The Role Of Galectin-14 In Regulating Trophoblast Migration And Invasion, And In The Pathogenesis Of Preeclampsia

Trophoblast is the main cellular component of the placenta.Together with the maternal decidua cells,they form the maternal-fetal interface.As the main component of placenta,trophoblast plays a major role in embryo implantation,placenta formation and development,the immune regulation at the maternal-fetal interface and in the full establishment of hemochorial circulation system in placenta.Dysregulation of trophoblast causes shallow placentation,dysregulation of immune tolerance and inadequate spiral artery remodeling.They further result in abnormal embryonic development,limited placentation,abnormal development and function of placenta and abnormal fetal growth and development,and thus cause the occurrence of pregnancy complications,like early pregnancy loss(EPL),pre-eclampsia(PE),fetal intrauterine growth restriction(FGR)and et al.The normal functions of trophoblast are controlled by multiple functional genes.Among the functional genes,there are several placental-specific expressing genes,such as PLAC1 and syncytin.These genes play important roles in regulating trophoblast migration,invasion,proliferation,apoptosis,and metabolism.Galectin-14 is one of the placental-specific expressing genes,which belongs to the galectin family.Recent years,several galectin family members were found to be dysregulated in pregnancy complications,including galectin-1,galectin-3,galectin-9 and galectin-13.Galectin-1 and galectin-13 have the lower expression in PE and EPL placenta,whereas the expression of galectin-3 and galectin-9 are increased in PE placenta.The diverse expression pattern of galectin family members in PE and EPL placenta indicate that galectin family may play a role in the occurrence of pregnancy complications.Further researches showed that galectin-1 and galectin-3 mainly regulate trophoblast migration and invasion,while galectin-9 and galectin-13 mainly regulate the functions of immune cells.Galectin-14 is one of the galectin family members.It is specifically expressed in placenta.It is considered to be primates-specific.Recent researches imply galectin-14 may be involved in the occurrence of pregnancy complications,such as PE,EPL and et al,and it may participate in the establishment and maintenance of pregnancy.However,little study has been reported the regulatory effects of galectin-14 on trophoblast.In this study,we compared the placental expression difference of galectin-14 m RNA and protein between severe preeclampsia and normal pregnancy by using RT-q PCR and Western blot assays.The results showed the expression level of galectin-14 was significant lower in the placental tissues of PE.The results imply the involvement of galectin-14 in the pathogenesis of PE.Then,by using primary trophoblast and trophoblast cell lines,we established the intracellular models of manipulating galectin-14 expression.We examined the effects of galectin-14 on trophoblast,and found that galectin-14 promoted trophoblast migration and invasion.Further,the results of Western blot showed that galectin-14 promoted the expression of N-cadherin and MMP-9,which are,respectively,the hallmark of epithelial-mesenchymal transition(EMT)and the gelatinase secreted by trophoblast.In the meantime,galectin-14 enhanced the activity of MMP-9.When looking into the signaling pathways which regulate the expression of N-cadherin and MMP-9,we found galectin-14 enhanced the expression of N-cadherin through promoting P38 phosphorylation,and enhanced the expression and activity of MMP-9through promoting Akt phosphorylation.Our results filled the gap in the mechanisms of the regulatory effects of galectin-14 on trophoblast.They may help to better understand the effects of trophoblast dysregulation on the pathogenesis of pregnancy complications.Part 1: The expression of galectin-14 in the placental tissues of severe preeclampsiaObjective:To investigate the placental expression difference of galectin-14 m RNA and protein between severe preeclampsia and normal pregnancy.Materials and methods:1.We collected placenta tissues from severe preeclampsia and normal pregnancy underwent Cesarean sections during 2017 and 2019 in Women’s Hospital,School of Medicine,Zhejiang University.This study included 20 severe preeclampsia cases and 20 normal pregnancy cases.2.The RNA and total protein were extracted from the placenta tissues.Real-time quantitative PCR and Western blot were used to analyze the expression levels of galectin-14 m RNA and protein.Results:1.Compared to the normal pregnancy group,the expression level of galectin-14 m RNA is lower in severe preeclampsia group,but the difference did not reach statistical significance.2.Compared to the normal pregnancy group,the expression level of galectin-14 protein is lower in severe preeclampsia group.Conclusions:The down-regulation of galectin-14 in severe preeclampsia placenta implies the involvement of galectin-14 in the pathogenesis of preeclampsia.Part 2: The role of galectin-14 on regulating trophoblast functionsObjective:To establish the intracellular models of manipulating galectin-14 expression,and examine the effects of galectin-14 on trophoblast.Materials and methods:1.The RNA was extracted from primary trophoblast,trophoblast cell lines HTR-8/SVneo,SWAN-71,JAR,JEG-3 and Be Wo.Reverse transcription-PCR and agarose gel electrophoresis were used to detect the galectin-14 m RNA expression in these cells.2.According to the results of agarose gel electrophoresis,si RNA transfection was performed in primary trophoblast to knock down galectin-14.Lentivirus-mediated overexpression was performed in HTR-8/SVneo to overexpress galectin-14.3.After knocking down or overexpressing galectin-14,cck-8 proliferation assay was used to analyze the cell proliferation ability.Annexin V-APC/7-AAD apoptosis kit and flow cytometry were used to analyze the cell apoptosis level.Transwell migration and invasion assays were used to analyze the cell migration and invasion ability.Results:1.The m RNA expression