Biological Role And Mechanism Of TXNIP In Placenta Invasion Of Gestational Diabetes Mellitus

Objective: Compare the expression levels of Thioredoxin-interacting Protein(TXNIP)in placental tissues of Gestational Diabetes Mellitus(GDM)patients and healthy parturients.The biological function of TXNIP gene in placental trophoblast cells was further clarified and its mechanism was explored.Method: 1.Submit the research proposal to the Ethics Committee of the People’s Hospital of Inner Mongolia Autonomous Region for ethical approval;The study was conducted after the enrolled patients and healthy controls signed informed consent.The placental samples of patients meeting the diagnostic criteria of GDM and normal puerpera were collected,the expression level of TXNIP m RNA in placental tissue was detected by RT-qPCR,and the differences of TXNIP in the placental tissues of GDM patients and healthy controls were compared.Western blot was used to compare TXNIP protein expression levels in GDM and control placenta samples.2.The expression of TXNIP in human chorionic trophoblast cell line HTR-8/SVneo and human chorionic cancer cell line Be Wo were detected by RT-qPCR.According to the expression results of TXNIP in the cell line,HTR-8/SVneo cell line was selected for subsequent experiments.Wild-type HTR-8/SVneo cells were used as Normal group.CRISPR/Cas9 was used to knock out TXNIP gene in HTR-8/SVneo cell line,and TXNIP stable knock out HTR-8/SVneo cell line was constructed,named KO-TXNIP group.By liposome transfection,TXNIP high expression plasmid and Control plasmid were transfected to construct TXNIP high expression HTR-8/SVneo cell line and Control group,which were denoted as OE-TXNIP group and Control group,respectively.RT-qPCR and Western blot were used to detect the expression levels of TXNIP m RNA and protein in each group to verify the effectiveness of knockout and overexpression,respectively.3.Long-term dynamic cell observation and functional analysis system(Incu Cyte)was used to observe and record the cell proliferation of KO-TXNIP group,Normal group,OE-TXNIP group and Control group.Scratch test and cell invasion test were performed to observe and compare the effects of TXNIP knockout and overexpression on HTR-8/SVneo cell phenotype.4.KO-TXNIP group and Normal group,selected by RT-qPCR,Western blot experiments in the detection of two groups of cells ectomesenchymal transformation of epithelial cells-EMT-related factors(epithelial-mesenchymal transition)(Vimentin,N-cadherin,e-cadherin),and compared the effects of TXNIP knockout on EMT-related factors in HTR-8/SVneo cell lines.5.The expression changes of matrix metalloproteinases(MMP-2 and MMP-9)in KO-TXNIP group and Normal group were detected by RT-qPCR and Western blot,and the effects of TXNIP knockout on MMP-2 and MMP-9 in HTR-8/SVneo cell lines were compared.Result:1.A total of 59 placenta samples were collected,including 18 from GDM patients and 41 from normal parturients.Excluding 11 placental samples affected by unqualified and other factors,a total of 16 GDM placental samples and 32 normal maternal placental samples were included in this study.RT-qPCR results showed that TXNIP m RNA expression in placental tissues of GDM patients was higher than that of Control group,and the difference was statistically significant(p < 0.05).In HTR-8/SVneo and Be Wo cell lines,TXNIP was highly expressed in HTR-8/SVneo cell lines(p < 0.05),and the difference was statistically significant.Based on this,HTR-8/SVneo cell lines were selected for subsequent experiments.2.TXNIP knockout HTR-8/SVneo cell lines were constructed,and confirmed by RT-qPCR and Western blot experiments,no TXNIP expression was found at m RNA level and protein level.Compared with the Control group,the expression of TXNIP at m RNA level increased about 350 times,the difference was statistically significant(p < 0.05).3.TXNIP knockout and overexpression showed no significant change in cell proliferation.Compared with the control group,the migration and invasion ability of TXNIP knockout cells were significantly reduced(p < 0.05).On the contrary,the migration and invasion ability of OE-TXNIP group were significantly enhanced compared with Control group,and the difference was statistically significant(p < 0.05).4.Compared with the control group,m RNA level and protein expression of Vimentin in EMT-related factors decreased with TXNIP knockout(p < 0.05).The m RNA level and protein expression of N-cadherin increased with TXNIP knockout(p < 0.05).The m RNA expression of E-cadherin decreased with TXNIP knockout(p < 0.05).5.Compared with the control group,the m RNA level and protein level of MMP-2 were up-regulated with TXNIP gene knockout.The m RNA and protein expression of MMP-9 decreased significantly with TXNIP gene knockout.Conclusion: TXNIP are highly expressed in placenta of GDM patients.TXNIP promotes the migration and invasion of trophoblast cells.TXNIP regulates trophoblast cell migration and invasion regulation by regulating the expression of EMT-related factors,MMP-2 and MMP-9 in HTR-8/SVneo cell lines TXNIP can be used as a potential indicator of placental biological function in GDM patients.