MicroRNA-144 Regulates Cardiomyocyte Proliferation and Apoptosis by Targeting TBX1 through the JAK2/STAT1 Pathway.

It is currently believed that the TBX1 gene is one of the core genes of congenital heart disease (CHD). However, there are few studies on the abnormal regulation of TBX1 gene expression. The purpose of this work was to investigate the role of miR-144 and TBX1 in cardiac development by studying the regulatory relationship and mechanism of miR-144 on TBX1/JAK2/STAT1 in cardiomyocytes. Cell proliferation was detected by MTT and clone formation assay and cell cycle and apoptosis by flow cytometry. The levels of miR-144 and TBX1 in H9c2 cells were assessed by qRT-PCR. Dual luciferase reporter assay was used to validate the direct targeting of TBX1 with miR-144. The protein expression levels of TBX1 and its downstream proteins were measured by Western blot analysis. miR-144 inhibited H9c2 cell proliferation by arresting cells in G1 phase. Furthermore, miR-144 induced H9c2 cell apoptosis and activated the JAK2/STAT1 signaling pathway. Bioinformatic predictions and luciferase reporter assay showed that miR-144 directly targets TBX1. Co-overexpression of miR-144 and TBX1 upregulated cell proliferation by accelerating G1 to S phase transition and downregulated cell apoptosis through inhibiting the JAK2/STAT1 signaling pathway. miR-144 acts as a proliferation inhibitor in cardiomyocytes via the TBX1/JAK2/STAT1 axis and is therefore a potential novel therapeutic target for CHD treatment.

miR-873 suppresses H9C2 cardiomyocyte proliferation by targeting GLI1.

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that regulate the expression of target genes. Previous studies have suggested that miRNAs are key regulators in cardiovascular systems. This study investigated the role of miR-873 in H9C2 cardiomyocytes by targeting glioma-associated oncogene 1 (GLI1). miR-873 was significantly up-regulated in serum samples from congenital heart disease (CHD) patients compared with those from normal individuals. Furthermore, miR-873 over-expression suppressed H9C2 proliferation and induced cell cycle arrest. Bioinformatic algorithms revealed a predicted target site for miR-873 in the 3'-untranslated region (3'UTR) of GLI1, which was verified using a dual-luciferase reporter assay. qPCR and western blot analysis also showed that miR-873 negatively regulated GLI1 mRNA and protein expression in H9C2 cells. Conversely, GLI1 over-expression partially reversed the growth-inhibitory effect of miR-873. To summarize, our data suggest that miR-873 is a novel miRNA that regulates H9C2 cell proliferation via targeting GLI1, and miR-873 may serve as a new potential biomarker diagnosis in CHD in the future.

Overexpression of DICER1 induced by the upregulation of GATA1 contributes to the proliferation and apoptosis of leukemia cells.

Dicer, a member of the RNase III family, is the key enzyme required for the biogenesis of microRNAs and small interfering RNAs. Recent evidence indicates that DICER1 expression levels vary among different solid tumors and decreased or increased DICER1 expression has been associated with aggressive cancers. In this study, we assessed DICER1 expression levels in acute myeloid leukemia (AML) and investigated its biological effects and transcriptional regulation in leukemia cell lines. We demonstrated that DICER1 was overexpressed in AML patients and leukemia cell lines by real-time quantitative PCR and western blot analysis. A functional assay demonstrated that the silencing of DICER1 inhibited cell proliferation and promoted apoptosis in leukemia cell lines. We also demonstrated that DICER1 was upregulated by the hematopoietic transcription factor, GATA1, through luciferase, electrophoretic mobility shift and chromatin immunoprecipitation assays. These data suggest that DICER1 plays an important role in AML and the finding that the upregulation of DICER1 is induced by GATA1 may provide a framework for the understanding of differential DICER1 expression levels in multiple types of cancer.

Mutations and down-regulation of CDX1 in children with anorectal malformations.

