Landscape Of LncRNAs And DNA Methylation In The Placentae And Their Association With Expression Pattern Change In Preeclampsia

BackgroundPreeclampsia is a pregnancy-specific disorder,presenting with hypertension and after 20 weeks of pregnancy,accompanied by proteinuria and maternal organ dysfunction.It is one of the leading causes of maternal and fetal death and one of the critical factors that lead to severe complications during the perinatal period,affecting the pregnancy outcomes and the prognosis of the parturient and brain development of the offspring.There is no effective therapy for this disorder but termination of the pregnancy.All the symptoms are alleviated soon after the placentae are delivered,suggesting that preeclampsia is a placenta-originated disorder.Many gene dysregulations have been reported in preeclampsia.Recently,we have searched the global gene expression change in preeclamptic patients’ placentae and found that about 3,000 genes are differentially expressed in the placentae of the patients with early-onset severe preeclampsia(EOSPE).The differentially expressed genes are mainly involved in functions such as immunity,metabolism,and transportation,providing resources for further study of the role of the placenta in this disorder.The key question now remains:what regulators control the gene expression pattern in the preeclamptic placenta?Although there are some anecdotal studies on the gene dysregulation in the placenta of preeclampsia,a systematic research on gene regulation is needed to illustrate the mechanism for changing the global gene expression pattern.Here,we present a landscape of the dysregulated lncRNAs in the placentae of patients with early-onset severe preeclampsia.We have further illustrated the role of these lncRNAs in the differential gene expression by constructing a"lncRNA-TF-target" hierarchical regulatory network.We also systematically searched differential DNA methylation to explore the possible epigenetic regulation in the placentae.Methods1.Transcrip tome sequencing and validation:We collected 32 placenta samples from 30 normal individuals and 9 placenta samples from 9 patients with EOSPE.We extracted total RNA for transcriptome sequencing.The differentially expressed genes(DEGs)were detected using DESeq2,and functional enrichment analysis was performed using clusterProfiler.Transcription-factor binding sites(TFBS)were predicted using HOMER.The lncRNA-protein interaction network was constructed using interaction data collected from databases and data predicted by catRAPID.The TF-target regulatory network was created using the transcription factors with significant TFBS predicted by HOMER.The triplexes of differential expression lncRNAs(DElncRNAs)and promoter regions of DEGs were predicted using TDF(Triplex domain finder).The"lncRNA-TF-target" hierarchical regulatory network was constructed by integrating the lncRNA-TF interaction network,TF-target regulatory network,and lncRNA-promoter interaction network.After the lncRNAs were ranked according to their importance in the network(including the number of targeting DEGs,fold change in EOSPE,and the biological function of its neighbourhood protein-coding genes),qRT-PCR was carried out to verify the change of FLNB-AS1 in EOSPE.The lncRNA pulldown experiment was performed,and mass spectrometry was used to detect proteins interacting with FLNB-AS1.Immunoprecipitation was performed to verify the interaction between JUNB and FLNB-AS1.2.DNA methylation analysis:We collected 25 placenta samples from 25 normal individuals and 10 placenta samples from 10 patients with EOSPE.We extracted genomic DNA samples for reduced represent bisulfite sequencing(RRBS).Differentially methylated sites/regions were detected using methylKit,and functional enrichment analysis was done using enrichR to search for enriched functions for genes with differentially methylated promoters.Results1.The role of DElncRNAs in differential gene expression in EOSPEIn RNA-sequencing data,we got 3,116 dysregulated genes,including 383 differentially expressed lncRNAs(DElncRNAs).We found that proteins interacting with DElncRNAs are enriched with TFs and coding DEGs in EOSPE,suggesting that DElncRNAs may regulate gene expression through interacting with TFs.And most of DElncRNAs can form a large number of triplex structures with different DNA regions,each interacting with the promoters of an average number of 1,453 DEGs,suggesting that DElncRNAs