Effect Of Rno-miR-33 And Rno-miR-455 Trageting MMP2/9 On Estrogen Induced Endometrial Vascular Permeability In Rat

Objectives: Dysfunctional uterine bleeding(DUB)with abnormal endometrial bleeding is closely related to the estrogen / progesterone imbalance and increase of matrix metalloproteinases(MMPs)levels can cause abnormal endometrial permeability,which is caused by the uterus molecular pathology of endometrial bleeding.In this study,MMP2 and MMP9 targeting study the role of regulation of MMP2 and MMP9 expression and activity miRs and their impact on vascular permeability changes in endometrial tissue and endometrial tissue tight junction protein ZO-1 level.Methods: In this study,first used online software Targetscan bioinformatics analysis and MMP2 and MMP9 targeted binding of mi Rs,with its combination of strength and conservative values of the size of the free energy filter to give the candidate miRs,and then to a luciferase reporter gene system analysis and verification of its combination of targeting;then the candidate rno-miRs transfected rats,12 h after gelatin zymography and ELISA analysis of endometrial tissue,plasma,liver MMP2 and MMP9 activity in qRT-PCR and western blot were used to detect mRNA and protein expression levels of endometrial tissue MMP2 and MMP9,and immunohistochemical methods to observe the expression and distribution of endometrial tissue of MMP2 and MMP9 protein;on this basis,detected by western blot tight junction protein ZO-1 expression levels,the last of endometrial tissue derived rats,frozen section,observed fluorescence intensity PI staining,fluorescence microscopy.All statistical analysis of the data are presented as mean ± standard deviation((?)± SD)represented by Graphpad Prism5.0.1 for data analysis and graphing,select the 95% confidence interval,P <0.05 was considered statistically significant.Results:(1)rat MMP2 of 3'-UTR has a strong conserved binding sites to rno-miR-33miR-23-3p,the 3'-UTR of rat MMP9 no strong conserved miR binding sites,rno-miR-455 is one of a weakly conserved binding sites,online software Bi Biserv2 RNAhybrid visible MMP2 mRNA 3'-UTR and rno-mi R-33,MMP9 mRNA 3'-UTR and freedom rno-miR-455 binding energy are lower threshold(-10 kcal / mol),described MMP2 mRNA 3'-UTR and rno-mi R-33,MMP9 mRNA 3'-UTR and rno-miR-455 has a stable bond.Wherein,MMP2 m RNA 3'-UTR and rno-miR-33 binding free energy value-25.7kcal / mol,MMP9 mRNA 3'-UTR and rno-miR-455 binding free energy value-17.0kcal / mol;(2)luciferase reporter gene analysis showed that: transfection rno-miR-33 mimics lead to a significant decline in wild type psiCHECK ?-2-MMP2-WT 3'UTR plasmid expression levels,while the mutant psiCHECK ?-2-expression of MMP2-Mut 3'UTR plasmid had no effect,rno-mi R-455 mimics make wild type psiCHECK ?-2-MMP9-WT expression levels 3'UTR plasmid significantly decreased,while the mutant psiCHECK ?-2-MMP9-Mut 3'UTR expression plasmid had no effect,which suggests that targeting MMP2 gene rno-miR-33's,MMP9 for the target gene of rno-miR-455,rno-mi R-33 and rno-miR-455,respectively,through the target MMP2 and MMP9 binding to the 3'UTR inhibit the expression of wild-type recombinant plasmid.(3)gelatin zymography results showed that estrogen-induced endometrial tissue can cause increased activity of MMP2 and MMP9,rno-miR-33 and rno-miR-455 transfection were inhibiting MMP2 and MMP9 activity increased,while plasma and liver control group,E2 group,E2 + rno-miR-33 group / E2 + rno-mi R-455 group MMP2 and MMP9 activity was no significant difference among the three groups;(4)qRT-PCR and western blot results showed that estrogen-induced causes endometrial tissue MMP2 and MMP9 mRNA and protein levels were elevated,while rno-miR-33 and rno-mi R-455 transfection were inhibiting MMP2 and MMP9 mRNA and protein levels increased;(5)western blot results showed that female hormone-induced endometrial tissue can cause decreased ZO-1 mRNA and protein levels,while rno-miR-33 and rno-miR-455 transfection can inhibit the decline;(6)frozen sections PI staining showed that uterine PBS-treated control group endometrial tissue was tiny dot fluorescence,and after E2 treatment,endoscopic fluorescence intensity increased dramatically,and there was a large lump,which shows their increased capillary permeability,but the transfected rno-miR-33 and after rno-miR-455,not only decreased the fluorescence intensity and fluorescence dot common,massive fluorescent rare,indicating rno-miR-33 and rno-mi R-455 by inhibiting the activity and expression of MMP2 and MMP9,so that the uterus membranous tissue capillary permeability decreases.But MMP2 and MMP9 inhibitors rno-miR-33 and rno-miR-455 does not make the fluorescence intensity of endometrial tissue response to the level of the control group,and a comparison between the two,there are still significant differences(P <0.05),indicating rno-miR-33 and rno-miR-455 did not completely inhibit MMP2 and MMP9 expression and activity.Conclusions: Rno-miR-33 and rno-mi R-455 can decrease the permeability of the endometrium induced by E2 through inhibiting expression and activity of MMP2 and MMP9 and suppressing the expression of tight junction protein ZO-1.