BACKGROUND: Anorectal malformations (ARMs) represent a variety of congenital disorders that involve abnormal termination of the anorectum. This study was to reveal relation between CDX1 and human ARMs phenotypes. METHODS: 108 Chinese patients and 120 Chinese controls were included in this study. We analyzed the relation between two by PCR, qRT-PCR, western blot and immunofluorescence. RESULTS: Four heterozygous mutations in CDX1 gene were identified in ARMs patients (3.7%, 4/108), no found in controls. CDX1 protein expression was significantly decreased in the ARMs compared with the control anorectum. All samples analyzed in ARMs group exhibited down-regulated CDX1 mRNA expression in comparison to matched normal group, demonstrated significant differences statistically. CONCLUSION: The findings represented the relation between CDX1 mutations and CDX1 genotype. Furthermore, it was suggested that the downregulation of CDX1 might be related to the development of ARMs.

Curcumin induces small cell lung cancer NCI-H446 cell apoptosis via the reactive oxygen species-mediated mitochondrial pathway and not the cell death receptor pathway.

Curcumin (diferuloylmethane), an active component of the spice turmeric, induces apoptosis in several types of malignancies. However, little is known about its anticancer activity in small cell lung cancer (SCLC). SCLC represents a highly malignant and particularly aggressive form of cancer, with early and widespread metastases and a poor prognosis. In this study, we found that curcumin does not activate caspase-8 cleavage or alter the expression of apoptotic receptors FAS and TRAIL in NCI-H446 cells, suggesting that curcumin-induced apoptosis is not associated with death receptor-mediated pathways in these cells. Instead, curcumin caused apoptosis by increasing Bax expression while decreasing the expression of Bcl-2 and Bcl-xL. Curcumin induced a rapid decrease in mitochondrial membrane potential and the release of cytochrome c into the cytosol, followed by activation of caspase-9 and caspase-3. In addition, curcumin-induced apoptosis was accompanied by an increase of intracellular reactive oxygen species (ROS) level. These results indicated that a ROS-mediated mitochondrial pathway played an important role in the process of curcumin-induced apoptosis of human SCLC NCI-H446 cells.

Reduced ACTC1 expression might play a role in the onset of congenital heart disease by inducing cardiomyocyte apoptosis.

BACKGROUND: The Cardiac α actin 1 gene (ACTC1) has been related to familial atrial septal defects. This study was set to explore a potential role of this gene in the formation of sporadic congenital heart disease (CHD). METHODS AND RESULTS: Assessment of cardiac tissue samples from 33 patients with sporadic CHD (gestational age (GA) 18 weeks-49 months) with real-time RT-PCR, Western blotting and immunohistochemistry has revealed a markedly decreased ACTC1 expression in the majority of samples (78.8%) compared with autopsied normal heart tissue from aged-matched subjects (GA 17 weeks-36 months). Also, as shown by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, the proportion of apoptotic cardiomyocytes in samples featuring down-regulated ACTC1 expression (Group 1) was significantly greater than those with normal expression (Group 2) and the controls (P<0.01). The proportion of apoptotic cells strongly correlated with the expression of ACTC1 (r=-0.918, P<0.01). A study of 2 essential genes involved in apoptosis, Caspase-3 and Bcl-2, confirmed that the former has significantly increased expression, whilst the latter has decreased expression in Group 1 than in the other groups (P<0.01). Transfection of a small interfering RNA targeting, Actc1 (Actc1-siRNA), to a cardiomyocyte cell line, H9C2, also detected more apoptotic cells. CONCLUSIONS: Reduced ACTC1 expression might play a role in the onset of CHD through induction of cardiomyocyte apoptosis.

[Teaching experience in integrated course of human development and genetics].

Establishment of integrated course system in human development and genetics is an important part of course reformation, and the improvement of this system is achieved by integrating the content of course, stabilizing teaching force, building teaching materials and applying problem-based learning. Integrity-PBL teaching model is founded and proved to be feasible and effective by teaching practice. Therefore, it maybe play an important role in improving teaching effect and cultivating ability of students to analyse and solve problems.

