Long non-coding RNA CTA sensitizes osteosarcoma cells to doxorubicin through inhibition of autophagy

Abstract

Recently, several long non-coding RNAs (lncRNAs) have been implicated in osteosarcoma (OS). However, the regulatory roles of lncRNAs in chemotherapy resistance of OS still remain unclear. This study aimed to screen a novel lncRNA that contributes to chemotherapeutic resistance of OS, and to explore the underlying mechanisms. Our data showed that lncRNA CTA was markedly downregulated in OS tissues compared to their matched non-tumor tissues, and low expression of lncRNA CTA was significantly associated with the advanced clinical stage and tumor size. In addition, OS patients with low lncRNA CTA levels showed a worse prognosis when compared with those with high expression of lncRNA CTA. Furthermore, we report that lncRNA CTA has an inverse relationship with miR-210 expression in OS tissues. LncRNA CTA could be activated by doxorubicin (DOX), and could promote OS cell apoptosis by competitively binding miR-210, while inhibit cell autophagy. On the other hand, lncRNA CTA was downregulated in DOX-resistant OS cells. Overexpression of lncRNA CTA reduced autophagy and subsequently overcame DOX resistance of OS in vitro and in vivo. Therefore, we demonstrate that lncRNA CTA is an essential regulator in DOX-induced OS cell apoptosis, and the lncRNA CTA-miR-210 axis plays an important role in reducing OS chemoresistance.

Keywords: autophagy; doxorubicin; long non-coding RNA; osteosarcoma; resistance.

Figure 1. The expression of miR-210 related lncRNAs in osteosarcoma tissues

qPCR was performed to analyze the expression of ten lncRNAs that were associated with miR-210 in osteosarcoma tissues and the match adjacent tissues. The expression of lncRNA CTC-255N20, lncRNA RP11-610P16, lncRNA CTA and lncRNA RP11-432I5 in osteosarcoma tissues was significantly higher than in the matched adjacent tissues.

Figure 2. The expression of CTA and miR-210 in OS tissues

(A) Real-time RT-PCR was performed to determine the relative CTA level in osteosarcoma and adjacent tissues. (B) Real-time RT-PCR was performed to determine the relative miR-210 level in osteosarcoma and adjacent tissues. ***P < 0.001.

Figure 3. Low lncRNA CTA predicts a poor prognosis in patients with osteosarcoma, and is associated with DOX-resistance in MG-63 cells

(A) ISH was performed to analyze the expression of lncRNA CTA in osteosarcoma tissue microarray (Left. cancer tissue, n = 92; adjacent tissue, n = 12), and quantification (Right). The positive signals for lncRNA CTA were stained in blue violet in osteosarcoma and normal osteoblast cells, while the bone matrix around the osteoblast is not colored. (B) Kaplan-Meier postoperative survival curve for patterns of patients with osteosarcoma and lncRNA CTA expression. Osteosarcoma patients with low lncRNA CTA expression showed a shorter survival time than those with high lncRNA CTA expression. (C) qPCR was performed to analyze the expression of four lncRNAs that were decreased in osteosarcoma tissues in DOX-resistant MG-63 cells and the parental cells. (D) qPCR was performed to analyze the expression of four lncRNAs that were decreased in osteosarcoma tissues in MG-63 cells stimulated with DOX. *P < 0.05, **P < 0.01.

Figure 4. LncRNA CTA sensitizes osteosarcoma cells to DOX

(A) LncRNA CTA was overexpressed in MG-63 and Saos-2 cells. (B) MTT was performed to measure the inhibition rate after indicated treatment. Upregulation of lncRNA CTA significantly increased the inhibition rate induced by DOX. (C) Flow cytometry was performed to measure the apoptosis rate after indicated treatment. Upregulation of lncRNA CTA significantly enhanced the apoptosis rate induced by DOX. (D) TUNEL was performed to measure the apoptotic cells after indicated treatment (left), and quantification (right). Upregulation of lncRNA CTA significantly increased the number of apoptotic cells induced by DOX. All the experiments were independently performed three times. Data were presented as mean ± standard deviation.*P < 0.05, **P < 0.01.

Figure 5. LncRNA CTA inhibits autophagy that induced by DOX

(A) Immunofluorescence was performed to detect the expression of LC3B and its location (left), and quantification of the red fluorescence intensity (right). (B) Western blot was performed to analyze the expression of the key molecules of autophagy signaling, including LC3BI, LC3BII, P62, cleaved caspase 3, Bcl-2, BNIP3 and BNIP3L, and quantification. All the experiments were independently performed three times. Data were presented as mean ± standard deviation. *P < 0.05, **P < 0.01.

Figure 6. LncRNA CTA inhibits osteosarcoma growth in vivo

(A) On 4 weeks after implantation, the nude mice in each group were sacrificed, and the xenografted osteosarcoma was obtained. (B) The tumor volume was calculated. (C) Western blot was performed to analyze the expression of the key molecules of autophagy signaling, including LC3B1, LC3BII, P62, cleaved caspase 3 and Bcl-2. (D) Quantification of the band of western blot in (C). Data were presented as mean ± standard deviation. *P < 0.05, **P < 0.01.

Figure 7. LncRNA CTA regulates miR-210′s targets by competitively binding to miR-210

(A) LncRNA CTA was inversely correlative with miR-210 in osteosarcoma tissues. (B) Real-time RT-PCR was performed to determine the relative miR-210 level in osteosarcoma MG-63 and Saos-2 cells transfected with lncRNA CTA, CTA-shRNA or scramble control. (C) The luciferase activity was notably decreased in osteosarcoma MG-63 and Saos-2 cells co-transfected with miR-210 mimics and CTA 3′UTR, but unaltered in osteosarcoma MG-63 and Saos-2 cells co-transfected with miR-210 mimics and Mut CTA 3′UTR. (D) Real-time RT-PCR was performed to determine the relative mRNA level of miR-210 targets in osteosarcoma MG-63 and Saos-2 cells transfected with lncRNA CTA, CTA-shRNA or scramble control. Data were presented as mean ± standard deviation. ns, no significance.*P < 0.05, **P < 0.01.