干扰多亮氨酸重复区免疫球蛋白样蛋白1促进人胶质母细胞瘤U251细胞株侵袭性的机制

Mechanism of RNA interference-mediated leucine.rich repeats and immunoglobulin-like domains 1 gene silencing promoting invasion of glioblastoma cell line U251

目的探讨干扰多亮氨酸重复区免疫球蛋白样蛋白1（LRIG1）基因表达促进人胶质母细胞瘤U251细胞株侵袭性的机制。方法用携带U6启动子的LRIG1特异性短发夹RNA（shRNA）序列的质粒载体pGenesil2-LRIG1-shRNA（siRNA）及含非特异shRNA编码序列的对照质粒pGenesil2-negativeshRNA（neg）转染U251细胞株，通过G418筛选，鉴定得到稳定转染细胞株。采用侵袭实验验证干扰LRIG1对U251侵袭性的影响，通过明胶酶谱实验检测基质金属蛋白激酶（MMP）-2、MMP-9的活性，Westernblot法检测表皮生长因子（EGFR）及其下游丝裂原激活蛋白激酶／细胞外信号调节激酶（MAPK／ERK）、磷脂酰肌醇3激酶／蛋白激酶B（P13K／AKT）信号通路蛋白的表达差异。结果含U6启动子LRIG1shRNA序列的质粒转染干扰组LRIG1mRNA降低至对照组（35．3％～39．2％，P〈0．05）。干扰LRIG1表达组U251细胞侵袭数[（159±15）-（188±9）／视野]，较空白对照组细胞侵袭数[（28±9）／视野]明显增多（P〈0．05）；干扰LRIG1激活EGFR通路传导；干扰LRIG1后干扰组MMP-2较对照组活性增加（1．66±0．11～1．96±0．12，P〈0．01），MMP-9干扰组较对照组活性增加（4．82±0．27～4．47±0．29）。结论LRIG1表达下调后MMP-2、MMP-9活性增强，从而促进U251细胞的侵袭性，可能通过激活EGFR信号通路引起。

Objective To explore the mechanism of RNA interference-mediated leucine-rich repeats and immunoglobulin-like domains 1 （ LRIG1 ） gene silencing promoting invasion of glioblastoma cell line U251. Methods The U6 promoter-driven plasmid pGenesil2-LRIGl-shRNA （siRNA） was transfected into U251 glioma cells by hpofectime 2000, and the cells （siRNA） that stably suppressed LRIG1 expression were selected by G418. The control cells were transfected with negative shRNA （neg）. The changes of invasive ability in the U251 cells were measured by cell invasion assay. The activities of the matrix metalloproteinases （MMP）-2, and -9 were detected by gelatin zymography. LRIG1, epidermal growth factor receptor （EGFR） and phosphorylated EGFR proteins were examined by using Western blotting. Results As compared with the negative shRNA-transfected cells, LRIG1 mRNA expression in the U6 promoterdriven plasmid pGenesil2-LRIGl-shRNA （siRNA） cells was silenced to 35.3% -39. 2% （P 〈 0. 05） , and knocking-down LRIG1 increased the mean invasion cell number of per field to （159 ± 15 ）-（ 188 ± 9 ）, compared to control group （ P 〈 0. 05 ）. Down-regulation of LRIG1 increased MMP-2 activity by （ 1.66 ± 0. 11 ） - （ 1.96 ± 0. 12 ） fold, and increased MMP9 activity by （ 4. 82 ± 0. 27 ）- （ 4. 47 ± 0. 29 ） fold, compared to control group （ P 〈 0. 01 ）. EGFR signaling pathway was activated after knocking down the LRIG1 expression. Conclusion Silencing of LRIG1 promotes invasion of glioblastoma cell line U251 via up-regu- lating the MMP-2, and -9 （P 〈0. 01 ）, which is possibly by enhancing activities of EGFR signaling path- way.