Transcriptional regulation of Fhl1 by estradiol in rat myoblastocytes.

Fhl1 (Four and a Half LIM domain 1) regulates muscle growth and development. In addition, skeletal myoblast growth is significantly affected by gender differences, implicating estrogen in the regulation of muscle development. We sought to determine if estrogen influences Fhl1 gene expression levels in rat L6GNR4 myoblastocytes that express the estrogen receptor beta (ERbeta), while luciferase assay, electrophoretic mobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) assay were employed to confirm the interaction between ERbeta and Fhl1. Treatment of L6GNR4 cells with physiological levels of 17beta-estradiol (E2) results in markedly decreased endogenous Fhl1 expression. Tamoxifen, an ER antagonist, partially reverses E2-mediated Fhl1 down-regulation in L6GNR4 cells. Furthermore, luciferase assay and EMSA identified a novel promoter region of Fhl1 that directly interacts with ERbeta. ChIP of the ERbeta-Fhl1 promoter complex from L6GNR4 cells confirmed that endogenous ERbeta interacts with this region. These data indicate that E2 down-regulates Fhl1 expression through its binding to the ERbeta. This is the first report of a small molecule that can affect Fhl1 expression. E2 may therefore be useful in developing new strategies for regulating Fhl1 expression and understanding the influence of estrogen on muscle growth and development.

[The mechanism of TBX5 abnormal expression in simple congenital heart disease].

To explore the mechanism of TBX5 abnormal expression in simple congenital heart disease (CHD), 100 CHD venous blood, 50 CHD heart tissues, and 5 non-CHD heart tissues were involved in this study. The mutation and methylation in the 1 200 bp region upstream of TBX5 gene were detected by high-performance liquid chromatography (DHPLC) and methylation-sensitive restriction endonuclease (MS-RE), respectively. The binding site of NKX2-5 to Tbx5 predicted by P-MATCH software was validated by EMSA (Electrophoretic mobility shift assay). Tbx5 gene expression in mouse cardiac muscle cell H9C2(2-1) transfected with NKX2-5 expression vector was evaluated. No mutation was found in all patients. Both non-CHD and CHD heart tissues had the same methylation in the two CpG islands. Exogenous Nkx2-5 efficiently activated the transcription of the endogenous Tbx5 gene in H9C2 (2-1) cells. EMSA showed that the special binding band appeared when Nkx2-5 existed. These results indicates that the down expression of TBX5 might not be caused by mutation and methylation in the 1 200 bp region upstream of gene, and might be regulated by abnormal expression of NKX2-5 gene in heart muscle of CHD.

[Effect of B-RAF-specific RNA interference on gastric cancer BGC823 cell line].

To investigate the influence of B-RAF-specific RNA interference on the proliferation and apoptosis of gastric cancer BGC823 cell line. B-RAF-siRNA and scramble-siRNA were synthesized and transfected into BGC823 cells by TransMessenger. The expression of B-RAF gene and Bcl-2 gene in BGC823 cells was detected by RT-PCR and the level of apoptosis was evaluated in transfected cells by flow cytometry. Results showed that TransMessenger could effectively transfect B-RAF-siRNA and scramble-siRNA into BGC823 cells. B-RAF-siRNA significantly inhibited the expression of B-RAF gene and Bcl-2 gene in BGC823 cells by more than 90% to 100%. B-RAF-siRNA inhibited BGC823 cell prolifera-tion and induced apoptosis (P < 0.01). In conclusion, B-RAF-siRNA can effectively inhibit the expression of B-RAF gene and Bcl-2 gene, induce cell apoptosis and inhibit the proliferation of gastric cancer BGC823 cells.

Association of the GLI gene with ventricular septal defect after the susceptibility gene being narrowed to 3.56 cM in 12q13.

