MicroRNA-205 Inhibits HMGB-1 Expression In Cholinergic Anti-inflammatory Pathway

ObjectivePrevious studies have found that, miRNA involvement in the cholinergic anti-inflammatory pathway (CAP) in the regulation of proinflammatory TNFa and IL-6, and the late inflammatory factors are the basis of sepsis progression. Therefore, the purpose of the present study is divided into the following:(1) Since cholinergic anti-inflammatory pathwayis a post-transcription regulator, act as the same way of miRNA regulation, we wander weather there are some unknowen miRNA in CAP regulate late inflammatory factor HMGB1, we screen them out with miRNA microarray and identify them.2) To verify the miRNA screened is targeting HMGB1 at cytological level.3) To determine wheather miRNA can play the same role in the body's CAP at histological level by animal experiment, and also study the substantial difference of target miRNA in different organs.Methods(1) At the time of late inflammatory cytokines peak, detecte the difference of miRNA expressed in sepsis and CAP group with microarray expression. Use the fold change value of multiple differences, identify the target miRNA expressed in CAPgroup increased two times more than in sepsis group. With GeneSpring 12.5 software and PITA, TargetScan, microRNA org database, topredict target gene which meet the requirements. Intersection of three databases is the genes screened, then analysis with GO and KEGG subsequently.(2) To verify the accuracy of miRNA Microarray with Real-time PCR. TransfectRAW264.7 with mimics and inhibitor. To verify targetgenes of miRNA by the different expression of miR-205, HMGB1 and HMGB1 mRNA in sepsis group and CAP group.(3) Detect different expression of miRNA, HMGB1 and HMGB1 mRNA in serum, brain, heart, lung, kidney, liver, spleen, colon in sepsis mice and CAP mice. Analysis anti-inflammatory effects of miRNAin tissues.Results(1) This study demonstrated optimal dose of LPS in sepsis model and GTS-21 dose in CAP model in RAW264.7 cells which are 50ng/ml and 8?g/ml respectively. Optimal drug dose in BALB-c mice are LPS 15mg/kg and GTS-214mg/kg.(2) To evaluate difference of miRNA by using the fold difference of fold change value, in the CAP group, expression of the miRNA increased more than two times greater than in the sepsis group is 38 in totally. Only four miRNA increased in CAP but other groups, they are miR0205,196a,193b,31.MiRNAspredict 2019 target gene distributed mainly in 66 pathway.(3) Select the maximum expression of miRNA in CAP group, miR-205 to carry out qRT-PCR. The result is consistent with microRNA array. RAW264.7 transfected with miR-205 mimics and inhibitor, HMGB1 expression in RAW264.7 transfected miR-205 mimics significantly lower compared with sepsis group after adding LPS; added LPS+GTS-21 in RAW 264.7 transfected with miR-205 inhibitor,the anti-inflammatory effects of GTS-21 disappeared, and HMGB1 expression was significantly increased. HMGB1 mRNA expression have not difference between two group. Which demonstrate that the mechanism of CAPdepress sepsis late inflammatory factor HMGB1 by miR-205 is in a way of post-transcription process.(4) To test miR-205 by qRT-PCR in Serum, lung, liver, spleen, colon, brain, kidney and heart in sepsis group and CAP group, observe the different expression of miR-205 in different tissues above. Expression of miR-205 in 8 organ tissues were detected significant differences. miR-205 in CAP group was significantly higher than in sepsis one. It express higher in serum, liver, colon of CAP group three times more than in sepsis group. miR-205 in brain and kidney tissue of CAP group was increased approximately 2-fold than in sepsis group.The difference were less than one doubled in myocardial tissue. P values:serum, brain, kidney, intestine<0.0001; liver= 0.0011; spleen= 0.0054; heart= 0.0049; lung= 0.0032. To detectHMGB 1 concentration of each tissue by ELISA, HMGB1 in CAP group was decreased than in sepsis group.P values:Serum= 0.0063, brain, kidney <0.0001, heart= 0.0561, colon= 0.0068, lung = 0.0010,spleen= 0.0090, liver= 0.0006.There was no significant difference HMGB1 mRNA expression in both groups (p> 0.05).Conclusions(1) Screening genome-wide differential miRNA with miRNA microarray, four miRNA, namely miR-193b, miR-196a, miR-205 and miR-31, were authenticated elevated expression in CAP anddecreased expression in sepsis.2019 kinds of target genes were predicted and 66 meaningful pathways werefound by KEGG analysis.miR-205 is selected as the target miRNA to study for itslargest fold difference expression. qRT-PCR results is consistent with miRNA microarray.(2) It is proved that miR-205 target HMGB1 in RAW264.7 by mimics and inhibitor transfected.(3) We has proved that regulation of miR-205 target at the posttranscription.(4) Multiples of miR-205 increased is not in equal proportion with multiples of HMGB1 in different tissues.