Construction of Pax-8 gene recombinant plasmid and study of its function

AIM: To investigate the effects of over-expression of Pax-8 gene on the proliferation and apoptosis of H9c2 cells(a cardiomyocyte cell line). METHODS: The full length of rat Pax-8 gene was restrictively digested by Kpn I and Not I from the pCMV sport6-Pax-8 vector, and then inserted into the eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)-Pax-8 was confirmed by restriction endonuclease digestion and sequencing. The pcDNA3.1(+)-Pax-8 was transfected into H9c2 cells. The expression of Pax-8 at mRNA and protein levels was identified after transfection by RT-PCR and Western blotting. The cell proliferation was measured by CCK-8. Cell apoptosis was induced by serum deprivation in H9c2 cells transfected with Pax-8 gene. The apoptosis rate of the cells was determined by flow cytometry with annexin V-FITC and propidium iodide double staining. The protein expression of activated caspase-3 was measured by Western blotting. RESULTS: The full length of Pax-8 gene was successfully cloned into pcDNA3.1(+) expression vector and over-expression of Pax-8 at mRNA and protein levels was observed in H9c2 cells transfected with Pax-8 gene as compared to the wild-type cells and the cells transfected with an empty vector (both P<0.05). Transfection of Pax-8 gene promoted the proliferation of the cardiomyocytes (P<0.05) and inhibited the apoptosis rates induced by serum deprivation (P<0.01). The expression level of activated caspase-3 was increased by serum deprivation and attenuated by Pax-8 transfection (P<0.01). CONCLUSION: The pcDNA3.1(+)-Pax-8 expression vector was successfully constructed and over-expression of Pax-8 gene in cardiomyocytes is obtained. Pax-8 gene acts as an anti-apoptotic factor in cardiomyocytes by promoting cell proliferation and inhibiting apoptosis.     

Pivotal role of microRNAs in cardiac development and heart diseases

MicroRNAs (miRNAs) are non-coding small RNAs, which bind to the 3'-UTR of target mRNAs and negatively regulate the gene expression. Accumulating evidence demonstrates that miRNAs are involved in many biological processes such as embryo development, cell proliferation, differentiation, apoptosis and tumorigenesis. Heart development and heart diseases are complex processes controlled by various signaling pathways. Recent researches indicate the importance of miRNAs in the process of cardiac development and heart diseases. In this review, the role of miRNAs in cardiac development and the pathogenesis of heart diseases are overviewed. The insight into the regulating miRNAs will significantly expand the cardiovascular therapeutic strategies beyond classical pharmacology.     

Expression of short-chain acyl-CoA dehydrogenase during heart development in rats and relationship with cardiac hypertrophy

AIM： To investigate the expression of short-chain acyl-CoA dehydrogenase during the heart deve-lopment in rats and to analyze the relationship between short-chain acyl-CoA dehydrogenase and cardiac hypertrophy in spontaneously hypertensive rats (SHR). METHODS：The expression and activity of short-chain acyl-CoA dehydrogenase in the hearts of Wistar rats with different ages were measured. Free fatty acids in serum and cardiac muscles were also determined. RESULTS：Compared with the fetal rats of 19 d, the expression and activity of short-chain acyl-CoA dehydrogenase in the postnatal rats of 1 d, 2 weeks, 6 weeks and 16 weeks were increased, and free fatty acids in the serum and myocardium were obviously decreased. The difference began in evidence from the age of 2 weeks. The expression of short-chain acyl-CoA dehydrogenase was significantly up-regulated with negative correlation to free fatty acids in the serum and myocardium during heart development. Systolic blood pressure was similar in 2-week-old SHR and WKY rats, which significantly increased in SHR of 6 weeks and 16 weeks old compared with the age-matched WKY rats. The ratio of left ventricular weight to body weight was markedly elevated in SHR of 2 weeks, 6 weeks and 16 weeks old compared with the age-matched WKY rats, indicating that the appearance of cardiac hypertrophy occurred before the development of hypertension in SHR. Compared with the age-matched WKY rats, the expression and activity of short-chain acyl-CoA dehydrogenase were decreased and free fatty acids in the serum and myocardium were obviously higher in SHR. The expression of short-chain acyl-CoA dehydrogenase was significantly down-regulated with a negative correlation to free fatty acids in the serum and myocardium of SHR. CONCLUSION：The expression of short-chain acyl-CoA dehydrogenase is increased during the heart development, which may be associated with the increase in cardiac fatty acid utilization. The down-regulated expression of short-chain acyl-CoA dehydrogenase in the hypertrophic heart may be responsible for the recapitulation of fetal energy metabolism.     

