The Study Of Effects And Mechanisms Of Long Non-Coding RNA NEAT1 Regulated By LIN28B In Preeclampsia

Objective: Preeclampsia is common diseases of obstetrics and gynecology,it is known that preeclampsia patients are treated by the ways of step-down,composed and diuretic which can temporarily relieve symptoms,but by far the most effective treatment is still a timely termination of pregnancy,maternal in the face of serious complications such as heart kidney at the same time,and therefore often leads to preterm labor and preterm birth associated increased perinatal morbidity and mortality.Trophoblast cells infiltrating in early pregnancy in bed too shallow,uterine spiral arteriolar recasting obstacle of preeclampsia is widely recognized by the cause mechanism,improve the early weeks of pregnancy in patients with preeclampsia "shallow bed",namely improve uterine spiral arteriolar recasting,to terminate the following endothelial cell injury and pathological physiological process is critical.LIN28 is a highly conserved RNA-binding protein discovered in 1997.With the regulation of RNA metabolism,LIN28 not only participates in the differentiation of embryonic stem cells,but also plays a key role in a variety of biological events such as cell proliferation,migration,tumgenesis and metastasis.The results of our previous studies suggest that LIN28 B is also expressed in human placental tissues,and the expression of LIN28 B in preeclampsia may have temporal differences and be related to trophoblast cell infiltration.Nuclear Paraspeckle Assembly transcript1(NEAT1)is a novel lnc RNA discovered in recent years,which is involved in the occurrence and development of a variety of tumors,including tumor cell proliferation,invasion and metastasis,cell cycle and apoptosis.NEAT1 is highly expressed and plays the role of oncogene in some solid tumors,while it is underexpressed and plays the role of tumor suppressor gene in some tumors.Such expression disorder may be a potential biological molecule in tumor diagnosis and prognosis.NEAT1 was expressed in villi and down-regulated in recurrent abortion.NEAT1 was also expressed in the placental villus trophoblast cells of full-term pregnancy,and its expression in the placenta of patients with fetal growth restriction was 4 times higher than the control group’ expression.NEAT1 is rarely reported in preeclampsia.Versican(VCAN)is a kind of chondroitin sulfate proteoglycan,which can promote tumor cell proliferation and apoptosis,and also has the effect of inhibiting tumor.Preeclampsia has a characteristic gene spectrum,and VCAN can be used as one of the central genes in the gene spectrum network to affect the pathogenesis of preeclampsia.VCAN expression is up-regulated in the umbilical artery and vein wall,but VCAN expression in blood and placenta has not been reported.This study intends to explore the relationship between LIN28B/NEAT1/miRNA-181d-5p /VCAN axis and preeclampsia through human placental tissue and in vitro cell experiments.Methods: 1.A total of 58 pregnant women who delivered in Glide Wing of Shengjing Hospital,China Medical University from January 2018 to May 2022 were selected as the study subjects(the early-onset preeclampsia group 15 cases,the early-onset control group 13 cases,the late-onset preeclampsia group 15 cases,the late-onset control group15 cases),and placental tissues were collected and processed.QRT-PCR and Western Blot were used to detect the expression of LIN28 B in the placenta of the study subjects.Further,LIN28 B was knocked down in HTR-8/SVneo cells to test the knock down efficiency.The effect of LIN28 B knock down on NEAT1 expression was detected by q RT-PCR.RIP detects whether NEAT1 and LIN28 B have targeted binding.The expression of NEAT1 was detected by q RT-PCR in the same placental tissues.2.NEAT1 was knocked down in HTR-8/SVneo cells to test the knock down efficiency,and the effect of NEAT1 on the biological behavior of HTR-8/SVneo was investigated by transwell experiment and scratch experiment.In the trophoblast cells co-transfected with