Objective To understand the relationship between GATA -4,-5,-6 gene mutations and con-genital heart disease(CHD),and to provide grounds for early prevention and genetic counseling of children with CHD. Methods GATA -4,-5,-6 coding regions exons and the flanking intron sequences in 1 98 CHD patients were screened,including 66 cases of the ventricular septal defects,84 cases of the atrial septal defects,and 48 cases of the nonsyndromic conotruncal heart defects patients.A total of 300 healthy subjects were selected as controls.The acquired sequences were aligned with which those publicized in GenBank by the aid of program BLAST.All exons and bilateral partial intron -exon boundaries of GATA -4,-5,-6 genes were amplified by the polymerase chain reaction (PCR). The PCR products were purified and directly sequenced by automatic DNA sequence equipment.The acquired GATA -4,-5,-6 gene sequences were compared with GenBank standard gene sequences with the aid of program BLAST. Results A heterozygous missense mutation in the GATA -4 gene was identified in a ventricular septal defect patient and a persistent truncus arteriosus patient.The mutation was located in c.799G >A(p.V267M)in exon 4 of GATA -4. Multiple aligenment of GATA -4 proteins across species demonstrated that altered amino acid was highly conserved. Transcription factor GATA -5,-6 screening showed no mutations in children with CHD in this study.Conclusions Transcription factor GATA -4 gene mutation may be associated with the occurrence of CHD.Transcription factor GATA -4 gene may be susceptible gene in human CHD.

Objective: To explore the molecular and genetic mechanism of transcription factor GATA-6 in nonsyndromic conotruncal defect (CTD) in order to provide evidence for early prevention and inheritance consultation of CTD. Methods: A total of 32 cases of patients with nonsyndromic CTD and 100 healthy individuals were enrolled in the study.A total of 7 exons and bilateral partial intron-exon boundaries of GATA-6 were amplified by means of polymerase chain reaction (PCR). The PCR products were purified and directly sequenced by using an ABI Genetic Analyzer 3100 Automatic DNA sequence equipment.The acquired GATA-6 gene sequence was compared with standard gene sequence published in National Center for Biotechnology Information database, as well as the healthy control group to observe the GATA-6 gene mutations.The mutations were introduced into pcDNA3.1(+ ) by site-directed mutagenesis PCR on the basis of pcDNA3.1(+ )-GATA-6 in order to generate the GATA6-G245R mutant constructs.Wild type GATA-6, GATA-6-G245R and atrial natriuretic factor-luciferase(ANF-luciferase) were cotransfected into HEK 293T cells in vitro, and the CMV-LacZ were cotransfected as internal reference.Luciferase and galactosidase activity were measured by using luminometer 24 h after transfection and detected in the downstream ANF-luciferase reporter gene. Results: A heterozygous missense mutation in the GATA-6 gene was identified in a patient with double outlets of the right ventricle.The mutation was located in Gly245Arg(G245R) in exon 2 of GATA-6.The mutation of pcDNA3.1(+ )-GATA-6 expression vectors were successfully constructed.Through the detection of luciferase reporter gene activity, it was found that GATA-6-G245R and wild-type GATA-6 decreased by 41.3%, and the comparison between them was statistically significant (P<0.001). Conclusions Transcription factor GATA-6 gene mutation is associated with the occurrence of nonsyndromic CTD.Transcription factor GATA-6 gene may be susceptible gene in human nonsyndromic CTD.

