Placental Transcriptome Analysis Of Hemoglobin Bart’s Hydrops Fetalis Syndrome And Functional And Mechanism Study Of Circular RNA Hsa＿circ＿0006687

CHAPTER 1:Placental transcriptome analysis in Hemoglobin Bart’s hydrops fetalis syndrome and the screening of differentially expressed circRNAsOBJECTIVE:Hemoglobin Bart’s hydrops fetalis syndrome(BHFS)is the most serious form of α-thalassaemia,with a high incident rate in South China,Southeast Asia and the Mediterranean region.It is a deadly disease threatening both mother and fetus.And it’s also a main challenge for birth defect prevention.Gross appearance and histological changes can be observed in the placenta of BHFS.However,few studies have been conducted on the molecular changes of BHFS.To our knowledge,there is no placenta transcriptome profile or circRNA study for this disease.The aim of this study was to define the placental transcriptional changes and find relevant circular RNAs in BHFS.METHODS:We performed high-throughput RNA sequencing to detect transcriptome alternation in placenta from BHFS fetuses(BHFS group,n=5)and normal fetuses(NF group,n=5).Bioinformatics methods including differential expression analysis,GO enrichment analysis,pathway enrichment analysis and gene set enrichment analysis(GSEA)were used for further analysis.RT-qPCR and Sanger sequencing were used to validate the differentially expressed circRNAs(BHFS group,n=22;NF group,n=11).GSEA was performed for the verified differentially expressed circRNAs.RESULTS:Our results showed 152 differentially expressed genes(DEGs),112 circRNAs,and 45 microRNAs that were differentially expressed.DEGs were found to be involved in Gene Ontology terms related to gas transport,cell adhesion,oxidative stress,organ development,hemopoiesis,etc.RT-qPCR results showed that hsa＿circ＿0003961 and hsa＿circ＿0006687 were upregulated(P<0.05),whereas the difference of hsa＿circ＿0008732,hsa＿circ＿0002810,and hsa＿circ＿0029872 was not significant.GSEA results for hsa＿circ＿0003961 and hsa＿circ＿0006687 showed that some gene sets associated with cell adhesion,hematopoietic system,angiogenesis and apoptosis were significantly enriched.CONCLUSION:Our study preliminarily explored the changes of the placental transcriptome of BHFS.The results showed that the changes in placental expression profile of BHFS may not only reflect the functional changes of the placenta,but also indicate the changes of the fetus and the mother.Two circRNAs(hsa＿circ＿0003961 and hsa＿circ＿0006687)in the placenta may be associated with BHFS and have biomarker potential.CHAPTER 2:Experiment research to explore the function of hsa＿circ＿0006687 in trophoblast and human umbilical vein endothelial cell linesOBJECTIVE:Placental transcriptomic study had revealed the differential expression of hsa＿circ＿0006687 in BHFS placenta.But the function of hsa＿circ＿0006687 has not been reported.In order to explore the potential effects of hsa＿circ＿0006687 on placenta,we investigated the function of hsa＿circ＿0006687 in cell proliferation,apoptosis,migration,invasion and angiogenesis in trophoblast and HUVEC cell lines,so as to understand the possible effects of hsa＿circ＿0006687 on placental function and pathophysiological processes of BHFS.METHODS:JEG-3,HTR-8/SVneo and HUVEC cell lines were used for function experiment(JEG-3 and HTR-8/SVneo cell lines were used for trophoblast proliferation,apoptosis,cell cycle,migration and invasion functions research;HTR-8/SVneo and HUVEC cell lines,which have tube formation ability,were used to conduct in vitro tube formation experiments).Subcellular localization of hsa＿circ＿0006687 was detected by nucleo-plasma isolation RNA extraction and RT-qPCR.The recombinant overexpression plasmids of hsa＿circ＿0006687 were constructed and transfected to the three cell lines,respectively.The effects of hsa＿circ＿0006687 on cell proliferation in JEG-3,HTR-8/SVneo and HUVEC cell lines were investigated by CCK-8 assay.The effects of hsa＿circ＿0006687 on apoptosis in JEG-3 and HTR-8/SVneo cells were investigated by TUNEL assay and flow cytometry.WB was performed to detect the apoptosis-related proteins Caspase3 and Caspase9.PI single staining method was used to detect the effect of hsa＿circ＿0006687 on the cell cycle in JEG-3 and HTR-8/SVneo cells.The effects of hsa＿circ＿0006687 on cell migration and invasion in JEG-3 and HTR-8/SVneo cells were detected by the transwell experiment.The effect of