The Preliminary Exploration Into The Influence Of The Aberation Of LncRNA MALAT1 On MSCs

Preeclampsia(PE)is a pregnancy-specific multi-organ dysfunctional disease and a leading cause of maternal and fetal morbidity and mortality worldwide.It is reported that insufficiency of angiogenesis and imbalanced immune system in the maternal-fetal interface are two main causes for the development of PE.Mesenchymal stem cells(MSCs)play important roles in regulating angiogenesis and immune balance.Our previous studies showed that both umbilical cord and decidua MSCs present slow proliferation,aberrant levels of cytokines,decreased angiogenesis-regulation ability and abnormal immunoregulation in PE.Furthermore,we used normal MSCs to treat PE-like mouse and got the ideal therapeutic effect.Other research groups also reported that MSCs treatment had beneficial effects in mice PE models.All these results indicate MSCs play an important role in the pathogenesis of PE.However,the molecular mechanisms responsible for the alteration of MSCs in PE are still unclear.Long non-coding RNAs(LncRNAs)are RNA species more than 200 nucleotides in length and are recently discovered to constitute a large proportion of the whole transcriptome.They were reported to be involved in many diseases,affecting cellular biological properties and cell function,such as proliferation,cell cycle,apoptosis and functions.LncRNAs were differentially expressed in the PE placentas,suggesting they may contribute to the pathogenesis of PE.MALAT1(Metastasis-Associated Lung Adenocarcinoma Transcript 1),also referred to as NEAT2(Nuclear-Enriched Abundant Transcript 2),is one of the first discovered cancer related IncRNAs and is considered as a prognostic factor for lung cancer metastasis.MALAT1 was reported to be downregulated in the placenta of PE patients and related with the proliferation,cell cycle,apoptosis,migration and invasion of trophoblast cells,suggesting it may be involved in the pathogenesis of PE.However,the roles of MALAT1 in MSCs are unknown.We reported that MSCs derived from PE patients grow much more slowly than MSCs derived from healthy pregnancies.Besides,the results we did before showed that lncRNA MALAT1 was lowly expressed both in placenta tissues and decidua-derived MSCs from PE patients compared with healthy pregnancies.Thus,in the present study,we are engaged to explore the possible roles of MALAT1 plays in the growth and function of MSCs to reveal the possible pathogenesis of abnormal proliferation and functions of MSCs in maternal fetal interface of PE.1.MALAT1 has no effect on the phenotype and differentiation of MSCs.In our previous experiments,we found that MALAT1 was lowly expressed in the placenta and decidua-MSCs derived from PE patients compared with healthy pregnancies,but the reason is still unknown.Based on this,we both upregulated MALAT1 and downregulated MALAT1 in MSCs derived from healthy pregnancies through transient transfection and tested the surface phenotype and differentiation ability.The results showed that MALAT1 has no effects on the phenotype and differentiation of MSCs.2.MALAT1 promotes proliferation,migration,invasion and capillary formation through VEGF.Through transiet transfection,we got MALAT1 overexpressing and downregulating MSCs.CCK-8,cell cycle and cell apoptosis tests showed that MALAT1-upregulation could promote cell viability and proliferation and inhibit cell apoptosis.We also tested the migration,invasion and capillary formation after changing the expression of MALAT1.We found that upregulation of MALAT1 could not only promote the migration of MSCs,the supernatants could also promote the migration and invasion of HTR-8/SVneo and tube formation of HUVEC.While downregulation of MALAT1 could inhibit the migration,invasion and capillary formation and promote apoptosis.To find out which MALAT1 affects MSCs through,we tested the expression of VEGF and found that VEGF was lowly expressed in PE.So we tested the expression after changing MALAT1 and found that VEGF could be upregulated through transfecting MALAT1 mimic plasmid and downregulated through interfering MALAT1.Then dual-luciferase assay was performed and identified the direct interaction between VEGF and MALAT1.We used VEGF neutralizing antibody(anti-V)or mouse derived IgG to neutralize VEGF in supernatants and tested the migration,invasion and tube formation,which certified that VEGF mediates the effects of MALAT1 on MSCs.3.MALAT1 enhances the regulatory effects of MSCs on macrophage polarization.MSCs are immune-modulatory and anti-inflammatory and imbalanced immune system in the maternal-fetal interface is regarded one of the main causes for the development of PE.Whether MALAT1 has an effect on the immune-modulatory and anti-inflammatory properties of MSCs is unknown.We cultured THP-1(pretreated with PMA for 72 hours)with conditioned MSCs supernatants for 48 hours,and then the polarization of THP-1 was analyzed using flow cytometry.The results showed that supernatants of MSCs overexpressing MALAT1 could promote the polarization of THP-1 towards M2 type,instead,interfering MALAT1 could inhibit the polarization of THP-1 towards M2 type.Furthermore,we tried to reveal the mechanism of MALAT1 affecting cell polarization.Firstly,we tested several cytokines which may affect the polarization of macrophages and high expressed in PE,such as IL-6,IDO and COX-2,and we found that MALAT1 could promote the expression of IDO significantly.As IDO could promote macrophages polarize towards M2,so we think IDO might mediate the effects of MALAT1 on the polarization of macrophages.4.MALAT1 is reduced by oxidative stress.In our present study,we found that MALAT1 was low-expressed in PE,and it had important effects on cell proliferation,migration,tube formation and macrophage polarization,but why it was low-expressed was unknown.As inflammation and excessive oxidative stress were reported to be involved in pathology of PE as well as the inadequate secretion of VEGF,we used LPS,IL-6,TGF-β1 and different concentration of H2O2(from 5 μM to 600μM)to stimulate MSCs and we found that H2O2 could reduce the expression of MALAT1 and VEGF in a dose-dependent manner,while LPS,IL-6 and TGF-β1 had no effects on MALAT1 expression.Above all,we found that MALAT1 promoted the growth,angiogenesis-regulating and immune-regulatory potential of MSCs.MALAT1-overexpressed MSCs promoted HTR-8/SVneo cells migration,invasion and HUVEC tube formation through inducing VEGF.MALAT1-overexpressed MSCs supernatants promoted polarization of macrophages towards M2 type.Conclusion:Aberation of lncRNA MALAT1 affects the proliferation and functions of MSCs.