Study Of The Effects Of KIF4A And Curcumin On Vascular Endothelial Cells

Background: The KIF4 A gene is a member of the kinesin 4 subfamily,which encodes an ATPdependent microtubule-based motor protein that regulates spindle formation and chromosome segregation during mitosis,affecting the cell cycle and cell division.k IF4 A is also involved in intracellular material transport.For example,the KIF kinesin superfamily is able to transport most of the proteins necessary for axon and synaptic terminals from the cytosol to the role of axon and synaptic terminals and is involved in the formation of myelin sheaths and the growth of nerve protrusions.Studies have shown that KIF4 A is associated with neurodevelopmental delay and advanced cognitive dysfunction Disorders associated with KIF4 A include mental retardation,motor retardation,X-linked related disorders,and renal disease with polycystic dysplasia.The vascular system consists of arteries,veins and interconnected capillaries,which transport oxygen,nutrients and metabolites and carry circulating blood cells to maintain human life.Studies have shown that abnormal vascular function often leads to nerve cell damage.In the basic theory of traditional Chinese medicine,blood vessels or "meridians" belong to the category of "pulse" and are the channels through which qi and blood flow.According to Nine Needles Theory,"Blood vessels are the cause of human birth"."Lingshu" also mentioned that "the blood vessels are unobstructed and the mind is normal." This shows that blood vessels and nerves are closely related.Vascular growth is mainly regulated by vascular growth factors.Vascular endothelial growth factor A(VEGF-A)belongs to the family of VEGF growth factors,which regulates angiogenesis and vascular development,including tumor angiogenesis,and maintains vascular stability under embryonic development,physiological and pathological conditions.It has been shown that KIF4 A silencing affects the expression of vascular endothelial growth factor receptor 1(VEGFR1)and VEGFA,and inhibits the activation of PI3K/Akt signaling pathway in mouse mononuclear macrophages(RAW264.7).Therefore,it is speculated that KIF4 A may regulate angiogenesis.However,the effect of KIF4 A on vascular endothelial cells and its mechanism is not clear.In this study,we used human brain microvascular endothelial cells h CMEC/D3 and human umbilical vein endothelial cells HUVEC to construct KIF4 A knockdown and overexpression stable cell lines,respectively,to study the effects of abnormal KIF4 A expression on vascular endothelial cell proliferation,migration,invasion,and tube formation ability,and to reveal the effect of KIF4 A on promoting angiogenesis by regulating VEGFA and reveal the mechanism of KIF4 A in promoting angiogenesis through the regulation of VEGFA,and to provide a scientific basis for exploring KIF4 A as a potential therapeutic target for neurovascular diseases.It has been reported that curcumin has the ability to inhibit the proliferation of vascular endothelial cells,however,the effect of curcumin on the proliferation of KIF4A overexpressing vascular endothelial cells is unclear.In this study,we investigated the effect of curcumin on the proliferation of KIF4 A overexpressing vascular endothelial cells,aiming to investigate the feasibility of using curcumin as a new anti-cancer drug for KIF4 A high-expression tumor diseases.Part Ⅰ.Effect of KIF4 A on the phenotype of vascular endothelial cellsMethods: Lentiviral packaging was used to construct KIF4 A knockdown or overexpressed h CMEC/D3 and HUVEC stable cell lines,respectively.Identification of stably transferred cell lines by q PCR)method and Western blot method.Cell Counting Kit-8(CCK-8)method was used to detect the effect of KIF4 A knockdown or overexpression on the proliferation viability of endothelial cells.wound healing assay was used to detect the effect of KIF4 A knockdown or overexpression on the migration ability of endothelial cells.Transwell invasion assay was used to detect the effect of KIF4 A knockdown or overexpression on the invasion ability of endothelial cells.The effect of KIF4 A knockdown or overexpression on the tube formation ability of endothelial cells was examined by the tube formation assay.Results: The results of western blot and q PCR showed that the KIF4 A knock-down and overexpression stably transformed cell lines were successfully constructed.Compared with the control group,the results of the CCK8 assay showed that