Study Of The Role Of MiRNA-145-3p/5p In The Pathogenesis Of Early Onset Preeclampsia

【Background】Early onset preeclampsia(EO-PE)is a kind of pregnancy complication which seriously threatens the health of mother and child.Poor placenta remodeling caused by insufficient trophoblast invasion plays a key role in its pathogenesis.Studies have reported that the posttranscriptional regulatory network mediated by micro RNA(micro RNA,miRNA)is widely involved in the pathogenesis of preeclampsia.Among them,miRNA-145-3p/5p may be involved in the pathogenesis of EO-PE by regulating trophoblast function and epithelial mesenchymal transition(EMT)pathway,but the evidence is very limited.【Objective】To identify the miRNA regulatory network related to EO-PE.To find the key miRNA that may play a key role in the pathogenesis of EO-PE and preliminarily illustrate its specific mechanism in order to provide a new perspective for revealing the pathogenesis of EO-PE.【Methods】1.Weighted gene co-expression network analysis combined with differential analysis to identify differentially expressed key genes and miRNAs in placenta of EO-PE.Constructting the miRNA regulatory network in the placenta of EO-PE through Target Scan,miRDB and miRTar Base databases and identifying potential key miRNA(miRNA-145-3p/5p)for subsequent study.2.The expression of miRNA-145-3p in EO-PE placenta was verified by stem-loop real-time quantitative PCR.Transfecting miRNA-145-3p mimic and inhibitor to construct overexpression and silencing trophoblast models in vitro.The abilities of cell proliferation,migration and invasion were detected by CCK-8 method,colony number counting tests,wound healing and transwell experiments.Western blotting experiments were used to detect the protein expression levels of EMT pathway to evaluate the effect of miRNA-145-3p on the EMT pathway.3.The expression of miRNA-145-5p in EO-PE placenta was verified by stem-loop real-time quantitative PCR.Transfecting miRNA-145-5p mimic and inhibitor to construct overexpression and silencing trophoblast models in vitro.The abilities of cell proliferation,migration and invasion were detected by CCK-8 method,colony number counting tests,wound healing and transwell experiments.Western blotting experiments were used to detect the protein expression levels of EMT pathway to evaluate the effect of miRNA-145-5p on the EMT pathway.4.Identifying the regulatory network of miRNA-145-3p and miRNA-145-5p in trophoblasts by high-throughput sequencing of cell transcriptomes to find the potential downstream gene HTRA1 for subsequent research.5.The expression of HTRA1 in the placenta of EO-PE was verified by real-time quantitative PCR and western blotting.The HTRA1 overexpression and silencing cell models were constructed by transfection of plasmid and siRNA respectively.The abilities of cell proliferation,migration and invasion were detected by CCK-8 method,colony number counting tests and transwell experiments.Western blotting experiments were used to detect the protein expression levels of EMT pathway.Mimics of miRNA-145-3p or miRNA-145-5p and HTRA1 overexpression plasmids were transfected simultaneously to block the negative regulation of miRNA-145-3p or miRNA-145-5p on HTRA1,in order to detect the changes of cell proliferation,migration and invasion abilities.Western blotting experiments were used to detect the protein expression levels of EMT pathway to evaluate the effect of HTRA1 gene on the regulation of miRNA-145-3p or miRNA-145-5p on EMT pathway.【Results】1.Identification of miRNA-145 as the key miRNA in the placenta of EO-PE.2.The expression of miRNA-145-3p was decreased in the placenta of EO-PE.Silencing of miRNA-145-3p inhibited the proliferation,migration and invasion abilities of trophoblast cells.Silencing of miRNA-145-3p inhibited the EMT transformation of trophoblast cells.3.The expression of miRNA-145-5p was decreased in the placenta of EO-PE.Silencing of miRNA-145-5p inhibited the proliferation,migration and invasion abilities of trophoblast cells.Silencing of miRNA-145-5p inhibited the EMT transformation of trophoblast cells.4.Cell transcriptome high-throughput sequencing identified 2180 and 2093 differential expressed genes in miRNA-145-3p and miRNA-145-5p overexpressing cell models,respectively,and HTRA1 was down-regulated in both cell models.5.HTRA1 was up-regulated in EO-PE,and overexpression HTRA1 inhibited trophoblast proliferation,migration and invasion abilities of trophoblast cells.Overexpression HTRA1 inhibited trophoblast EMT transformation.Both miRNA-145-3p and miRNA-145-5p could negatively regulate the expression of HTRA1.The cotransfection of HTRA1 plasmid with miRNA-145-3p or miRNA-145-5p mimics could relieve the inhibitory effect of miRNA-145-3p or miRNA-145-5p on HTRA1 expression,and reverse the promotion effect of miRNA-145-3p or miRNA-145-5p on trophoblast proliferation,migration and invasion abilities and EMT.【Conclusions】Overexpression of miRNA-145-3p and miRNA-145-5p may play protective roles by negatively regulating the expression of HTRA1 to promote the proliferation,migration and invasion of trophoblast cells.The low expression of miRNA-145-3p and miRNA-145-5p promoted the occurrence and development of EO-PE by regulating HTRA1.