Analysis And Vadidation Of Flk1's Response To Hypoxia In Different Tissues Of Lasiopodomys Mandarinus

Flk1 is a key gene in angiogenesis and neurogenesis.RNA-Seq studies have shown that the Flk1(Kdr)gene in the brain tissue of Lasiopodomys mandarinus(mandarin vole)is up-regulated by hypoxia and should be involved in the process of angiogenesis.At present,there are few researches on Flk1 related to subterranean rodents,such as the gene expression in the brain and muscle tissue of Spalax and sequence homologous analysis for this gene.L.mandarinus as a typical subterranean rodent has evolved many features to adapt to hypoxia.Although studies on EPO/EPOR,p53 and hemoglobin genes of mandarin vole have been reported,Flk1 has not been deeply studied.Thus,we aim to study the expression of different tissues,sequence analysis and experimental verification of Flk1 under hypoxic environment.In this paper,we take L.mandarinus as our research object and Kunming mice as the control.The expression of Flk1 gene in brain,liver,heart,lung and muscle was investigated under the condition of hypoxia mimicked by a hypoxic chamber in the lab.And then,the sequence conservation of Flk1 gene was analyzed by bioinformatics methods such as phylogenetic analysis,selective pressure analysis and functional annotation of variation sites.RT-q PCR was used to detect the expression of angiogenesis-related genes in the brain tissue of L.mandarinus after hypoxia treatment.CCK-8 was used to detect the effect of Flk1 on cell viability after hypoxia treatment.Through the detection of the expression of Flk1 and its related genes in different tissues,sequence structure analysis and cell viability assays,this paper aims to characterize the hypoxic response of Flk1 gene in different tissues of mandarin voles and then to provide new insights into the mechanism of hypoxia adaptation in subterranean rodents.The main results are as follows:1.Expression of Flk1 gene in different tissues of mandarin voles exposed to hypoxia.After hypoxia treatment,there were significant differences in the expression patterns of Flk1 gene in different tissues of L.mandarinus.More specifically,the expression of Flk1 gene in brain(P<0.001)and muscle tissue(P<0.01)of mandarin vole was significantly up-regulated about 20-fold upon hypoxia.The expression of Flk1 gene in heart and liver was also significantly up-regulated(P<0.05)although the fold change was much lower than that in brain and muscle.However,there was no significant difference in the expression of Flk1 gene in lung of mandarin vole.The expression pattern of Flk1 gene in different tissues of Kunming mice was different from that of mandarin vole.After hypoxia treatment,the expression of Flk1 gene in the brain of Kunming mice was up-regulated about 3-fold(P<0.05),and skeletal muscle was upregulated about 15-fold(P<0.01)and lung was about 1.5-fold(P<0.05),but there was no significant difference among liver and heart.2.Bioinformatics analysis of Flk1 gene in mandarin voleThe CDS of Flk1 gene of L.mandarinus is 4032 bp,encoding 1343 aa,which encodes a protein composed of seven IG-like homologous domains,one transmembrane domain and one tyrosine kinase domain.The sequence of FLK1 is highly conserved,and no convergence evolution between mandarin vole and other subterranean rodents was found.Multi-sequence alignment and function prediction of variant site of FLK1 found that the 4 variant sites shared by L.mandarinus,Nannospalax galili,Fukomys damarensis and Heterocephalus glaber may affect the binding of FLK1 to ligands or signal transduction,of which I450 N and P453 L are located in the fifth IG-like domain,G1145 E is located in the tyrosine kinase domai.It is worth noting that mandarin voles also have two unique variation sites may affect the binding of FLK1 to ligands,in which K183 T is located in the second IG-like domain and T542 I is located in the fifth IG-like domain.In addition,the mandarin vole has 103 mutation sites compared with FLK1 in mice,but these mutations will not have a significant effect on function.3.Expression of angiogenesis-related genes in brain tissue of mandarin voles exposed to hypoxia.After hypoxia treatment,the expression of eight angiogenesis-related genes in the brain tissue of mandarin voles was detected,including VEGFA,MMP2,MMP9,Ecadherin,N-cadherin,VE-cadherin,CD31 and CD34.It was found that the expression patterns of these genes were significantly different in the two species.Although proliferation and migration of vascular endothelial cells was induced by VEGFA upon hypoxia in both mandarin voles and Kunming mice,the genes involved in regulating other stages of angiogenesis may be different in two species.Among them,MMP2,as the key gene of basement membrane degradation process was significantly upregulated in mandarin voles(P<0.05),while MMP9 was significantly up-regulated in Kunming mice(P<0.05).The angiogenesis marker gene CD34 was significantly upregulated in mandarin voles(P<0.05),while CD31 was significantly up-regulated in Kunming mice(P<0.05).In addition,the expression of VE-cadherin and CD31,a mechanosensory complex that can activate VEGFR2 signal,has no significant difference in mandarin vole.It is worth noting that after hypoxia treatment,the expression of E-cadherin in the brain tissue of mandarin voles was significantly downregulated(P<0.05),while that of N-cadherin was significantly up-regulated(P<0.01),which was contrary to the results in Kunmming mice.4.The effect of Flk1 gene of mandarin vole on the survival of HEK293 T cells exposed to hypoxiaThe Flk1 gene of the mandarin vole is conducive to the survival of cells in a hypoxic environment.CCK-8 results showed that compared with untreated HEK293 T cells,the survival rate of cells transfected with empty plasmid or Kunming mice Flk1 overexpression vector under hypoxic conditions did not change significantly,while the cell survival rate with the mandarin vole Flk1 overexpression was significantly upregulated(P<0.05).In addition,compared with cells transfected with empty plasmid,the survival rate of cells transfected with Flk1 overexpression vector of mandarin vole was also significantly increased(P<0.05).Main conclusion:Angiogenesis is important for skeletal muscles to respond to hypoxia.After hypoxia treatment,the upregulation of Flk1 expression level in the skeletal muscles of the mandarin vole and Kunming mice is very similar with relatively high fold change.Mandarin vole could uses the substantially elevated expression of Flk1 gene for angiogenesis or neuroprotection.And sequence analysis did not find that FLK1 has effective structural variation although hypoxia significantly induced the upregulation of Flk1 expression in the brain.The mandarin voles and Kunming mice have different angiogenesis patterns in which the mandarin voles could initiate the angiogenesis process more efficiently through the EMT pathway.This study preliminarily revealed the role of Flk1 gene in the hypoxic adaptation of the mandarin vole.