BACKGROUND: Our previous research has suggested that genes around D12S1056 in 12q13 may confer susceptibility to ventricular septal defect (VSD) in humans. The present study was to define the chromosome region assignment by transmission disequilibrium test (TDT), and to identify the important candidate gene by family-based association study and haplotype analysis. METHODS: Surrounding D12S1056, ten microsatellite markers including D12S329, D12S305, D12S1662, D12S1056, D12S1293, D12S334, D12S102, D12S83, D12S1655 and D12S1691 were chosen, and TDT was performed in 62 nuclear family trios each consisting of an affected child and two healty parents. Subsequently, the GLI gene, a positional candidate gene that maps to the target region, was selected for further analysis. Three single nucleotide polymorphisms (SNPs), G11888C, G11388A, and G11625T, were selected for family-based association study and haplotype analysis. RESULTS: VSD was significantly associated with all selected markers except D12S1691 [72.2 centi morgen (cM)] and D12S1700 (75.76 cM). VSD was also significantly associated with G11888C (chi(2) = 5.918, P = 0.015), G11388A (chi(2) = 8.067, P = 0.005), and G11625T (chi(2) = 11.842, P = 0.001). Haplotype analysis showed a strong linkage disequilibrium between G11888C and G11388A (D' = 0.999), but in significant (chi(2) = 1.035, df = 2, P > 0.05). CONCLUSIONS: The susceptibility gene of VSD was mapped to 3.56 cM in 12q13 by TDT, and the GLI gene, an important candidate in the target region, was associated with VSD.

Cloning of TBX5, a key gene during heart formation and its expression in rat embryonic heart.

To study the expression of TBX5, a key gene during heart formation, full-length TBX5 cDNA was cloned from Wistar rat embryonic heart (GenBank Accession No. AY859491). The cDNA and predicted amino acid sequences were different from those previously reported in GenBank. The expression profile of TBX5 in rat tissues was studied by RT-PCR and Northern blot. TBX5 was expressed in many rat tissues as a single transcript, and the highest level of expression was found in the heart. For subcellular localization, TBX5 was expressed as an EGFP fusion protein in rat hepatocarcinoma cells. Results showed that TBX5 was nuclear. In addition, TBX5-GST fusion protein was obtained by prokaryotic expression. These findings provide a good basis for further identification of TBX5-related transcription factors and protein-protein interaction studies among each other.

Analysis of single nucleotide polymorphisms and haplotypes in HOXC gene cluster within susceptible region 12q13 of simple congenital heart disease.

OBJECTIVE: In the candidate region 12q13 of simple congenital heart disease(CHD), four single nucleotide polymorphisms(SNPs) in HOXC4 gene were chosen in order to investigate the distribution of SNP and haplotypes in simple CHD patients and normal people. METHODS: The genotype of 4 SNPs in 108 simple CHD patients and 200 normal people were analyzed by restriction fragment length polymorphism(RFLP) and denaturing high-performance liquid chromatography(DHPLC). The statistical contingency table method was used to analyze SNP genotype frequency and gene frequency in patients and control group; then, the haplotypes were established and their frequencies in the two groups were assessed by PHASE software. RESULTS: C16476T polymorphism was not detected; A17860G located in 3' flanking sequence of HOXC5 gene displayed significant difference between the two groups. The G allele frequency in simple CHD patients was higher than that in healthy controls(P < 0.05); the distribution of frequencies of 4 haplotypes showed significant difference(P < 0.01). CONCLUSION: The A17860G located in 3'flanking sequence of HOXC5 gene is associated with simple CHD; the risk of CHD in the persons with G17860 is higher than that in those with A17860. the haplotype of 3 SNPs may be linked with the susceptible gene of simple CHD.

[Analysis and application of SCA1 and SCA3/MJD gene CAG repeats in Han population in Northeastern China].

