 improves long-term outcomes in ischemic mice and proved to be a potential novel therapeutic target for stroke.

Statin Function as an Anti-inflammation Therapy for Depression in Patients With Coronary Artery Disease by Downregulating Interleukin-1β.

It is well known that inflammation contributes to the development of coronary artery disease (CAD) and depressive symptoms. Previous studies have shown that long-term application of statin reduces the occurrence of depression in patients with CAD. However, the mechanism remains unclear. We hypothesized that inflammation contributes to depression in patients with CAD and statin function as an anti-inflammation therapy for those depressive patients. Patients with confirmed CAD hospitalized in the Department of Cardiology of Tongji Hospital in Shanghai, China, were enrolled. Depression was identified as none (ND), mild (MiD), moderate (MoD), or severe (SD) on the basis of scores of the patient health questionnaire with 9 items. Inflammatory factors in peripheral blood were measured using a chemiluminescence immunoassay and Bio-plex. Luciferase expression level was detected using the Dual-Luciferase Reporter Assay System for IL-1β or NF-κB expression by transfection in human umbilical vein endothelial cells, and patient serum was added. Data obtained from 217 patients with CAD were analyzed. The IL-1β level of CAD with SD was 14.70, which was significantly higher than that of CAD with ND 7.52, MiD 7.73, or MoD 8.63. Luciferase reporter gene analysis showed that IL-1β or NF-κB expression level was upregulated by the serum of CAD and depression patients. After the addition of atorvastatin, IL-1β or NF-κB luciferase reporter expression level decreased. It suggested that depression in patients with CAD is associated with inflammation. Statin may function as an anti-inflammation therapy for depression in patients with CAD by downregulation of IL-1β.

Promoter analysis of bovine cardiomyopathy-associated protein 1 gene.

The CMYA1 (cardiomyopathy-associated protein 1) is an actin-binding protein that plays a vital role in cardiac morphogenesis. CMYA1 is expressed specifically in the myocardial and skeletal muscle and is up-regulated in injured muscle. We therefore speculated that the bovine CMYA1 promoter might be muscle-specific. In this study, the promoter (+20/-1135) region of the bovine CMYA1 gene was cloned into a pEGFP-1 vector, and we found that the EGFP was observed only in C2C12 and myoblast cells. Thus, the CMYA1 promoter is muscle-specific. Thereafter, eight pGL3-basic vectors with various truncated CMYA1 promoter fragments were transfected into C2C12 cells, to identify the core promoter region using a Dual-Luciferase Reporter Assay System. The results showed that the promoter region from -457 to +20 bp was essential for CMYA1 to maintain the promoter activity, implying that this region may be the CMYA1 core promoter. We thus illustrate that the core promoter is muscle-specific. To evaluate the activity of the CMYA1 core promoter, the CMYA1 core and muscle creatine kinase (MCK) promoters were cloned into a pcDNA3.1 vector. The expression levels of their target genes were measured in C2C12 cells using real-time polymerase chain reaction. The CMYA1 promoter drove the expression of the target gene six times higher than did the MCK promoter. The results thus suggest that the CMYA1 promoter could be an effective muscle-specific promoter, which may be useful in further studies of cardiomyopathy treatment and transgenic animal research.

Maternal betaine supplementation during gestation modifies hippocampal expression of GR and its regulatory miRNAs in neonatal piglets.

Methyl donor nutrients are critical for embryonic development of brain. Hippocampus is the most susceptible brain region to various factors including prenatal supply of methyl donors. Glucocorticoid receptor (GR) expressed in hippocampus is involved in the regulation of energy homeostasis and stress sensitivity. Hippocampal GR expression is highly susceptible to epigenetic regulation, yet the effect of maternal methyl donor supplementation on epigenetic regulation of GR transcription in offspring hippocampus remains unclear. In this study, we fed sows with betaine (3 g/kg) throughout the gestation and analyzed the hippocampal expression of GR mRNA and its variants, as well as the CpG methylation status of the promoter and the microRNAs predicted to target 3’ UTR of porcine GR gene in neonatal piglets. Total GR mRNA (P<0.01) and its variants GR 1-4 (P<0.05) and 1-9,10 (P<0.01), were significantly higher in the hippocampus of betaine-treated piglets, while the content of GR protein was not significantly changed. The CpGs located in the –1650 ~ –1515 segment of GR gene were hypermethylated (P<0.05). The hippocampal expression of miR-130b (P<0.05), miR-181a (P<0.05) and miR-181d (P<0.01) was significantly up-regulated. The targeting efficacy of miR-130b and miR-181d was validated in vitro using dual-luciferase reporter assay system. Our results demonstrate that maternal betaine supplementation during gestation enhances GR mRNA expression in offspring hippocampus, which involves alterations in miRNAs expression.