信号转导和转录激活因子3信号通路在多亮氨酸重复区免疫球蛋白样蛋白1诱导U251细胞凋亡中的作用

Roles of signal transducers and activators of transcription 3 signaling pathway in leucine-rich re- peats and immunoglobulin-like domains 1-induced apoptosis of U251 cells

目的探讨信号转导和转录激活因子3 （STAT3）信号通路在多亮氨酸重复区免疫球蛋白样蛋白l（LRIGl）诱导人脑胶质瘤细胞株U251细胞凋亡中的作用及机制。方法传代培养U251细胞，随机分为对照组、空载体组及实验组，应用脂质体介导的基因转染技术分别将磷酸盐缓冲液（PBS）、PEGFP-N1质粒体、PEGFP-LRIG1质粒体转染入各组细胞，应用细胞计数试剂盒（CCK-8）法检测细胞体外生长活性，膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）/碘化丙锭（PI）法经流式检测各组细胞凋亡率，逆转录-聚合酶链反应（RT-PCR）法测定各组细胞中STAT3的mRNA表达，Westernblot法测定各组细胞中STAT3、磷酸化STAT3（p-STAT3）、B淋巴细胞/白血病-2（ bcl-2）、bax的蛋白表达。结果成功转染LRIG1，实验组细胞中LRIG1基因表达明显上调（P〈0.05）;实验组细胞增殖显著受抑，48 h抑制率为（45. 12 ±0. 68）%，72h抑制率为（52. 24 ± 1. 77）%，总凋亡率由（2. 54 土0. 43）%增高至（22. 51 ±2. 12）%（P〈0. 05） ； RT-PCR检测显示实验组细胞中STAT3 mRNA表达明显降低;Western blot检测显示实验组细胞中STAT3蛋白表达及磷酸化水平明显降低，bcl-2表达明显降低，bax表达明显升高。结论LRIG1可促进U251细胞凋亡，其机制可能是通过下调STAT3信号通路，进而抑制STAT3激活的抗凋亡途径。

Objective To investigate the effects of signal transducers and activators of transcrip-tion 3 （ STAT3） signaling pathway on leucine-rich repeats and immunoglobulin-like domains 1 （ LRIG1 ）-induced apoptosis of U251 cells and the mechansim. Methods U251 cells were cultured and randomly di-vided into three groups： control group, N1-U251 and LRIG1-U251, which were respectively transfectedwith PBS, PEGFP-N1 plasmid and PEGFP-LRIG1 plasmid by liposome transfection. Cell counting kit-8（CCK-8） assay was used to assess the proliferation of U251 cells, and the apoptosis rate by flow cytometryAnnexin V-FITC/PI. The mRNA expression of STAT3 was detected by using reverse transcription-polymerasechain reaction （ RT-PCR） , and the protein expression of STAT3, p-STAT3, B lymphocytes/leukemia-2（bcl-2） and bax by using Western blotting. Results LRIG1 gene was transfected into U251 cells, and theprotein expression level of LRIG1 was significantly upregulated （ P 〈 0. 05）. After transfection withLRIG1,proliferation of U251 cells was obviously inhibited with the inhibition rate at 48 and 72 h being（45. 12 ±0. 68）% and （52. 24 ± 1. 77）% respectively, and the total apoptosis rate was increased from（2. 54 ± 0. 43 ） % to （ 22. 51 ± 2. 12 ） % （ P 〈 0. 05 ） ; The mRNA expression level of STAT3 was signifi-cantly downregulated. The protein expression level of STAT3,phosphorylated STAT3 and Bcl-2 was signifi-cantly downregulated, and the protein expression level of Bax was significantly upregulated. ConclusionLRIG1 can downregulate the STAT3 signaling pathway,resulting in block of the antiapoptotic way,to pro-mote apoptosis of U251 cells.