| BackgroundPreeclampsia (pre-eclampsia, PE),a group of disease about pregnancy along with ahigh blood pressure,which seriously affects maternal and child health, is one of the leadingcauses of elevated maternal and perinatal mortality.However,the pathogenesis ofpreeclampsia is not yet completely clear,which hinders the progress of the prevention andtreatment of the disease.Therefore,further study of the development mechanism ofpreeclampsia is currently the top priority.Small RNA(microRNA, miRNA), a kind ofsmall RNA which can adjust the protein expression but not encoded protein, can targetgene mRNA degradation or hinder its translation,plays an important role in regulatinggene expression.Studies have shown that miRNAs play an important role in the regulationof cell cycle and growth and development of life.Imbalance miRNAs express in theplacenta may lead to preeclampsia and other pregnancy-related disease[1].Our group in theprophase study stage using gene chip technology and real-time PCR technology hasconfirmed that miR-30a-3p express specificity increases in the placenta of patients with preeclampsia, and has carried out in vitro experiments,researching the influence ofmiR-30a-3p expression level to HTR-8/SVneo cell function,which showed thatmiR-30a-3p expression level and HTR-8/SVneo cell apoptosis rate were positivelycorrelated,we speculate that high expression of miR-30a-3p in the placenta is associatedwith the onset of preeclampsia. However,miR-30a-3p regulates which target genesspecifically?And what role relevant target genes play in the pathogenesis of preeclampsia?To solve these problems,we carry out further prediction and validation studies ofmiR-30a-3p related target genes of preeclampsia.Objects1. To predict and screening miR-30a-3p target genes.2. Study the regulation relationship and acting sites between miR-30a-3p and targetgenes.Methods1. Using bioinformatics software predicted target genes of miR-30a-3p, screeningtarget genes according to the scores in the candidate target genes by bioinformaticssoftware and its relation with preeclampsia.2. Using Liposome transfection technique in HTR-8/Svneo cell establish miR-30a-3poverexpress and inhibition model,using real-time fluorescent quantitative PCR andWestern-blot technique to detect the cell model of miR-30a-3p and expression level oftarget genes.3. Building purpose luciferase report carrier of target genes,using double carrierluciferase report system test the relationship between miR-30a-3p and target genes andbinding sites.Results1. Bioinformatics prediction results of the prediction software show that IGF-1is oneof the theory target genes for miR-30a-3p,IGF-13′UTR region contains five sites whichcould theoretically combine with miR-30a-3p. 2. Real-time PCR and Western-blot results show that the expression of miR-30a-3psignificantly increases (P<0.05) in the over expression group,and the expression of mRNAand protein levels of IGF-1decrease significantly (P<0.05);in the inhibition expressiongroup, the expression of miR-30a-3p is significantly decreased (P<0.05), the expression ofIGF-1on mRNA and protein levels is significantly increased (P<0.05).3. Dual luciferase report system results show that miR-30a-3p and IGF-1a hasprecise targeting relations;IGF-13’UTR region has four possible sites which couldcombine with miR-30a-3p.ConclusionsIGF-1is one of miR-30a-3p target genes,in HTR-8/Svneo cells miR-30a-3p haveobvious negative regulation function on IGF-1on the mRNA and protein levels, highexpression of miR-30a-3p in placenta reduced the expression levels of IGF-1gene,whichleads to increased cell apoptosis rate of trophoblastic cells,decreased invasion ability,thateventually leading to the onset of preeclampsia. |
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