| Part one Expression and localization of MALAT1 and PFKFB3 in placental tissues from patients with early-onset preeclampsia PurposeTo detect the expression and localization of MALAT1 and PFKFB3 in human placental tissues,and compare the expression differences of MALAT1 and PFKFB3 in early-onset preeclampsia patients with normal pregnant patients.Methods1.Experimental grouping: According to the experimental design,it was divided into earlyonset preeclampsia group and normal control group,and collect the placental tissues of the pregnant women in the third trimester of the two groups.2.q RT-PCR and(or)Western Blot were used to detect the expression of MALAT1 and PFKFB3 in the placenta tissues of the two groups of pregnant women.3.Fluorescence in situ hybridization(FISH)assay was used to detect the expression and localization of MALAT1 in placental tissues and immunohistochemistry(IHC)was used to detect the expression and localization of PFKFB3 in placental tissues.Results1.The m RNA level of MALAT1 and the m RNA and protein levels of PFKFB3 in placental tissues from patients with early-onset preeclampsia were significantly reduced compared with normal placental tissues.2.MALAT1 and PFKFB3 were predominately localized in the cytoplasm in placental tissues of early-onset preeclampsia patients and normal pregnant women.ConclusionThe MALAT1 and PFKFB3 levels were significantly decreased in placental tissues from patients of early-onset preeclampsia compared with normal placental tissues,which suggests that MALAT1 and PFKFB3 are involved in early-onset preeclampsia pathogenesis.Part twoPFKFB3 regulates glycolytic activity and endothelial cell function PurposeTo explore the effect of PFKFB3 on glycolytic activity and endothelial cell function.Methods1.The expression of PFKFB3 was upregulated by transfection of PFKFB3-overexpressed plasmid: the PFKFB3 overexpression group(transfected with PFKFB3 overexpression plasmid to endothelial cells),and the negative control group(transfected with vector)were set up at the same time.The transfection efficiency was detected by q RT-PCR and Western Blot.2.The expression of PFKFB3 was downregulated by transfection of PFKFB3 si RNA: the PFKFB3 knockdown group(transfected with PFKFB3 si RNA to endothelial cells),and the negative control group(transfected with si-NC)were set up at the same time.The transfection efficiency was detected by q RT-PCR and Western blot.3.The glycolytic activity of endothelial cells were detected after overexpression or downregulation of PFKFB3: the cellular levels of ATP,NADPH,reactive oxygen species(ROS),and extracellular lactate production were detected by corresponding kits.4.The effect of overexpression / downregulation of PFKFB3 on endothelial cell function were detected: CCK-8,flow cytometry,Transwell,tubule-formation and cytoskeleton staining experiments were used to study the effects of PFKFB3 on endothelial cell proliferation,cell cycle progression,migration,tube formation and filopodia and lamellipodia formation.Results1.The expression of PFKFB3 in endothelial cells was significantly increased in PFKFB3 overexpression group.2.The expression of PFKFB3 in endothelial cells was significantly decreased in PFKFB3 knockdown group,and si RNA-3 was identified as exhibiting the best PFKFB3 knockdown efficacy and was used in subsequent experiments.3.The cellular levels of ATP,NADPH and extracellular lactate were increased in PFKFB3 overexpression group,while the ROS levels were decreased in PFKFB3 overexpression group.Whereas the exact opposite effects could be seen in PFKFB3 knockdown group.4.PFKFB3 overexpression promoted the proliferation,cell cycle progression,migration,tube formation and filopodia and lamellipodia formation of endothelial cells,while PFKFB3 knockdown had the opposite effect.ConclusionPFKFB3 overexpression increased glycolytic activity and the proliferation,migration,and tube formation of endothelial cells.PFKFB3 overexpression increased the proliferation of endothelial cells by accelerating the cell cycle and increased the motility of endothelial cells by promoting the formation of cell protrusions.PFKFB3 knockdown exerted the opposite effects.Part threeMALAT1 functions as a ce RNA of mi R-26 a and mi R-26 b and regulates the expression of PFKFB3 in endothelial cells PurposeTo elucidate that MALAT1 functions as a ce RNA of mi R-26 a and mi R-26 b and regulates PFKFB3 expression in endothelial cells.Methods1.The expression