The Role And Mechanism Of MiR-139-5p By Targeting SFlt-1 In Preeclampsia

Preeclampsia(PE)is termed as an obstetric issue that is characterized by hypertension,proteinuria and organ damage.PE is one of the main causes of high morbidity and mortality in pregnant women and perinatal children.Currently,the exact etiologies and pathogenesis for the preeclampsia are unknown.There are several mechanisms that contribute to the development of PE: spiral arterial remodeling failure,placental shallow implantation,placental oxidative stress,endothelial cell damage and excessive inflammatory responses.Earlier studies have found that the expression of soluble fms-like tyrsine kinase-1(s Flt-1)in peripheral blood and placental trophoblasts of PE patients is abnormally increased,which plays an important role in the occurrence and development of PE.s Flt-1 can interact with vascular endothelial growth factor(VEGF)and placental growth factor(PLGF),thereby blocking the biological activity of VEGF and PLGF,leading to angiogenesis disorders and vascular endothelial damage,then induce preeclampsia.However,the molecular mechanism that regulates the expression of s Flt-1 is not yet clear.Micro RNA(mi RNA)is a class of non-coding small RNA which contains 21-25 nucleotides.mi RNA plays an important regulatory role in the development of early fetal embryos,cell differentiation,apoptosis,cell proliferation and tumors.Multiple mi RNAs can be abnormally and differentially expressed in peripheral blood and placenta of PE patients.mi RNAs in PE patients play a major role in cell adhesion,signal transduction and cell cycle,immunity,cardiovascular development,etc.,which are critical signal pathways involved in the pathogenesis of PE.mi R-139-5p is located in the 11q13.4 region of human chromosome.It is a common mature mi RNA with a length of 23 nt.It is currently a well-known tumor suppressor gene.A significant reduction in the expression of mi R-139-5p in the placenta of PE patients has also been reported,but its role and mechanism in preeclampsia is not clear.Earlier studies have showed that the expression level of s Flt-1 is regulated by mi RNA.In addition,it has been reported that mi R-139-5p mimics can effectively inhibit the expression of Flt-1 in glioma cells,and s Flt-1 is a splicing of the m RNA precursor encoded by Flt-1 gene precursor encoded.Target Scan(www.targetscan.org)analyzed that mi R-139-5p and s Flt-1 have predicted binding sequences,therefore,we speculated that mi R-139-5p may be related to s Flt-1 and plays an important role in preeclampsia.This study aimed to investigate the expression and the mechanism of mi R-139-5p and s Flt-1 in s PE.It provides a theoretical basis for studying the pathogenesis of preeclampsia and treating preeclampsia.Part One Expression pattern of s Flt-1 and mi R-139-5p in s PE patientsObjective: To detect the expression levels of s Flt-1 and mi R-139-5p in the placenta of s PE patients,and to analyze the correlation between them.Methods: 1.Peripheral blood and placenta were obtained from patients diagnosed with s PE(46 cases)and pregnant women(57 cases)without any pregnancy complications,and culture the placental trophoblasts.2.ELISA was used to detect the expression level of s Flt-1 in the serum of the subject,and q RT-PCR was used to detect the expression level of s Flt-1 m RNA and mi R-139-5p in the placenta of the subject.3.Transfect s PE trophoblasts with mi R-139-5p mimics and its negative control plasmid,use q RT-PCR and Western Blot to study the effect of mi R-139-5p overexpression on s Flt-1 expression level.4.The sequences of s Flt-1 including the wild type binding sites of mi R-139-5p(WT-s Flt-1)and the mutated binding sites(MUT-s Flt-1)were subcloned into p MIR reporter vector and cotransfected into HEK-293 T cells along with the mi R-139-5p mimics.The direct interaction between s Flt-1 and mi R-139-5p was determined by Dual-luciferase reporter assay.Results: 1.The expression level of s Flt-1 in serum and placental tissue of s PE patients was significantly increased.ELISA and q RT-PCR results showed that compared with normal pregnancy,the expression level of s Flt-1 in serum and s Flt-The expression level of 1m RNA was significantly increased.2.The expression of mi R-139-5p in placental tissue of s PE patients was significantly down-regulated q RT-PCR results showed that compared with the normal pregnancy group,the expression of mi R-139-5p in placental tissue of s PE patients was significantly down-regulated.3.Spearman's analysis shows that mi R-139-5p was negatively correlated with the expression of s Flt-1 in the placental tissue of s PE patients.4.Study on the targeting relationship between mi R-139-5p and s Flt-1 Transfect the trophoblasts of s PE patients with mi R-139-5p mimics and its negative control plasmid.q RT-PCR and Western Blot results showed that the excessive expression of s Flt-1 in s PE patients was downregulated by the overexpression of mi R-139-5p mimics.Dual luciferase assay results showed that a significant decrease in luciferase activity was observed when WT-s Flt-1 were co-expressed with mi R-139-5p mimics,while MUT-s Flt-1 showed no remarkable difference in the luciferase activity.Summary: 1.mi R-139-5p is down-regulated in s PE placenta tissue.2.mi R-139-5p targeted regulation of s Flt-1 and play an important role in the development of preeclampsia.Part Two Effects of mi R-139-5p targeted regulation of s Flt-1 on the biological function of trophoblast cells in s PE.Objective: To study the effects of mi R139-5p and s Flt-1 on the biological function of s PE trophoblast cells.Methods: 1.Transfect the placental trophoblasts of s PE patients with mi R-139-5p mimics and its negative control plasmid.2.The CCK-8 analysis was used to detect the proliferation of trophoblasts,and q RT-PCR