| Objective To investigate the differential expressed microRNA expression in the hearts of sepsis mice and normal mice by using microarray chip with sepsis induced by cecal ligation and puncture and to find the relevant target genes of microRNA.Methods(1)To establish the model of mice with sepsis and to isolate the hearts.The clean 8-week-old male C57BL/6 mice were selected.The method of cecal ligation and puncture was used to establish the model of mice with sepsis.Three survival mice were randomly selected in 24 h as the experimental group.At the same time,three male C57BL/6 health mice were selected as the control group.After weighing and anesthesia,the beating hearts were exposed by surgery.The needle was passed through cardiac apex and left ventricle into the artery.Meanwhile the right auricle was cut open.The mice hearts were irrigated by the normal saline and then taken out rapidly.The surrounding tissues were removed and the liquid in the hearts were disposed by filter paper.We placed the hearts in the precooling tube,put it in the liquid nitrogen at least 5 min and stored in the-80℃ refrigerator.(2)To extract the total RNA and to do the quality test.About 100mg~200mg myocardial tissue was needed to extract the total RNA.The mir Vana? microRNA Isolation Kit(AM1561)was used to purify and quantify RNA.We took 1μg purified RNA to do the quality test.The ultraviolet spectrometry was applied to test the purity and concentration of RNA and the integrality of samples was analyzed by 1.3% agarose gel electrophoresis.(3)About 2μg purified RNA was used for hybridization,staining and scanning with Flash Tag? Biotin RNA labeling kit.The differential expressed microRNA were selected using the cluster analysis and SAM analysis.(4)With the analysis of the target gene prediction by the database of software,the relevant target gene of the differential expressed microRNA would be predicted.Result(1)The sepsis mice model had been established.The dead and survival were equalamong the 10 sepsis mice in 24 h and the mortality reached to 50%.The experimental mice showed fur on end,conjunctivitis,diarrhea,lethargy and so on.(2)The results of quality test for total RNA were as followings.The purity A260/280 was equal or greater than 1.90.The amounts of RNA were equal or greater than 1μg.The brightness of bands 28S:18S r RNA was equal or greater than 2:1.Above all,the purity,amounts and integrality of the samples were suitable for microarray experiments and the next step could go on.(3)According to the standard of significantly expressed microRNA(q-value≤5% and Fold Change≥2 or≤0.5),the result of microarray experiment was as following.Compared with the control group,6 microRNA were signaificantly expressed in the sepsis group among 675 microRNA.The expression of mmu-mi R-5620-3p,mmu-mi R-3968 and mmu-mi R-215-5p in sepsis group was significantly higher and the expression of mmu-mi R-16-1-3p,mmu-mi R-200c-3p and mmu-mi R-122-5p was significantly lower in sepsis group than that of control group by microarray experiment.(4)We selected the target gene of the differential expressed microRNA with the predicted software of the target gene.CDKN1A、ZEB2 and CTNNBIP1 were the target genes of mi R-215.BCL2、CCND1、CCNE1 and WNT3 A were the target genes of mi R-16.The target gene of mi R-200 was ZEB1.BCL2、SOX11 were the target genes of mi R-122.Conclusion(1)The model of mice with sepsis by cecal ligation and puncture is successful.(2)Differential microRNA are expressed in the hearts of sepsis mice,which are up-regulated or down-regulated.(3)The target genes of differential expressed microRNA are closely related to cell proliferation,differentiation and apoptosis,which can contribute to the mechanism of sepsis and will lay a solid experimental and theoretical foundation for the further research and therapy of sepsis. |
