Department of Joint Disease II, Zhengzhou Orthopedics Hospital, Zhengzhou City, Henan Province, P.R. China.
Eur Rev Med Pharmacol Sci. 2020 Dec;24(24):12655-12666. doi: 10.26355/eurrev_202012_24163.
To explore the regulatory mechanism of microRNA-122-5p (miR-122-5) targeting tumor protein p53 (TP53) gene to mediate PI3K-Akt-mTOR signaling pathway on the proliferation and apoptosis of osteosarcoma (OS) cells.
With the collection of osteosarcoma and normal adjacent tissues, the mRNA of miR-122-5p, TP53, PTEN, PI3K, Akt, mTOR, Bim, Bax, and Bcl-2 was detected by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), followed by the detection of the protein expression by Western blot. The target relationship between miR-122-5p and TP53 gene was verified. The third generation osteosarcoma cells were divided into Blank group, miR-122-5p mimic negative control (NC) group, miR-122-5p mimic group, miR-122-5p inhibitor NC group, miR-122-5p inhibitor group, rapamycin group and miR-122-5p inhibitor + rapamycin group. Furthermore, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect the proliferation ability, cell cycle distribution and apoptosis of each group after transfection.
The expression level of miR-122-5p in osteosarcoma was lower than that in normal tissues (p < 0.05), TP53, PTEN, Bim and Bax expression levels were decreased (all p < 0.05), while the expression levels of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR and Bcl-2 were highly upregulated (all p < 0.05). TP53 had the lowest expression in osteosarcoma cell line U-2OS (p < 0.05), which was selected for subsequent cell experiments. TP53 was the target gene of miR-122-5p. Compared with Blank group, miR-122-5p mimic group had increased expression of miR-122-5p (all p < 0.05); besides, there were significantly increased expression of TP53, PTEN, Bim, and Bax in miR-122-5p mimic group and rapamycin group, while remarkably decreased expression of PI3K, p-PI3K, Akt, p-Akt, mTOR, p-mTOR, and Bcl-2 (all p < 0.05), accompanied by increased proportion of cells in G0/G1 phase, decreased cell proportion in S phase, increased cell apoptosis and inhibited cell proliferation (all p < 0.05). The opposite trends were found in miR-122-5p inhibitor group relative to miR-122-5p mimic group and rapamycin group (all p < 0.05). Meanwhile, no significant difference was found in miR-122-5p inhibitor+rapamycin group when compared with that in Blank group (all p > 0.05) except for significantly decreased miR-122-5p expression (p < 0.05).
Upregulation of miR-122-5p may inhibit the proliferation and promote the apoptosis of osteosarcoma cells by inhibiting the activation of PI3K-Akt-mTOR signaling pathway, which may be related to the targeted up-regulation of TP53 expression.
探讨 microRNA-122-5p（miR-122-5）靶向肿瘤蛋白 p53（TP53）基因调控机制，介导 PI3K-Akt-mTOR 信号通路对骨肉瘤（OS）细胞增殖和凋亡的影响。
收集骨肉瘤和正常相邻组织，采用实时定量聚合酶链反应（qRT-PCR）检测 miR-122-5p、TP53、PTEN、PI3K、Akt、mTOR、Bim、Bax 和 Bcl-2 的 mRNA 表达，Western blot 检测蛋白表达，验证 miR-122-5p 与 TP53 基因的靶向关系。将第三代骨肉瘤细胞分为空白组、miR-122-5p 模拟阴性对照（NC）组、miR-122-5p 模拟组、miR-122-5p 抑制剂 NC 组、miR-122-5p 抑制剂组、雷帕霉素组和 miR-122-5p 抑制剂+雷帕霉素组。转染后，采用 3-(4,5-二甲基噻唑-2-基）-2,5-二苯基四氮唑溴盐（MTT）法和流式细胞术检测各组细胞增殖能力、细胞周期分布和凋亡情况。
骨肉瘤组织中 miR-122-5p 的表达水平低于正常组织（p<0.05），TP53、PTEN、Bim 和 Bax 的表达水平降低（均 p<0.05），而 PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR 和 Bcl-2 的表达水平显著升高（均 p<0.05）。骨肉瘤细胞系 U-2OS 中 TP53 表达最低（p<0.05），因此选择其进行后续细胞实验。TP53 是 miR-122-5p 的靶基因。与空白组相比，miR-122-5p 模拟组中 miR-122-5p 的表达增加（均 p<0.05）；此外，miR-122-5p 模拟组和雷帕霉素组中 TP53、PTEN、Bim 和 Bax 的表达显著增加，而 PI3K、p-PI3K、Akt、p-Akt、mTOR、p-mTOR 和 Bcl-2 的表达显著降低（均 p<0.05），同时 G0/G1 期细胞比例增加，S 期细胞比例减少，细胞凋亡增加，细胞增殖受到抑制（均 p<0.05）。与 miR-122-5p 模拟组和雷帕霉素组相比，miR-122-5p 抑制剂组呈现相反的趋势（均 p<0.05）。此外，miR-122-5p 抑制剂+雷帕霉素组与空白组相比，除 miR-122-5p 表达明显降低（p<0.05）外，差异无统计学意义（均 p>0.05）。
上调 miR-122-5p 可能通过抑制 PI3K-Akt-mTOR 信号通路的激活来抑制骨肉瘤细胞的增殖并促进其凋亡，这可能与靶向上调 TP53 表达有关。
