The Reserch Of MiR-146a Regulation In Smoke Inhalation Injury

Objective:Smoke inhalation injury have high fatality rate,its nosogenesis is complex,which is mainly NF-κB signaling pathway on inflammatory cells membrane in lung activated,can lead the release of a large amount of cytokines,prompting uncontrolled inflammatory response,all this caused the ALI/ARDS.Mi RNA is a kind of non coding single stranded RNA, through identifying and combing target m RNA 3’-UTR,resulting in m RNA degradation or inhibition of translation.mi R-146 a block NF-κB activation by targeting TRAF6 and IRAK1,negatively regulating inflammation.Each mi RNA could control many target genes,in the development of smoke inhalation injury,whether mi R-146 a to relieve the inflammation response in lung by targeting TMEM33, it has not been reported in domestic and foreign.Based on it, according to the prediction targets of mi R-146 a,constructing recombinant plasmid of luciferase reporter gene and testing its function,investigating the influence of mi R-146 a upon the inflammatory cytokine and protein expression by the mouse model of smoke injury, trying to verify mi R-146 a reduce lung inflammatory response by targeted TMEM33,and raising the therapeutic effect of ALI/ARDS.Methods:1. The use of biology software Target Scan or mi Randa predict TMEM33 m RNA3ˊ-UTR may be a target of mi R-146 a and constructe the fluorescence protein reporter gene carrying TMEM33 m RNA 3ˊ-UTR.Dual luciferase report system detects and proves the target relationship between mi R-146 a and TMEM33;2. Select 20 C57BL/6 mice in SPF grade,male and female not required.establishing an mouse model of smoke injury,models were randomly divided into 4 groups:control group,smoke injured+normal saline group,smoke injured+mi R-146 a group,smoke injured+mi R-control group,5 in each group.24 h after smoke injury,the mice were killed and the lung was taken.The right lung used hematoxy lin-eosin staining to exam the lung injured degree, using Taq Man RT-q PCR to exam the relative quantitative expression of mi R-146 a, and using RT-q PCR to test TNF-α,IL-1 and TMEM33 m RNA of each group.the left lung used Western Blot to test COX-2 and TMEM33 proteins of each group.Results:1. Target Scan predictive software show there exists complementary binding-site between TMEM33 m RNA 3ˊ-UTR and mi R-146 a,constructed luciferase reporter gene vector were correctly identified by restriction enzyme digestion and sequencing.Dual luciferase report system shows,compared with p MIR-TMEM33+mi R-control group,relative luciferase activity of p MIR-TMEM33+mi R-146 a mimics group was reduced by 62 percent, and the difference was statistically significant(P<0.01);Relative luciferase activity of p MIR-TMEM33-mu+mi R-146 a group had no significant difference(P>0.01).2. HE stained indicated there was no pulmonary obvious abnormality in the control group,the lung of smoke injured group showed medium and severe inflammatory cell infiltration and bleeding.The result of Taq Man RT-q PCR indicated,compared with mi R-146 a expression of mi R-control group(0.829±0.060),mi R-146 a group(11.640±0.190)raised about 10 times(P<0.01).The result of RT-q PCR showed,TNF-α and IL-1 expression of mi R-146 a group was substantially below NS groups and mi R-control group(P<0.01);However,TMEM33 expression of mi R-146 a group had no significant difference(P>0.05).The result of Western Blot showed,compared with saline groups and mi R-control group,COX-2 and TMEM33 expression of mi R-146 a group were relatively low.Conclusions:1. mi R-146 a might down regulate the expression of TMEM33 protein on translation by combining with TMEM33 m RNA 3ˊ-UTR.2. mi R-146 a reduce lung inflammatory cytokines by targeted TMEM33,declining the ALI.