LIN28B-SI3 and NEAT1-SI3,the changes of cell infiltration and migration ability were detected again to verify whether NEAT1-SI3 could reverse the effects of LIN28B-SI3 on HTR-8/SVneo biological behavior.3.The miRNA expression profile was screened by chip after LIN28 B was knocked down in HTR-8/SVneo cells,and several miRNAs with obvious differences were selected and verified in expanded tissue samples by quantitative real-time polymerase chain reaction(q RT-PCR)technology.We detected mir-181d-5p expression in the placental tissues by q RT-PCR.Further,mir-181d-5p was knocked down or overexpressed in HTR-8/SVneo cells to test the efficiency,and transwell and scratch experiment were used to explore the effect of mir-181d-5p on HTR-8/SVneo biological behavior.In the trophoblast cells co-transfected with LIN28B-SI3 and mir-181d-5p minics,the changes of cell infiltration and migration ability were detected again to verify whether mir-181d-5p minics could reverse the effects of LIN28B-SI3 on HTR-8/SVneo biological behavior.4.According to the "competing endogenous RNA(ce RNA)" hypothesis,we used the bioinformatics database to predict and verified the targeted regulation of mir-181d-5p/VCAN axis by NEAT1。Double luciferase assay was used to verify whether NEAT1 had targeted binding with mir-181d-5p and whether mir-181d-5p had targeted binding with VCAN.In HTR-8/SVneo,q RT-PCR and WB were used to detect the effect of NEAT1/mir-181d-5p on VCAN expression.We detected VCAN expression in the placental tissues by q RT-PCR and WB.Further,VCAN was knocked down in HTR-8/SVneo cells to test the efficiency,and transwell and scratch experiment were used to explore the effect of VCAN on HTR-8/SVneo biological behavior.NEAT1-SI3 and mir-181d-5p inhibitor,VCAN-SI1 and mir-181d-5p inhibitor were co-transfected into HTR-8/SVneo cells respectively,to observe the effects on the biological behavior of cells.To verify whether mir-181d-5p inhibitor could reverse NEAT1-SI3 and whether VCAN-SI1 could reverse the effect of mir-181d-5p inhibitor on HTR-8/SVneo biological behavior.In HTR-8/SVneo,q RT-PCR and WB were used to detect the effect of LIN28 B on VCAN expression.Results: 1.The LIN28B’s expression in early-onset and late-onset preeclampsia’s placental tissues was obviously lower than that of the corresponding control group by q RT-PCR and WB.LIN28 B was knockdown and transfected into HTR-8/SVneo cells.QRT-PCR and WB experiments showed that the knockdown effect was obvious(P<0.05).LIN28 B knockdown inhibit HTR-8/SVneo’s invasion and migration capabilities.QRT-PCR showed that the expression of NEAT1 increased after LIN28 B knockdown.The targeted interaction between LIN28 B and NEATI was confirmed by RIP experiment.The expression of NEAT1 in placenta of preeclampsia was significantly higher than that of normal pregnant women at the same gestational week.2.The NEAT1 knockdown sequence was transfected into HTR-8/SVneo cells,q RT-PCR experiments showed that the knockdown effect was obvious(P<0.05).NEAT1 knockdown increases HTR-8/SVneo’s invasion and migration capabilities.NEATI-SI3 can reverse LIN28B-SI3’s ability to invade and migrate trophoblast cells,and NEAT1 may play an important role in the pathogenesis of trophoblast cells in preeclampsia.3.Mi RNA expression profiles of HTR-8/SVneo cells with LIN28 B knocked down were detected and 29 down-regulated miRNAs and 2 up-regulated miRNAs(∣log2FC∣≥1,P<0.05)were found at the same time,among which 1 up-regulated and 9 down-regulated miRNAs’ target genes with differential expression were predicted,and 3down-regulated miRNAs whose predicted target genes ≧3 times in the database were selected for validation by q RT-PCR in clinically expanded tissue samples.The experimental results were consistent with the sequencing results.QRT-PCR experiments showed that the expression of mir-181d-5p in preeclampsia’s placental tissues was obviously lower than the control group(P<0.05).The mir-181d-5p overexpressed or knockdown sequence were transfected into HTR-8/SVneo cells,and we verified the effect of transfection