EV71感染横纹肌肉瘤细胞与MAPK信号通路分子基因差异表达的相关性

Correlation of differential gene expressions of MAPK signaling pathway molecules with enterovirus 71 infection in rhabdomyosarcoma cells

目的探讨肠道病毒71型(EV71)感染横纹肌肉瘤(RD)细胞后丝裂原活化蛋白激酶(MARK)信号通路分子的变化及作用机制。方法按感染复数(MOI)=5的病毒量感染RD细胞,用台盼蓝染色观察不同时间的细胞存活率,annexin V-FITC/PI染色法检测凋亡细胞形态学变化,ELISA法检测感染后不同时间细胞因子(IL-4、IL-10和TNF-α)水平变化,PCR芯片技术检测感染后8 h和12 h MAPK信号通路相关基因的差异表达情况。结果台盼蓝染色结果显示,RD细胞存活率随EV71感染时间延长明显下降,感染组IL-4、IL-10和TNF-α浓度随感染时间延长明显增加,与对照组差异有统计学意义(P<0.05)。EV71感染RD细胞8 h时,88种MAPK相关基因中,FOS、JUN分别上调3.34和3.19倍,而IL-1A、IL-1B、MAP2K3、IRAK1、NGF、IL-4、MAP3K1、MAP3K10、MAP3K11、MAP3K14、MAPK3下调2～5倍。EV71感染RD细胞20 h时,ELK1、FGF1、FOS、JUN、IRAK1、NGF、IL4、MAP3K1、MAP3K10、MAP3K11、MAP3K14、MAPK3、PIK3CG、STAT1和TNF明显上调。结论 EV71感染激活RD细胞MAPK信号通路,产生炎症细胞因子,与细胞凋亡有关。

Objective To explore the effects and mechanism of mitogen-activated protein kinase （MAPK） signaling pathway molecules in enterovirus 71 （EV71）-infected rhabdomyosarcoma ceils. Methods RD cells were infected with EV71 at multiplicity of infection （MOI） of 5. The cell survival rates at different times were observed by trypan blue staining and the apoptosis of cells was determined by Annexin V-FITC/PI staining. The levels of cytokines were tested by ELASA. The differential gene expressions of MAPK signaling pathway molecules in RD ceils were detected by PCR array at the 8 and 20 hours following EV71 infection respectively. Results The trypan blue staining revealed that the survival rates of RD cells significantly decreased with the extension of EV71 infection. Compared with the control group, the concentrations of IL-4, IL-10 and TNF-α significantly increased in the supernatants of EV71-infected RD cell group （P 〈 0.05）. After 8 hours of EV71 infection 88 MAPK-related genes in RD cells were analyzed. The expressions of FOS and JUN genes were up-regulated by 3.34 and 3.19 folds, respectively, while those of IL-1A, 1L-1B, MAP2K3, IRAKI NGF, IL-4, MAP3K1, MAP3K10, MAP3Kll, MAP3K14 and MAPK3 genes were down-regulated 2 to 5 folds. At the 20 hours after infection, how- ever, the expressions of ELK1, FGF1, FOS, JUN, IRAK1, NGF, IL-4, MAP3K1, MAP3KIO, MAP3Kll, MAP3K14, MAPK3, PIK3CG, STAT1 and TNF genes were significantly up-regulated in EV71-infected RD cells. Conclusion EV71 infection may activate MAPK signaling pathway in RD cells, which results in the secretions of inflammatory cytokines and may be associated with apoptosis.