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

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si RNA 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si RNA 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。

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.