of them by q RT-PCR(P<0.05).Transwell and scratch experiments proved that overexpression of mir-181d-5p increased the invasion and migration ability of HTR-8/SVneo,and knockdown of mir-181d-5p inhibited the invasion and migration ability of HTR-8/SVneo.The invasion and migration of trophoblast cells by LIN28B-SI3 can be reversed by mir-181d-5p minics.4.Starbase predicted that NEAT1 could silence mir-181d-5p through ce RNA mechanism and target regulation of VCAN,and we verified it.We verified the targeted binding between NEAT1 and mir-181d-5p by dual luciferase assay,and the same way bwtween mir-181d-5p and VCAN.In HTR-8/SVneo trophoblast cells,the expression of VCAN were significantly decreased by q RT-PCR and WB after mir-181d-5p minics and NEAT1 knocking,and was significantly increased after mir-181d-5p inhibitor.QRT-PCR experiments showed that the expression of VCAN was obviously higher than that by q RT-PCR and WB(P<0.05),and the expression of VCAN in early-onset preeclampsia was significantly higher than that in late-onset preeclampsia.The VCAN knockdown sequence were transfected into HTR-8/SVneo cells,and we verified the effect of transfection of them by q RT-PCR and WB(P<0.05).Knocking down VCAN increases HTR-8/SVneo’s invasion and migration capabilities.Mir-181d-5p inhibitor could reverse the invasion and migration of NEAT1-SI3 to trophoblast cells,and VCAN inhibitor could reverse the invasion and migration of mir-181d-5p inhibitor to trophoblast cells.QRT-PCR and WB experiments demonstrated that the expression of VCAN increased after LIN28 B knocking,Conclusions: 1.LIN28 B expression could be detected in normal/preeclampsia placental tissues of pregnant women.In the placental tissues of preeclampsia,the expressions of LIN28 B was obviously lower than the control group,which can inhibit the infiltration and migration of trophoblast cells.In norma/preeclampsial placental tissues of pregnant women NEAT1 expression could be detected.The NEAT1’s expression in placental tissues of preeclampsia was obviously higher than the control group in the same gestational week.LIN28 B can negatively regulate NEAT1 expression,and LIN28 B has targeted interaction with NEAT1 by RIP.LIN28 B may be involved in the pathogenesis of preeclampsia by regulating NEAT1.2.In HTR-8/SVneo trophoblast cell,NEAT1 knockdown promoted trophoblast cell migration and invasion.Knocking down of NEAT1 can reverse the ability of LIN28B-SI3 to infiltrate and migrate trophoblast cells.LIN28 B participates in trophoblast infiltration by regulating NEAT1.3.LIN28 B microarray results showed that multiple miRNAs were differentially expressed in HTR-8/SVneo cells with LIN28 B knockdown.The expression of mir-181d-5p can be detected in the placental tissues of normal and preeclampsia pregnant women.The expression of mir-181d-5p in placental tissues of preeclampsia was significantly lower than that of normal pregnant women in the same gestational week,In HTR-8/SVneo trophoblast cell,mir-181d-5p overexpression promoted trophoblast cell migration and invasion,knockdown reversly.LIN28 B inhibitor’s infiltration and migration ability of trophoblast cells could be reversed when cotransfected with mir-181d-5p minics.4.The ce RNA interaction mechanism of NEAT1/mir-181d-5p /VCAN axis is predicted by starbase and verified in the pathogenesis of preeclampsia.The expression of VCAN can be detected in the placental tissues of normal and preeclampsia pregnant women.The expression of VCAN in early-onset preeclampsia was significantly higher than that in late-onset preeclampsia.In HTR-8/SVneo trophoblast cell,VCAN knockdown promoted trophoblast cell migration and invasion.Rescue assay could restore the biological function of invasion and migration of trophoblast cells in NEAT1/ mir-181d-5p /VCAN axis.Changes in LIN28 B expression affect VCAN expression,and there is a negative regulatory mechanism.The NEAT1/ mir-181D-5p /VCAN axis regulated by LIN28 B is involved in the invasion and migration of trophoblast cells and affects the pathogenesis of preeclampsia.