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

Zhou Y, Huang Z, Wu S, Zang X, Liu M, Shi J - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, China. zcy1958@medmail.com.cn.

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.

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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.
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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.

Mentions: We next examined the effects of miRNA-33a on TWIST expression in human OS cells. As shown in Figure 5, miR-33a was highly expressed in Saos-2 cells, which had a low constitutive expression of TWIST at both the mRNA and the protein levels. In contrast, MG-63 cells had a constitutive low expression of miR-33a, and a high expression of TWIST at both the mRNA and the protein levels (Figure 5). Thus, overexpression and knockdown of TWIST were respectively performed in the two cell lines to approach the study objectives. As shown in Figure 6A, inhibition of miR-33a by antagomir-33a increased TWIST expression by over 1.5 fold in Saos-2 cells. On the other hand, overexpression of miR-33a decreased TWIST expression by about 30%. Overexpression of TWIST led to an approximately two-fold increase of TWIST expression in Saos-2 cells, which was largely reversed by overexpression of miR-33a and doubled by antagomir-33a. As shown in Figure 6B, overexpression of miR-33a decreased TWIST expression by nearly 70% in MG-63 cells, while antagomir-33a increased TWIST expression by 0.4 fold. Knockdown of TWIST by shRNA resulted in an approximately 80% decrease of endogenous TWIST expression in MG-63 cells, which was partially reversed by antagomir-33a.


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

Zhou Y, Huang Z, Wu S, Zang X, Liu M, Shi J - J. Exp. Clin. Cancer Res. (2014)

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.
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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.
Mentions: We next examined the effects of miRNA-33a on TWIST expression in human OS cells. As shown in Figure 5, miR-33a was highly expressed in Saos-2 cells, which had a low constitutive expression of TWIST at both the mRNA and the protein levels. In contrast, MG-63 cells had a constitutive low expression of miR-33a, and a high expression of TWIST at both the mRNA and the protein levels (Figure 5). Thus, overexpression and knockdown of TWIST were respectively performed in the two cell lines to approach the study objectives. As shown in Figure 6A, inhibition of miR-33a by antagomir-33a increased TWIST expression by over 1.5 fold in Saos-2 cells. On the other hand, overexpression of miR-33a decreased TWIST expression by about 30%. Overexpression of TWIST led to an approximately two-fold increase of TWIST expression in Saos-2 cells, which was largely reversed by overexpression of miR-33a and doubled by antagomir-33a. As shown in Figure 6B, overexpression of miR-33a decreased TWIST expression by nearly 70% in MG-63 cells, while antagomir-33a increased TWIST expression by 0.4 fold. Knockdown of TWIST by shRNA resulted in an approximately 80% decrease of endogenous TWIST expression in MG-63 cells, which was partially reversed by antagomir-33a.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Orthopaedics, The Third Xiangya Hospital, Central South University, 138 Tongzipo Road, Changsha, Hunan 410013, China. zcy1958@medmail.com.cn.

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.

Show MeSH
Related in: MedlinePlus