| ObjectiveNeuroblastoma is the most common tumor diagnosed in children and infants,with high recurrence and poor prognosis.At present,the main treatment methods of neuroblastoma include surgery,chemotherapy,radiotherapy,stem cell transplantation and biotherapy.Chemotherapy is still dominant,but the side effects of chemotherapy are great.Therefore,it is necessary to find an effective but low toxic treatment of neuroblastoma.Angelicasinensis polysaccharide(AP)whose average molecular weight is 72,900-78,000 Da possesses various bioactivities and its anti-tumor activity has attracted much attention.However,its therapeutic effect and mechanism on neuroblastoma is still unclear.We aimed to explore the effects of AP on neuroblastoma SH-SY5Y cells as well as the underlying mechanisms.MethodIn this study,neuroblastoma SH-SY5Y cell line was cultured in vitro.1.The evaluation method of angelica polysaccharides on proliferation,apoptosis,migration and invasion of SH-SY5Y cell line.1)Cells without angelica polysaccharide were used as control group,and cells with concentration of angelica polysaccharide of 100,200,300,400 and 500μg/ml were used as experimental group.The effect of angelica polysaccharide on SH-SY5Y cell line activity was detected by cell counting KIT-8(CCK-8),and the concentration of angelica polysaccharide was determined.2)Cells without angelica polysaccharide stimulation were used as control group and cells stimulated by angelica polysaccharide at a concentration of 300μg/ml were used as experimental group.The effects of angelica polysaccharide on proliferation,apoptosis,migration and invasion of SH-SY5Y cell line were evaluated by BrdU cell proliferation assay,Annexin V-FITC/PI double staining and 24-hole Transwell system.Western blot was used to detect the effects of Angelica polysaccharide stimulation on SH-SY5Y cell line.Proliferation and apoptosis-related protein expression in SH-SY5Y cell line.2.Evaluation of the effects of microRNA-675(miR-675)overexpression on proliferation,apoptosis and migration of SH-SY5Y cell line induced by angelica sinensis polysaccharide.1)qRT-PCR was used to compare the expression of long-chain lncRNA-H19 and miR-675 in normal human dorsal root ganglion cells and SH-SY5Y cell lines.The expressions of lncRNA-H19 and miR-675 in cells of Angelica sinensis polysaccharide group and 300μg/ml angelica sinensis polysaccharide group were compared.2)Scamble miRNAs and miR-675 mimic were transfected into SH-SY5Y cell line by Lipofectamine~?3000.300μg/ml angelica polysaccharides stimulated transfected cells.Scamble miRNAs cells were used as control group and miR-675mimic cells were used as experimental group.The effects of over-expression of miR-675 on proliferation,apoptosis,migration and invasion of SH-SY5Y cells stimulated by angelica polysaccharides were analyzed by BrdU cell proliferation assay,Annexin V-FITC/PI double-staining,24-well system and Western blot.3)The phosphorylation levels of key kinases in PI3K/AKT and JAK/STAT signaling pathways were compared by Western blot in cells without Angelica polysaccharide and 300ug/ml angelica polysaccharide.At the same time,angelica polysaccharide were stimulated to transfect cells,and phosphorylation levels of key kinases in PI3K/AKT and JAK/STAT signaling pathways were detected in scramble miRNAs transfected cells and miR-675 mimic transfected cells.3.Validation of the target of miR-675.1)Predicting KIF1Bβon-line by TargetScan software may be the target of miR-675.KIF1Bβ-wt was co-transfected with scramble miRNAs and miR-675 mimic respectively.KIF1Bβ-mut and scramble miRNAsand miR-675 mimic were transfected into 293T cells respectively.Using scramble miRNAs+KIF1Bβ-wt(KIF1Bβ-mut)as control group and miR-675 mimic+KIF1Bβ-wt(KIF1Bβ-mut)as experimental group,the effect of miR-675 overexpression on luciferase activity in KIF1Bβ-wt or KIF1Bβ-mut cells was evaluated by double luciferase reporter gene detection system.2)Scamble miRNAs and miR-675 mimic were transfected into SH-SY5Y cells by Lipofectamine~?3000.The expression of KIF1Bβin scramble miRNAs cells and miR-675 mimic cells was compared.3)SH-SY5Y cell lines were transfected with miR-675 mimic,scramble miRNAs+pEX-2 and miR-675 mimic+pEX-KIF1Bβrespectively.After 300μg/ml angelica polysaccharide stimulation,scramble miRNAs+pEX-2 and miR-675mimic+pEX-KIF1Bβwere used as control group,respectively.BrdU cell proliferation test,transxin V-FITC/PI double-staining,and Western blot analysis were used.The effect of over-expression of KIF1Bβon proliferation,apoptosis,migration and invasion of SH-SY5Y cells stimulated by Angelica sinensis polysaccharide induced by over-expression of miR-675.Result1.The effects of Angelica polysaccharides on the activity,proliferation,apoptosis,migration and invasion of SH-SY5Y cell line.1)The effect of angelica polysaccharide on the activity of SH-SY5Y cell line and the determination of the concentration of angelica polysaccharide for experiment:When the concentration of angelica polysaccharide was 200,300,400 and 500μg/ml,the survival rate of SH-SY5Y cell line was significantly lower than that of control group(P<0.05,<0.01,<0.01,<0.001).Similarly,when the concentration of angelica polysaccharide was 300μg/ml,normal human epidermal fibroblasts and