may contribute to the global gene expression change in EOSPE.Previous studies show that some lncRNAs can act as a scaffold for lncRNA-DNA triplex structure to mediate or stabilize the interaction between TFs and promoters.We predicted 18 significantly enriched TF-binding motifs in the DEGs of EOSPE using HOMER.A total of 2,479 DEGs(79.56%of total DEGs)were found to have the binding motifs at their promoters for these 18 TFs.However,most of these TFs(14 of 18)did not show a significant change in EOSPE,indicating that some other factors may modulate the function of the transcription factors.Therefore,we hypothesized DElncRNAs might contribute to the dysregulation of most of the DEGs through interacting with these 18 TFs.To investigate the role of DElncRNAs in the regulation of differentially expressed genes in EOSPE,we then constructed a hierarchical regulatory network of "DElncRNA-TF-target" by integrating the DElncRNA-protein physical interactions,TF-target regulatory interactions,and interactions between DElncRNAs and promoters of the DEGs.This"lncRNA-TF-target" hierarchical regulatory network contains 287 DElncRNAs,18 TFs,and 2,357 DEGs(75.64%of total DEGs),which can explain most of the dysregulated genes in the placentae of patients with EOSPE,suggesting that DElncRNAs can affect gene expression in preeclamptic placentae.We further ranked the DElncRNAs based on the number of targeting genes,their expression fold change in EOSPE,and the biological functions of the protein-coding genes in their neighbourhoods on the chromosomes.We chose a top-ranked DElncRNA,FLNB-AS1,as an example for further functional studies in the downstream experiments.We conducted RNA-pulldown coupled with mass spectrometry and found that FLNB-AS1 can interact with 27 transcription factors,including JUNB.JUNB may be one of the key transcription factors which regulate differentially expressed genes in EOSPE.The differentially expressed genes of JUNB targeting were significantly enriched in a known pathway involved in preeclampsia,the HIF-1 signaling pathway.We further performed RNA immunoprecipitation to validate the interaction between JUNB and FLNB-AS1.Moreover,we overexpressed FLNB-AS1 in HTR8/SVneo cells.We found JUNB did not show a significant change in expression,but the expression level of its regulatory targets in the HIF-1 signaling pathway was significantly changed,consistent with what we found in EOSPE patients.However,it remains to be further illustrated whether FLNB-AS1 is involved in the pathogenesis of preeclampsia through interacting with JUNB.2.The role of differential DNA methylation in differential gene expression in EOSPEWe detected 6,886 differentially methylated cytosine sites and 173 differentially methylated promoter regions between EOSPE and normal.Furthermore,DNA methylation is negatively correlated with the gene expression level,suggesting that DNA methylation may contribute partially to the gene expression change in EOSPE.We further performed functional analysis on the genes associated with differentially methylated promoter regions and found that they were notably involved in the biological process of apoptotic.For example,GDF15(Gene growth differentiation 15)reported to be associated with hypoxia and inflammation is one of the up-regulated genes in EOSPE,and its promoter region is hypomethylated.We further checked the expression level of DNA methyltransferases in EOSPE.Only DNMT3A(DNA methyltransferase 3 alpha)was down-regulated in EOSPE.Since DNMT3A functions in de novo methylation,the decreased expression of DNMT3A in EOSPE may lead to changes in the DNA methylation pattern.Further studies are needed to explore the role of DNMT3A in the DNA methylation of the placenta of EOSPE patients and its contribution to gene dysregulation.DiscussionWe have provided a landscape of placental DElncRNAs in EOSPE and constructed a "lncRNA-TF-target" hierarchical regulatory network by integrating the lncRNA-protein interaction network,TF-target regulatory network,and lncRNA-promoter interaction network.We found that differential expression of lncRNAs may contribute to most of the differential expression of the transcriptome in EOSPE by interacting with TFs.We have also provided a landscape of differential DNA methylation in the placentae of EOSPE patients and found that DNA methylation change in promoter regions may partially contribute to the global gene dysregulation found in the placentae of EOSPE patients.