KIF4 A knockdown inhibited the proliferation ability of endothelial cells and KIF4 A overexpression promoted the proliferation ability of endothelial cells(p<0.05).The results of the wound healing assay showed that KIF4 A knockdown inhibited the migration ability of endothelial cells and KIF4 A overexpression promoted the migration ability of endothelial cells(p<0.05).The results of transwell invasion assay showed that KIF4 A knockdown The results of transwell invasion assay showed that KIF4 A knockdown inhibited endothelial cell invasion and KIF4 A overexpression promoted endothelial cell invasion(p<0.05).Tube formation assay showed that KIF4 A knockdown inhibited endothelial cell angiogenesis in vitro and KIF4 A overexpression promoted endothelial cell angiogenesis in vitro(p<0.05).Conclusions:The KIF4 A knock-down and overexpression stably transformed cell lines were successfully constructed.KIF4 A knockdown inhibits the proliferation,migration,invasion,and tube formation of vascular endothelial cells.KIF4 A overexpression promotes the proliferation,migration,invasion and tube formation of vascular endothelial cells.Part Ⅱ.Mechanistic studies on the regulation of vascular endothelial cell function by KIF4AMethods: The lentiviral packaging system was used to construct KIF4 A knockdown or overexpressed h CMEC/D3 and HUVEC stable cell lines,respectively.q PCR was used to detect the effect of aberrant KIF4 A expression on VEGFA m RNA level,Western blot was used to detect the effect of aberrant KIF4 A expression on VEGFA protein level,the pull-down method was used to pull down method to verify the interaction between exogenous KIF4A(KIF4A plasmid was transferred into human embryonic kidney 293 T cells using liposomes)and VEGFA,Co-immunoprecipitation(Co-IP)method to verify the interaction between endogenous KIF4 A and VEGFA protein(wild type of h CMEC/D3 and HUVEC),immunofluorescence(IF)analyses to detect the endogenous KIF4 A and VEGFA protein cell co-localization.Results: Compared with the control group,m RNA and protein expression levels of VEGFA were decreased in KIF4 A knockdown h CMEC/D3 and HUVEC cell lines,m RNA and protein expression levels of VEGFA were increased in KIF4 A overexpressing h CMEC/D3 and HUVEC cell lines,the results of co-immunoprecipitation and immunofluorescence analyses indicated that VEGFA interacted with protein KIF4 A and was co-localized with KIF4 A in the nucleus.Conclusion: On the one hand,KIF4 A promotes the function of vascular endothelial cells by regulating the expression levels of VEGFA m RNA and protein.On the other hand,KIF4 A interacted with protein VEGFA and was co-localized with VEGFA in the nucleus.Part Ⅲ.Effect of curcumin on KIF4 A overexpressing vascular endothelial cellsMethods: The CCK-8 method was used to detect the effects of curcumin at different concentrations(20 μmol/L,40 μmol/L and 80 μmol/L)and action time(12h,24 h and 48h)on the cellular activity of KIF4 A overexpressing vascular endothelial cells(h CMEC/D3 and HUVEC).Specifically,cells in logarithmic phase were divided into experimental groups(1)Control group: endothelial cell complete medium +DMSO+ endothelial cells(Con or OE),(2)20 μmol/L dosing group: endothelial cell complete medium +20 μmol/L curcumin+endothelial cells(Con or OE),(3)40 μmol/L dosing group: endothelial cell complete medium+40 μmol/L curcumin+endothelial cells(Con or OE),(4)The 80 μmol/L dosing group: endothelial cell complete medium +80 μmol/L curcumin+endothelial cells(Con or OE).Results: Curcumin can inhibit the cell activity of endothelial cells.With the increase of drug concentration and the extension of action time,the inhibitory effect of curcumin on endothelial cell activity increased.In this study,20 μmol/L curcumin for 12 h significantly inhibited the cellular activity of h CMEC/D3 and HUVEC control group(Con).For the h CMEC/D3 and HUVEC(OE)overexpressed with KIF4 A,the promotion effect of KIF4 A on endothelial cell cellular activity was reduced by prolonging the action time of curcumin(24–48 h)or increasing the concentration of curcumin(40–80 μmol/L).Conclusion: Curcumin can inhibit the cellular activity of endothelial cells.The inhibitory effect of curcumin on endothelial cells increased with increasing drug concentration and duration of action.For KIF4 A overexpressing endothelial cells,the promotion of cell cellular activity by KIF4 A could be inhibited by prolonging the duration of curcumin action or increasing the curcumin concentration.