Expression Of Plasma MicroRNA-301b In Patients With Hypertensive Disorder Complicating Pregnancy And Its Clinical Significance

Hypertensive disorder complicating pregnancy(HDCP)is a group of diseases that are unique to pregnancy and accumulated in multiple systems.It is the main cause of maternal and perinatal morbidity and mortality.The prevalence of preeclampsia(PE)is high,and it has the greatest effect on mothers and babies.Hypertension with or without proteinuria is the main clinical manifestation after 20 weeks of gestation.Once diagnosed,the entire remaining gestational weeks have been deteriorating state until delivery.Clinical therapeutic intervention usually ends with early delivery.Although this intervention protects the mother,the neonatal morbidity and mortality are significantly higher.So looking for its cause,early diagnosis and intervention is our goal.The pathogenesis involves shallow placenta implantation,placental ischemia and hypoxia,vascular endothelial cell damage,genetic inheritance,maternal-infant immune imbalance,nutritional deficiencies,and insulin resistance.There is no single theory to explain its cause and pathogenesis.The mechanism,despite the overall improvement in medical standards worldwide,still lacks accurate early diagnostic indicators and effective treatment methods.Most pregnant women have found complicationswhen they have high blood pressure,even affect the fetus,and missed the golden intervention period.The current methods for diagnosis of HDCP are mainly based on elevated blood pressure,urinary protein positive and headache,abdominal pain and other clinical manifestations and Biomarkers such as PP13,PIGF,s Flt-1,but these methods have hysteresis or limitations.Fortunately,the discovery of miRNAs opens another window of opportunity for the early diagnosis of HDCP because plasma miRNAs are non-invasive biomarkers,such as screening for miRNAs with high sensitivity and specificity for the diagnosis of HDCP,early detection and prevention HDCP will become possible.There is increasing evidence that miRNAs are abnormally expressed in placental tissue and plasma in patients with hypertensive disorders complicating pregnancy and are involved in the development of hypertensive disorders during pregnancy.However,the understanding of the expression pattern of miRNAs and their biological function in the occurrence and progression of hypertensive disorders during pregnancy,especially in HDCP,is still unclear.Research at home and abroad shows that uterine spiral artery arterial trophoblastic remodeling disorders in early pregnancy make "placenta shallow implantation",leading to placental ischemia,hypoxia is the key to the pathogenesis of HDCP,more and more evidence shows that many miRNAs passed Hypoxia reacts differentially and participates in the occurrence and development of HDCP.At present,the most studied miR-210 is one of theproducts of cell expression in a hypoxic environment and can be specifically induced by the hypoxia-inducible factor HIF-a.Hypoxia-induced miR-210 can down-regulate the expression of hepatocyte receptor ligand Ephin-A3 and homeobox gene A9(Hox9)and participate in trophoblast migration,invasion and vascular remodeling.Zhang et al.confirmed that activated Toll-like receptor 3 can bind to and induce expression of the promoter of miR-210 through HIF-1a and nuclear factor k B p50 protein(NF-k B p50),thereby targeting inhibition of IL-4 and transcription activation The factor 6(IL-4/STAT6)signaling pathway plays a role in the pathogenesis of HDCP.miR-301 b is one of the micro RNAs that are differentially expressed in response to hypoxia.It has been reported that miR-301 b is involved in vasoconstriction in pulmonary arterial hypertension.miR-301 b is aberrantly expressed in a variety of solid cancers,such as up-regulated expression in relatively hypoxic pancreatic,lung,colorectal,and oral cancer tissues.Recent studies have found that the abnormal expression of miRNA-301 b involves the occurrence and development of hypertensive disorders in pregnancy.The expression of miR-301 b can be induced by hypoxia and play an important role in trophoblast differentiation,invasion,and vascular remodeling.However,the expression and biological functions of miR-301 b in hypertensive disorders during pregnancy are not fully understood.In this study,we found that miR-301 b is differentially expressed in the plasmaIn this study,we found that miR-301 b is differentially expressed