method was used to detect the nuclear antigens(PCNA and Ki67)which can reflect the cell proliferation status.3.Transwell assay was used to analyze the invasion function of trophoblasts.q RT-PCR was used to detect matrix metalloproteinases(MMP-2,MMP-9)and their inhibitors(TIMP-1).4.Overexpression of s Flt-1 and mi R-139-5p mimics in s PE trophoblasts,CCK-8 analysis was used to detect the proliferation of trophoblasts,and Transwell assay was used to analyze the invasion function of trophoblasts.Results: 1.mi R-139-5p promotes the proliferation of trophoblast cells in s PE patients CCK-8 analysis showed that the cell proliferation of trophoblast cells was significantly decreased in s PE patients,while overexpression of mi R-139-5p partially reversed this effect.q RT-PCR results showed that the expression levels of PCNA m RNA and Ki67 m RNA in s PE placental trophoblast cells were significantly reduced.The decreased expression of PCNA and Ki67 in s PE patients was upregulated by transfection of mi R-139-5p mimics.mi R-139-5p promotes the proliferation of trophoblast cells in s PE patients.2.mi R-139-5p promotes the invasion of trophoblast cells in s PE patients Transwell analysis showed that mi R-139-5p significantly increased the number of the invasive cells,which was decreased in s PE patients.q RT-PCR results showed that the decreased expression of MMP-2,MMP-9,TIMP-1 in s PE patients was remarkably upregulated by transfection of mi R-139-5p mimics.mi R-139-5p promotes the invasion of trophoblast cells in s PE patients.3.Overexpression of s Flt-1 attenuates the effects of mi R-139-5p CCK-8 analysis revealed that the promoting effects of mi R-139-5p on cell proliferation were inhibited by the co-transfection with s Flt-1.Transwell analysis showed that the cell invasion of trophoblast cells was significantly decreased when transfected with mi R-139-5p mimics,while overexpression of s Flt-1 partially reversed this effect.Overexpression of s Flt-1 attenuates the effects of mi R-139-5p.Summary:mi R-139-5p promoted the proliferation and invasion of trophoblast cells by directly targeting s Flt-1 in s PE.Part Three Study on the mechanism of mi R-139-5p and mangiferin in PEObjective: To establish a mouse model of PE and explore the relationship between mi R-139-5p and the PI3 K /Akt/m TOR pathway.To study the effect of mangiferin on the expression of mi R-139-5p and its mechanism in PE.Methods: 1.The PE mouse model was established by the injection of PS/PC.The peripheral blood and placental tissues of the subjects were collected.2.ELISA was used to detect the expression of s Flt-1 in peripheral serum of normal pregnancy mice,PE mice and PE + mi R-139-5p mimics mice.q RT-RCR method was used to detect s Flt-1 m RNA in placental tissue of the three groups.Western Blot method was used to detect the phosphorylation levels of PI3 K,Akt and m TOR in placental tissues of the three groups.3.The mi RNA microarray chip was used to analyze the mi RNA expression profile of in mice.q RT-PCR was used to detect mi R-139-5p expression in placental tissues of normal pregnancy mice,PE mice and PE + mangiferin 40 mg/kg mice.4.ELISA method was used to detect the expression level of s Flt-1 in the serum of normal pregnancy mice,PE mice and PE + different doses of mangiferin mice.q RT-RCR was used to detect the expression level of s Flt-1 m RNA in placental tissue of each group.Western blot was used to detect the phosphorylation levels of PI3 K,Akt and m TOR in placental tissues of each group.5.To study the effect of mangiferin on systolic blood pressure,proteinuria and fetal weight in PE mice.Results: 1.Overexpression of mi R-139-5p inhibited the expression of s Flt-1 ELISA and q RT-RCR results showed that overexpression of mi R-139-5p significantly reduced the expression level of s Flt-1 in serum and placental tissue of PE mice.2.Overexpression of mi R-139-5p activated PI3K/Akt/m TOR pathway Western Blot results showed that the protein expression levels of p-PI3 K,p-Akt and p-m TOR in the placenta of PE mice were significantly reduced compared with the control group.Overexpression of mi R-139-5p can significantly increase the protein expression levels of p-PI3 K,p-Akt,p-m TOR in the placenta of PE mice.3.Mangiferin increased the expression of mi R-139-5p Analysis of mi RNA microarray chip and q RT-PCR showed that mangiferin could significantly increase the expression level of mi R-139-5p.4.Mangiferin reduced the expression of s Flt-1 ELISA and q RT-PCR results showed that mangiferin could significantly reduced the expression level of s Flt-1 in PE mice.The upregulated s Flt-1 expression was rescued by mangiferin treatment in a dose-dependent manner.5.Mangiferin can activate the PI3K/Akt/m TOR pathway Western blot results showed that compared with the control group,the protein levels of p-PI3 K,p-Akt and p-m TOR in the placenta of the PE mice were significantly reduced.After the treatment of mangiferin,p-PI3 K,p-Akt and p-m TOR were all rescued in a dose-dependent manner.6.Mangiferin treatment can improve the pregnancy outcome of PE mice.The results showed that mangiferin treatment efficiently decreased the up-regulated SBP and proteinuria level as well increased fetal weight in PE mice in a dose-dependent manner.Summary: 1.PC/PS can successfully establish a PE mouse model.2.mi R-139-5p plays a role in PE mice by activates PI3K/Akt/ m TOR pathway.3.Mangiferin up-regulated mi R-139-5p level can improve pregnancy outcome in PE mice.Conclusion: 1.mi R-139-5p is down-regulated in s PE placenta tissue.2.mi R-139-5p promoted the proliferation and invasion of trophoblast cells by directly targeting s Flt-1 in s PE.3.mi R-139-5p plays a role in PE mice by activates PI3K/Akt/ m TOR pathway.4.Mangiferin can up-regulate mi R-139-5p to improve pregnancy outcome in PE mice.