The Role Of LncRNA MEG3 On Disintegration Of Vascular Smooth Muscle Cell In Spiral Artery Remodeling

Part one.The role of MEG3-induced VSMC biological functions in spiral artery remodelingObjectiveInvestigating the expression of MEG3 in placental and decidual tissues from preeclampsia patients,and studying the role of MEG3 in spiral artery remodeling by regulating VSMC functions.Methods1.The MEG3 expression was detected in the placenta and decidua from preeclampsia pregnancy and controls using q RT-PCR.2.Localizing the expression of MEG3 in maternal decidua of early gestation by in-situ hybridization.3.VSMC were classified into five groups:(1)VSMC were transfected with sh-MEG3 plasmid(sh MEG3 group);(2)VSMC were transfected with MEG3 overexpression plasmid(H-MEG3 group);(3)VSMC without transfection(blank control group);Then,VSMC were transfected with empty vector as negative control groups.4.Applying q RT-PCR to testify the efficiency of MEG3 in VSMC at m RNA level;5.Examining cell proliferation by CCK-8 assay.6.Flow cytometry assays was used to test VSMC apoptosis by.7.Examining cell migration ability by Transwell models.Results1.The MEG3 expression level of preeclampsia placenta and decidua was much lower than controls.MEG3 was expressed in VSMC layer of spiral artery and extracellular matrix.2.Transfection efficiency was confirmed by q RT-PCR.Expression of MEG3 m RNA substantially increased in MEG3 over-expression group and decreased in inhibition group compared to negative control groups.3.Compared with negative controls,the m RNA and protein levels of p53 and MMP-2 was increased in overexpression group and decreased in inhibition group,respectively.4.Compared with negative controls,increased apoptosis level and migration capacity,decreased cellular activity was observed in MEG3 overexpression group.However,MEG3 silence contributed to the opposite effects on VSMC.ConclusionMEG3 might involve in the pathogenesis of preeclampsia by damaging the VSMC-mediated spiral artery remodeling.Part Two.MEG3 as a competitive endogenous RNA regulates the function of VSMC by sponging mi R-21ObjectiveTo clarify the effect of mi R-21 on the biological function of VSMC,and explore the mechanism of MEG3 regulating mi R-21.Methods1.The mi R-21 expression was detected in the placenta and decidua from preeclampsia pregnancy and controls using q RT-PCR.2.VSMC were classified into five groups:(1)VSMC were transfected with mi R-21 inhibitor;(2)VSMC were transfected with mi R-21 mimic;(3)VSMC without transfection(blank control group);Then,VSMC were transfected with empty vector as negative control groups.3.Applying q RT-PCR to testify the efficiency of mi R-21 in VSMC at m RNA level;4.Examining cell proliferation by CCK-8 assay.5.Flow cytometry assays was used to test VSMC apoptosis by.6.Examining cell migration ability by Transwell models.7.Applying q RT-PCR to testify the expression of mi R-21 after VSMC transfected with MEG3 overexpression or si RNA.Results1.The mi R-21 expression level of preeclampsia placenta and decidua was much lower than controls.2.Transfection efficiency was confirmed by q RT-PCR.Expression of mi R-21 m RNA substantially increased in mi R-21 mimic group and decreased in mi R-21 inhibitor group compared to negative control groups.3.Compared with negative controls,increased apoptosis level and migration capacity,decreased cellular activity was observed in mi R-21 inhibitor group.However,mi R-21 mimic contributed to the opposite effects on VSMC.4.The expression of mi R-21 was decreased in MEG3 overexpression group,and increased when MEG3 was down-regulated.ConclusionMEG3 might as a competitive endogenous RNA regulates VSMC apoptosis and migration by sponging mi R-21.Part three.d NK cells-derived TGF-β1 modulated VSMC apoptosis and migration via the induction of MEG3ObjectiveTo clarify the role of TGF-β1 secreted by d NK cells and its relationship with MEG3 in VSMC.Methods1.Examing the purity of primary d NK cells by flow cytometry assays.2.According to experimental treatment,VSMC