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目的探讨miR-181b在氧糖剥夺(OGD)致N2As神经瘤细胞缺血损伤中的作用,及其对热休克蛋白(HSP)A5表达的调节。方法应用N2A细胞OGD模型模拟神经细胞缺血损伤,MTT比色法检测N2A细胞生存率,免疫印迹法检测HSPA5蛋白表达水平,实时定量PCR法检测miR-181b和HSPA5 mRNA表达水平,荧光素酶报告基因技术检测miR-181b对HSPA5 mRNA的直接调控作用。结果 miR-181b在OGD致N2A细胞缺血损伤中表达水平明显降低(n=5);在OGD致N2A细胞缺血损伤过程中,通过上调或抑制miR-181b的表达水平可以显著影响N2A细胞的生存率(n=6);而在非OGD条件下,miR-181b表达水平的改变对N2A细胞活力无影响;miR-181b表达水平的改变可显著影响HSPA5蛋白表达水平(n=3),而非HSPA5的mRNA水平;共转染miR-181b前体(premiR-181b)或miR-181b抑制剂(anti-miR-181b)可显著抑制或增高含有HSPA5 mRNA 3’-UTR的荧光素酶报告基因的活性(n=5)。结论 miR-181b通过负性调节HSPA5的蛋白表达水平,在OGD致N2A神经细胞缺血性损伤中发挥重要作用。
Objective To investigate the role of miR-181b in ischemic injury induced by oxygen glucose deprivation (OGD) in N2As neuroblastoma and its regulation of heat shock protein (HSP) A5 expression. Methods The N2 cell OGD model was used to simulate the ischemia injury of neurons. The survival rate of N2A cells was detected by MTT colorimetric assay. The expression of HSPA5 protein was detected by Western blotting. The expression of miR-181b and HSPA5 mRNA was detected by real- Gene technology to detect miR-181b direct regulation of HSPA5 mRNA. Results The expression of miR-181b was significantly decreased in N2A-deficient cells induced by OGD (n = 5). In OGD induced N2A ischemia-reperfusion injury, miR-181b expression was significantly up-regulated in N2A cells (N = 6). However, under the condition of non-OGD, the change of miR-181b expression had no effect on the viability of N2A cells. The change of miR-181b expression significantly affected the expression of HSPA5 protein (n = 3) Co-transfected miR-181b precursor or miR-181b inhibitor (miR-181b) can significantly inhibit or increase the luciferase reporter gene containing 3’-UTR of HSPA5 mRNA Activity (n = 5). Conclusion miR-181b plays an important role in ischemic injury induced by OGD in N2A neurons by negatively regulating the protein expression of HSPA5.