Expression And Regulation Of The Fibrinogen Gene Of Gansu Zokor In The Heart Under Hypoxia

Myospalax cansus(E.cansus),belong to the genus Myospalax family of Rodentia,Spalacidae.During the long-term evolution,it was adapted to the underground environment with hypoxia and high carbon dioxide.A series of adaptive characteristics can be observed in Myospalax cansus,which is considered as one of ideal species for the study of hypoxia adaptation.In front of hypoxia,animals will compensatorily increase blood viscosity,which will increase blood circulation resistance.The results caused damage in the cardiovascular system,but not observed in subterranean rodents.At present,many studies focused on the heart of E.cansus under hypoxia at the level of physiological and biochemical.In order to explore the molecular mechanism of hypoxia adaptation in the subterranean rodents,we analysised the transcriptomes of E.cansus.Eighteen individuals of E.cansus were captured from Yan,an,which were separated randomly into three groups,containing six individals equally,and exposed under three conditions:21%O2for 7 days,10.5%O2 for 6hours,6.5%O2 for 44hours.The RNA of heart samples were performed for RNA sequencing.After assembly of clean read data,unigenes were annoted with the GO function and KEGG metabolic pathway.Different expressed genes(DEGs)were identified between different conditions,and enriched with GO terms.Then real-time polymerase chain reaction(PCR)were used to validate DEGs'expression,like FG and its related genes,including TFPI,TF,FV,FX and F?.After this,we amplified the cDNA sequence of FG.The main results were showed as follows:1.34,988 unigenes were acquired after assembly of E.cansus heart trnscriptomes.274 different expression genes(DEGs)were obtained.GO term enrichment analysis of DEGs were carried out for those DEGs The most of DEGs and metabolic pathways were enriched for 10.5%vs 6.5%,suggesting that the environment of 6.5%oxygen concentration has more significant influence on E.cansus,suggesting that the acute hypoxia could threated its normal life and E.cansus adapt the hypoxia environment by changing many genes' expression pattern.2.RNA-seq experiments showed that the DEG FG was downregulated in E.cansus heart at 6.5%O2 on.Real time quantitative PCR was used to validate its mRNA expression in E.cansus and SD rat too.The results showed that FG was downregulated in10.5%and 6.5%oxygen concentration in E.cansus,but upregulated in SD rat.Compared with SD rats,the relative expression of fibrinogen gene mRNA in E.cansus was significantly decreased at 10.5%and 6.5%oxygen concentration.These results could reflect the mechanism of protection to the heart under hypoxia.It was inferred that FG downregulation under hypoxia could reduce red blood cell number and platelet aggregation in plasma,which prevent excessive blood sticky,and it lead to more smooth for blood circulation,and heart will pump enough blood for body,and reduce the possibility of thrombus formation.3.Here,partial cDNA sequence of FG(FG A,FGB and FGG)were first amplified in Myospalax cansus.The length of cDNA sequence of FGA,FGB and FGG is 1442bp,902bp,432bp,respectively homology in Nannospalax galili shows the highest identity of 89%using BLAST,and the protein homology with 71%idenitity,The results showed that our sequences of FG were the real cDNA sequences of E.cansus.4.Other DEGs in the pathways of fibrinogen were also validated.The relative expression of TFPI mRNA was significantly up-regulated at 10.5%and 6.5%,and KNG was significantly down-regulated,which showed the consistent in both transcriptional sequencing results and real-time quantitative results.We speculated that the upregulation of TFPI reduced the expression level of TF and FX,and then downregulate the expression of FG.The upregulation KNG under hypoxia may reduce the intrinsic pathway of blood coagulation and then result in the downregulation of FG.5.Real time quantitative PCR was also conducted on other genes(TF,F?,F? and F?)related with FG,which aimimg fot the understand of the regulatory mechanism of FG in E.cansus.These results suggested that:1)No significant differencial expression was observed for TF between 21%and 10.5%,21%and 6.5%.6.5%O2 cause an upregulation of TF compared with 10.5%oxygen concentration.It was speculated that 6.5%oxygen concentration has more influence on blood coagulation.2)Hypoxia(10.5%and 6.5%)causes an downregulation of FX,FV and FII.The variation trends of these factors all contributed to the expression changes of fibrinogen gene,and regulated blood coagulation process together.In our study,the differential expression gene fibrinogen was firstly obtained by transcriptome analysis.The fibrinogen was down-regulated in hypoxia and it was mainly regulated followed by two distinct routes.On the one hand it was regulated by the up-regulation of TFPI and the down-regulated of TF in the exogenous pathway;on the other hand it was regulated by the down-regulation of KNG in endogenous pathway.The combination of the two ways leads to the down-regulation of FX?FV and FII.Ultimately,the fibrinogen mRNA expression was down-regulated,which can reduce red blood cells and platelet aggregation,reduce blood circulation resistance;and lessen the incidence of cardiovascular disease for the adaption of hypoxia environment.