of MALAT1 was downregulated by transfection of MALAT1 knockdown plasmid: MALAT1 knockdown group(transfected with MALAT1 knockdown plasmid to endothelial cells),and the negative control group(transfected with sh-NC)were set up at the same time.The transfection efficiency and the expression of mi R-26 a / 26 b and PFKFB3 were detected by q RT-PCR and(or)Western Blot.2.The expression of mi R-26a/26 b was upregulated by transfection of mi R-26a/26 b overexpressed plasmid: mi R-26a/26 b overexpression group(transfected with mi R-26a/26 b overexpression plasmid to endothelial cells),and the negative control group(transfected with mimic-NC)were set up at the same time.The transfection efficiency and the expression of PFKFB3 were detected by q RT-PCR and Western Blot.3.RNA hybrid software was used to predict the possible binding sites for mi R-26a/26 b on MALAT1 and verified them by luciferase reporter assay and RNA pull-down assay in endothelial cells.4.RNA hybrid software was used to predict the possible binding sites for mi R-26a/26 b on PFKFB3 and verified them by luciferase reporter assay and RNA pull-down assay in endothelial cells.5.sh-MALAT1 and mi R-26a/26 b inhibitors were co-transfected into endothelial cells,and then the expression of PFKFB3 was detected by q RT-PCR and Western Blot.Results1.sh RNA-5277 displayed the best MALAT1 knockdown efficacy and was used in subsequent experiments.MALAT1 knockdown in endothelial cells significantly downregulated PFKFB3 expression at the m RNA and protein levels,while regulating the mi R-26a/26 b m RNA levels2.In mi R-26a/26 b overexpression group,the expression of mi R-26a/26 b was significantly increased,and the m RNA and protein levels of PFKFB3 were significantly decreased.3.The mi R-26a/26 b mimic reduced the luciferase activity of wild-type but not mutant MALAT1,and mi R-26a/26 b was pulled down by sense MALAT1.The results manifested that mi R-26a/ 26 b directly binds to MALAT1.4.The mi R-26a/26 b mimic reduced the luciferase activity of wild-type but not mutant PFKFB3,and the results showed that mi R-26a/ 26 b directly binds to PFKFB3.5.The inhibition of mi R-26a/26 b in MALAT1-silenced cells reversed the decrease in PFKFB3 m RNA and protein expression.ConclusionIn endothelial cells,MALAT1 might regulate PFKFB3 expression by sponging mi R-26a/26 b.Part fourMALAT1 regulates glycolytic activity and endothelial cell function through PFKFB3PurposeTo determine the effect of MALAT1 on glycolytic activity and endothelial cell function through PFKFB3.Methods1.Cell experiment grouping:sh-NC: negative control group.sh-MALAT1: MALAT1 knockdown plasmids were transfected into endothelial cells.sh-NC + PFKFB3 plasmid: The negative control plasmid and PFKFB3 overexpression plasmid were co-transfect into endothelial cells.sh-MALAT1 + PFKFB3 plasmid: MALAT1 knockdown plasmid and PFKFB3 overexpression plasmid were co-transfect into endothelial cells.2.q RT-PCR and(or)Western Blot were used to detect the transfection efficiency.3.The glycolytic activity of endothelial cells after transfection were measured: the cellular levels of ATP,NADPH,reactive oxygen species(ROS),and extracellular lactate production were detected by corresponding kits.4.The different effects after transfection on endothelial cell function were detected: CCK-8,flow cytometry,Transwell,tubule-formation and cytoskeleton staining experiments were used to study the effects after transfection on endothelial cell proliferation,cell cycle progression,migration,tube formation and filopodia and lamellipodia formation.Results1.Cells were divided into four groups and transfected with the sh-NC,sh-MALAT1,sh-NC+ PFKFB3,or sh-MALAT1 + PFKFB3 plasmids,which effectively upregulated or downregulated the expression of MALAT1 and PFKFB3.2.Knockdown of MALAT1 significantly decreased the cellular ATP,NADPH,and extracellular lactate levels but increased the ROS levels,whereas PFKFB3 overexpression in MALAT1-silenced cells reversed these effects.3.Overexpression of PFKFB3 reversed the inhibitory effects on proliferation,cell cycle progression,migration,tube formation,and filopodia and lamellipodia formation induced by MALAT1 knockdown.ConclusionMALAT1 knockdown reduces glycolytic activity and contributes to inhibiting the angiogenesis of endothelial cells through the glycolytic rate-limiting enzyme PFKFB3,whereas overexpression of PFKFB3 reverses these changes. |
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