干扰LRIG2通过调控PDGFRβ信号通路抑制人胶质母细胞瘤细胞增殖

LRIG2 knockdown inhibited the proliferation of glioblastoma cells through modulating the PDGFRβ signaling pathway

目的探讨干扰富含亮氨酸重复序列免疫球蛋白样蛋白2(LRIG2)基因表达对人胶质母细胞瘤细胞U87增殖的影响及其机制。方法构建LRIG2干扰(LRIG2sh)及对照(scr)慢病毒载体质粒并感染U87细胞,筛选获得稳定细胞株; CCK8检测细胞增殖及软琼脂克隆形成实验检测细胞克隆形成能力;流式细胞术检测细胞周期;裸鼠皮下成瘤实验检测在体成瘤能力; Western Blotting检测信号通路蛋白表达。结果 LRIG2干扰组中LRIG2 mRNA水平及蛋白表达水平均明显低于对照组,免疫荧光示干扰组中PDGFRβ荧光强度明显弱于对照组。PDGF-BB刺激下LRIG2干扰组细胞增殖率明显低于对照组(P <0. 05),且呈浓度和时间依赖性。PDGF-BB刺激下LRIG2干扰组中软琼脂克隆形成能力明显低于对照组,G0/G1期细胞数百分比明显高于对照组,S期及G2/M期细胞数百分比明显低于对照组(P<0. 01)。LRIG2干扰组裸鼠皮下成瘤体积和重量明显小于对照组(P <0. 01)。PDGF-BB刺激下LRIG2干扰组中p PDGFRβ及其下游p Akt、p Stat3表达水平明显低于对照组,且LRIG2干扰后PDGFRβ抑制剂对其信号通路的抑制水平明显减弱。结论 LRIG2下调通过抑制PDGFRβ信号通路抑制胶质母细胞瘤细胞离体和在体增殖,LRIG2有望成为胶质细胞瘤治疗的新靶点。

Objective The effects of leucine-rich repeats and immunoglobulin-like domains2(LRIG2)knockdown on the proliferation of glioblastoma U87 cells and the underlying mechanisms were investigated.Methods The LRIG2 targeted shRNA and the scramble RNA were constructed and stably transduced into U87cells.CCK8and soft agar colony formation assay were used to detect the in vitro and anchorage-independent cell proliferation,respectively.Flow cytometric analysis was used to investigate the cell cycle.In vivo tumor formation in nude mice was performed and the Western Blotting was used to examine the signaling pathway proteins.Results The mRNA and protein expression levels of LRIG2,as well as the protein level of PDGFRβ,were all significantly decreased in the LRIG2 knockdown cells.LRIG2 knockdown markedly inhibited the PDGF-BB-induced proliferation of U87 cells in a concentration-and time-dependent manner(P<0.05).The PDGF-BB-induced anchorage-independent growth of glioblastoma cells was abrogated in the LRIG2 knockdown U87 cells.The percentage of cells in the G0/G1phase significantly increased and the percentage in the S or G2/M phase dramatically decreased in the PDGF-BB-induced LRIG2 downregulation U87cells(P<0.01).The tumor volumes and tumor weights were markedly decreased in mice injected with LRIG2knockdown U87cells(P<0.01).The levels of PDGF-BB-induced phosphorylation of PDGFRβand its downstream phosphorylation of Akt and Stat3were all markedly decreased in LRIG2knockdown U87cells.Conclusion LRIG2 knockdown inhibits the proliferation of glioblastoma cells in vitro and in vivo through modulating the PDGFRβsignaling pathways,validating LRIG2 as a promising potential therapeutic target for treatment of glioblastoma.