miR-33a is up-regulated in chemoresistant osteosarcoma and promotes osteosarcoma cell resistance to cisplatin by down-regulating TWIST

Abstract

Background: miRNAs are involved in osteosarcoma (OS) chemoresistance, and TWIST reportedly enhances cisplatin-induced OS cell apoptosis by inhibiting multiple signaling pathways. In this study, we profiled miRNAs differentially expressed in chemoresistant OS, with a focus to identify miRNAs that regulate TWIST expression and OS chemoresistance.

Methods: OS patients who showed <90% tumor necrosis after neochemotherapy were defined as poor responders (chemoresistant), and those who showed ≥90% tumor necrosis were defined as good responders (control). miRNA microarray analysis was carried out with a discovery cohort (n = 12) of age-, sex- and tumor stage-matched chemoresistant and control OS patients.

Results: Among the up-regulated miRNAs in chemoresistant OS samples, miR-33a was verified to down-regulate TWIST expression, which was supported by an inverse miRNA-33a/TWIST expression trend in the validation cohort (n = 70), target-sequence-specific inhibition of TWIST-3' untranslated region-luciferase reporter activity by miR-33a, and alteration of TWIST expression by overexpression or inhibition of miR-33a in human OS cell lines. In Saos-2 cells treated with cisplatin, inhibition of miR-33a by antagomir-33a markedly increased cell apoptosis, which was enhanced by overexpression of TWIST. The apoptosis-inducing effect of TWIST overexpression was reversed by overexpression of miR-33a. In MG-63 cells, overexpression of miR-33a significantly decreased cisplatin-induced cell apoptosis, which was enhanced by knockdown of TWIST. Antagomir-33a significantly increased cisplatin-induced cell apoptosis, which was reversed by knockdown of TWIST.

Conclusions: We have demonstrated in this study that miR-33a is up-regulated in chemoresistant OS and that the miR-33a level is negatively correlated with the TWIST protein level in OS. Our in vitro data indicate that miR-33a promotes OS cell resistance to cisplatin by down-regulating TWIST; on the other hand, inhibition of miR-33a by antagomir-33a enhances cisplatin-induced apoptosis in OS cells by up-regulating TWIST expression. The findings suggest that inhibition of miR-33a/TWIST signaling could be a potential new strategy to enhance neoadjuvant chemotherapy for OS.

Figure 1

Western blot analysis of TWIST expression in chemoresistant and control osteosarcoma (OS) tissues. (A) OS tissue lysates from the chemoresistant OS and the non-chemoresistant control groups (n = 6 each) were subject to western blot analysis for TWIST expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Lanes 1, 3, 5, 7, 9, 11 were samples from the chemoresistant OS group. Lanes 2, 4, 6, 8, 10, 12 were samples from the control group. (B) Density of the TWIST blots was normalized against that of GAPDH to obtain a relative blot density. The relative TWIST blot density of the non-chemoresistant control group was expressed as fold changes to that of the chemoresistant OS group (designated as 1). *p < 0.05 vs chemoresistant OS.

Figure 2

Effects of selected miRNAs on TWIST 3′-untranslated region (UTR). Twelve miRNAs potentially able to regulate TWIST 3′-UTR were selected based on TargetScan prediction software and individually co-transfected with a TWIST-3′UTR-luciferase reporter in MG-63 cells. Among the selected miRNAs, miR-33a, miR-25 and miR-363 were differentially expressed between chemoresistant and control osteosarcoma tissues based on results of the microarray analysis. miRNAs that significantly reduced renilla luciferase activity compared to the control miR-Vec (hTR) (designated as 1, cut off value: 0.8) were selected for validation (light bars). *p < 0.05 vs 0.8 (cut off value).

Figure 3

Regulation of TWIST 3′-untranslated region (UTR) by miR-33a. (A) MG-63 cells were co-transfected with a TWIST-3′UTR-luciferase reporter and miR-33a or control miR-Vec (hTR). The relative luciferase activity in cells co-transfected with hTR was designated as 1. (B) Schematic presentation of generation of a TWIST-mut33-luciferase reporter (mut33) by site-directed mutagenesis of the predicted binding sequence of miR-33a in TWIST 3′-UTR. (C) MG-63 cells were co-transfected with the TWIST-3′UTR-luciferase reporter or TWIST-mut33-luciferase reporter and miR-33a or control miR-Vec (hTR). The relative luciferase activity in cells co-transfected with the TWIST-3′UTR-luciferase reporter (wild type) and hTR was designated as 1. *p < 0.05 vs hTR.

