MiRNA Expression Profiling Involved In Preterm Birth Placenta And Preliminary Study Of MiR-182-5p Function In Preterm Birth

Preterm birth(PTB) is defined as any birth before 37 weeks of gestation, or less than 259 days of pregnancy calculated from the first day of the last menstrual period. The incidence of PTB in our country is about 8%, and is increasing. PTBs are responsible for 75% of perinatal mortality and more than half of the long-term morbidity, which are now the most common cause of perinatal mortality and morbidity. Now PTB is thought to be a syndrome, initiated by multifactors, with the precise mechanism of preterm labour remaining to be identified. Still, there are many risk factors that have been associated with PTB, including clinical genetics,infection, a low progesterone level, multiple pregnancy, a short cervix, placental aberrations, age, nutritional status, inflammation and coagulation factors.In recent years, the expression of microRNA(miRNA) and sequence polymorphisms of 3’-UTR of miRNA biding genes are found to be related to preterm birth, preeclampsia, miscarriage and adverse pregnancy outcomes. The objective of this research is focused on establishing the expression profile of mi RNA in preterm placentae, screening preterm birth related miRNA, and preliminarily researching the function which mi RNA played in PTB.Methods:Placental tissue samples were collected soon after the placenta was deliveried from 34 pregnant women from Feb 2013 to Oct 2013. Among them there are 16 preterm patients(PTB group), and 18 normal pregnant women as controls(control group). Microarray of stacking hybridization-based universal tag(SHUT) assay was used to screen the differentially expressed miRNA and real time-PCR method was used to confirm. And construct the wild and mutant vectors of Zinc finger E-box binding homeobox 2(ZEB2)and Factor XIII A(F13A1)using psi CHECK2. Through the use of luciferase and in situ hybridization technique to investigate the function miR-182-5p plays in PTB.Results:1. The result of microarray showed that there were 1138 miRNAs expressed in all the placentae, among which, miR-4251,miR-517c-3p and mi R-23b-3p with the highest expression, mi R-1292-3p,mi R-4632-5p and miR-5787 with the lowest expression. 44 mi RNAs were differentially expressed in preterm placentae compared to control group, among which 33 were up-regulated, and 11 were down-regulated, miR-493-3p was the most down-regulated miRNA with foldchange of 2.77, miR-1322 was the most up-regulated mi RNA with foldchange of 9.81. The RT-PCR results were consistent with microarray result, confirming the accuracy of microarray result.2. miR-182-5p was differentially up-regulated in preterm placentae by RT-PCR results, and mi R-182-5p was expressed universally in cytoplasm of placentae by in situ hybridization method.3. The target genes ZEB2 and F13A1 were predicted by online software. Through Luciferase experiment miR-182-5p was proved to repress ZEB2 and F13A1 directly in HTR-8/SVneo cell line. But there was no statistic difference of mRNA expression of ZEB2 and F13A1 through RT-PCR experiment.Conclusions:1. MiRNAs were differentially expressed in preterm placentae, The role of these miRNAs played in PTB need to be researched in the future.2. Mi R-182-5p was up-regulated in preterm placentae, ZEB2 and F13A1 were repressed by miR-182-5p in vitro, but in miR-182-5p overexpressed preterm placentae, mRNA expression of ZEB2 and F13A1 was not different significantly. So in placentae, miR-182-5p was involved in PTB not by degrading m RNA expression of ZEB2 and F13A1.