miR-143在氧糖剥夺/再灌注引起的星形胶质细胞活化中的作用机制研究

Study on the Mechanism of miR-143 in the Activation of Astrocytes Induced by Oxygen-glucose Deprivation/Reperfusion

关键词:  氧糖剥夺/再灌注  miR-143  星形胶质细胞  炎症反应

Key words:  oxygen-glucose deprivation/reperfusion  miR-143  astrocyte  inflammation

摘要:

目的 探讨miR-143/HIPK2轴在氧糖剥夺/再灌注（oxygen-glucose deprivation/reperfusion，OGD/R）引起的星形胶质细胞活化中的作用。方法 星形胶质细胞OGD/R处理0，3，6，12 h，应用Western blotting检测星形胶质细胞的活化标志物GFAP和蛋白激酶HIPK2表达，实时荧光定量PCR检测miR-143水平变化，免疫荧光染色检测OGD/R处理6 h后GFAP表达。对照组、OGD/R模型组给予Anti-Con和Anti-miR-143干预，应用Western blotting检测GFAP表达。采用荧光素酶报告基因技术验证HIPK2是否为miR-143的靶标分子。miR-143和miR-Con以及Anti-miR-143和Anti-miR-Con分别转染星形胶质细胞，应用Western blotting检测HIPK2表达。离体培养星形胶质细胞分为5个处理组，分别为Anti-Con干预的对照组、Anti-Con干预的OGD/R模型组、Anti-miR-143干预的OGD/R模型组、Anti-miR-143和siRNA Con共同干预的OGD/R模型组以及Anti-miR-143和HIPK2 siRNA共同干预的OGD/R模型组，应用Western blotting检测GFAP表达。结果 与对照组相比，OGD/R 6 h处理的星形胶质细胞GFAP表达增高至峰值（P<0.01），该结果进一步被免疫荧光染色验证，HIPK2表达下调至最低值（P<0.001），miR-143表达增加（P<0.01）。与Anti-Con干预的对照组相比，Anti-Con干预的OGD/R模型组星形胶质细胞GFAP表达上调（P<0.01）；与Anti-Con干预的OGD/R模型组比，Anti-miR-143干预的OGD/R模型组星形胶质细胞GFAP表达下调（P<0.05）。miR-143能明显抑制HIPK2基因活性，但对其结合位点突变体没有显著抑制作用。与Anti-miR-143和siRNA Con共同干预的OGD/R模型组比，Anti-miR-143和HIPK2 siRNA共同干预的OGD/R模型组星形胶质细胞GFAP表达水平上调（P<0.05）。结论OGD/R通过miR-143/HIPK2轴引起星形胶质细胞活化。

Abstract:

OBJECTIVE To explore the effect of miR-143/HIPK2 axis on the astrocyte activation induced by oxygen-glucose deprivation/reperfusion(OGD/R). METHODS The expression of GFAP, an astrocyte specific activation marker, and HIPK2 protein kinase were detected by Western blotting, and the level of miR-143 was detected by real time PCR in astrocyte with OGD/R treatment for 0, 3, 6, 12 h. GFAP expression detected by immunofluorescence staining after OGD/R treatment for 6 h. The control group was transfected with Anti-Con and Anti-miR-143, also the OGD/R model group was transfected with Anti-Con and Anti-miR-143. The expression of GFAP was detected by Western blotting. The method of luciferase reporter gene was used to verify whether HIPK2 was the target of miR-143. Astrocytes were transfected with miR-143/miR-Con and Anti-miR-143/Anti-miR-Con followed by HIPK2 examination using Western blotting. Astrocytes cultured in vitro were divided into five treatment groups:control group with Anti-Con intervention, OGD/R model group with Anti-Con intervention, OGD/R model group with Anti-miR-143 intervention, OGD/R model group with Anti-miR-143 and siRNA Con cointervention, and OGD/R model group with Anti-miR-143 and HIPK2 siRNA cointervention. GFAP expression was detected by Western blotting. RESULTS Compared with the control group, GFAP expression in astrocytes increased to the peak value(P<0.01), confirmed by immunofluorescent staining HIPK2 expression decreased to the lowest value (P<0.001), the expression of miR-143 in astrocytes increased after OGD/R 6 h treatment(P<0.01). Compared with control group with Anti-Con intervention, GFAP expression in OGD/R group with Anti-Con intervention was upregulated(P<0.01). Compared with OGD/R group with Anti-Con intervention, GFAP expression was downregulated in OGD/R group with Anti-miR-143 intervention(P<0.05). miR-143 significantly inhibited HIPK2 gene activity, but had no significant inhibitory effect on its binding site mutants. Compared with OGD/R group with Anti-miR-143 and siRNA Con cointervention, GFAP expression level in astrocytes in OGD/R group with Anti-miR-143 and HIPK2 siRNA cointervention was upregulated(P<0.05). CONCLUSION OGD/R induces activation of astrocytes through miR-143/HIPK2 axis.