HOTAIRM1靶向miR-433对MPP+诱导的SK-N-SH细胞损伤的影响

The effect of HOTAIRM1 on injury of MPP-induced SK-N-SH cells by targeting miR-433

目的探讨同源异型盒A转录本反义RNA1(Homeobox A transcript antisense RNA myeloid-specific 1,HOTAIRM1)对1-甲基-4-苯基吡啶离子(1-Methyl-4-phenylpyridine, MPP+)诱导的SK-N-SH细胞损伤的影响及作用机制。方法体外培养SK-N-SH细胞,分为对照组(Con组)、Model组、乱序无意义阴性序列(Out-of-order nonsense negative sequence, si-NC)组、HOTAIRM1小干扰RNA (HOTAIRM1 small interfering RNA,si-HOTAIRM1)组、si-HOTAIRM1+抑制剂阴性序列(Inhibitor negative sequence, anti-miR-NC)组和si-HOTAIRM1+miR-433抑制剂(MiR-433 inhibitor, anti-miR-433)组;流式细胞仪检测细胞周期和细胞凋亡;Western Blot检测兔抗人B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)和B淋巴细胞瘤-2相关蛋白(Bcl-2-associated X protein, Bax)蛋白表达水平;试剂盒检测丙二醛(Malonaldehyde, MDA)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-Px)和超氧化物歧化酶(Superoxide dismutase, SOD)水平;实时荧光定量反转录-聚合酶链反应(Real-time quantitative polymerase chain reaction, RT-qPCR)检测HOTAIRM1和微小RNA-433(microRNA-433,miR-433)表达水平;双荧光素酶报告基因实验验证miR-433与HOTAIRM1靶向关系。结果与Con组比较,Model组SK-N-SH细胞的细胞周期G0-G1期延长(P<0.05),S期缩短(P<0.05),细胞凋亡率、Bax蛋白、MDA和OTAIRM1表达水平升高(P<0.05),Bcl-2蛋白、GSH-Px和SOD活性及miR-433表达水平降低(P<0.05);与Model组比较,si-HOTAIRM1组SK-N-SH细胞的细胞周期G0-G1期缩短(P<0.05),S期延长(P<0.05),细胞凋亡率、Bax蛋白、MDA和OTAIRM1表达水平降低(P<0.05),Bcl-2蛋白、GSH-Px和SOD活性及miR-433表达水平升高(P<0.05);与si-HOTAIRM1+anti-miR-NC组比较,si-HOTAIRM1+anti-miR-433组SK-N-SH细胞的细胞周期G0-G1期延长(P<0.05),S期缩短(P<0.05),细胞凋亡率、Bax蛋白和MDA水平升高(P<0.05),Bcl-2蛋白、GSH-Px水平和SOD活性降低(P<0.05);Model组与si-NC组、si-HOTAIRM1组与si-HOTAIRM1+anti-miR-NC组各检测指标水平比较均无显著差异(P>0.05)。HOTAIRM1可与miR-433靶向结合。结论下调HOTAIRM1可靶向上调miR-433表达来抑制MPP+诱导的SK-N-SH细胞氧化应激和凋亡,进而减轻细胞损伤。

Objective To investigate the effect of HOTAIRM1 on MPP+-induced damage in SK-N-SH cells and its mechanism.Methods SK-N-SH cells were cultured and divided into control group(Con group), Model group, si-NC group, si-HOTAIRM1, si-HOTAIRM1+anti-miR-NC group and si-HOTAIRM1+anti-miR-433 group, then cell cycle and apoptosis were detected by flow cytometry. The protein expressions of Bcl-2 and Bax were detected by Western Blot. The expressions of MDA, GSH-Px and SOD were quantified by detection kit. The expressions of HOTAIRM1 and miR-433 were detected by RT-qPCR. The dual luciferase reporter assay was conducted to verify the relationship between miR-433 and HOTAIRM1.Results Compared with the Con group, G0-G1 phase of SK-N-SH cells in the Model group was prolonged(P<0.05),while S phase was shortened(P<0.05). Furthermore, apoptosis level and expressions of Bax, MDA, and OTAIRM1 were increased(P<0.05), while the levels of Bcl-2 and miR-433 and the activity of GSH-Px and SOD were decreased(P<0.05). Compared with the Model group, G0-G1 phase of SK-N-SH cells in the si-HOTAIRM1 group was shortened(P<0.05), while S phase was prolonged(P<0.05). Apoptosis level and of Bax, MDA, and OTAIRM1 were decreased(P<0.05), while expressions of Bcl-2 and miR-433 and activities of GSH-Px and SOD were increased(P<0.05). Compared with the si-HOTAIRM1+anti-miR-NC group, G0-G1 phase of SK-N-SH cells in the si-HOTAIRM1+anti-miR-433 group was prolonged(P<0.05), while S phase was shortened(P<0.05). Apoptosis level and expressions of Bax and MDA were increased(P<0.05), while Bcl-2 expression and the activities of GSH-Px and SOD were decreased(P<0.05). However, there were no significant differences among the detection indicators of Model group and the si-NC group or the si-HOTAIRM1 group and the si-HOTAIRM1+anti-miR-NC group(P>0.05). HOTAIRM1 could target miR-433 binding.Conclusion Downregulating HOTAIRM1 could inhibit MPP+-induced oxidative stress and apoptosis in SK-N-SH cells by upregulation of miR-433, thereby alleviating cell injury.