The Roles And Mechanisms Of LncRNA MEG3-mediated MiR-106a-5p/SIRT3 Axis In Postoperative Cognitive Dysfunction

Part I LncRNA improves cognitive function by inhibiting inflammatory factors in POCD miceObjective: Postoperative cognitive dysfunction(POCD)is a common clinical postoperative complication,which can significantly delay the discharge of patients,increase the economic burden of patients,and have a significant negative impact on the prognosis and long-term quality of life of patients.It takes up a lot of available resources from both family and society.Although there are many studies on POCD,its pathogenesis is complex and the specific pathogenesis is still unclear.Therefore,there is currently a lack of effective prevention and treatment measures for POCD.Long non-coding RNA(LncRNA)is a class of non-coding RNA with a length of more than 200 nucleotides.In recent years,some scholars have tried to elucidate the role and mechanism of lncRNA in POCD,but it is still unknown what function lncRNA MEG3 plays in POCD and what molecular mechanism it affects POCD.This study aims to construct a POCD mouse model to investigate the expression of lncRNA MEG3 in POCD and the effect of overexpression of MEG3 on animal cognitive function,inflammatory factor secretion,and microglial activation in POCD mice.Methods: In this study,C57BL/6 male mice were used to establish a POCD mouse model by osteotomy surgery.The control group mice underwent sham surgery.LncRNA MEG3 was overexpressed in mice model using a lentiviral expression vector of MEG3(LV-MEG3)by tail vein injection of LV-MEG3 before POCD modeling,with LV-NC as a negative control.RT-qPCR was used to detect the expression of MEG3 in mouse hippocampal tissue.After one week of animal modeling,the cognitive function of the animals was tested using a water maze experiment.IBA-1 fluorescence staining was used to detect the activation of microglia.The expression of inflammatory factors TNF-α and IL-1β in hippocampus homogenate of POCD mice was tested by enzyme linked immunosorbent assay(ELISA).Results: 1)In the hippocampus of POCD mice,the expression of MEG3 decreased by nearly 50% compared to the control group,with a significant difference between the two groups(P=0.0024).2)The results of the water maze experiment showed that the time of POCD model mice in the target quadrant was significantly reduced compared to the control mice,with a statistically significant difference(P<0.001).However,after overexpression of MEG3.At the same time,the frequency of POCD mice crossing the platform within 60 seconds was significantly lower than that of control mice(P<0.001).However,after overexpression of MEG3 in mice,the frequency of mice crossing the platform within 60 seconds in the lv-MEG3 overexpression group was significantly higher than that in the lv-NC group,with a statistically significant difference(P=0.0074).3)The expression of TNF-α and IL-1β was significantly higher in hippocampal homogenate of POCD mice than that of the control mice,with a statistically significant difference(P<0.001).However,after overexpression of MEG3 in POCD mice,compared with the lv-NC negative control group,with a statistically significant difference(P<0.05).4)In the hippocampus of POCD mice,the fluorescence intensity of IBA-1increased significantly,with a statistically significant difference compared to the control group(P<0.01).However,after injecting MEG3 overexpressed lentivirus plasmid LV-MEG3 into the tail vein,the fluorescence intensity of IBA-1 in the hippocampus of the lv-MEG3 group was significantly lower than that of the lv-NC negative control group,with a statistically significant difference(P<0.01).Conclusion:1.MEG3 was decreased in POCD mice.2.Overexpression of MEG3 improves the cognitive function of POCD mice.3.Overexpression of MEG3 decreased the secretion of inflammatory factors in the hippocampus of POCD mice and inhibited the inflammatory activation of microglia.Part II LncRNA MEG3 regulates inflammatory response,oxidative stress and miR-106a-5p/SIRT3 axis in LPS-induced BV-2 cellsObjective: The inflammatory response induced by microglia plays a crucial role in the development of POCD.Inflammatory activation of microglia can release a large number of inflammatory factors,activate both inflammatory and oxidative stress pathways,ultimately causing