in the plasma of patients with HDCP.Differentially expressed miR-301 b plays an important role in trophoblast differentiation,invasion,and vascular remodeling.Objectives: This study was designed to detect the expression level of miR-301 b in plasma and to explore the differences in the expression and clinical significance of miR-301 b in hypertensive disorder complicating pregnancy patients and healthy pregnant women.Materials and methods1.Clinical data Recruited 66 patients with hypertensive disorder complicating pregnancy from January to November 2017 in the obstetric department of our hospital and Guangzhou Women’s and Children’s Hospital.The prenatal group was divided into the following three groups:(1)Pregnancy Hypertension group(A,n=20;SBP ≥140 mm Hg and(or)DBP ≥90 mm Hg after 20 weeks of gestation,normal blood pressure within 12 hours after delivery);(2)Mild preeclampsia group(B,n=21 20 weeks after pregnancy SBP ≥140mm Hg and(or)DBP ≥90mm Hg with proteinuria >0.3g/24 h,or random urinary protein(+),blood pressure returned to normal within 12 W after birth);(3)Severe preeclampsia group(C,n=25;SBP ≥160 mm Hg and/or DBP ≥110 mm Hg,proteinuria≥5 g/24 h or random proteinuria≥(+++)after 20 weeks of gestation,normal blood pressure within 12 W after delivery;gestational period Hypertensive disease group subgroups postpartum(n=9,13,20,respectively).In the same period,20 patients in the normal early pregnancy group(NGT1)and the normal late pregnancy group(NGT2)who were examined in this hospital were selected as the control group.All patients involved in this study were clinically confirmed and met the eighth edition diagnostic criteria for obstetrics and gynecology textbooks.Exclusion criteria:acute and chronic infections,autoimmune diseases,hematological diseases,tumors and any other obstetric complications.At the same time,24-hour urinary protein,blood pressure,gestational age,BMI,neonatal weight,blood routine,and coagulation function were recorded.2.Method Prenatal components of pregnant women with hypertensive disorders during pregnancy were pregnancy-induced hypertension(A),mild preeclampsia group(B),severe preeclampsia group(C),and normal early pregnancy group(NGT1)and normal late pregnancy group(NGT2),and some blood samples were collected from postpartum 2nd day of hypertensive disorder complicating pregnancy,and were classified as their postpartum group.The plasma miR-301 b expression was detected by q PCR.And analyze the relationship between HDCP and blood pressure,24 h urinary protein,age,gestational age,BMI,white blood cells,platelets,coagulation function.2.1 Separation and storage of plasma: All selected subjects were randomly assigned to take 10 ml of venous blood in the early morning.The specimens were centrifuged at 4°C,3000 r/min,and centrifuged for 10 min within 1 h after collection.The plasma was extracted and transferred to RNase-free sterile Eppendorf.The tubes(EP tubes)were centrifuged at 4°C and16000×g/min for 10 minutes,transferred to a new RNase-free sterile EP tube,and frozen at-80°C until use.2.2 Extraction of plasma total RNA: Plasma total RNA was extracted using a kit for miRNA rapid extraction kit(spin column type)III(Bioteke Corporation),and the total amount and purity of plasma RNA were determined by ultraviolet spectrophotometry.2.3 miRNA primer design and synthesis: Design and synthesis of miR-301 b and internal reference U6 gene primers were provided by Gene Copoeia(Cat.No.HmiRQP0379).2.4 Detection of miR-301 b expression: Real-time fluorescent quantitative PCR method was used with U6 as the internal reference.The first strand c DNA was synthesized using Gene Copoeia’s All-in-One TM miRNA q RT-PCR Detection Kit(Cat.No.QP015)and q PCR was used to quantify miRNAs.Each c DN sample was tested three times at a time and the mean value of the CT values was taken for analysis..3.Statistical method SPSS 13.0 software was used for statistical analysis.The measurement data was represented by mean ± SD.One-way ANOVA was used for comparison among multiple groups.Multiple comparisons between groups were performed using LSD method.Welch test was used for inconsistent variance.Multiple comparisons were performed using Dunnett’s T3 method;q PCR results were analyzed using U6 as an internal reference gene and 2-ΔΔCT method was used to calculate the relative expression of the target gene;the prenatal and postpartum group was compared using the paired sample t-test(satisfying positive