dermal fibroblasts were also The survival rate of human umbilical vein endothelial cells was also significantly reduced(all P<0.01),so the following experiment used 300μg/ml as the concentration of angelica polysaccharide.2)Effects of angelica polysaccharides on proliferation,apoptosis,migration and invasion of SH-SY5Y cell line:Compared with the control group,the percentage of BrdU-positive cells in the experimental group decreased significantly(P<0.01),the expression of cyclin D1 decreased significantly,and the expression of cyclin-dependent kinase inhibitor p21 increased significantly(all P<0.05).Meanwhile,Angelica polysaccharides down-regulated the expression of Bcl-2 and up-regulated the expression of Bax,lysed caspase-3 and lysed caspase-9.Cell migration and invasion in the experimental group were significantly reduced(P<0.05).2.Effects of over-expression of miR-675 on proliferation,apoptosis and migration of SH-SY5Y cell line induced by angelica sinensis polysaccharide1)Effects of angelica polysaccharides on the expression of lncRNA-H19 and miR-675 in SH-SY5Y cell lines:The expression levels of lncRNA-H19 and miR-675in SH-SY5Y cell lines were significantly higher than those in normal dorsal root ganglion cells(P<0.001),and the expression levels of lncRNA-H19 and miR-675 in angelica polysaccharides group were significantly lower than those in untreated group(P<0.05).2)The effect of over-expression of miR-675 on SH-SY5Y cell line induced by angelica sinensis polysaccharide:In experimental group,over-expression of miR-675significantly promoted cell proliferation(P<0.05),up-regulation of cyclinD1expression(P<0.05),down-regulation of p21 expression(P<0.01);over-expression of miR-675 significantly reduced cell apoptosis(P<0.05),up-regulation of Bcl-2expression,and up-regulation of cyclinD1 expression.The expression of Bax,lysed caspase-3 and lysed caspase-9 was up-regulated,and cell migration and invasion were significantly reduced in the experimental group(all P<0.05).2.Effects of over-expression of miR-675 on proliferation,apoptosis and migration of SH-SY5Y cell line induced by Angelica sinensis polysaccharide1)Effects of angelica polysaccharides on the expression of lncRNA-H19 and miR-675 in SH-SY5Y cell lines:The expression levels of lncRNA-H19 and miR-675in SH-SY5Y cell lines were significantly higher than those in normal dorsal root ganglion cells(P<0.001),and the expression levels of lncRNA-H19 and miR-675 in angelica polysaccharides group were significantly lower than those in untreated group(P<0.05).2)The effect of over-expression of miR-675 on SH-SY5Y cell line induced by angelica sinensis polysaccharide:In experimental group,over-expression of miR-675significantly promoted cell proliferation(P<0.05),up-regulation of cyclinD1expression(P<0.05),down-regulation of p21 expression(P<0.01);over-expression of miR-675 significantly reduced cell apoptosis(P<0.05),up-regulation of Bcl-2expression,down-regulation of Bax,lysis caspase-3,lysis caspase-9 expression;Overexpression of 675 significantly increased cell migration(P<0.05)and invasion(P<0.05).3)Effects of angelica polysaccharides on PI3K/AKT and JAK/STAT pathways:Phosphorylation levels of PI3K,AKT,JAK1 and STAT3 in Angelica polysaccharides group were significantly lower than those in non-angelica polysaccharides group(P<0.05 or P<0.01);Overexpression of miR-675 could significantly increase the phosphorylation levels of these kinases(P<0.01 or P<0.001).3.Verification of the target of miR-675.1)Luciferase activity:In KIF1Bβ-wt transfected cells,over-expression of miR-675 significantly reduced luciferase activity(P<0.05).2)The effect of over-expression of miR-675 on the expression of KIF1Bβ:Compared with scramble miRNAs group,the expression of KIF1Bβin the miR-675mimic group decreased significantly(P<0.05).3)The effect of overexpression of KIF1Bβon SH-SY5Y cell line induced by over-expression of angelica sinensis polysaccharide after stimulation by miR-675:Compared with the miR-675 mimic group,the percentage of BrdU-positive cells in the miR-675 mimic+pEX-KIF1Bβgroup decreased significantly(P<0.01),the expression of cyclinD1 decreased significantly,and the expression of p21 protein increased significantly(P<0.05),and the percentage of apoptotic cells increased significantly(P<0.01),the same as that in the miR-675 mimic+pEX-KIF1Bβ.The expression of Bcl-2 was down-regulated,while the expression of Bax,lysed caspase-3and lysed caspase-9 was up-regulated,and cell migration and invasion were significantly reduced(all P<0.05).conclusion1.Angelica polysaccharide can inhibit proliferation,migration and invasion of SH-SY5Y cell line and induce apoptosis.2.Overexpression of miR-675 reverses the effect of Angelica polysaccharide on the biological behavior of SH-SY5Y cell line and up-regulates the phosphorylation levels of PI3K/AKT and JAK/STAT pathway kinases,suggesting that angelica polysaccharide plays a role in the down-regulation of PI3K/AKT and JAK/STAT signaling pathways.3.Overexpression of KIF1Bβcan reverse the effect of miR-675 overexpression on the biological behavior of SH-SY5Y cell line stimulated by angelica sinensis polysaccharide,suggesting that KIF1Bβmay be the target of miR-675. |
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