The Mechanisms Of Preeclampsia Serum Increasing The Permeability Of HRGECs Through MiRNAs-CAV1 Pathway

ObjectiveThis study is to predict miRNAs targetingon CAV1 and screening miRNAs related tothepathogenesis of proteinuria in preeclampsia.Then explore the effects of the predicted miRNAs on the expression of CAV1 and permeability of human renal glomerular endothelial cells(HRGECs)to evaluate thetargeting functions of miRNAs to CAV1.Methods1.Predict targeted miRNAs of CAV1 by using bioinformatic methods and screening miRNAs associated with pathogenesis of preeclampsia.2.Collect serum samples of patients with severe preecalmpsia and normal pregnancies.Measure the expression of miR-199a-5p,miR-199b-5p,miR-204 in HRGECs after treated with serumfrom severe preecalmpsia patient and normal pregnancy women.Detect CAV1 protein expression of each group by Western blot.The permeability of HRGECs to high molecular weight protein in each group were measured by Evans-blue conjuncted BSA in Transwell chambers.3.MiRNA mimics,inhibitors and their negative controls were transfected into HRGECs respectively.After 48 hours of transfection,detect CAV1 mRNA and protein expression of each group by q PCR and Western blot.Measure the permeability of HRGECs to high molecular weight protein in each group by Evans-blue conjuncted BSA in Transwell chambers.4.Select the most possible targeted miRNA and construct plasmid vector with the binding sites of CAV1 3'UTR.Detect the relationship between the selected miRNA and CAV1 by using dual-luciferase reporter assay system test.Results1.miR-199a-5p,miR-199b-5p,miR-204 were selected as research objects according to the prediction results.2.The level of CAV1 protein as well as the premeability of HRGECs were increased in PE group(p<0.05).QPCR indicated that severe preeclampsia serum decreased the expression of miR-199a-5p,miR-199b-5p,miR-204 in HRGECs significantly(p<0.05).3.QPCR showed that there is no obvious difference in CAV1 mRNA expression inmiR-199a-5p,miR-199b-5p,miR-204 upregulated groups and all of the miRNA-down regulated groups(p>0.05).Western blot showed that miR-199b-5p and miR-204 mimics inhibited the expression of CAV1 protein(p < 0.05),while There is no dramatic difference in CAV1 protein expressioninmiR-199a-5pupregulated groups and NC groups.(p>0.05).For all the miRNA-downregulated groups,CAV1 protein expression all increased significantly compared with NC group.4.The results of permeability of HRGEC monolayer show that compared with NC group,the HRGEC monolayer has lower permeability in miR-199b-5pand miR-204 mimic groups and no obvious difference in miR-199a-5p mimic group.(p<0.05)Transfection of the 3 miRNA inhibitors all increased the HRGEC monolayer permeabilitysignificantly.(p<0.05)5.Dual-luciferase reporter assay system test demonstratedmiR-199b-5p and miR-204 have precise targeting relationship with CAV1.ConclusionsThis study shows miR-199b-5p andmiR-204 increased the permeability of HRGECs by inhibiting the process of CAV1 translation directly.MiR-199a-5p may participate in the regulation of CAV1 in indirect pathways.MiR-199a-5p,miR-199b-5p,miR-204-CAV1 pathway may contribute to the pathogenesis of proteinuriain preecalmpsia.