| Background:Preeclampsia is one of the major causes of perinatal death,premature delivery and intrauterine growth restriction.At present,the specific pathogenesis of preeclampsia is still not clear.It is generally believed that it is caused by a variety of pathological and physiological processes,including poor placenta implantation,vascular endothelial dysfunction and systemic inflammatory reaction.Studies have found that poor placental implantation in early pregnancy is particularly related to the pathogenesis of preeclampsia,which is caused by the decrease of placental blood flow due to the insufficient remolding of uterine spiral arteriolae,resulting in placental ischemia and hypoxia,and then releasing various placental factors,promoting the activation of maternal systemic inflammatory response.These biological behaviors influence each other and eventually lead to the occurrence and development of preeclampsia.A number of studies in recent years have found that miR-146 a plays an important role in immune regulation,angiogenesis,inflammatory response and other aspects.Studies have shown that miR-146 a promotes melanoma migration and invasion by targeting Smad4.Previous studies have shown that the expression of miR-146 a is decreased in preeclampsia,but the specific role of miR-146 a in the pathogenesis of preeclampsia and its effect on trophoblast cells are still unclear.Smad4 is a member of the Smad family,which mediates signaling in the TGFβpathway that inhibits epithelial cell growth.Smad4 acts as a tumor suppressor gene to regulate the occurrence and progression of many cancers,such as prostate cancer and melanoma.Smad4 is also a key factor in TGFβ1-induced epithelial mesenchymal transformation(EMT),growth inhibition,and apoptosis.Vascular endothelial growth factor(VEGF),as a downstream gene of Smad4,has been widely accepted to play an important role in the pathogenesis of preeclampsia.VEGF is not only involved in angiogenesis and uterine spiral arteriolar recasting,but also regulates trophoblast cell invasion,proliferation and differentiation.A number of studies have shown that the expression of VEGF is significantly reduced in placental tissue and serum of patients with preeclampsia.Currently,there are few studies on miR-146 a and Smad4 in preeclampsia.Therefore,this study takes this as the starting point to explain the changes in the expression of miR-146 a and Smad4 in the placenta of preeclampsia and the relationship between the two.In addition,trophoblast cells HTR-8/ SVneo cell lines were used to verify the effects of hypoxia on miR-146 a,as well as the effects of miR-146 a transfection on the proliferation,invasion and apoptosis of HTR-8/SVneo cells and the expression of Smad4 and VEGF.Objective1.To explore the expression of miR-146 a and Smad4 in the placental tissues of preeclampsia patients and their relationship;And the effects of miR-146 a on the proliferation,invasion and apoptosis of trophoblast cells,as well as the effects of miR-146 a on the expression of Smad4 and VEGF2.To explore whether trophoblast proliferation ability was affected in the simulated hypoxia environment,and whether the expression of miR-146 a was changed in the hypoxia environment.Experimental methods1.Select research objects and collect specimensTissue samples were collected from patients in the severe preeclampsia group according to the diagnostic criteria of the newly released Guidelines for the Diagnosis and Treatment of Hypertension Diseases in Pregnancy(2020).This study was divided into severe pre-eclampsia(SPE)group and Normal group(N),with 30 samples in each group.Normal group were healthy pregnant women with term pregnancy in our hospital.Exclusion criteria were chronic hypertension,hypothyroidism,gestational diabetes mellitus,autoimmune diseases,chronic hepatitis,assisted reproduction and twin pregnancy.Tissue samples used in this study were all collected from the Biobank of Scientific Research Center of the Third Affiliated Hospital of Zhengzhou University by Biobank staff from May 2018 to December 2019.2.The mRNA expressions of miR-146 a and SMAD4 in the placental tissues of the severe preeclampsia group and the normal group were detected by RT-PCR,and the data were analyzed by 2-△△ CT relative quantitative method.3.Western blot was used to detect the protein expression of Smad4 in the placental tissues of the two groups,and image analysis software was used for relevant quantitative analysis.4.Immunohistochemical method was used to detect the protein expression and localization of Smad4 in the placental tissues of the two groups,and Image analysis software Image J was used for relevant semi-quantitative analysis of the images.5.The trophoblast cells were transfected with miR-146 a mimics,miR-146 a inhibitors,small interfering RNA of SMAD4 and negative control,and the transfection efficiency of miR-146 a and SMAD4 was observed by RT-RCR and Western blot.6.After successful transfection,the effect of miR-146 a on trophoblast proliferation ability was observed by CCK8 assay,the effect of miR-146 a on trophoblast invasion ability was observed by Transwell assay,and the apoptosis of trophoblast cells was detected by flow cytometry,as well as the effect of miR-146 a on downstream genes Smad4 and VEGF,and the effect of Smad4 silencing on VEGF;7.After simulating anoxic environment with cobalt chloride,RT-PCR was used to detect the expression of miR-146 a and whether the proliferation ability of trophoblast cells was affected under anoxic conditions under different concentrations of cobalt chloride.8.Statistical analysis: Statistical software SPSS21.0 was used to analyze the obtained data.All the data were expressed as mean ± standard deviation.0.05 is statistically significant.Result1.Comparison of the basic clinical data of the two groups of pregnant women.There were 30 pregnant women in the normal group(Normal),with an average age of(31.03±5.32),an average gestational age of(38.65±1.18)weeks,and an average prepregnancy BMI of(23.63±3.88)Kg /m2,the