neuronal damage in the microenvironment,leading to nerve damage and cognitive dysfunction.However,up to now,it is unclear which molecular pathway regulates the inflammatory activation of microglia in POCD.In the previous study,we clarified the improvement of cognitive function of mice by lncRNA MEG3 in the POCD mouse model and its inhibitory effect on inflammatory reactions and inflammatory activation of microglia in the mouse hippocampus.In this part of the study,we planned to use BV-2 microglia to establish an inflammatory induced cell model induced by LPS,and explore the regulatory effects of MEG3 on the miR-106a-5p/SIRT3 axis,inflammatory factor secretion,and oxidative stress in the LPS-induced microglia model.Methods: The study used lipopolysaccharide(LPS)to induce BV-2 cells to establish an inflammatory model.pc DNA3.1-MEG3 was transfected into LPS-induced BV-2 microglial cells to overexpress MEG3.RT-qPCR was used to detect the expression of MEG3,miR-106a-5p,and SIRT3.Western blotting was used to detect the protein expression of SIRT3.IBA-1 fluorescence staining was used to detect microglia activation.ELISA was used to test the expression of TNF-α and IL-1β.The contents of malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione peroxidase(GSH-PX)were detected using commercially available kits.The binding sites between miR-106a-5p and MEG3 were predicted using the bioinformatics analysis platform Starbase.The binding relationship between lncRNA MEG3 and miR-106a-5p was detected by dual luciferase report analysis.Results: The expressions of inflammatory factors tumor necrosis factor-α and interleukin-1β were significantly increased in lipopolysaccharids-guided vas-2 cells.2)RT-qPCR results showed that the expression of MEG3 in BV-2 cells transfected with pc DNA3.1-MEG3 significantly increased(P<0.001),indicating that the cell transfection successfully established a model of MEG3 overexpression.ELISA results showed that LPS induction significantly increased the expression of TNF-α and IL-1β(P<0.001).After overexpression of MEG3 in cells,the expression of TNF-α and IL-1β was significantly decreased compared with the negative control group of LPS+pc DNA3.1(P<0.001).3)In LPS induced BV-2 cells,the fluorescence intensity of IBA-1 increased significantly,with a fluorescence intensity of IBA-1 in LPS-induced BV-2 cells,which was statistically significant compared to the negative control group of LPS+pcDNA3.1(P<0.001).4)The expression of oxidative stress factor MDA was significantly increased in LPS induced BV-2 microglial cells,with a statistically significant difference compared to control cells(P<0.001).However,after overexpression of MEG3 with pc DNA3.1-MEG3,the expression of MDA was significantly reduced compared to the LPS+pc DNA3.1 negative control group,with a statistically significant difference(P<0.001).The expression of antioxidant stress SOD and GSH-PX in BV-2 microglia induced by LPS was significantly reduced.After overexpression of MEG3 with pc DNA3.1-MEG3,the expression of SOD and GSH-PX was significantly increased,and the difference was statistically significant compared to the LPS+pc DNA3.1negative control group(P=0.0142).5)The bioinformatics analysis platform Starbase predicted the binding sites between MEG3 and miR-106a-5p.In 293 T cells with MEG3-WT wild-type,luciferase activity was significantly decreased when miR-106a-5p was overexpressed(P<0.05),while overexpression of miR-106a-5p in MEG3-MUT mutant cells had no significant effect on luciferase activity.The results of RT-qPCR and Western blotting showed that overexpression of MEG3 significantly decreased the expression of miR-106a-5p in BV-2 cells(P<0.05),while overexpression of MEG3 significantly increased the m RNA and protein expression of SIRT3(P<0.001).Conclusion:1.Overexpression of MEG3 can inhibit LPS-induced inflammatory responses in BV-2 microglia.2.Overexpression of MEG3 can inhibit LPS-induced oxidative stress in BV-2microglial cells.3.MEG3 can bind to miR-106a-5p and negatively regulate the has-miR-106a-5p/SIRT3 axis.Part III miR-106a-5p reverses the suppressive effects of lncRNA MEG3 on inflammation and oxidative stress in LPS-induced BV-2cells by regulating SIRT3Objective: Although some studies have reported the regulatory effects of lncRNAs on the secretion of inflammatory factors and