Homogeneity and homogeneity of variance; using multiple linear regression analysis of the factors affecting pregnancy-induced hypertension,when P <0.05,the difference was considered statistically significant.Result1.Comparison of basic clinical data There was no significant difference in the age,white blood cells,hemoglobin,and platelets among the groups(P>0.05);gestational age was less than 13 weeks in the early pregnancy group,which was not comparable with gestational age and BMI in other groups.There was no significant difference in gestational age between the early pregnancy group and the normal group(P>0.05).The BMI in the mild preeclampsia group was higher than that in the normal late pregnancy group,the gestational hypertension group,and the severe preeclampsia group.Significance(P<0.05);Differences in D-dimer and fibrinogen(Fib)between normal late pregnancy group,gestational hypertension group,mild preeclampsia group,and severe preeclampsia group There was statistical significance(P<0.05);SBP,DBP,and urinary protein in the severe preeclampsia group were higher than those in the gestational hypertension group,mild preeclampsia group,normal pregnancy early and late group;normal pregnancy early and late group The SBP,DBP,and urinary protein levels were lower than those in the gestational hypertension group and the mild preeclampsia group(P<0.05).The weight of the newborns in the normal and early pregnancy group was heavier than that of pregnancy-induced hypertension.Group,mild preeclampsia group and Preeclampsia group,the difference was statistically significant(P <0.05);Differences in the expression of miR-301 b between the hypertensive disorder complicating pregnancy and the normal pregnancy control group,hypertensive disorder complicating pregnancy and its subgroups postpartum,compared with the normal early pregnancy group.Plasma miR-301 b expression in the normal late pregnancy group,mild preeclampsia group and severe preeclampsia group was down-regulated,and was negatively correlated with the severity of the disease(P<0.05);and the normal late pregnancy group Compared with the hypertensive group,plasma miR-301 b was highly expressed in the hypertensive group,there was a statistically significant difference(P<0.05).Mild preeclampsia group,severe preeclampsia group plasma miR-301 b expression decreased,there are statistically significant differences(P<0.05);miR-301 b expression levels in hypertensive disorders during pregnancy were significantly lower in the severe preeclampsia group than in the gestational hypertension group,mild preeclampsia group,and severe preeclampsia group.Plasma miR-301 b was down-regulated,with statistically significant differences(P<0.05);postpartum plasma miR-301 b expression was increased in the mild preeclampsia group and severe preeclampsia group compared with prenatal.Difference(P<0.05),pregnancy-induced hypertension group After plasma decreased expression of miR-301 b,no significant difference(P> 0.05);Multiple linear regression analysis of age,gestational age,systolic blood pressure,24 h urinary protein,leukocytes,platelets and fibrinogen are the influencing factors of pregnancy-induced hypertensive disease,and the effect is 24 h urinary protein,systolic blood pressure,white blood cell in descending order.Gestational age,platelets,fibrinogen,age.Conclusion1.Plasma miR-301 b expression is highest in normal early pregnancy patients.With the prolonged pregnancy cycle,plasma miR-301 b in the late normal pregnancy group showed a low expression trend,the difference was statistically significant(P<0.05),but the normal late pregnancy The plasma miR-301 b was highly expressed in patients with gestational hypertension compared with gestational age(P>0.05),and low miR-301 b expression was observed in patients with mild preeclampsia and severe preeclampsia.There was a statistically significant difference(P<0.05)and a negative correlation with the severity of HDCP.The plasma miR-301 b expression in the hypertensive disorder group after delivery had converged to a normal value,suggesting plasma miR-301 b It may participate in the occurrence and development of HDCP,and it has certain guiding significance for the clinical diagnosis of HDCP.2.Plasma miR-301 b may be a promising biomarker for the early diagnosis of HDCP.3.Multiple linear regression analysis showed that miR-301 b is the primary influencing factor of HDCP,followed by systolic blood pressure,neutrophils,24 h urine protein.