Expression Changes And Significance Of MiR-155 And CXCR4 In Placenta Tissues From Patients With Preeclampsia

Preeclampsia(PE)is a common medical complications during pregnancy--a type of hypertensive disorders complicating pregnancy.PE is more common in obstetrics.The diagnosis of PE is pregnant women systolic blood pressure?140mm Hg and/or diastolic blood pressure ?90mm Hg manifested after 20 weeks of gestation for the first time combined with one of the following new-onset conditions: proteinuria(?300 mg/day or random urine protein+);or maternal organ dysfunction(including renal insufficiency,liver damage,neurological or haematological complications);or uteroplacental dysfunction(potential result in fetal growth restriction).From a global perspective,more than one million women die each year from pregnancy-related disease in low-and middle-income countries.Approximately 10% of women have high blood pressure during pregnancy and the concurrent preeclampsia accounted for 2% to 8%.Considering the placenta between maternal and fetal is the important organ which maintain the fetus intrauterine growth and development.Based on clinical symptoms of preeclampsia quickly mitigate or heal after delivery of the placenta,speculated that the onset of preeclampsia is closely related to pathological and physiological changes of the placenta.Most scholars believe that the structure of the placenta is relatively complex and trophoblast cells are the main types of it.Dysfunction of trophoblast cells can lead to blood vessels invasion obstacle of the pregnant women,as well as uterine spiral arterial remodeling is insufficient,result in reducing utero-placental perfusion,placental hypoxia/ischemia and release some cytokines into the maternal circulation which further damage the maternal endothelium.Pathogenesis theory also relates to genetics,nutrition,inflammation and insulin resistance.But so far,the precise pathogenesis of preeclampsia remains unclear.MicroRNAs(miRNAs)are a kind of highly conserved noncoding-protein RNA molecules(about 19~22 nucleotides in length)that are key players in the inhibition of messenger RNA(m RNA)expression or to its degradation mainly at the post-transcriptional level.Nowadays miRNAs databases have been established since the researchers have successfully identified approximately 3707 novel mature miRNAs.Bioinformatics analysis showed that more than one thirds of human genes are regulated by miRNAs.Recently,accumulating evidence has indicated that the abnormal expression of miRNAs in the placenta is associated with the pathogenesis of preeclampsia.Chemokine receptor 4(CXCR4)is a specific receptor of chemokine stromal cell-derived factor-1(SDF-1,also known as CXCL12),which have strong affinity and often forming SDF-1/CXCR4 signaling pathway.Several studies have confirmed that the SDF-1/CXCR4 signaling pathway not only affects the activation,migration and recruitment of immune cells,but also involves in some processes including angiogenesis,hematopoietic function,embryogenesis,as well as tumor invasion and metastasis.In addition,another scholar in cancer research found that CXCR4 may be a target gene of miR-155,miR-155 affects the angiogenesis of cancer and the function of cancer cells by regulating the expression of CXCR4 and then participate in the occurrence of cancer.However,the expression level of miR-155 and CXCR4 and how to participate in the pathogenesis of preeclampsia has not been reported.Objective The objective of this study were to using real-time fluorescent quantitative PCR(Real-time PCR)experiment method,detection the expression level of miR-155 and CXCR4 m RNA of severe preeclampsia group and normal control group in placenta tissue.Using Streptavidin-biotin-peroxidase complex(SABC)immunohistochemical methods and high-throughput detection tool�tissue microarray to analyse CXCR4 protein's localization and expression in preeclampsia group and normal control group.In order to investigate the expression level of miR-155 and CXCR4 in placenta tissue and relationship with the pathogenesis of preeclampsia.Materials and Methods 1 Materials 1.1 Research object of Real-time PCR The placenta tissue of 30 pregnant women in cesarean section delivery were selected as the experimental group,who were diagnosed as severe preeclampsia from November 2015 to February 2016 Maternal and Child Care of Henan Province,and the placenta tissue of 30 healthy pregnant women ungergo cesarean section delivery were selected as normal control group at the same period.The placenta tissue in both experimental group and normal control group were used for detection the expression of m RNA.1.2 Research object of Placenta Tissue Microarray The placenta tissue of 80 pregnant women in cesarean section delivery who were diagnosed as severe preeclampsia and the placenta tissue of 58 healthy pregnant women undergo cesarean section delivery from December 12,2007 to December 31,2010 in the Third Affiliated Hospital of Zhengzhou University were used for constructing a