茯苓提取物以miR-649/Akt1通路调控AngⅡ诱导血管平滑肌细胞的增殖、迁移和侵袭机制

Effects of tuckahoe extract on the proliferation,migration and invasion of AngⅡ-induced vascular smooth muscle cells via miR-649/Akt1 pathway

目的探讨茯苓提取物对血管紧张素Ⅱ(AngⅡ)诱导的血管平滑肌细胞(VSMCs)增殖、迁移和侵袭的影响及作用机制。方法AngⅡ诱导VSMCs细胞,茯苓提取物作用后,四甲基噻唑蓝染色法(MTT)检测细胞增殖,Transwell检测细胞的迁移和侵袭能力,Western Blot检测细胞周期蛋白D1(CyclinD1)、p21、上皮性钙粘附蛋白(E-cadherin)、基质金属蛋白酶-2(MMP-2)、丝氨酸/苏氨酸蛋白激酶1(Akt1)和磷酸化的Akt1(p-Akt1)蛋白表达,实时荧光定量PCR检测miR-649表达。转染miR-649模拟物至VSMCs细胞,上述方法检测miR-649过表达对AngⅡ诱导VSMCs细胞增殖、迁移和侵袭及相关蛋白表达的影响。双荧光素酶报告基因实验验证miR-649与Akt1靶向关系。结果AngⅡ诱导可促进VSMCs细胞增殖、迁移和侵袭,Cyclin D1和MMP-2蛋白、miR-649表达(P<0.05),抑制P21、E-cadherin、Akt1和p-Akt1蛋白表达(P<0.05)。茯苓提取物可抑制AngⅡ诱导的VSMCs细胞增殖、迁移和侵袭及Cyclin D1和MMP-2蛋白、miR-649表达(P<0.05),促进P21、E-cadherin、Akt1和p-Akt1蛋白表达(P<0.05)。过表达miR-649与茯苓提取物具有相似的功能,可抑制AngⅡ诱导的VSMCs细胞增殖、迁移和侵袭及Cyclin D1和MMP-2蛋白表达(P<0.05),促进P21和E-cadherin蛋白表达(P<0.05)。miR-649在VSMCs细胞中靶向负调控Akt1表达,抑制miR-649表达逆转了茯苓提取物对AngⅡ诱导的VSMCs细胞增殖、迁移和侵袭及相关蛋白Cyclin D1、P21、E-cadherin、MMP-2和Akt1、p-Akt1蛋白表达的影响。结论茯苓提取物可抑制AngⅡ诱导的VSMCs的增殖、迁移和侵袭,其可能通过调控miR-649/Akt1发挥作用。

Objective To investigate the effects of tuckahoe extract on the proliferation,migration and invasion of angiotensinⅡ(AngⅡ)-induced vascular smooth muscle cells,and to explore its action mechanism.Methods VSMCs were induced by AngⅡ,then which were treated with tuckahoe extract.The cell proliferation was detected by MTT assay,and the cell migration and invasion abilities were detected by Transwell assay,and the expression levels of CyclinD1,p21 and E-cadherin,matrix metalloproteinase-2(MMP-2),serine/threonine protein kinase 1(Akt1)and phosphorylated Aktl(p-Akt1)were measured by Western Blot,and the expression levels of miR-649 were detected by quantitative real-time PCR.Moreover the miR-649 simulant was transfected into VSMCs cells.The effects of miR-649 overexpression on the proliferation,migration and invasion of AngⅡ-induced VSMCs and on the expression of related proteins were detected by the methods mentioned above.In addition the dual luciferase reporter gene assay was used to verify the targeting relationship between miR-649 and Aktl.Results AngⅡinduction could significantly promote the proliferation,migration and invasion of VSMCs,and the expression levels of Cyclin D1,MMP-2 protein and miR-649(P<0.05),which could significantly inhibit the expression levels of P21,E-cadherin,Aktl and p-Akt1 proteins(P<0.05).The tuckahoe extract could inhibit the proliferation,migration and invasion of AngⅡ-induced VSMCs and the expressions of Cyclin D1,MMP-2 and miR-649(P<0.05),which could promotethe expressions of P21,E-cadherin,Aktl and p-Akt1 protein(P<0.05).In addition the overexpression of miR-649 had similar effects with those by tuckahoe extract(P<0.05).Moreover iR-649 targeted negatively regulated the Aktl expression in VSMCs,and inhibited the miR-649 expression to reverse the effects of tuckahoe extract on the proliferation,migration and invasion of AngⅡ-induced VSMCs and on the expressions of related proteins including Cyclin D1,P21,E-cadherin,MMP-2 and Aktl,p-Akt1.Conclusion The tuckahoe extract can inhibit the proliferation,migration and invasion of AngⅡ-induced VSMCs,which may play a role through regulating miR-649/Akt1.