Figure 4

miR-33a and TWIST levels in osteosarcoma (OS) tissues from chemoresistant and control OS patients in the validation cohort. Real-time RT-PCR and Western blot analysis were performed to determine (A) miR-33a and (B) TWIST protein levels in OS tissues from the chemoresistant OS and control groups in the validation cohort (n = 35 each group), respectively. Data are shown in scatter plots. The mean miR-33a and TWIST protein levels are marked by a horizontal bar in each group.

Figure 5

miR-33a and TWIST levels in osteosarcoma (OS) cells. Real-time RT-PCR and Western blot analysis were performed to determine (A) miR-33a and (B) TWIST mRNA and (C) TWIST protein levels in Saos-2 and MG-63 human OS cells. The miR-33a expression level and the relative TWIST mRNA level and protein blot density in MG-63 cells were designated as 1, respectively. *p < 0.05 vs MG-63.

Figure 6

TWIST expression in osteosarcoma cells with overexpression or knockdown/inhibition of TWIST and/or miR-33a. (A) In Saos-2 cells, TWIST expression was determined in normal control cells (NC, lane 1), cells stably transfected with empty pcDNA3 vector (VC, lane 2), cells overexpressing TWIST (lane 3), cells overexpressing miR-33a (lane 4), cells transfected with antagomir-33a (lane 5), cells overexpressing TWIST and miR-33a (lane 6), and cells overexpressing TWIST plus transfection of antagomir-33a (lane 7). (B) In MG-63 cells, TWIST expression was determined in normal control cells (NC, lane 1), cells stably transduced with scramble control shRNA (SC, lane 2), cells stably expressing TWIST-shRNA (T-shRNA, lane 3), cells overexpressing miR-33a (lane 4), cells transfected with antagomir-33a (lane 5), cells stably expressing T-shRNA and overexpressing miR-33a (lane 6), and cells stably expressing T-shRNA plus transfection of antagomir-33a (lane 7). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) blotting was used as a loading control. Density of the TWIST blot was normalized against that of GAPDH to obtain a relative blot density, which was expressed as fold changes to the relative TWIST blot density of NC cells (designated as 1). In (A) Saos-2 cells, ^ap < 0.05 vs NC and VC; ^bp < 0.05 vs TWIST; ^cp < 0.05 vs miR-33a; ^dp < 0.05 vs antagomir-33a. In (B) MG-63 cells, ^ap < 0.05 vs NC and SC; ^bp < 0.05 vs T-shRNA; ^cp < 0.05 vs miR-33a; ^dp < 0.05 vs antagomir-33a; ^ep < 0.05 vs T-shRNA + miR-33a.

Figure 7

Cisplatin-induced apoptosis in osteosarcoma cells with overexpression or knockdown/inhibition of TWIST and/or miR-33a. In Saos-2 cells, TUNEL (terminal deoxynucleotidyl transferase mediated nick-end labeling) assays were performed in normal control cells (NC), cells stably transfected with empty pcDNA3 vector (VC), cells overexpressing TWIST, cells overexpressing miR-33a, cells transfected with antagomir-33a, cells overexpressing TWIST and miR-33a, and cells overexpressing TWIST plus transfection of antagomir-33a. In MG-63 cells, TUNEL assays were performed in normal control cells (NC), cells stably transduced with scramble control shRNA (SC), cells stably expressing TWIST-shRNA (T-shRNA), cells overexpressing miR-33a, cells transfected with antagomir-33a, cells stably expressing T-shRNA and overexpressing miR-33a, and cells stably expressing T-shRNA plus transfection of antagomir-33a. (A) The cells were under normal culture conditions for 8 hours. The cell apoptosis rate at 8 hours was expressed as fold changes to that of the Saos-2 NC cells (designated as 1). (B) Saos-2 and (C) MG-63 cells were treated with 15 nM of cisplatin for 8 hours. The cell apoptosis rates at 4 hours and 8 hours were shown as the percentage of TUNEL positive cells in total cells.