hsa_circ_0008039和miR-484在SK-N-SH细胞氧糖剥夺-复糖复氧损伤中的作用:与Fis1的关系

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目的:评价hsa_circ_0008039和miR-484在人神经母细胞瘤(SK-N-SH)细胞氧糖剥夺-复糖复氧(OGD/R)损伤中的作用及其与线粒体分裂蛋白1(Fis1)的关系。方法:体外培养SK-N-SH细胞至对数生长期,采用随机数字表法分为5组(n n=25):对照组(C组)、OGD/R组、hsa_circ_0008039 siRNA组(S组)、hsa_circ_0008039过表达组(E组)和hsa_circ_0008039 siRNA+miR-484抑制剂组(S+I组)。C组细胞在正常条件下培养;S组、E组和S+I组分别转染has_circ_0008039 siRNA、has_circ_0008039过表达载体、has_circ_0008039 siRNA和miR-484抑制剂后,氧糖剥夺12 h复糖复氧24 h制备OGD/R损伤模型。于复糖复氧24 h时采用CCK-8法检测细胞活力和LDH漏出量,采用RT-qPCR法检测hsa_circ_0008039、miR-484和Fis1 mRNA的表达,Western blot法检测Fis1表达;双荧光素酶报告验证hsa_circ_0008039与miR-484的靶向关系。n 结果:与C组比较,其余4组细胞活力降低,LDH漏出量增加,OGD/R组和E组hsa_circ_0008039和Fis1表达上调,miR-484表达下调,S组hsa_circ_0008039和Fis1表达下调,miR-484表达上调,S+I组hsa_circ_0008039和miR-484表达下调,Fis1表达上调(n P<0.05);与OGD/R组比较,E组和S+I组细胞活力降低,LDH漏出量增加,S组细胞活力升高,LDH漏出量减少,E组hsa_circ_0008039和Fis1表达上调,miR-484表达下调,S组hsa_circ_0008039和Fis1表达下调,miR-484表达上调,S+I组hsa_circ_0008039和miR-484表达下调,Fis1表达上调(n P<0.05);与S组比较,S+I组细胞活力降低,LDH漏出量增加,miR-484表达下调,Fis1表达上调(n P<0.01)。双荧光素酶报告实验表明:has_circ_0008039靶向结合miR-484。n 结论:hsa_circ_0008039靶向结合miR-484参与了SK-N-SH细胞氧糖剥夺-复糖复氧损伤的过程,与上调Fis1表达有关。“,”Objective:To evaluate the role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured n in vitro to logarithmic growth stage and divided into 5 groups (n n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of On 2-glucose supply to develop the OGD/R model.At 24 h of restoration of On 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484.n Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I (n P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I (n P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I (n P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically.n Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.
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