TPX2 regulated by miR-29c-3p induces cell proliferation in osteosarcoma via the AKT signaling pathway

Abstract

The present study aimed to investigate the significance of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression in osteosarcoma. First, the TPX2 expression and survival analysis data were evaluated from The Cancer Genome Atlas (TCGA) database. Next, reverse transcription-quantitative PCR was used to explore the expression of TPX2 in osteosarcoma tissues. The observed potential target relationship between TPX2 and microRNA (miR)-29c-3p was verified using TargetScan and luciferase reporter assays. Kaplan-Meier survival analysis was used to determine associations between TPX2 expression levels and survival prognosis. TPX2 small interfering RNA was successfully constructed and transfected into osteosarcoma cell lines. The effects of TPX2 on osteosarcoma cell proliferation were then detected by MTT assay. In addition, the expression levels of AKT signaling pathway-associated proteins were identified by western blot analysis. The expression of TPX2 was upregulated and the expression of miR-29c-3p was downregulated in osteosarcoma. High expression of TPX2 was linked to a poor prognosis. Using luciferase assay and the miRNA mimic and inhibitors, miR-29c-3p was able to target and repress TPX2, and siRNA knockdown of TPX2 resulted in the inhibition of osteosarcoma cell proliferation by affecting the AKT pathway. Overall, the study showed that miR-29c-3p could inhibit the proliferation of osteosarcoma cells via TPX2 downregulation, and that TPX2 and miR-29c-3p may serve as promising prognostic indicators.

Keywords: AKT pathway; TPX2; osteosarcoma; pediatrics; proliferation.

Figure 1.

TPX2 and miR-29c-3p expression levels in osteosarcoma and the associated prognosis. (A) TPX2 expression at the mRNA level was high in 262 tumor tissues and low in 2 normal tissues (data from GEPIA). (B) miR-29c-3p expression level in osteosarcoma (data from OncoLnc). (C) Osteosarcoma patients with high TPX2 expression had poor overall survival (data from Kaplan-Meier plotter). (D) Osteosarcoma patients with high miR-29c-3p expression had favorable overall survival (data from Kaplan-Meier plotter). *P<0.05. T, tumor; N, normal; miR, microRNA; TPX2, targeting protein for Xenopus kinesin-like protein 2.

Figure 2.

TPX2 is upregulated in osteosarcoma cell lines and tissues, and is associated with a poor prognosis in patients with osteosarcoma. (A) The expression level of TPX2 in the osteosarcoma samples was higher than that in the pair-matched adjacent tissues, as determined by RT-qPCR. (B) The expression level of miR-29c-3p in the osteosarcoma samples was lower than that in the pair-matched adjacent tissues, as determined by RT-qPCR. (C) TPX2 expression at the mRNA level was high in osteosarcoma cell lines vs. the human osteoblast cells, as determined by RT-qPCR. (D) miR-29c-3p expression level was low in the osteosarcoma cell lines vs. the human osteoblast cells, as determined by RT-qPCR. (E) Regression analysis of the correlation between miR-29c-3p and TPX2 expression. (F) The cut-off value of TPX2 was evaluated in the patients with osteosarcoma. (G) Survival curves for patients with osteosarcoma with regard to TPX2 expression. *P<0.05, **P<0.01 and ***P<0.001. miR, microRNA; TPX2, targeting protein for Xenopus kinesin-like protein 2; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; AUC, area under the curve.

Figure 3.

Knockdown of TPX2 inhibits the proliferation of osteosarcoma cells. (A) RT-qPCR analysis of the transfection efficiency of si-TPX2 in the MG63 cell line. (B) RT-qPCR analysis of the transfection efficiency of si-TPX2 in the U2OS cell line. (C) Western blotting analysis of the transfection efficiency of si-TPX2 in the MG63 and U2OS cell lines. (D) MTT analysis of the effects of TPX2 knockdown on cell proliferation in the MG63 cell line. (E) MTT analysis of the effects of TPX2 knockdown on cell proliferation in the U2OS cell line. The NC was a non-targeting (scramble) siRNA sequence. **P<0.01 and ***P<0.001. si, small interfering; KD, knockdown; TPX2, targeting protein for Xenopus kinesin-like protein 2; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density.

Figure 4.

miR-29c-3p suppresses the proliferation of osteosarcoma cells. miR-29c-3p expression in MG63 cells transfected with (A) miR mimics and (B) miR inhibitor, as measured by RT-qPCR. miR-29c-3p expression in U2OS cells transfected with (C) miR mimics and (D) miR inhibitor, as measured by RT-qPCR. MTT analysis of the effects of (E) miR-29c-3p mimics and (F) miR inhibitor on cell proliferation in MG63 cell lines. MTT analysis of the effects of (G) miR-29c-3p mimics and (H) miR inhibitor on cell proliferation in U2OS cell lines. The NCs for the miRNA mimics and inhibitors were non-targeting sequences. **P<0.01 and ***P<0.001. miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density.

Figure 5.

miR-29c-3p downregulates TPX2 expression level by binding to its 3′UTR directly. (A) The target site of miR-29c-3p is in the TPX2 3′UTR. (B-E) Luciferase reporter gene assays were performed to detect the fluorescence activities of the TPX2 3′UTR in MG63 and U2OS cells, which were co-transfected with wt TPX2 3′UTR or mut TPX2 3′UTR and miR-29c-3p mimics/inhibitors, respectively. TPX2 expression in transfected (F) MG63 and (G) U2OS cells, as determined by RT-qPCR and western blot analysis. TPX2 mRNA expression level was detected by RT-qPCR in (H) MG63 cells and (I) U2OS cells co-transfected with TPX2 siRNA and miR-29c-3p inhibitor. An MTT assay was performed to detect proliferation abilities of (J) MG63 and (K) U2OS cells co-transfected with TPX2 siRNA and miR-29c-3p inhibitor. *P<0.05, **P<0.01 and ***P<0.001. si/siRNA, small interfering; KD, knockdown; TPX2, targeting protein for Xenopus kinesin-like protein 2; UTR, untranslated region; miR, microRNA; NC, negative control; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; OD, optical density; wt, wild-type; mut, mutant; ns, not significant; CDS, coding sequence.

Figure 6.

TPX2 exhibits a significant positive correlation with the AKT signaling pathway in osteosarcoma. (A) The AKT signaling pathway. (B-D) Pearson's rank correlation analysis of the correlation of TPX2 expression with (B) HSP90, (C) AKT and (D) BRCA1 in osteosarcoma. (E) Knockdown of TPX2 inhibits the AKT signaling pathway. (F) TPX2 regulated by miR-29c-3p induces cell proliferation in osteosarcoma via the AKT signaling pathway. *P<0.05, **P<0.01 and ***P<0.001. KD, knockdown; TPX2, targeting protein for Xenopus kinesin-like protein 2; miR, microRNA; NC, negative control.