of galectin-14 was detected in primary trophoblast but not in trophoblast cell lines HTR-8/SVneo,SWAN-71,JAR,JEG-3 and Be Wo.2.After knocking down galectin-14 in primary trophoblast or overexpressing galectin-14 in HTR-8/SVneo,the efficiency of knock-down or overexpression was examined by using real-time quantitative PCR,Western blot and cell immunofluorescence.3.After knocking down galectin-14 in primary trophoblast or overexpressing galectin-14 in HTR-8/SVneo,the m RNA expression of the other galectin family members was examined by using real-time quantitative PCR,to verify the specificity of knockdown or overexpression.4.After knocking down galectin-14 in primary trophoblast,the cell proliferation ability and apoptosis level had no significant change,but the migration and invasion abilities were significantly reduced.After overexpressing galectin-14 in HTR-8/SVneo,the cell proliferation ability and apoptosis level had no significant change as well,but the migration and invasion abilities were significantly enhanced.Conclusions:Galectin-14 was expressed by primary trophoblast.In trophoblast,galectin-14 promoted cell migration and invasion.Part 3:The mechanisms involved in galectin-14 regulating trophoblast migration and invasionObjective:To clarify the effects of galectin-14 on the physiological processes which affect trophoblast migration and invasion,and on the related signaling pathways.Materials and methods:1.After knocking down galectin-14 in primary trophoblast or overexpressing galectin-14 in HTR-8/SVneo,the expressions of hallmarks of epithelial-mesenchymal transition N-cadherin,E-cadherin,VE-cadherin,the expression of the gelatinases secreted by trophoblast MMP-2 and MMP-9,and their tissue inhibitor TIMP-2 and TIMP-1 was examined by using Western blot.The activities of MMP-2 and MMP-9 were examined by gelatin zymography assay.2.According to the results of Western blot and gelatin zymography assay,chose the molecules whose expression was affected by galectin-14,examined their effects on trophoblast migration and invasion by using si RNA knock-down.3.According to the results,chose the related signaling pathways.After knocking down galectin-14 in primary trophoblast or overexpressing galectin-14 in HTR-8/SVneo,the phosphorylation levels of key regulatory factors were examined by using Western blot.4.By using corresponding agonists and inhibitors,the molecules regulated by the chosen signaling pathways were examined by using Western blot.The effects of chosen signaling pathways on cell migration and invasion were examined by Transwell migration and invasion assays.Results:1.After knocking down galectin-14 in primary trophoblast,the expression of Ncadherin and MMP-9 significantly decreased and the activity of MMP-9 significantly decreased,but the expression of VE-cadherin,MMP-2,TIMP-1,TIMP-2 had no significant change,the activity of MMP-2 had no significant change.After overexpressing galectin-14 in HTR-8/SVneo,the expression of N-cadherin and MMP-9 significantly increased and the activity of MMP-9 significantly increased,but the expression of VE-cadherin,MMP-2,TIMP-1,TIMP-2 had no significant change,the activity of MMP-2 had no significant change.The expression of E-cadherin was not detected in primary trophoblast and HTR-8/SVneo.2.In galectin-14-over-expressed HTR-8/SVneo,after knocking down N-cadherin,cell migration and invasion were significantly inhibited,and no significant change was detected in cell proliferation ability.After knocking down MMP-9,cell migration and invasion were significantly inhibited,and no significant change was detected in cell proliferation ability.3.The MAPK/P38,ERK,SMAD2/SMAD3 and Akt signaling pathways are related to the expression of N-cadherin and MMP-9 in trophoblast.After knocking down galectin-14 in primary trophoblast,the expression of p-Akt and p-P38 significantly decreased,the ratios of p-Akt/Akt,p-P38/P38 significantly decreased.No significant change was detected in the expression of p-ERK,p-SMAD2,p-SMAD3,and in the ratios of p-ERK/ERK,p-SMAD2/SMAD2,p-SMAD3/SMAD3.After overexpressing galectin-14 in HTR-8/SVneo,the expression of p-Akt and p-P38 significantly increased,the ratios of p-Akt/Akt,p-P38/P38 significantly increased.No significant change was detected in the expression of p-ERK,p-SMAD2,p-SMAD3,and in the ratios of p-ERK/ERK,pSMAD2/SMAD2,p-SMAD3/SMAD3.4.In galectin-14-over-expressed HTR-8/SVneo,after using the agonist of Akt phosphorylation,SC79,the expression of p-Akt and the ratio of p-Akt/Akt significantly increased.Even though the cell proliferation ability was significantly inhibited,the cell migration and invasion were significantly enhanced.In the meantime,the expression and activity of MMP-9 were significantly increased,no significant change was detected in the expression of N-cadherin.After using the inhibitor of Akt phosphorylation,LY294002,the expression of p-Akt and the ratio of p-Akt/Akt significantly decreased.The cell proliferation ability was not affected,and the cell migration and invasion were significantly inhibited.In the meantime,the expression and activity of MMP-9 were significantly decreased,no significant change was detected in the expression of N-cadherin.5.In galectin-14-over-expressed HTR-8/SVneo,after using the inhibitor of P38 phosphorylation,SB202190,the expression of p-P38 and the ratio of p-P38/P38 significantly decreased.The cell proliferation ability was not affected,and the cell migration and invasion were significantly inhibited.In the meantime,the expression and activity of N-cadherin were significantly decreased,no significant change was detected in the expression of MMP-9.Conclusions:In trophoblast,galectin-14 promoted the expression of MM-9 and N-cadherin,activated the activity of MMP-9.The elevated expression of MMP-9 and N-cadherin,elevated activity of MMP-9,promoted trophoblast migration and invasion.Galectin-14 promoted the expression and activity of MMP-9 through activating the Akt signaling pathway,while it promoted the expression of N-cadherin through activating the MAPK/P38 signaling pathway.