OBJECTIVE: To investigate the normal range of (CAG)n in spinocerebellar ataxia type 1 (SCA1) gene and spinocerebellar ataxia type 3 (SCA3/MJD) gene in 110 normal subjects of Han population in Northeastern China, to assess the genotypes for clinically diagnosed spinocerebellar ataxia(SCA) individuals including 25 patients from 8 families and 6 sporadic patients, and to make presymptomatic and prenatal diagnosis. METHODS: DNA fragments from the normal subjects and the patients were detected by fluorescence-PCR. Homozygosities were selected for DNA sequencing. RESULTS: The normal ranges of (CAG)n of SCA1 and SCA3/MJD were 20-39 and 14-38 repeats respectively, SCA1 was found mostly to be 26 and 27 repeats, allele frequency 34.09% and 20.91%; heterozygosity was 84.55%, SCA3/MJD was found mostly to be 14 repeats, allele frequency 39.55%, heterozygosity was 78.18%.(CAG)(68) of SCA3/MJD gene of one affected individual had been found in a family but no CAG mutative expansion in related members was observed. CONCLUSION: The normal ranges of CAG repeats vary with areas and races. SCAs genotyping is the first choice in presymptomatic and prenatal diagnosis.

[Analysis on genetic polymorphism of 14 short tandem repeat loci on chromosome 7p14-15 and 12q13 in Chinese north Hans].

OBJECTIVE: To analyze the genetic polymorphism of 6 short tandem repeat (STR) loci on chromosome 7p14-15 and 8 STR loci on chromosome 12q13 in Chinese north Hans. METHODS: Fluorescence-labeling polymerase chain reaction and capillary electrophoresis were used to analyze the genetic polymorphism of 100 randomly selected individuals from Chinese north Han nationality at 6 STR loci (D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528) on chromosome 7p14-15 and 8 STR loci(D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102) on chromosome 12q13. RESULTS: In the Chinese north Han population, 7 alleles and 24 genotypes, 8 alleles and 27 genotypes, 7 alleles and 22 genotypes, 4 alleles and 10 genotypes, 6 alleles and 17 genotypes, 5 alleles and 13 genotypes were observed at D7S1808, D7S2250, D7S2251, D7S683, D7S656 and D7S528. The heterozygosities at the above 6 STR loci were 86%, 88%, 83%, 79%, 85% and 80%, respectively. Five alleles and 15 genotypes, 5 alleles and 15 genotypes, 8 alleles and 29 genotypes, 6 alleles and 17 genotypes, 6 alleles and 17 genotypes, 6 alleles and 19 genotypes, 5 alleles and 13 genotypes, 7 alleles and 24 genotypes were observed at D12S1056, D12S1293, D12S83, D12S1655, D12S1662, D12S334, D12S137 and D12S102. The heterozygosities at the above 8 STR loci were 86%, 84%, 87%, 82%, 84%, 85%, 81% and 89%, respectively. CONCLUSION: The distributions of allele frequencies of 6 STR loci on chromosome 7p14-15 and of 8 STR loci on chromosome 12q13 were consistent with the Hardy-Weinberg equilibrium. The highly genetic polymorphism was observed in Chinese north Han population.

[Studies on the mutation and expression of TBX5 gene in human simple congenital heart disease].

This work is to investigate the mutation and expression of TBX5 gene in human simple congenital heart disease. The mutations of eight exons of TBX5 gene in 61 CHD family members (a total of 216 individuals including 65 patients and 151 normal relatives) were examined by PCR-DGGE. Using beta-actin as internal control, the differential expression between 34 myocardium samples from simple congenital heart disease patients and three normal controls was conducted by RT-PCR. There is no mutation detected in all samples; The mRNA expression levels of TBX5 gene show descent tendency in samples of simple congenital heart disease compared with normal controls. The mutations in coding region of TBX5 gene do not cause human simple congenital heart disease, but the abnormality in transcription level of TBX5 gene maybe a kind of mechanism causing human simple congenital heart disease.