Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Method...Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Methods:In an in vivo experiment,the expression levels of IRF-3 and miRNA146a in peripheral blood of 45 children with infectious mononucleosis and 34 healthy controls were detected by real-time PCR.The in vitro experiment was performed to examine the effect of miRNA146a on IRF-3 expression levels by transfecting miRNA146a mimics and their inhibitors.Results:There was significant difference in IRF-3 gene expression in peripheral blood between healthy controls and children with infectious mononucleosis(t=30.340,P<0.001)while miRNA146a expression was significantly increased compared with healthy controls(t=34.659,P<0.001),and there was a negative correlation between the two groups(r=-0.960,P<0.05).In HeLa cells,transfection with miRNA146a mimics significantly decreased the expression of IRF-3 mRNA(t=8.270,P<0.001)and protein(t=46.170,P<0.001),while miRNA146a inhibitor significantly up-regulated the expression of IRF-3 mRNA(t=8.582,P<0.001)and protein(t=25.891,P<0.001).Conclusion:miRNA146a may be involved in the down-regulation of IRF-3 gene expression by Epstein-Barr virus in children with infectious mononucleosis and then involved in the pathogenesis of infectious mononucleosis.展开更多
Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells...Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction （PCR）. The PCR product was separated by electrophoresis on 1% agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMP- miRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5a strain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5a via transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cells with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection efficiency was ob- served under fluorescence microscopy and cell counting kit-8 assay was conduced to evaluate the effect of miRNA-451 on SGC-7901 cell proliferation. Results： Our results showed that pLMP-miRNA-451 expression vector was not only constructed successfully and effectively infected SGC-7901 cells, but also could repress the SGC-7901 cell proliferation. Conclusion： The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cell lines.展开更多
基金Youth Medical Talents Project of Science,Education and Health of Jiangsu Province(No.QNRC2016615)Maternal and Child Health Key Talents Project of Jiangsu Province(No.FRC201736)。
文摘Objective:To investigate the expression levels of interferon regulatory factor 3(IRF-3)and miRNA146a in peripheral blood of children with infectious mononucleosis and the effect of miRNA146a on IRF-3 expression.Methods:In an in vivo experiment,the expression levels of IRF-3 and miRNA146a in peripheral blood of 45 children with infectious mononucleosis and 34 healthy controls were detected by real-time PCR.The in vitro experiment was performed to examine the effect of miRNA146a on IRF-3 expression levels by transfecting miRNA146a mimics and their inhibitors.Results:There was significant difference in IRF-3 gene expression in peripheral blood between healthy controls and children with infectious mononucleosis(t=30.340,P<0.001)while miRNA146a expression was significantly increased compared with healthy controls(t=34.659,P<0.001),and there was a negative correlation between the two groups(r=-0.960,P<0.05).In HeLa cells,transfection with miRNA146a mimics significantly decreased the expression of IRF-3 mRNA(t=8.270,P<0.001)and protein(t=46.170,P<0.001),while miRNA146a inhibitor significantly up-regulated the expression of IRF-3 mRNA(t=8.582,P<0.001)and protein(t=25.891,P<0.001).Conclusion:miRNA146a may be involved in the down-regulation of IRF-3 gene expression by Epstein-Barr virus in children with infectious mononucleosis and then involved in the pathogenesis of infectious mononucleosis.
基金supported by a grant from the Natural Science Foundation of China (No. 8100098)
文摘Objective： The aim of the study was to construct miRNA-451 expression vector pLMP-miRNA-451 which could help identify the functions of miRNA-451 in SGC-7901 cell. Methods： Total RNA was extracted from SGC-7901 cells to synthesized cDNA. The synthesized cDNA encoding pre-miRNA-451 was amplified by polymerase chain reaction （PCR）. The PCR product was separated by electrophoresis on 1% agarose gel and then recovered and purified. The purified cDNA fragments of miRNA-451 precursor sequence was then ligated with vector pLMP for 1 h by using DNA ligase to form pLMP- miRNA-451 plasmid. After that, the pLMP-miRNA-451 plasmid was transformed into E. coli DH5a strain expression system to clone and amplificate. The purified pLMP-miRNA-451 extracted from E. coli DH5a via transformation and clone screening was identificatied with restriction enzyme digestion and DNA sequencing. At last, pLMP-miRNA-451 was transfected into SGC-7901 cells with lip2000. Real-time PCR was used for detection of the miRNA-451, the transfection efficiency was ob- served under fluorescence microscopy and cell counting kit-8 assay was conduced to evaluate the effect of miRNA-451 on SGC-7901 cell proliferation. Results： Our results showed that pLMP-miRNA-451 expression vector was not only constructed successfully and effectively infected SGC-7901 cells, but also could repress the SGC-7901 cell proliferation. Conclusion： The constructed plasmid pLMP-miRNA-451 could used for further studies of miRNA-451 in SGC-7901 cell lines.
