| Objective:To investigate the expression of miRNA-6786 in peripheral blood of patients with sepsis,to stimulate the model of macrophages by LPS in vitro,to investigate the role of miRNA-6786 in the inflammatory response through the regulation of the target gene FRAT-2,and to explore its regulatory mechanisms as wnt / ?-catenin and NF-KB signal transduction pathway.Methods:1.Clinical study:(1)30 cases each in sepsis group(sepsis group)and healthy control group(NC group).Sepsis group was admitted to hospital d1,NC group took peripheral blood during physical examination,and detected miRNA by RT-PCR method.-6786 expression.(2).Using the Mi RDB database to predict which target genes and signal pathways are most relevant for miR6786.2.In vitro cell experiments:(1)THP-1 mononuclear macrophages are induced into macrophages and divided into 6 groups:(1)normal macrophage group(group C);(2)LPS-stimulated macrophage group(group L): 10 ug / ml LPS stimulated macrophages;(3)miR-6786 mimic transfection group(L + M group): miR-6786 mimic transfection of macrophages 10 ug / ml LPS stimulation;(4)miR-6786 mimic transfection control group(L + MC group)): Garbled nonfunctional miR6786 mimic transfection of macrophages 10 ug / ml LPS stimulation;(5)miR-6786 inhibitor transfection group(L+ IN-M group): miR-6786 inhibitor transfection of macrophages 10 ug / ml LPS stimulation;6786inhibitor transfection control group(L + IN-MC group): garbled and nonfunctional miR6786 inhibitor was stimulated with 10 ug / ml LPS after transfection into macrophages.(2)RT-PCR was used to determine the expression of FRAT-2,?-catanin and NF-KB m RNA in macrophages.(3)Western Blot was used to determine the expression of FRAT-2 protein in macrophages.(4)The inflammatory factors of IL-1?,IL-6 and TNF-? were measured by ELISA.Results: 1.Clinical research results: The expression of miRNA-6786 in NC group and sepsis group determined by RT-PCR was:(1.34 ± 0.026,7.350 ± 1.418).The expression of miRNA-6786 in sepsis group was significantly higher than that in NC group(t = 18.92,P <0.0001).2.In vitro cell experiments:(1)RT-PCR detection of miR-6786 results: The expression level of miRNA-6786 in group L was higher than that in group C(t = 44.5,P <0.0001),L + MC group,L + IN There was no significant difference in miRNA-6786 expression between the-MC group and the L group(P> 0.05).The expression level of miRNA-6786 in the L + M group was significantly higher than that in the L + MC group(t = 49.5,P <0.0001).The expression of miRNA-6786 in the IN-M group was significantly lower than that in the L + IN-MC group(t = 14.8,P <0.0001).RT-PCR detection of FRAT-2,?-catanin,and NF-KB m RNA results: There was no significant difference between group L and group C FRAT-2 m RNA(P> 0.05),and the expression levels of ?-catanin and NF-KB m RNA increased significantly High(t = 20.9 and 5.00,P <0.05).There was no significant difference in FRAT-2,?-catanin,and NF-KB m RNA expression in the L +MC group and L + IN-MC group compared with the L group(P> 0.05).Compared with the L + MC group,the + M group had no significant difference in FRAT-2m RNA(P> 0.05),and the expression levels of ?-catanin and NF-KB m RNA were significantly reduced(t = 11.5 and 15.6,P <0.001).Compared with the L + IN-MC group,the + IN-M group had no significant difference in FRAT-2 m RNA(P> 0.05),and the expression levels of ?-catanin and NF-KB m RNA increased significantly(t =40.0 and 9.098,P <0.001).(2)Western Blot detection of FRAT-2 protein results: The gray value of the L group was lower than that of the C group(t = 10.3,P <0.0001).The L + MC group and L + IN-MC group were grayer than the L group protein.There was no significant difference in values(P> 0.05).Compared with the L + MC group,the gray value of the FRAT-2 protein was significantly lower in the L + M group(t =17.7,P <0.0001).The L + IN-M group and L + Compared with the IN-MC group,the gray value of FRAT-2 protein was significantly increased(t = 15.2,P <0.0001).(3)ELISA detection of IL-1?,IL-6,TNF-? Results: The expression levels of IL-1? and IL-6,TNF-? in group L were higher than those in group C(t = 17.3,11.8 and 19.9,P< 0.001),there was no significant difference in the expression levels of IL-1?,IL-6and TNF-? in the L + MC group and L + IN-MC group compared with the L group(P> 0.05).Ratio,the expression levels of IL-1?,IL-6 and TNF-? were significantly reduced(t = 12.5,8,20,and 7.72,P <0.05).Compared with the L + IN-MC group,the L + IN-M group,The expressions of IL-1?,IL-6 and TNF-? were significantly increased(t = 5.45,5.91 and 19.2,P <0.05).Conclusion: 1.This study found that mi-R6786 is highly expressed in peripheral blood of patients with sepsis.2.LPS-stimulated macrophage experiments show that mi-R6786 inhibits the production of FRAT2 protein,affects the Wnt / ?-catenin pathway of macrophages,and weakens the transcriptional activity of NF-KB,thereby reducing IL-1?,IL-6 and Proinflammatory factors such as TNF-? are released.3.LPS stimulation of macrophages can increase miR-6786 to a certain extent,but there is no obvious anti-inflammatory effect.After miR-6786 transfection,it can significantly increase the expression of miR-6786 and significantly reduce IL-1?,IL-6 and The release of pro-inflammatory factors such as TNF-? may have a dose-effect relationship with miR-6786. |
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