The Research Of The Function Of Preeclampsia-related MiR-181a

Preeclampsia is a systemic maternal disease, characterized by hypertension andproteinuria, it can involve many organs and is a leading cause of considerable maternalmorbidity and perinatal morbidity and mortality worldwide. Although the cause of thiscondition is poorly understood, many studies have shown that the disease is mainly relatedto placental trophoblast cell dysfunction.MicroRNAs (miRNAs, miRs) are an abundantclass of small (~22nucleotides), non-coding RNAs that play important regulatory roles indiverse bioprocesses including differentiation, development, apoptosis and cellproliferation. The low complementarity required between the sequences of a miRNA andits target mRNA enables a single miRNA to act on a large range of targets. Thus miRNAshave an intersecting complex effect that spans a multiplicity of pathways and processes.miR-181is important in cellular development and differentiation, evidenced by itsaberrant expression in cancers and autoimmunity, which highlights its responsibility forabnormal cellular functions in such disorders.Zhu XM found that miR-181a have specificexpression enhancement in the placenta tissue of patients with preeclampsia by using gene chip technology,and we speculated that miR-181a may be involved in thepathophisiological prosess of preeclampsia.We found that Estrogen receptor α（ESR1/ERα） was the target gene of miR-181a through target gene predicting. Estrogenreceptor α plays an important role in the growth, hormonal, and behavior of women by theregulation of estradiol（E2）. Research has shown that the expression of estrogen receptorα in the placenta tissue may be associated with PE.Therefore it is imperative to know therole which miR-181a plays and its function.There is requirement to study miR-181a andthe correlation of ERα and clarify the target relationship of them.This project slectedhuman trophoblast tumor cell line(JEG-3)for the cell model. Preliminary clarify thefunction of preeclampsia related miR-181a by transient transfection,real time PCR andcellular fuction testing technology.Objective1. To investigate the different expression of miR-181a and its target gene estrogenreceptor α in normal placenta comared with PE placenta,further to validate therelationship of miR-181a, estrogen receptor α and PE.2. To build the construction of vector and study the targeted relationship of miR-181aand estrogen receptor α. Understanding the regulation of miR-181a and estrogenreceptor α in transcription and translation level.3. To understand the regulation function of miR-181a in JEG-3cell apoptosis,proliferation and invasion.Methods1. Real time PCR was putted into use on the tissue of the normal and PE groups to investthe gene expression of miR-181a and estrogen receptor α (ESR1).2. MiR-181a mimics and miR-181a inhibitor was transfected in JEG-3cell, then weevaluated the expression of miR-181a.3. Using biology information forecast software and dual luciferase report system tovalidate the target relationship between miR-181a and estrogen receptor α(ESR1).Evaluating the expression of estrogen receptor α in transfected JEG-3cell with miR-181a mimics and miR-181a inhibitor by real time PCR and Western Blottechnology.4. Flow cytometry and transwell technology were used to study the effect on thefunction of JEG-3cell (apoptosis, proliferation and invasion) after transfected withmiR-181a mimics and miR-181a inhibitor.Results1. Real time PCR results presented that,the expression of miR-181a in normal placentawas obviously higher than that in PE placenta（P＜0.05）,the expression of estrogenreceptor α (ESR1) in normal placenta was obviously lower than that in PE placenta（P＜0.05）.2. Real time PCR results showed that the expression of miR-181a displayed a markedincrease after transfected with miR-181a mimics in JEG-3cell（P＜0.05）; theexpression of miR-181a showed a marked decline after transfected with miR-181ainhibitor in JEG-3cell（P＜0.05）3. Dual luciferase report system result showed that there is clear targrt relationshipbetween miR-181a and estrogen receptor α (ESR1);real time PCR results showedthat,after transfected with miR-181a mimics,the expression of estrogen receptor α(ESR1) was decreased in JEG-3cell;and after transfected with miR-181a inhibitor,theexpression of estrogen receptor α (ESR1) was increased in JEG-3cell（P＜0.05); theresult of Western Blot consistented with the result of PCR（P＜0.05).4. Flow cytometry results showed that there was no significant difference in transfectiongroup compared with control group in JEG-3cell apoptosis and proliferation(P＞0.05);transwell result showed that the invasive ability was obviously decreased aftertansfected with miR-181a inhibitor（P＜0.05).Conclusion1. The expression of miR-181a in normal placenta was obviously higher than that in PEplacenta,the expression of ERα in normal placenta was obviously lower than that inPE placenta.As a target gene of miR-181a, estrogen receptor α was negatively regulated by miR-181a in mRNA and protein level in JEG-3cell.2. The suppression of miR-181a in JEG-3cell resulted in the declined invasion ability,therefore, the high expression of miR-181a may participate in the development of PEby involving the invasion function of trophpblast.