抑制信号转导和转录激活因子-3活化对恶性胶质瘤细胞LN229运动能力的影响

Effect of signal transducer and activators of transcription 3 on migration of human glloblastoma cells

目的观察抑制信号转导和转录激活因子-3（STAT3）的活化对调控人恶性胶质瘤LN229细胞运动能力的影响。方法应用JAK／STAT3信号通路抑制剂AG490处理LN229细胞株，Westernblot检测STAT3、磷酸化STAT3（P—STAT3）的表达。噻唑蓝（MTT）比色法筛选能够阻断LN229细胞STAT3活化的最适AG490浓度；划痕实验和趋化实验检测AG490给药后，LN229细胞运动能力的变化；F-肌动蛋白（F-actin）实验检测AG490对LN229细胞肌动蛋白聚合能力的影响。结果AG490可有效抑制LN229细胞STAT3的持续活化。50μmol／L浓度的AG490在不影响LN229细胞存活时（t=1．862，P〉0．05），能够有效抑制STAT3的磷酸化，对照组和50wmol／LAG490处理组的p-STAT3／STAT3的密度比值分别为0．530±0．040和0．291±0．036（t=4．821，P〈0．01）。伤口愈合实验可见50μmol／LAG490处理的LN229细胞非定向运动能力降低，24h时对照组运动的距离为（0．290±0．049）mm；实验组为（0．150±0．054）mm（P〈0．05）。体外趋化实验显示50μmol／LAG490处理的12q229细胞定向运动能力降低（P〈0．01），表皮细胞生长因子（EGF）浓度为10μg/L时对照组穿过膜的细胞数为93．92±9．52；实验组为24．52±3．04。50μmol／L浓度的AG490抑制LN229细胞肌动蛋白聚合能力（P〈0．05）。结论STAT3信号转导通路参与调控胶质母细胞瘤LN229的细胞运动过程，阻断STAT3的活化可以抑制恶性胶质瘤的运动能力。

Objective To investigate the role of signal transducer and activators of transcription 3 （STAT3） signaling pathway in giioblastoma cell migration. Methods Janus kinase 2 selective inhibitor AG490 was used to inhibit the activation of STAT3 signaling pathway. The expression of STAT3 and p-STAT3 proteins in gIiohIastoma LN229 cells was detected by Western blotting. The optimal concentration of AC,490 which can inhibit the activation of STAT3 in LN229 cell was chosen by methyl thiazol tetrazoIium （MTT） assay. The migration ability of LN229 ceils was evaluated by using wound-healing assay and chemotaxis assay. The cellular actin polymerization in LN229 ceils was detected by the F-actin polymerization assay. Results When the LN229 ceils were pretreated with AC,490 at 50μmol/L, there was no cytotoxic against LN229 ceils （ t = 1. 862, P 〉 0. 05 ）, however, the activation of p-STAT3 was suppressed （ t = 4. 82 1, P 〈 0. 01 ）. The relative intensity of p-STAT3 was 0. 291±0. 036 in the 50 p,mol/L AG490-pretrea- ted ceils and 0. 530 ±0. 040 in the control ceils. At 24th h after wounding, the migrating distance of the 50 p, moL/L AG490-pretreated cells was （ 0. 150 ± 0. 054 ） mm and that of the control cells was （ 0. 290 ± 0. 049） mm （P 〈0. 05）. After administration of 50μmoL/L AC,490, the motility ability of LN229 ceils was decreased in contrast to the control group in the chemotaxis assay （ P 〈 0. 05 ）. When the 10 ttg/L EGF was added in the low well, the trans-membrane ceil number in the 50 ttmoL/L AC-490-pretreated group was 24. 52±3.04 and that in the control group was 93.92 ±9. 52 respectively. Meanwhile, F-actin polymerization of LN229 ceils was also significantly impaired with AG490 treatment （ P 〈 0. 05 ）. Conclusion STAT3 signaling pathway plays a critical role in migration of glioblastoma ceils. Inhibition of STAT3 signaling by AC,490 treatment suppresses the migration ability of gliohlastoma LN229 cells.