Résumé
Objective To explore the of miR-10b cordribution in patients with non-small cell lung cancer (NSCLC) and adjacent tissues,and to investigate the effect of miR-10b on the malignant change of lung cancer cell A549 by regulating the expression of Kruppel-like factor 4 (KLF4).Methods Fourty patients with NSCLC were selected with lung cancer and in miR-10b expression adjacent tissues of lung cancer cell A549 transfected miR-10b mimics,changes of CCK-8 assay were used to detect the proliferation of lung cancer cells;real time PCR and Western blot were used to examine the cell KLF4 mRNA and protein levels;soft agar colony formation assay was used to detect the expression of miR-10b on the proliferation of lung cancer cell A549 tumor malignant.Results miR-10b expression in lung cancer A549 cells and lung cancer tissue were higher than that of normal lung epithelial 16HBE cells and cancer adjacent tissues;overexpression of miR-10banalogue in A549 cells,KLF4 protein levels significantly decreased,KLF4 mRNA was not changed significantly;miR-10b expression significantly increased the growth of A549 cells.Conclusions The distribution of miR-10b in different cell types and tissues may be different,which may promote the proliferation and malignancy of lung cancer cells by inhibitingthe expression of KLF4.
Résumé
In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.
Sujets)
Séquence d'acides aminés , Protéines bactériennes , Chimie , Chromatographie , Dexaméthasone , Chimie , Masse moléculaire , Pseudomonas alcaligenesRésumé
Objective To investigate the role of a transcription factor p 53 in dengue virus infec-tion.Methods A plasmid expressing siRNA specific for p 53 gene was constructed and then used to prepare HepG2 cell line with a suppressed expression of p 53 protein.The expression of p53 protein was detected by Western blot assay .A wild type control group and a siRNA group were set up by infecting wildtype HepG 2 cells and p53 low expressing HepG2 cells with type 2 dengue viruses,respectively.The virus titers in two dif-ferent cells were determined by plaque forming assay using Vero cells .Indirect immunofluorescence assay was performed to detect virus multiplication .The apoptosis of virus infected cells were analyzed by flow cytome-try.ELISA was performed to analyze the levels of IFN-βsecreted by infected cells from two groups .Results Compared with wildtype control group ,the cells in siRNA group showed a suppressed expression of p 53 pro-tein,suggesting that the HepG2 cell line with low p53 protein expression was successfully established .The vi-rus titer in supernatants of the cells from siRNA group was about 100-fold higher than that of wildtype control group at 24 hours after viral infection .Fluorescence activated cell sorting analysis showed that the numbers of green fluorescence labeled cells were remarkably increased in siRNA group .We speculated that p53 protein might play a role in the inhibition of dengue virus infection as indicated by the observed results .The numbers of apoptotic cells showed no significant difference between two groups .However,the level of IFN-βsecreted by wildtype HepG2 cells was six times higher than that of the cells in siRNA group .Conclusion p53 pro-tein might inhibit dengue virus infection through the activation of type Ⅰ interferon signaling pathway rather than enhance cell apoptosis .