Biliverdin Treatment With Rats Cerebral Ischemia Reperfusion Injury Related MicroRNAs, MRNA Screening And Functional Identification

Inflammation plays an important role in the occurrence and development of CIR.Biliverdin can active anti-inflammatory effects in the process of the occurrence and development of many diseases, but its role in the pathophysiology and mechanisms are still poorly understood. Our previous research found: BV has protective effect on the brain after CIR in rats, and the release of inflammatory factors in cerebral cortex is reduce. High-throughput sequencing provides a powerful tool for our comprehensive analysis of the occurrence and development of disease. To investigate the mechanism of biliverdin play a protective role in CIR, we used miRNAs microarray, mRNA microarray, qPCR, bioinformatics analysis, RNA interference,luciferase,WB,Elisa technology.To investigate the anti-inflammatory effect of Litaf in CIR and the regulatory mechanism of miR-27a at the molecular and cellular level.Part ? Biliverdin treatment of cerebral ischemia reperfusion injury related microRNAs and quantitative analysis of candidate microRNAs[Objective]Through microRNAs microarray to detected the normal cerebral cortex tissue,ischemia cerebral cortex tissue, and the cerebral ischemic cortex tissue treatment of biliverdin?To screen related microRNAs and quantitative analysis of candidate microRNAs[Methods]Through microRNAs microarray obtain expression profile of microRNAs,Among the different trends after ischemia and n biliverdin treatment to screen related microRNAs with Fold Change?2 as screening criteria. QPCR technology was used to verify the candidate microRNAs for subsequent screening.[Results]1. Compared with S group, C group rno-miR-181b-5p;rno-miR-124-5p;rno-miR-27a-3p;rno-miR-29b-3P expression were significantly reduced, The difference was statistically significant (P<0.05);Compared with C group, BV group rno-miR-181 b-5p;rno-miR- 126a-5p;rno-miR-124-5p;rno-miR-27a-3p;rno-miR-29 b-3p expression were significantly increased, The difference was statistically significant (P<0.05).2. Compared with S group, C group rno-miR-3072; rno-miR-150-5p;mo-miR-539-3p expression were significantly increased, The difference was statistically significant ( P < 0.05 ) ; Compared with C group, BV group rno-miR-3072; rno-miR-3573-3p ; rno-miR-150-5p; rno-miR-935 ;rno-miR-539-3p expression were significantly reduced,The difference was statistically significant (P<0.05)[Conclusions]1. The microRNAs expression of the normal cerebral cortex tissue, ischemia cerebral cortex tissue, and the cerebral ischemic cortex tissue are different.2. There are total 13 microRNAs QPCR quantitative analysis results matching with microRNAs microarray and they may related to the anti-inflammatory effect of biliverdin in CIR.Part II Bioinformatics function prediction of candidate micro RNAs[Objective]To screen out the related to the anti-inflammatory effect of biliverdin in CIR.we predicted potential target genes and their corresponding signal transduction pathway of candidate micro RNAs by bioinformatics.[Methods]TargetScan biological databases, miRmap biological databases and Bioinformatics analysis tools (self-developed by Shanghai Bo Hao) were used to predict potential target enes of candidate micro RNAs. Selected the three databases intersection target genes for GO analysis and Pathway enrichment analysis predicting the potential biological functions of target genes and the potential signaling pathways.[Results]Candated miRNAs predicted total 1040target gene for following analysis. GO analysis GO results suggest: these target genes are mainly associated with ubiquitin ligase complex, neuron to neuron synapse, asymmetric synapse, artery morphogenesis,artery development, memory .Pathway enrichment analysis results suggest these target genes are mainly associated with: : Rap1, cAMP, MAPK, FoxO, cGMP-PKG,Ras signaling pathway,and cancer related signaling pathway.[Conclusions]Through the miRNA chip we selected the candidate microRNAs, and target gene prediction suggests that a miRNA can regulate multiple target genes,a target gene and may also be regulated by multiple miRNA, visible miRNA control mechanism is very complex. Bioinformatics analysis showed that the differential expression of target gene miRNA predicted are widely involved in many biological processes, we can speculate that the abnormal expression of miRNA may be the target gene regulation of specific functions, to participate in an important signal transduction pathway in biliverdin treatmen of CIR. The need for further research to understand these differences of miRNA in in biliverdin treatmen of CIR.Part ? Biliverdin treatment of cerebral ischemia reperfusion injury related mRNA and quantitative analysis of candidate mRNA[Objective]Through mRNA microarray to detected the normal cerebral cortex tissue, ischemia cerebral cortex tissue, and the cerebral ischemic cortex tissue treatment of biliverdin?To screen related mRNA and quantitative analysis of candidate mRNA.Using the candidate microRNAS made "miRNA- target protein" network analysis, to find the potential "miRNA- target protein" of biliverdin in CIR.