Progress of embryonic stem cells during directional differentiation regulated by epigenetic modification

Embryonic stem cells undergo extensive self-renewal and have the capacity to differentiate along multiple cell lineages. Research on totipotency and directional differentiation of embryonic stem cells in order to treat intractable disease, such as cancer, heart failure, atherosclerosis by tissue regeneration and cell transplantation are investigated. Epigenetic modification, including DNA methylation, chromatin restructure, and non-coding RNA-mediated regulatory events, regulate the differentiation of embryonic stem cells without detectable genetic changes. These mechanisms are often associated with starting-up and maintenance of epigenetic silence. The achievement and focuses on the molecular mechanism of embryonic stem cells during directional differentiation regulated by epigenetic modification are reviewed.     

西瓜果实发育过程中转录组的初析

<正>目的与意义:目前关于西瓜果实在分子水平上发育和成熟的信息非常少,栽培西瓜是一种非呼吸越变型水果。到目前为止,只有一份报告描述了西瓜果实发育过程中一小部分基因的表达,对西瓜进行了大规模的转录组测序,以期对西瓜果实发育过程中的分子基础有更多了解。     

Relationship between MAP-2, nestin and development of muscular layer of human embryonic esophagus

AIM: To explore the relationship between microtubule-associated protein 2 (MAP-2), nestin and development of esophageal muscular layer in human embryos. METHODS: At the second to fourth month of gestation, the protein expression of MAP-2 and nestin was observed using the PV method of immunohistochemistry in the human embryonic esophageal tissues. RESULTS: At the second month, the third month and the fourth month of gestation, MAP-2 positive expression in the neural cells of the intermuscular nerve plexus, and negative expression in the muscle cells of human embryonic developing esophageal muscle were observed. Nestin positive expression was found in the muscle cells at the second month to the third month of gestation. However, no nestin expression in the muscle cells of the esophageal muscle at the fourth month of gestation was observed. At the second month to the fourth month of gestation, nestin showed negative or weak positive expression in the neural cells of the intermuscular nerve plexus of human embryonic esophageal tissues. CONCLUSION: MAP-2 and nestin might regulate the formation of esophageal muscular layer in human embryos.     

授粉对板栗胚胎细胞发育的影响

【目的】通过研究板栗花粉直感效应对胚胎发育的影响,为下一步开展板栗杂交育种提供理论基础。【方法】以云南板栗品种‘云富’和‘云良’,北方板栗品种‘燕山红’及‘燕龙’4个板栗品种为材料,通过控制授粉实验,观察不同授粉组合胚胎发育情况。【结果】在同一正交组合中,以果实成熟早的品种的花粉授粉的胚胎发育也早。不同授粉组合在不同时期胚乳核细胞的横纵径差异皆达显著。不同组合同一发育时期子叶细胞横纵径差异不大,且子叶细胞横纵径的发育进程与成熟时坚果的大小无明显的线性关系。同一母本的授粉组合中,杂交授粉的板栗坚果大小均大于自交授粉。【结论】板栗杂交优势明显,在生产实践中可用早熟品种进行授粉使果实成熟早,还可以利用南北方果实口感及大小来改良果实品质,提高板栗整体的经济价值。     

Effect of IL-8 on mitochondrial fusion and fission gene expression in human lung adenocarcinoma cell line A549 during cell migration

AIM: To investigate the changes of migration of lung adenocarcinoma cells promoted by IL-8 and the inner and outer mitochondrial membrane dynamic changes during this process.METHODS: Human lung adenocarcinoma cell line A549 was divided into control group and IL-8 group. Cell migration was analyzed by scratch detection and Transwell assay. The secretion of endogenous IL-8 was detected by ELISA. The protein levels of mitochondrial cytochrome C (Cyt C) and mitochondrial outer membrane protein Tom20 was detected by Western blot. The mRNA expression of mitochondrial fusion genes Mfn1, Mfn2 and OPA1 and fission genes Fis1, Drp1 and MTP18 was detected by RT-PCR. The morphological changes of mitochondria were observed by MitoTracker Red CMXRos dye staining and confocal microscopy.RESULTS: The migratory rate of A549 cells and endogenous secretion of IL-8 in A549 cells were higher than those in SPC-A-1 cells. The migratory rate of A549 cells was improved by IL-8 in a time-dependent manner. Compared with control group, the Tom20 protein expression was increased (P<0.05), and the Cyt C protein expression was decreased (P<0.05). The expression of mitochondrial outer membrane fusion genes Mfn1 and Mfn2 was increased (P<0.05), and the expression of mitochondrial inner membrane fusion gene OPA1 was decreased (P<0.05). The expression of fission genes Drp1 and MTP18 were decreased (P<0.05), while the expression of Fis1 was no change (P>0.05). Under confocal microscope, the punctate aggregates in the mitochondria of the A549 cells treated with IL-8 were observed.CONCLUSION: The migratory rate of A549 cells is increased by IL-8, which is related to the changes of mitochondrial fusion genes and the fission genes.     

