Overexpressed circPVT1, a potential new circular RNA biomarker, contributes to doxorubicin and cisplatin resistance of osteosarcoma cells by regulating ABCB1

Abstract

Circular RNAs (circRNAs) represent a widespread class of non-coding RNAs generated from back-splicing, with a circular loop structure. Many circRNAs have been reported to play essential roles in cancer development and have the potential to serve as a novel class of biomarkers for clinical diagnosis. However, the role of circRNA in osteosarcoma (OS) remains largely unknown. In the current study, we examined the expression level of circular RNA PVT1 (circPVT1), previously screened and identified the oncogenic role in gastric cancer, in OS and found that circPVT1 was significantly up-regulated in the OS tissues, serums and chemoresistant cell lines, correlated with poor prognosis of OS patients. Besides, ROC curve demonstrated that circPVT1 may be a better diagnostic biomarker than alkaline phosphatase (ALP) in OS with more sensitivity and specificity. In addition, functional assays revealed that circPVT1 knockdown by siRNA could weaken the resistance to doxorubicin and cisplatin of OS cells through decreasing the expression of classical drug resistance-related gene ABCB1. These findings may provide a new insight into the role of circPVT1 as a biomarker for the diagnosis and treatment target of OS.

Keywords: biomarker; chemoresistance.; circPVT1; circular RNA; osteosarcoma.

Figure 1

CircPVT1 is up-regulated and correlated with poor clinical outcomes in OS. (A) Expression level of circPVT1 in 80 paired OS and paracancerous tissues. (B) Expression level of circPVT1 in OS tissues of lung metastasis and lung non-metastasis group. (C) Expression level of circPVT1 in OS tissues of chemoresistant and chemosensitive group. (D) Patients with increased circPVT1 expression had a reduced overall survival time than those with low level of circPVT1 expression.

Figure 2

Serum circPVT1 may be a better diagnostic biomarker than ALP in OS. (A) Expression level of circPVT1 in serum from 50 OS patients, 20 benign bone tumor patients and 20 age- and sex-matched healthy individuals. (B) ROC curves of the serum circPVT1 and ALP in 50 newly diagnosed patients and 20 healthy donors.

Figure 3

CircPVT1 knockdown partly reverses the doxorubicin and cisplatin resistance of OS cells in vitro. (A) Expression level of circPVT1 in seven human OS cell lines and normal osteoblast cell line hFOB1.19, including three paired chemoresistant and chemosensitive OS cell lines. (B) RT-qPCR analysis of the effect on knockdown of circPVT1 expression by siRNA in the MG63R and U2OSR cell lines. si-circPVT1-1 was chosen for the siRNA used in the study owing to the highest knockdown efficiency. (C-D) CCK-8 assay showed that the viability and IC50 value of MG63R (U2OSR) cells in the si-circPVT1 group when exposed to doxorubicin or cisplatin were reduced compared with the control groups. (E-F) Cell colony formation assay showed that the clone numbers of MG63R (U2OSR) cells in the si-circPVT1 group were reduced compared with the si-NC group when exposed to 8ug/ml doxorubicin or 3ug/ml cisplatin for 14 days. *P < 0.05, **P < 0.01.

Figure 4

CircPVT1 knockdown reduces the expression of classical multidrug resistance related gene-ABCB1 in OS cells. (A) The mRNA level of ABCB1 was detected by qPCR in the MG63R (or U2OSR) transfected with si-circPVT1 or si-NC and the controlled MG63 (or U2OS) cells. (B) The protein level of ABCB1 was detected by WB in the MG63R (or U2OSR) transfected with si-circPVT1 or si-NC and the controlled MG63 (or U2OS) cells. *P < 0.05, **P < 0.01.