Objective: To explore the action mechanism of miR-139-5p inhibiting proliferation and invasion of epithelial ovarian cancer (EOC) cells by targetedly regulatingNotch1.Methods: A total of 24 pairs of EOC tissues and its corresponding para-cancerous tissues from patients, who underwent surgical resection in the DepartmentofGynecology,Nanyang Central Hospital of Henan Province, were collected for this study; in addition, human ovarian cancer cell lines (SKOV3, ES2, HEY-T30) and human ovarian epithelial cell line IOSE80 were also collected. Real-time quantitative PCR (qPCR) was applied to detectmRNAexpressionofmiR-139-5pandNotch1 in EOC tissues and cell lines. The miR-139-5p over-expression vector and recombinant plasmid pLV-Notch1 were transfected into SKOV3 cells. Blank control group (Ctrl group) and negative control group (NC group) were set up. Dual luciferase reporter gene assay was applied to verify the targeting relationship between miR-139-5p and Notch1 3'-UTR. CCK-8, Transwell and Scratch healing experiments were applied to detect cell proliferationinvasionandmigration, respectively. Western blotting was applied to detect expressions of proliferation and migration related proteins in cells. Results: Compared with para-cancerous tissues and IOSE80 cells, the expression of miR-139-5p was significantly decreased in EOC tissues and cell lines, while the expression of Notch1 mRNA was significantly increased (all P<0.01). The results of Dual luciferase reporter showed that Notch1 was the downstream target gene of miR-139-5p. Compared with NC group, cell proliferation, invasion and migration ability, expression levels of Notch1, NICD, Cyclin D1, Cyclin A1, Snail1, β-catenin and N-cadherin were all significantly decreased on 3 d in miR-139-5p mimic group (all P<0.01), while expression of E-cadherin was significantly increased (P<0.01); meanwhile, over-expression of Notch1 could reverse the inhibitory effect of miR-1395p on proliferation, invasion and migration of SKOV3 cells. Conclusion: miR-139-5p can targetedly regulate Notch1 to inhibit proliferation, invasion and migration of EOC cells, which may be related to its down-regulation of NICD, Cyclin D1, Cyclin A1, Snail1, βcatenin and N-cadherin, and up-regulation of E-cadherin.

ObjectiveMetabolomics was used to reveal the mechanism of Aconiti Lateralis Radix Praeparata（ALRP） in attenuating toxicity by processing from the aspects of amino acid metabolism， oxidative stress and energy metabolism by analyzing multiple metabolic pathways. MethodTwenty-four rats were randomly divided into control group， raw group and processed group， 8 rats in each group. The raw and processed group were given with 0.64 g·kg^-1 of raw ALRP and processed ALRP respectively every day， the control group was given with an equal amount of normal saline once a day. After continuous administration for 7 days， the urine， serum and heart tissue of rats were collected. Pathological examination of the heart was carried out using hematoxylin-eosin（HE） staining， and the activities of lactate dehydrogenase（LDH） and creatine kinase-MB（CK-MB） in serum and cardiac tissues were detected by microplate assay and immunoinhibition assay. The effects of ALRP on rat heart before and after processing were compared and analyzed. Ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS） was used to perform urine metabolomics analysis， and multivariate statistical analysis was used to screen for differential metabolites related to ALRP in attenuating toxicity by processing， and pathway enrichment analysis was carried out to explore the processing mechanism. ResultHE staining showed that no obvious pathological changes were observed in the heart tissue of the control group， while obvious infiltration of inflammatory cells such as plasma cells and granulocytes was observed in the heart tissue of the raw group， indicating that the raw ALRP had strong cardiotoxicity. There was no significant difference in HE staining of heart tissue between the processed group and the control group， indicating that the toxicity of ALRP was significantly reduced after processing. Compared with the control group， the activities of LDH and CK-MB were significantly increased in serum and heart tissue of the raw group， and those were significantly decreased in serum and heart tissue of the processed group， suggesting that the myocardial toxicity of processed ALRP was reduced. A total of 108 endogenous differential metabolites associated with the raw ALRP were screened using multivariate statistical analysis in positive and negative modes， of which 51 differential metabolites were back-regulated by the processed ALRP. Biological analysis of the key regulatory pathways and associated network changes showed that the pathways related to toxicity of ALRP mainly included tryptophan metabolism， arginine and proline metabolism， phenylalanine metabolism， aminoacyl-tRNA biosynthesis， alanine， aspartate and glutamate metabolism， etc. The metabolic pathways related to the attenuation of processed ALRP mainly included aminoacyl-tRNA biosynthesis， tryptophan metabolism， phenylalanine， tyrosine and tryptophan biosynthesis， phenylalanine metabolism and caffeine metabolism. ConclusionThe processing technology of ALRP in Guilingji can significantly attenuate the cardiotoxicity of raw products， the mechanism mainly involves amino acid metabolism， oxidative stress and energy metabolism， which can provide experimental bases for the research related to the mechanism of toxicity reduction of ALRP by processing and its clinical safety applications.