average systolic blood pressure was(111.00±7.37)mm Hg,and the average diastolic blood pressure was(75.10±4.43)mm Hg;30 pregnant women(sPE)in the severe preeclampsia group,the average age was(31.23±5.00),and the average gestational week of delivery It was(34.23±2.88),the average pre-pregnancy BMI was(23.29±3.52)Kg/m2,the average systolic blood pressure was(165.20±14.00)mm Hg,and the average diastolic blood pressure was(103.13±9.29)mm Hg.The results of the two groups showed that there was no statistically significant difference in the age of delivery and pre-pregnancy BMI of pregnant women between the severe preeclampsia group and the normal group;the difference between the two groups of pregnant women’s gestational week,systolic blood pressure,diastolic blood pressure and 24 h urine protein quantitative difference There is statistical significance,P<0.01.2.Comparison of the two groups of newborns.The average newborn weight of pregnant women in the normal group(Normal)is(3393.00±414.81)g,and the placental weight is(614.00±87.00)g.The average newborn weight of pregnant women in the severe preeclampsia group(sPE)is(1813.66±727.92)g,placental weight was(401.33±119.50)g.The results of the two groups showed that the difference between the weight of newborns and the weight of the placenta between the severe preeclampsia group and the normal group was statistically significant,P<0.01.3.The expression of miR-146 a and SMAD4 in the placenta tissue of the two groups of pregnant women was at the mRNA level.Compared with the normal group,the expression of miR-146 a in the placenta tissue of the severe preeclampsia group(sPE)was lower(p<0.01),the expression of SMAD4 is higher,and the two are negatively correlated,and the difference is statistically significant;at the protein level,compared with the normal group,the expression of SMAD4 protein in the severe preeclampsia group(sPE)is higher,and the difference is statistically significant.Significance(p<0.01);immunohistochemistry results show that SMAD4 protein is expressed in both cytoplasm and nucleus,and Image J analysis shows that the expression of Smad4 protein in severe preeclampsia group(sPE)is higher,and there are differences Statistically significant(p<0.01).4.The expression of miR-146 a,Smad4 and VEGF after transfection of miR-146aCompared with the miR-146 a mimic negative control group(miR-146 a mimics NC),RT-PCR results showed that the expression of miR-146 a in the miR-146 a mimics group was increased;and compared with the miR-146 a inhibitor NC group.Compared with the miR-146 a inhibitor group,the expression of miR-146 a was greatly reduced,and the difference was statistically significant.Compared with the negative control group(miR-146 a mimics NC),the miR-146 a mimics group transfected to make the downstream gene expression corresponding The expression of Smad4 in the miR-146 a mimics group was significantly reduced at both the mRNA and protein levels,while the expression of VEGF was increased at both the mRNA and protein levels.The difference was statistically significant;the performance of the miR-146 a inhibitor group was similar to that in the miR-146 a inhibitor group.On the contrary,compared with the negative control group(miR-146 a inhibitor NC),the expression of SMAD4 in the miR-146 a inhibitor group was significantly increased at both the mRNA and protein levels,while the expression of VEGF was reduced at both the mRNA and protein levels.The difference was statistically significant.5.The effect of silencing Smad4 on the expression of VEGF.Three Smad4 small interfering RNAs were transfected.RT-PCR results showed that the silencing efficiency of No.03 was the best,reaching 80%.Therefore,the subsequent experiments were all selected to use No.03 Smad4 small interfering RNA.Compared with the negative control group(Smad4-si-NC),after silencing Smad4,the expression of Smad4 at both the mRNA and protein levels was significantly reduced,while the expression of VEGF was increased at both the mRNA and protein levels,and the difference was statistically significant.significance.6.The effect of miR-146 a transfection on cell proliferation,invasion and apoptosis.Compared with the negative control group,after overexpression of miR-146 a,the proliferation ability of HTR-8/SVneo cells was increased by CCK8 method.The Transwell experiment showed that HTR-8/SVneo.The cell invasion ability is improved,and the apoptosis of HTR-8/SVneo cells is reduced by flow cytometry;on the contrary,after miR-146 a is silenced,the proliferation ability of HTR-8/SVneo cells is reduced,the invasion ability is reduced,and the apoptosis is increased.The differences are statistically significant.7.The effect of simulated hypoxia on cell proliferation and miR-146 a expression.After adding cobalt chloride to the cells to simulate hypoxic environment,the results of CCK8 showed that compared with the blank control group,as the concentration of cobalt chloride gradually increased,And with the gradual prolongation of the action time,the proliferation activity of HTR-8/SVneo cells at24 h,48h,and 72 h gradually decreased,which also proved that the proliferation of HTR-8/SVneo cells was inhibited in the hypoxic environment;compared with the blank control group,when chlorine When the concentrations of cobaltide were50μmol/L,125μmol/L,250μmol/L,and 500μmol/L,RT-PCR results showed that the expression of miR-146 a in HTR-8/SVneo cells gradually decreased,and the difference was statistically significant.Conclusion:(1)The expression of miR-146 a decreased in placental tissues of patients with severe preeclampsia,while the expression of Smad4 increased,and the two were negatively correlated;(2)Under hypoxia,trophoblast cell proliferation was inhibited and miR-146 a expression was decreased;(3)The down-regulation of miR-146 a can affect the TGFβ-1/ Smad4 pathway,regulate the expression of VEGF,inhibit the proliferation and invasion of trophoblast cells,and promote the apoptosis of trophoblast cells. |
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