miRNA-296-5p介导PTEN/PI3K/AKT信号通路抑制神经细胞SK-N-SH中EV71病毒复制的作用机制

The mechanism of miRNA-296-5p inhibiting EV71 virus replication in nerve cells SK-N-SH by targeting PTEN/PI3K/AKT signaling pathway

目的探讨miRNA-296-5p介导第10号染色体缺失的磷酸酶张力蛋白同源物/磷脂酰肌醇-3激酶/丝氨酸蛋白激酶(PTEN/PI3K/AKT)信号通路发挥抑制神经细胞SK-N-SH中肠道病毒71型(EV71)病毒复制的作用机制。方法收集临床EV71病毒感染手足口病患儿及正常体检儿的血清样本,采用酶联免疫吸附(ELISA)法检测两组儿童血清炎症因子降钙素原(PCT)、CRP、TNF-α、IL-1β、IL-6和IL-13的表达变化。取对数生长期经EV71病毒感染的人神经母细胞瘤SK-N-SH细胞,设置对照组、miRNA-296-5p mimic组和miRNA-296-5p inhibitor组,根据Lipofectamine■2000■细胞转染试剂进行转染。实时荧光定量PCR(qRT-PCR)和蛋白免疫印迹(Western blot)法观察三组细胞中EV71-VP1病毒基因mRNA和蛋白以及PTEN/PI3K/AKT信号通路相关分子表达。结果ELISA检测结果表明EV71病毒感染手足口病患儿血清炎症因子PCT、CRP、TNF-α、IL-1β、IL-6和IL-13的水平明显高于正常体检儿(P<0.05)。qRT-RCR和Western blot结果显示EV71病毒感染后神经细胞SK-N-SH中miRNA-296-5p和PTEN的mRNA和蛋白水平明显降低,而EV71-VP1和PTEN/PI3K/AKT信号通路相关分子的mRNA和蛋白水平明显升高(P<0.05);miRNA-296-5p mimic组中PTEN表达明显升高,同时抑制EV71-VP1的表达和PI3K/AKT信号通路的活化,而miRNA-296-5p inhibitor组可逆转上述作用(P<0.05)。结论miRNA-296-5p通过靶向PTEN/PI3K/AKT信号通路抑制神经细胞SK-N-SH中EV71病毒的复制,同时降低细胞炎症反应,靶向miRNA-296-5p及其下游PTEN/PI3K/AKT信号通路有望为临床EV71病毒导致的手足口病治疗提供可行的治疗靶点。

Objective To investigate whether miRNA-296-5p can inhibit enterovirus 71(EV71)virus replication in neural cells SK-N-SH by targeting the phosphatase and tensin homology deleted on chromosome 10(PTEN)/phosphatidylinositol 3-kinase(PI3K)/serine/threonine kinases(AKT)signaling pathway.Methods Serum samples were collected from patients with EV71 virus-infected hand-foot-mouth disease and normal physical examination,and the expressions of serum inflammatory factors procalcitonin(PCT),C-reactive protein(CRP),tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,and IL-13 were detected by enzymelinked immunosorbent assay(ELISA).Human neuroblastoma SK-N-SH cells infected by EV71 virus in logarithmic growth phase were set up as control group,miRNA-296-5p mimic group and miRNA-296-5p inhibitor group.The transfection was carried out according to the Lipofectamine 2000tm cell transfection reagent.The expression of EV71-VP1 gene mRNA and protein and PTEN/PI3K/Akt signal pathway related molecules in three groups of cells was observed by real-time fluorescent quantitative polymerase chain reaction(qRT-PCR)and Western blot.Results ELISA test results showed that the levels of serum inflammatory factors PCT,CRP,TNF-α,IL-1β,IL-6 and IL-13 in patients with EV71 virus-infected hand,foot and mouth disease were significantly higher than those in normal physical examination(P<0.05).The results of qRT-PCR and Western blot showed that the mRNA and protein levels of mirna-296-5p and PTEN in SK-N-SH were significantly decreased after EV71 virus infection,while the mRNA and protein levels of EV71-VP1 and molecules related to PI3K/AKT signaling pathway were significantly increased(P<0.05).The expression of PTEN was significantly increased in the miRNA-296-5p mimic group,and the expression of EV71-VP1 and the activation of the PI3K/AKT signaling pathway were inhibited,while the effect was reversed in the miRNA-296-5p inhibitor group(P<0.05).Conclusions MiRNA-296-5p inhibits the replication of EV71 virus in neural cells SK-N-SH by targeting the PTEN/PI3K/AKT signaling pathway,while reducing the cellular inflammatory response,targeting miRNA-296-5p and downstream PTEN/PI3K/AKT The signal pathway is expected to provide therapeutic targets and theoretical basis for the treatment of hand-foot-mouth disease caused by clinical EV71 virus.