hsa＿circ＿0006687 on the angiogenesis of HTR-8/SVneo and HUVEC cells was investigated by angiogenesis experiment.RESULTS:The results of ucleo-plasmic RNA isolation and RT-qPCR showed that hsa＿circ＿0006687 expressed more in cytoplasm than in nuclear.CCK-8 assay showed that overexpression of hsa＿circ＿0006687 inhibited the proliferation of JEG-3 and HTR-8/SVneo cells,but no effect was observed in HUVEC cells.The results of flow cytometry and TUNEL assay showed that the apoptotic cells in JEG-3 and HTR-8/SVneo cells increased after overexpression of hsa＿circ＿0006687.The apoptotic proteins Caspase3 and Caspase9 were upregulated in the overexpression group of hsa＿circ＿0006687.The results of PI single staining method showed that there was no significant change in the proportion of cells in each period of the cell cycle among three groups.The results of the transwell experiment indicated that the migration and invasion ability of cells decreased after overexpression of hsa＿circ＿0006687.Angiogenesis assay results showed that no significant changes in the cell tube formation ability were observed after overexpression of hsa＿circ＿0006687 in HTR-8/SVneo and HUVEC cell lines.CONCLUSIONS:Hsa＿circ＿0006687 can inhibit the proliferation,migration and invasion and induce the apoptosis of trophoblast cells in vitro,suggesting that hsa＿circ＿0006687 has adverse effects on placental function.It may be involved in the pathophysiological process of hypoxia,edema,abnormal development in BHFS fetus and maternal complications by affecting the development of placental villi and the invasion ability of trophoblasts.CHAPTER 3:Mechanism exploration of hsa＿circ＿0006687 in BHFS placentaOBJECTIVE:In order to explore the possible mechanism of hsa＿circ＿0006687,which inhibits the proliferation,migration and invasion,and promots apoptosis in trophoblast cells,we performed these experiment.METHODS:To predict the molecules that might interact with hsa＿circ＿0006687,we performed the ceRNA network and co-expression network analysis starting with hsa＿circ＿0006687.The predicted downstream factors hsa-miR-625-5p and SOX6 expression changes were verified using RTqPCR and immunohistochemistry methods in the placenta of the NF group and BHFS group.After overexpression of hsa＿circ＿0006687 in vitro,the expression of predicted downstream molecules hsa-miR-625-5p and SOX6 were detected by RT-qPCR and WB.The potential binding sites(hsa＿circ＿0006687 to hsamiR-625-5p and hsa-miR-625-5p to SOX6)were predicted using the online website Circular Interactome and TargetScan.Hsa＿circ＿0006687 and SOX6 wild-type and mutated dual-luciferase reporter plasmids were constructed,respectively.293T cells were co-transfected in 96-well plates.The cells were divided into 6 groups(EV+NC,EV+miRNA mimics,WT+NC,WT+miRNA,MUT+NC,MUT+miRNA),with 3 replicates in each group.Fluorescence intensity was detect 24 hours after transfection.RESULTS:The results of CeRNA network analysis showed that hsa-miR625-5p and hsa＿circ＿0006687 had the same MRE,and interacted with 14 coding genes potentially.Co-expression network analysis showed that hsa＿circ＿0006687 was highly correlated with 51 genes in the sequencing data.The genes in the co-expression network were intersected with those in the ceRNA network.It was found that a total of 5 genes both in the ceRNA network and the co-expression network,among which SOX6 gene had been reported that it can inhibit proliferation,migration,invasion and promote apoptosis in cells.Cricular Interactome and TargetScan websites predicted that they had potential binding sites.RT-qPCR and immunohistochemistry results showed that the expression of hsa-miR-625-5p was down-regulated,while SOX6 was upregulated in the BHFS placenta group.After the overexpression of hsa＿circ＿0006687 in JEG-3 and HTR-8/SVneo cells,the expression level of hsa-miR-625-5p was decreased,while the level of SOX6 was increased.The results of dual luciferase assay indicated that the relative fluorescence intensity of 293T cells was significantly decreased after co-transfection of hsa＿circ＿0006687 or SOX6 wild-type plasmids with hsa-miR-625-5p mimics compared to the control group.CONCLUSIONS:Hsa＿circ＿0006687 may inhibit the proliferation,migration and invasion and induce the apoptosis of trophoblast cells through hsa-miR-625-5p/SOX6,thus participating in the pathophysiological changes of fetal hypoxia,edema,malformationg and maternal complication in BHFS.