CircTLK1通过miR-424-5p/FBXO3轴对氧糖剥夺/复氧诱导的神经细胞损伤的影响

目的 探讨环状RNA TLK1(CircRNA TLK1,CircTLK1)对氧糖剥夺/复氧(Oxygen glucose deprivation/Reoxygenation,OGD/R)诱导的神经元HT22损伤的影响以及对微小RNA(microRNA,miR)-424-5p/F-box蛋白3(F-box protein 3,FBXO3)的调控作用。方法 将HT22细胞分为对照组、OGD/R组、sh-NC组、沉默环状RNA TLK1(Silencing circular RNA tlk1,sh-circTLK1)组、sh-circTLK1+抑制剂NC组、sh-circTLK1+miR-424-5p抑制剂组、sh-circTLK1+miR-424-5p抑制剂+sh NC组、sh-circTLK1+miR-424-5p抑制剂+sh FBXO3组,除对照组外其余各组细胞均行OGD/R操作,细胞计数试剂盒8(Cell counting Kit 8,CCK-8)法测定HT22细胞活力; 脂连蛋白V-异硫氰酸荧光素(Adiponectin V-fluorescein isothiocyanate,Annexin V-FITC)细胞凋亡试剂盒测定HT22细胞凋亡; 测定HT22细胞乳酸脱氢酶(Lactate dehydrogenase,LDH)漏出率; 实时荧光定量聚合酶链反应(Real time fluorescent quantitative polymerase chain reaction,RT-qRCR)法测定HT22细胞miR-424-5p,FBXO3 mRNA水平; 蛋白免疫印记法(Western Blot)检测B淋巴细胞瘤-2关联基因X(B lymphoma-2 gene association X,Bax)、活化半胱天冬酶-3(Cleaved caspase-3)、FBXO3水平; 双荧光素酶测定CircTLK1与miR-424-5p以及miR-424-5p与FBXO3靶向关系,并使用RNA下拉实验验证CircTLK1与miR-424-5p关系。结果 与对照组比较,OGD/R组miR-424-5p,HT22细胞活力降低(P<0.05),circTLK1,FBXO3 mRNA水平,HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率升高(P<0.05); 与OGD/R组比较,sh-circTLK1组HT22细胞活力增加(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平,LDH漏出率降低(P<0.05); 与sh-circTLK1组比较,sh-circTLK1+miR-424-5p抑制剂组细胞活力降低(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率升高(P<0.05); 与sh-circTLK1+miR-424-5p抑制剂组比较,sh-circTLK1+miR-424-5p抑制剂+sh FBXO3组细胞活力升高(P<0.05),HT22细胞凋亡率、Bax,Cleaved caspase-3水平、LDH漏出率降低(P<0.05); CircTLK1与miR-424-5p以及miR-424-5p与FBXO3均存在靶向关系。结论 CircTLK1沉默可能通过调控miR-424-5p/FBXO3对OGD/R诱导的HT22细胞损伤来发挥保护作用。

Effects of CircTLK1 on oxygen-glucose deprivation/reoxygenation-induced neuronal injury via miR-424-5p/FBXO3 axis

ObjectiveTo explore the effect of CircTLK1 on oxygen-glucose deprivation/reoxygenation(OGD/R)-induced neuronal HT22 cell injury and its regulatory effect on miR-424-5p/FBXO3.Methods HT22 cells were divided into control group, OGD/R group, sh-NC group, sh-circTLK1 group, sh-circTLK1+inhibitor NC group, sh-circTLK1+miR-424-5p inhibitor group, sh-circTLK1+miR-424-5p inhibitor+sh NC group, and sh-circTLK1+miR-424-5p inhibitor+sh FBXO3 group, except for the control group, the cells in the other groups were subjected to OGD/R operation. The viability of HT22 cells was determined by CCK-8 kit. Annexin V-FITC apoptosis kit was used to measure the apoptosis of HT22 cells. The LDH leakage rate of HT22 cells was measured. RT-qRCR method was used to measure the miR-424-5p and FBXO3 mRNA levels in HT22 cells. Western Blot was used to detect the expression levels of Bax, Cleaved Caspase-3, and FBXO3. The targeting relationship between CircTLK1 and miR-424-5p and between miR-424-5p and FBXO3 was determined by dual luciferase, and the relationship between CircTLK1 and miR-424-5p was verified by RNA pull-down experiment.Results Compared with the control group, the miR-424-5p level and viability of HT22 cells in the OGD/R group decreased(P<0.05), while themRNAlevels of circTLK1 and FBXO3, the apoptosis rate of HT22 cell,the expression levels of Bax and cleaved caspase-3, and the LDH leakage rate increased(P<0.05). Compared with OGD/R group, the viability of HT22 cells in sh-circTLK1 group increased(P<0.05), the HT22 cell apoptosis rate, the expression levels of Bax andcleaved caspase-3, and LDH leakage rate decreased(P<0.05). Compared with the sh-circTLK1 group, the cell viability in the sh-circTLK1+miR-424-5p inhibitor group decreased(P<0.05), the HT22 cell apoptosis rate, the expression levels of Bax and cleaved caspase-3, and LDH leakage rate increased(P<0.05). Compared with the sh-circTLK1+miR-424-5p inhibitor group, the cell viability in the sh-circTLK1+miR-424-5p inhibitor+sh FBXO3 group increased(P<0.05), the apoptosis rate of HT22 cell, the expression levels of Bax andcleaved caspase-3, and LDH leakage rate decreased(P<0.05). There was a targeting relationship between CircTLK1 and miR-424-5p and between miR-424-5p and FBXO3.Conclusion CircTLK1 silencing may play a protective effect on OGD/R-induced HT22 cell injury by regulating miR-424-5p/FBXO3, and provide a target for the treatment of stroke-like diseases.