Expression level of JT8 gene decreased in coronary artery disease

AIM: To confirm the expression level of the gene which corresponding to JT8 tag decreased in coronary artery disease (CAD). METHODS: The validity and reliability of the results gotten by serial analysis of gene expression method was verified by RT-PCR and Northern blot. The expression level of the gene which corresponding to JT8 tag was compared with the expression level of GAPDH and β-actin in JT and WY, while according to SAGE results that the gene expression level of JT8 gene was 8 times higher in JT than in WY. RESULTS: It was found that the results of RT-PCR and Northern bolt were identified with the results of SAGE. The expression level of JT8 gene decreased in CAD. CONCLUSION: These results verified the validity of SAGE method and made a good foundation for further discovery of new candidate genes.     

Experimental study of a new hypertension-related gene Slc7a8

AIM: To investigate the disease related genes in SHR.METHODS: The total RNA samples were obtained from second-order mesenteric arteries and kidney of SHR and WKY.Microarray containing over 10 000 genes was used to determine the level of mRNA expression in two groups.The genes were identified using real time quantitative RT- PCR.RESULTS: 19 down-regulated genes were determined by microarray,which were classified as chaperones,transport,growth factors,signal transduction,nuclear factor and lipoprotein.The result was confirmed by the method of real time quantitative RT- PCR.It was found that the Slc7a8 gene was up-regulated 9.3 fold in SHR.CONCLUSION: Slc7a8 gene may relate to hypertension.Further study on the Slc7a8 gene and its function would help us wholly understand the mechanism of hypertension and provide new clue to hypertension causes.     

Function of Shh gene in the development of embryos

Mammals have three genes with homology to the hh gene. These comprise Sonic hedgehog (Shh), Indian hedgehog (Ihh), and Desert hedgehog (Dhh). Shh has been shown to play a crucial role in embryogenesis and the development of ectoderm. Shh has the great relationship with the forming all the system. Shh-Patched-Smoothened signal pathway will lead the embryogenesis in the normal way. Otherwise, the abnormal embryogenesis, malformation, and tumor will be happened.     

Construction of mouse pdx-1 gene eukaryotic expression vector and its expression in embryonic stem cells

AIM: To clone mouse pdx-1 gene and construct its eukaryotic expression vector for expression of pdx-1 in mouse embryonic stem cells.METHODS: Mouse pdx-1 cDNA fragment was amplified with polymerase chain reaction (PCR) from mouse pancreatic cDNA. The purified fragment was recombinated with a eukaryotic expression vector carrying enhanced green fluorescent protein, pEGFP-N1. The pdx-1 cDNA fragment was inserted into the multi-clone sites of the vector to construct a new plasmid, pEGFP/pdx-1. E.colli strain DH5α was transfected with the new recombinant plasmid to expand it. Plasmid DNA extracted from the expanded DH5α was identifed by cutting with Hind Ⅲ, BamHⅠ nuclease and by DNA sequencing. Identified plasmid DNA was transfected into mouse embryonic stem cell line MESPU13 by carrying with liposome. RESULTS: A 876 bp cDNA fragment was amplified from mouse pancreatic cDNA by PCR and it was inserted into the vector pEGFP-N1 correctly. The fragment was defined to be pdx-1 gene by nuclease digestion and DNA sequencing. Mouse embryonic stem cell line MESPU13 was transfected with the new recombinant plasmid DNA. The green fluorescent protein report gene and pdx-1 gene expressed in transfected mouse embryonic stem cells within 24 h. CONCLUSION: Mouse pdx-1 gene is cloned and its recombinant eukaryotic expression vector carrying green fluorescent protein is constructed successfully. It provides a useful tool for further research on the function of pdx-1.     

