The Role Of MiR-133 In The Pathogenesis Of Preeclampsiaon

Pre-eclampsia(pre-eclampsiaon,PE)is a multi-system progressive disease unique to pregnancy.It is characterized by new onset of hypertension and proteinuria after 20 weeks of pregnancy,or new onset of hypertension and terminal organ dysfunction accompanied with or without proteinuria.The incidence is about 4-6%over the world,and it is divided into early-onset preeclampsia and late-onset preeclampsia according to the time of onset(with a 34-week boundary).Those with an onset of less than 34 weeks are early onset preeclampsia and also named severe preeclampsia.PE is caused by a variety of factors,including spiral arterial remodeling,placental oxidative stress,endothelial dysfunction,and systemic inflammation.Regardless of the initial cause,it is known that the placenta plays a key role in the occurrence and development of PE Removal of the placenta will cause the clinical symptoms of PE to cease.Therefore,to elucidate the pathogenesis of PE,it is important to understand how the gene regulatory network functions in the placenta during PE.In recent years,microRNAs(miRNAs),a non-coding RNA with a length of about 19-25 nt,have attracted much attention as a new class of gene expression regulators.miRNAs can regulate various cellular processes involved in normal development and disease onset,including cell cycle,development,metabolism,various immune responses and other processes,and participate in the occurrence and development of various diseases,such as:developmental disorders,tumors,Alz Heimer disease and PE.Several studies have shown that miRNA expression in the placenta of PE patients is significantly abnormal,and a variety of differentially expressed miRNAs play an important role in the development of PE.The miR-133 gene is dysregulated in many human diseases,including bladder cancer,lung cancer,gastric cancer,pituitary tumors,and various cardiovascular diseases.At present,there is no research on the expression and related functions of miR-133 in peripheral blood and trophoblasts in PE patients.This project intends to study the expression of miRNA-133 in early-onset preeclampsia,its biological function,and its possible mechanism of action.Part Ⅰ:Expression and biological function of MiR133 in Preeclampsia ObjectiveThe aim of this study was to investigate the expression of miRNA133 in peripheral blood and placenta-derived primary trophoblasts from early onset PE patients,and to explore its possible biological functions.Materials and Methods:Patients with eraly onset pre-eclampsia(PE group)and normal pregnancy(NC group)were tested for blood pressure,24-hour urine protein,and liver function and renal function.The expression of miR133 in peripheral blood of PE group and NC group was detected by RT-PCR.Trophoblast were separated from placental tissue by density gradient centrifugation.miR-133 Mimics and inhibitors were constructed to overexpress or knock down the expression of miR-133 in trophoblast.The cytokine changes before and after transfection were detected by ELISA.The TUNEL method was used to detect the apoptosis of trophoblast cells before and after transfectionResults:1.Patients with early-onset preeclampsia(PE group)systolic blood pressure(146.8±2.4mmHg VS 112.5±3.8mmHg),diastolic blood pressure(108±4.3 mmHg VS 75.8±3.9mmHg),and 24-hour urine protein(7.25±0.42 g VS 0.01g)was significantly higher than the normal control group,and liver and kidney function of PE patients were significantly abnormal.2.The expression of miR-133 in peripheral blood of patients with early-onset preeclampsia(PE group)was significantly increased3.The concentration of inflammatory factors(TNF-α,IL-6,IL-1)increased after miR-133 upregulated,while the concentration of inflammatory factors decreased after miR-133 was knocked down4.Apoptosis of trophoblasts was significantly increased after overexpression of miR-133,while apoptosis of trophoblasts was significantly reduced after miR-133 was knocked down.Part Ⅱ:Research on miR133 induced trophoblast apoptosis related mechanismObjective:To explore the mechanism of miR-133 induced trophoblast apoptosis.Materials and Methods:miR-133 Mimics and inhibitors were constructed to overexpress or knock out the expression of miR-133.RT-PCR was used to detect the expression of apoptosis-related genes and RHO\ROCK-related genes.Western blot was used to verify the expression of related genes.Results:1.Upregulated miR-133 promoted the expression of Caspase-3 at the RNA level and suppressed the expression of apoptosis-inhibiting gene BCL-2,while knockdown of miR-133 inhibited the expression of Caspase3 and elevated BCL-2 expression.The Rho/ROCK pathway-related protein ROCKI was highly expressed in the Mimics group,while ROCKI was low expressed in the Inhibitors group.2.Overexpressed miR-133 promoted the expression of apoptotisis-inducing gene Caspase-3 and Rho/ROCK pathway-related protein ROCKI,while reverse effect was observed while konckdow of miR-133.It is suggested that MiR-133 may regulate trophoblast apoptosis through the Rho\ROCK signaling pathway.Conclusion:1.Compared with normal pregnant women,the expression of mir-133 in peripheral blood of patients with early-onset eclampsia was significantly increased.Therefore,mir-133 may be used as a new marker for the diagnosis of PE.2.Overexpression of mir-133 in primary trophoblast cells promoted the production of inflammatory cytokines and trophoblast cell apoptosis,and inhibited the production of inflammatory cytokines and trophoblast cell apoptosis after knockout.In addition,after overexpression of mir-133,the pro-apoptotic gene caspase-3 was highly expressed at RNA and protein levels,while the expression of apoptotic gene bcl-2 was inhibited.The opposite phenomenon was observed after knockout of mir-133.This suggests that mir-133 plays a role in the occurrence of PE by regulating trophoblast apoptosis.3.After overexpression of mir-133,Rho/ROCK pathology-related protein ROCKI was highly expressed at RNA and protein levels,while after knockdown of mir-133,ROCKI was poorly expressed at RNA and protein levels.This suggests that mir-133 may play an important role in early preeclampsia by regulating trophoblast apoptosis by activating the Rho/ROCK signaling pathway.