[Methods]Through mRNA microarray obtain expression profile of mRNA, among the different trends after ischemia and n biliverdin treatment to screen related microRNAs with Fold Change?2 as screening criteria. When the differentially expressed mRNA and the candidate microRNAs target genes were met, the target gene was reserved for subsequent screening. The overlapping target genes were retrieved from the Pubmed target sites in the ischemic and hypoxic diseases. The genes related to the disease were screened and analyzed by qPCR. According to the results of quantitative analysis of mRNA qPCR combined with the results of microRNAs microarray analysis, we screened out the target protein which is in accordance with the "miRNA- target protein" network regulation theory.[Results]6. The 49 differentially expressed mRNA and the candidate microRNAs target genes were met7. 33 genes that may be involved in the process of the disease were screened for qPCR validation8. Compared with S group, C group Sox7; Fbxo33; Ets1; Gcnt2; Csrnp1; En2;Rgs1; Litaf; Rnd3; Zc3hl2d; P4hb; Mthfd2; Mafk; B4galt1; Slc25a25;Esm1; Vps37c; Zmiz1; Rhoq; Tagln2; Plp2; Zkscan1; Guca1b; Pnrclexpression were significantly increased , The difference was statistically significant (P <0.05); Compared with C group, BV group Sox7; Fbxo33; Ets1 ; Gcnt2; Csrnp1;En2; Rgs1; Litaf; Rnd3; Zc3h12d; P4hb; Mthfd2; Mafk; B4galt1; Slc25a25;Esm1; Vps37c; Zmizl; Rhoq; Tagln2; Plp2; Zkscan1; Gucalb; Pnrcl xpression were significantly reduced, the difference was statistically significant (P<0.05);9. Compared with S group, C group Map2k6 ; Scn4bexpression were significantly reduced, The difference was statistically significant (P<0.05) ; Compared with C group, BV group Map2k6 xpression were significantly, the difference was statistically significant increased (P<0.05);[Conclusions]1. The mRNA expression of the normal cerebral cortex tissue, ischemia cerebral cortex tissue, and the cerebral ischemic cortex tissue are different.2. There are total 26 mRNA QPCR quantitative analysis results matching with the"miRNA- target protein" regulatory network theory and they may related to the anti-inflammatory effect of biliverdin in CIR.3. Speculating rno-miR-27a-3p and it's target gene may related to the anti-inflammatory effect of biliverdin in CIR.Part ? Effects of Litaf on the release of TNF- alpha in cortical neurons after oxygen glucose deprivation reoxygenation[Objective]Explorated of Litaf and miR27a whether through inhibition of inflammatory factor TNF- alpha release in biliverdin treatment of CIR play a protective role.[Methods]PC 12 cell line qPCR, the original generation of cortical neurons in rats,bioinformatics analysis, RNA interference, luciferase,WB,Elisa technology.To investigate the anti-inflammatory effect of Litaf in CIR and the role of Litaf in CIR and the regulatory mechanism of miR-27a at the molecular and cellular level.[Results]1. 2ug/mlbiliverdin treatment can increase cell viability after OGD/R, the difference was statistically significant increased (P<0.01);2. After OGD/R the Litaf expression level increased significantly, biliverdin treatment Litaf expression level decreased significantly; miR-27a variation trend on the contrary of Litaf.3. Compared with group 1, group 2 the Litaf ? TNF-aexpression level increased significantly, the difference was statistically significant (P<0.01) ; Compared with group 2, group 6 the Litaf ? TNF-aexpression level decreased significantly the difference was statistically significant (P<0.01).4. Compared with group 1, group 2 the Litaf ? TNF-aexpression level increased significantly, the difference was statistically significant (P<0.01) ; Compared with group 2, group 3 the Litaf?TNF-aexpression level decreased significantly the difference was statistically significant (P<0.01).5. Compared with group 1, group 2 the Litaf ? TNF-a expression level increased significantly, the difference was statistically significant (P<0.01); Compared with group 2, group 6 the Litaf ? TNF-aexpression level decreased significantly the difference was statistically significant (P<0.01). Compared with group 2,group 7 the Litaf?TNF-?aexpression level decreased significantly the difference was statistically significant (P<0.01).6. Except group 6, miR-27a variation trend contrary to Litaf, Compared with group 6,group7?8?9 the difference was statistically not significant (P>0.05).[Conclusions]1. Biliverdin treatment can increase cell viability after OGD/R.2. Biliverdin?Litaf SiRNA can obvious inhibition the TNF-arelease after cortical neurons OGD/R.3.