high-throughput detection tool�tissue microarray to analysis CXCR4protein's localization and expression.The tissue microarray was constructed by Maternal and child health translational medicine engineering laboratory of Henan Province.1.3 The diagnosis and inclusion criteria of research object Preeclampsia and its different classification's diagnosis criteria were strictly according to the eighth edition of the �Obstetrics and Gynecology� which published by the People's Medical Publishing House.Blood pressure below 140/90 mm Hg throughout pregnancy,no urine protein,without any complications such as autoimmune diseases,obstetric complications and fetal anomalies were incorporated into the normal control group.All the research subjects enrolled into this study were single pregnancies,cesarean section delivery.2 Methods The expression level of miR-155 and CXCR4 m RNA of experimental group and normal control group in placenta tissue was detected by real-time fluorescent quantitative PCR.Using the Spearman method to analyse the correlation between miR-155 and CXCR4 m RNA inexperimental group.Using Streptavidin-biotinperoxidase complex(SABC)immunohistochemical methods and high-throughput detection tool�tissue microarray to analyse CXCR4 protein's localization and expression in preeclampsia group and normal control group.The placental villous cytotrophoblast(VCT)tissue microarray contains 42 cases normal control placenta samples and 56 cases preeclampsia placenta samples.Similarly,29 cases normal control placenta samples and 47 cases preeclampsia placenta samples on placental extravillous cytotrophoblast(EVCT)tissue microarray.3 Statistics methods The experimental data was analyzed with the statistics software SPSS 22.0.Measurement data are expressed as mean�standard deviation((?)�s).The 2-??Ct method was exploited for analyzing the Real-time PCR data.After the inspectionnormality and variance homogeneity of data,both of which are consistent with the use of two independent sample t-test.Using the Spearman method to analyse the correlation between miR-155 and CXCR4 m RNA inexperimental group.Using nonparametric rank test to compare Immunohistochemical results.With inspection level ?=0.05.Results 1 The comparison of clinical features between the experimental group and normal control group Experimental group(s PE group)pregnant women aged between 22~35 years old,with an average of 27.7�2.8 years old.Normal control group(N group)pregnant women aged between 20~32 years old,with an average of 28.8�2.5 years old.Body mass index(BMI)at delivery,gestational age,systolic blood pressure,diastolic blood pressure,mean arterial pressure,the birth weight,maternal urine protein of s PE group were respectively 21.2�2.9 kg/m2,37.2�2.7 weeks,169.5�17.6 mm Hg,112.4�15.7 mm Hg,131.5�15.6 mm Hg,2210.6�905.1g,5.6�2.6 g/L.And normal control group was respectively 22.2�2.4 kg/m2,38.7�0.8 weeks,112.6�9.4 mm Hg,71.8�7.1mm Hg,85.6�7.5 mm Hg,3650.5�403.6g,0 g/L.In terms of average age,BMI at delivery and gestational age,no statistically significant differences between the two groups(P> 0.05).In terms of systolic and diastolic blood pressure,mean arterial pressure and urine protein,the experimental group were significantly higher than the normal control group(P< 0.05).In terms of birth weight,the experimental group was lower than the normal control group(P< 0.05).2 The expression levels of miR-155 and CXCR4 m RNA in the experimental group and the normal control group The relative expression levels of miR-155 and CXCR4 m RNA in the experimental group and the normal control group in turn were1.53�0.92,0.87�0.73;0.54�0.38,1.53�0.73.And the difference between the two groups was statistically significant(P< 0.05).3 The correlation between miR-155 and CXCR4 m RNA in experimental group The relative expression levels of miR-155 in the experimental group was 1.53�0.92.And the relative expression levels of CXCR4 m RNA was 0.54�0.38.Using the Spearman method to analyse the correlation between miR-155 and CXCR4 m RNA in experimental group.The results showed that there was significant negative correlation(r=?0.773,P< 0.05).4 The localization and expression of CXCR4 protein on placental VCT and EVCT tissue microarrays CXCR4 proteins were expressed in cytomembrane and cytoplasm of villous cytotrophoblast and extravillous cytotrophoblast.The results showed that the positive expression rate of CXCR4 protein of preeclampsia group on VCT tissue microarray was 48.21%(27/56).And the normal control group was 83.33%(35/42).The difference between the two groups was statistically significant(U=715.5,P< 0.05).On EVCT tissue microarray,the positive expression rate of CXCR4 protein of preeclampsia group was 48.94%(23/47),the normal control group was 79.31%(23/29).The difference between the two groups was statistically significant(U=362.0,P< 0.05).Conclusions MiR-155 in placental tissue with severe preeclampsia might regulate the expression level of CXCR4 m RNA and proteininvolved in the pathogenesis of preeclampsia.