Effects of rs35100176 polymorphism in IPO8 promoter on gene expression

AIM: To investigate the effect of rs35100176 CCT insertion/deletion polymorphism in the promoter region of importin 8 (IPO8) gene on its mRNA expression. METHODS: A 342-bp fragment of IPO8 gene promoter containing the rs35100176 polymorphism was amplified from 49 DNA samples and sequenced. The IPO8 promoter fragments containing CCT 3-nucleotide insertion or deletion were amplified using the corresponding homozygote DNA samples. The PCR products were sequenced and inserted into the luciferase reporter vector pGL3-Basic. Recombinant vectors were transfected into the cells by Fugene 6.0 and the expression of the reporter gene was detected by a dual-luciferase reporter assay system. The mRNA expression level of IPO8 was detected by real-time PCR in 3-nucleotide insertion or deletion homozygote cells. RESULTS：The sequencing results showed that there were 3 kinds of genotypes in the rs35100176 polymorphism, CCT/CCT,CCT/- and -/-, and the gene frequencies were 1837%, 5510% and 2653%, respectively. The recombinant expression vectors pGL3-3N Insertion and pGL3-3N Deletion were successfully constructed. The luciferase assay showed that pGL3-3N Insertion produced significantly lower luciferase activity than that by pGL3-3N Deletion. Real-time PCR showed that HEK293 cells with 3-nucleotide insertion homozygote expressed relative lower IPO8 mRNA than Saos-2 cells with 3-nucleotide deletion homozygote. CONCLUSION：The CCT 3-nucleotide insertion variant decreases the promoter activity of IPO8, thus affecting the gene expression.     

Effect of P53 binding site on regulation of human NOD8 gene

AIM: To investigated the characterization and biological function of P53 binding element in the regulation of NOD8 gene. METHODS: Using the method of bioinformatics, we found a completely preserved P53 binding site in NOD8 core promoter both in humans and chimpanzees. NOD8 gene was amplified by PCR from human cDNA and correctly connected into the vector p NOD8 (760 bp)-EGFP-C2/ mp NOD8 (750 bp)-EGFP-C2. The constructed plasmids p NOD8 (760 bp)-EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were transiently transfected into the cell line HEK293 by JetPei^TM and treated with pifithrin alpha (PFT-α,an inhibitor of P53) at different concentrations for 24 h. The mRNA and protein expression levels of NOD8 were detected by RT-PCR and Western blotting. In addition, chromatin immunoprecipitation (ChIP) assay was performed to determine the binding of P53 to the NOD8 promoter after recombinant plasmid p NOD8 (760 bp)-EGFP was transfected into HEK293 cells. RESULTS: The plasmids p NOD8 (760 bp) -EGFP- NOD8 and mp NOD8 (750 bp)-EGFP- NOD8 were successfully constructed and conformed by restriction digestion and sequence analysis. The results of ChIP confirmed that P53 bound to the promoter of NOD8 . The mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 was stronger than that in the cells transfected with control vector pEGFP-C2 (P<0.05). Furthermore, the mRNA expression of NOD8 was reduced in HEK293 cells transfectnt with the mutant plasmid mp NOD8 (750 bp)-EGFP- NOD8 compared with the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 . Meanwhile, PFT-α inhibited the mRNA expression of NOD8 in the cells transfected with p NOD8 (760 bp)-EGFP- NOD8 in a concentration-dependent manner, and PFT-α at concentration of 90 μmol/L was the most effective in inhibiting NOD8 mRNA expression (P<0.01). As expected, the protein expression of NOD8 in pNOD8 (760 bp)-EGFP- NOD8 group significantly increased compared with that in pNOD8 -C2 group, the protein expression of NOD8 in mp NOD8 (750 bp)-EGFP- NOD8 group or pNOD8 (760 bp)-EGFP- NOD8 + PFT-α group was dramatically decreased compared with that in p NOD8 (760 bp) -EGFP- NOD8 group. CONCLUSION: The results suggest that the P53 binding site is critical for the regulation of NOD8 gene and there is positive feedback regulation between P53 binding site and NOD8 , which may maintain efficient balance between defense and self-inflicted injury in response to the invasion of pathogen.     

Differential expression of genes in the rat heart during centrifuge training

AIM: Centrifuge training can improve forward acceleration (+Gz) endurance. This study was to analyzed the gene expression of rat heart affected by centrifuge, and to research the molecular mechanism of improving+Gz endurance by centrifuge training. METHODS: Differential expressed genes between high+Gz endurance (+16Gz) rats, of test group after trained12 d and control were screened using suppression subtractive hybridization (SSH) and dot blot hybridization. The obtained expressed sequence tags (ESTs)were used as probes to perform RNA slot hybridization with heart total RNA isolated from each gruop of centrifuge training and high+Gz endurance and low+Gz endurance (+12Gz) rats, respectively. The positive ESTs were sequenced and analyzed using BLAST(nr) at NCBI.RESULTS: Three down-regulated ESTs were obtained from heart samples, all of them are new, and their expression levels were decreasing during centrifuge training. CONCLUSION: Centrifuge training can significantly affect the special gene expressions of rat heart, and the expression changes of these genes may be ralated to the mechainism that+Gz endurance can be improved by centrifuge training.