oxidative stress in microglia,it is unclear which molecular signaling pathways are involved in the process,thereby affecting the development of POCD.Mi R-106a-5p has been shown in some studies to promote inflammatory processes,while its downstream target m RNA SIRT3 has been found to have anti-inflammatory and antioxidant effects.However,currently,no research has focused on the interaction of miR-106a-5p/SIRT3 pathway with MEG3 in POCD and its impact on the inflammatory and oxidative stress of microglia in POCD.In this part of the study,we investigated the expression of miR-106a-5p/SIRT3 in POCD and BV-2 inflammatory cells through animal models of POCD and LPS-induced BV-2 cells,and explored the reverse effect of overexpression of miR-106a-5p on MEG3 affecting BV-2 cells,while clarifying the role of SIRT3 in this role.Methods: The study used C57BL/6 male mice to establish a POCD mouse model through osteotomy surgery.The control group mice underwent sham surgery.An in vitro model of BV-2 microglia was established by LPS induction.RT-qPCR was used to detect the expression of miR-106a-5p and SIRT3 in the hippocampus of POCD mice and in LPS-induced BV-2 microglia.ELISA was used to evaluate the contents of TNF-α and IL-1β in cell supernatants.The levels of MDA,SOD,GSH-PX were detected using commercially available kits.Targetscan,a bioinformatics analysis platform,was used to predict the binding sites between miR-106a-5p and SIRT3.Double luciferase report analysis detected the binding of miR-106a-5p to SIRT3 in 293 T cells.Results: 1)In the hippocampus of POCD mice,the expression of miR-106a-5p was significantly higher than that of control mice,and the difference was statistically significant(P=0.0021).The expression of SIRT3 in the hippocampus of POCD mice was significantly lower than that of control mice(P=0.0025).In LPS-induced BV-2microglial cells,the expression of miR-106a-5p was also significantly increased,and the difference was statistically significant compared to the control group(P=0.0247).However,the expression of SIRT3 in LPS-induced BV-2 microglia was significantly reduced compared to control mice(P=0.0076).2)Overexpression of MEG3 in LPS-induced BV-2 cells significantly reduces the expression of TNF-α and IL-1β compared with the negative control group,the difference was statistically significant(P<0.001).Transfection of BV-2 cells with miR-106a-5p mimics while overexpressing MEG3 significantly increased the expression of TNF-α and IL-1β,the difference was statistically significant compared to overexpression of MEG3 alone(P<0.001).3)Overexpression of MEG3 in LPS-induced BV-2 cells significantly reduced the level of MDA expression,with a statistically significant difference compared to the negative control group(P<0.001).Transfecting BV-2 cells with miR-106a-5p mimics while overexpressing MEG3 significantly increased the expression of MDA,with a statistically significant difference compared to overexpressing MEG3 alone(P<0.001).On the contrary,overexpression of MEG3 in LPS-induced BV-2 cells significantly increased the expression levels of antioxidant factors SOD and GSH-PX,with a statistically significant difference compared to the negative control group(P<0.001).Transfection of BV-2 cells with miR-106a-5p mimics while overexpressing MEG3 significantly reduced the expression of SOD and GSH-PX,with a statistically significant difference compared to overexpressing MEG3 alone(P<0.05).4)Target Scan,a bioinformatics analysis platform,predicted the binding effect of miR-106a-5p with SIRT3.In 293 T cells with SIRT3-WT wild-type,luciferase activity was significantly decreased when miR-106a-5p was overexpressed(P<0.05),while overexpression of miR-106a-5p in SIRT3-MUT mutant cells had no significant effect on luciferase activity.The results of RT-qPCR and Western blotting showed that overexpression of miR-106a-5p significantly decreased the expression of SIRT3 in BV-2 cells(P<0.05).Conclusion:1.The expression of miR-106a-5p was significantly increased in the hippocampus of POCD mice and LPS-induced BV-2 microglia,while the expression of SIRT3 was significantly decreased.2.miR-106a-5p reverses the inhibitory effects of MEG3 on inflammatory factor secretion and oxidative stress in LPS-induced BV-2 microglia.3.miR-106a-5p can bind to SIRT3 and negatively regulate its expression.