were classified into seven groups: blank control group;sh NC group;sh MEG3 group;TGF-β1 group(VSMC treated with recombinant human TGF-β1);TGF-β1+sh MEG3 group(recombinant human TGF-β1 was added to transfected VSMC);u NK group;u NK+TGF-β1 Ri group(u NK supernatant was added to VSMC with TGF-β1-receptor inhibitor blocking the TGF-β1 receptor).3.Applying q RT-PCR to testify the expression of MEG3 at transcriptional level.4.Applying q RT-PCR and western blotting to testify the expression of MMP-2.5.CCK-8 was used to exam VSMC viability.6.Flow cytometry assays was used to detect VSMC apoptosis.7.Examining cell migration ability by Transwell models.Results1.The purity of the cells was tested: the result showed that the purity of CD56+CD3-d NK cells was more than 92%.2.Compared with the control group,d NK cell supernatant and TGF-β1 exhibited reduced and significantly increased rates of proliferation and apoptosis.Moreover,both the d NK cell supernatant and TGF-β1 promoted VSMC migration.Conversely,when TGF-β1 signaling was abrogated by treatment with the TGF-β1-receptor inhibitor,treatment with the d NK cell supernatant had no significant effect on VSMC proliferation,apoptosis nor migration.Similarly,TGF-β1 treatment did not promote VSMC apoptosis nor migration in the MEG3-silenced cells compared to the sh NC.3.Compared to control group,MEG3 and MMP-2 expression levels were significantly increased after treatment with the d NK cells supernatant(d NK group) or human recombinant TGF-β1(TGF-β1 group).Moreover,blocking TGF-β1 signaling(with the utilized TGF-β1-receptor inhibitor;d NK + TGF-β1-receptor inhibitor group)prevented treatment with the d NK cell supernatant from inducing these observed increases.4.TGF-β1 treatment failed to upregulate MEG3 and MMP-2 expression when MEG3 was down-regulated compared to the negative control group,such that no significant difference in either MEG3 or MMP-2 expression was observed between the sh MEG3 and TGF-β1+ sh MEG3 groups.Conclusiond NK cells-derived TGF-β1 stimulated MEG3 and MMP-2 expression and further regulated VSMC apoptosis and migration,which may be an important molecular mechanism of spiral artery remodeling.Part four.The role of MEG3 on spiral artery remodeling in PDC systemObjectiveWe applied placental-decidual co-culture(PDC)system to further investigate the effect of MEG3 knock-down on spiral artery remodeling.Methods1.Decidual and villus tissues from the same pregnant woman was collected to establish the PDC system.It was classified into two groups: inhibition group(cells transfected with a lentiviral vector carrying a short interfering RNA targeting MEG3)and negative control group(cells transfected with an empty lentiviral).2.Applying q RT-PCR to testify the expression of MEG3 in tissues.3.Testifying EVT invasion and maternal spiral arteries in ex vivo mode of artery remodeling by immunohistochemistry staining.4.Tissue apoptosis was detected by TUNEL.Results1.q RT-PCR showed that the expression of MEG3 in MEG3 inhibition group was significantly decreased compared to the negative control group.2.The IHC results showed that,compared with negative control group,the uterine spiral artery remodeling in MEG3 knocked-down group was at an earlier stage with less EVTs in decidua tissue and more superficial penetration,and the spiral artery VSMC layer was not sufficiently damaged,as well.3.TUNEL results showed that the apoptotic level of tissue cells in MEG3 inhibitory group was decreased.Apoptotic cells could be seen in the extracellular matrix of the tissue in MEG3 inhibition group,but there was no apoptosis in the spiral artery myometrium.There were more apoptotic cells in the negative control group.Apoptotic VSMC could be seen in the myometrium,which indicated that the expression of MEG3 in silent tissue might mainly inhibit the invasive function of EVT,leading to the disorder of spiral artery remodeling.ConclusionMEG3 may positively modulate the biological function of EVTs in VSMC separation during trophoblast-dependent Sp A remodeling.