Transcriptome Analysis Of Placenta And Kidney In SFLT-1 Induced PE Mice Model

ObjectiveThrough analyzing the differentially expressed genes(DEGs)in kidney or placenta tissues of over-expressing sFlt-1 preeclampsia(PE)mice model and normal pregnant mice,screening the DEGs for functional annotation family classification and information regulatory pathway analysis,analyzing the main functions and signal pathways of genes that are significantly changed in the pathological process of placenta and kidney of over-expressing sFlt-1 PE mice model,it provides evidence-based bases for exploring possible molecular bases and regulatory mechanisms in pathological process of kidney and placenta in PE,and new targets for PE prevention and clinical intervention.Methods1.Construction of over-expressing sFlt-1 PE mice model:In this study,the overexpressing sFlt-1 PE mice model were constructed by tail vein injection of sFlt-1recombinant adenovirus(Ad-sFlt-1).First,pregnant mice were divided into 5 groups of normal saline control group(NS)and different PFU Ad-sFlt-1 treatment groups(1×10~9,2×10~9,3×10~9,4×10~9).NS/4×10~9 PFU Ad-sFlt-1 was injected through tail vein at the 9.5 day of pregnancy.The data of sFlt-1 serum concentration,blood pressure,urinary microalbumin/creatinine ratio(ACR)and the weight of fetal mice of each group were compared to determine the optimal injection dose of the overexpressed sFlt-1 PE mice model.2.Model building experimental grouping:The formal experiment were divided into three groups:the sFlt-1 recombinant adenovirus group(Ad-sFlt-1),adenovirus no-load control group(Ad-EGFP),and normal saline control group(NS).Ad-sFlt-1/Ad-EGFP/NS was injected to compare the sFlt-1 serum concentration,blood pressure,ACR,the weight of fetal mice,and renal pathological changes in the three groups.3.Over-expressing sFlt-1 PE mice model’s placenta and kidney differential gene screening and functional mapping:placenta and kidney tissue RNAs of three mice in each group were extracted for transcriptome analysis,DEGs of placenta and kidney samples were counted,gene sets were constructed,GO and KEGG enrichment analysis and PPI network analysis were performed.Then some genes were selected for q RT-PCR verification.Results1.With the gradual increase of the tail-vein injection dose,blood pressure,serum sFlt-1 levels,and urinary ACR levels in mice also gradually increased.The 3×10~9 PFU and 4×10~9 PFU groups were significantly different from the NS group(P<0.05),there was no difference between the two groups(P>0.05).So we choose 3×10~9 PFU as the tail vein injection dose for construction of over-expressing sFlt-1 PE mice model:.2.Compared with the other two groups,the Ad-sFlt-1 group had significantly increased sFlt-1 serum concentration,blood pressure,and ACR(P<0.05),significantly reduced fetal mice weight(P<0.05),and pathological changes of the kidney under light and electronmicroscope.It showed typical PE glomerular changes such as increased glomerular volume,narrowed capillary lumen,endothelial cell proliferation and swelling,and partial foot process fusion.3.The results of placenta DEGs analysis showed that 106 genes were significantly up-regulated and 9 genes were significantly down-regulated in over-expressing sFlt-1 PE mice model’s placenta.Genes related to inflammation,immunity,and invasion mechanisms were significantly up-regulated.GO and KEGG enrichment analysis suggested extracellular matrix components interactions,complement and coagulation cascades,glycerophospholipid biosynthesis and other pathways were significant.PPI network analysis screened out key genes such as Apoe,Acta2 and Epcam.The q RT-PCR verification results of Lcn2,Sftpd,Hsd3b1,Acta2,Epcam Apoe,Tagln,Tnnt2,Cnn1 and Myh11 were consistent with the trend of sequencing data.4.The results of kidney DEGs analysis showed that 156 genes were significantly up-regulated and 14 genes were significantly down-regulated in over-expressing sFlt-1PE mice model’s kidney.Genes related to dyslipidemia,inflammation and podocyte injury were significantly up-regulated.GO and KEGG enrichment analysis showed significant changes in complement and coagulation cascades,and glycerophospholipid metabolism pathways.PPI network analysis screened out key genes such as Fgg and Fgb.The q RT-PCR verification results of Pcsk9,Adgrg3,Clcf1,Tia1,Leng8,Kcnc3,Plcd3,Zfp692,Fgg and Fgb were consistent with the trend of sequencing data.Conclusion1.High levels of sFlt-1 in the circulation can cause PE-like symptoms in pregnant mice,and within a certain range,the symptoms become worse with the increase of sFlt-1 serum concentration.3×10~9PFU Ad-sFlt-1 can successfully establish over-expression PE clinical mice model.2.Overexpression of sFlt-1 may participate in the pathological process of placenta in mice model with clinical characteristics of PE by initiating inflammation,immunity and trophoblast invasion through activating the pathways of extracellular matrix components interactions,complement and coagulation cascades and glycerophospho-lipid biosynthesis.3.Overexpression of sFlt-1 may participate in the pathological process of kidney in mice model with clinical characteristics of PE by initiating dyslipidemia,inflammation and podocyte injury through activating the pathways of complement and coagulation cascades,and glycerophospholipid metabolism pathways.