Mechanism of miR-122-5p regulating the activation of PI3K-Akt-mTOR signaling pathway on the cell proliferation and apoptosis of osteosarcoma cells through targeting TP53 gene.

Abstract

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.