Liu Min, Wang Shuo-Min, Jiang Zheng-Xuan, Lauren Hennein, Tao Li-Ming
Department of Ophthalmology, the Second Affiliated Hospital of Anhui Medical University, Hefei 230601, Anhui Province, China.
Department of Ophthalmology, the Fourth Affiliated Hospital of Anhui Medical University, Hefei 230032, Anhui Province, China.
Int J Ophthalmol. 2018 Oct 18;11(10):1600-1607. doi: 10.18240/ijo.2018.10.05. eCollection 2018.
To explore the effect of miR-22 on viability, migration, invasion and apoptosis in retinoblastoma (RB) Y79 cells and to further detect the potential mechanism.
Plasmids were constructed to change the expression level of miR-22 in Y79 cells. Real-time reverse transcription polymerase chain reaction (RT-PCR) was conducted to test the expression level of miR-22. After changing the expression of miR-22, the mRNA and protein levels of high-mobility group box 1 (HMGB1) were investigated using RT-PCR and Western blotting. The effect of miR-22 on viability was analyzed by using cell counting kit-8 (CCK-8) assay and the effect on apoptosis was detected by the flow cytometry. Wound healing migration assay and Transwell invasion assay were used to detect the effects of miR-22 on cell motility.
miR-22 inhibited viability, migration and invasion, while promoting apoptosis, in RB Y79 cells. The inhibition rate of miR-22 overexpression group at 12, 24, 48h was 11.71%±2.54%, 21.36%±1.39% and 29.44%±1.15%, respectively. Cellular apoptosis was higher in miR-22 overexpression group (17.00%±0.39%) compared with negative control (4.38%±0.38%). miR-22 negatively mediated the expression of HMGB1. Furthermore, decreased HMGB1 significantly attenuated viability, migration and invasion, while promoting apoptosis. Enforced expression of HMGB1 partially rescued the effects of miR-22 overexpression on cell viability, migration, invasion and apoptosis. Moreover, the phosphorylated protein kinase B (p-AKT) was significantly downregulated in the HMGB1 shRNA group and miR-22 overexpression group and elevated in the HMGB1 overexpression group compared with the normal control.
miR-22 inhibites viability, migration and invasion and increases apoptosis in Y79 cells by targeting HMGB1. These findings may provide a therapeutic strategy for RB.
探讨微小RNA-22（miR-22）对视网膜母细胞瘤（RB）Y79细胞活力、迁移、侵袭及凋亡的影响，并进一步检测其潜在机制。
构建质粒以改变Y79细胞中miR-22的表达水平。采用实时逆转录聚合酶链反应（RT-PCR）检测miR-22的表达水平。改变miR-22表达后，运用RT-PCR和蛋白质印迹法检测高迁移率族蛋白B1（HMGB1）的mRNA和蛋白水平。使用细胞计数试剂盒-8（CCK-8）法分析miR-22对细胞活力的影响，通过流式细胞术检测其对凋亡的影响。采用伤口愈合迁移试验和Transwell侵袭试验检测miR-22对细胞运动能力的影响。
miR-22抑制RB Y79细胞的活力、迁移和侵袭，同时促进其凋亡。miR-22过表达组在12、24、48小时的抑制率分别为11.71%±2.54%、21.36%±1.39%和29.44%±1.15%。与阴性对照组（4.38%±0.38%）相比，miR-22过表达组的细胞凋亡率更高（17.00%±0.39%）。miR-22负向介导HMGB1的表达。此外，HMGB1表达降低显著减弱细胞活力、迁移和侵袭，同时促进凋亡。HMGB1的强制表达部分挽救了miR-22过表达对细胞活力、迁移、侵袭和凋亡的影响。此外，与正常对照组相比，HMGB1短发夹RNA（shRNA）组和miR-22过表达组中磷酸化蛋白激酶B（p-AKT）显著下调，而HMGB1过表达组中p-AKT升高。
miR-22通过靶向HMGB1抑制Y79细胞的活力、迁移和侵袭并增加其凋亡。这些发现可能为RB提供一种治疗策略。
