ZBTB7A Enhances Osteosarcoma Chemoresistance by Transcriptionally Repressing lncRNALINC00473-IL24 Activity

Abstract

Chemoresistance remains a major drawback to osteosarcoma treatment. ZBTB7A, a member of the POK transcription repressor family, was shown to play an important role in tumorigenesis. However, the effect of ZBTB7A on osteosarcoma chemoresistance is completely unknown. In this study, we found that ZBTB7A is increased in cisplatin-resistant osteosarcoma cells and that elevated ZBTB7A inhibits cisplatin-induced apoptosis by repressing LINC00473 expression. Further mechanistic studies revealed that ZBTB7A directly binds to the promoter and suppresses the transcription of LINC00473. Additionally, our data indicate that LINC00473 interacts with the transcript factor C/EBPβ, facilitating its binding to the promoter of IL24, leading to decrease chemoresistance. Thus, these findings indicate that the ZBTB7A-mediated LINC00473-C/EBPβ-IL24 pathway is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Figure 1

ZBTB7A inhibited cisplatin-induced apoptosis. (A) The protein levels of ZBTB7A were analyzed in U2OS and U2OSR cells. (B-E) U2OS and U2OSR cells were treated with cisplatin as indicated, and cell lysates were then subjected to Western blotting analysis using the indicated antibodies. (F and G) U2OSR and U2OS cells with or without knocked down ZBTB7A were treated with cisplatin as indicated, and the cell lysates were analyzed by Western blotting with the indicated antibodies. (H) U2OS cells with or without overexpressing ZBTB7A were treated with cisplatin as indicated, and the cell lysates were then subjected to Western blotting analysis using the indicated antibodies. (I and J) U2OS cells with or without knocked down ZBTB7A were treated with cisplatin as indicated. The percentage of cell apoptosis was analyzed by flow cytometry. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (K and L) U2OS cells with or without overexpressedZBTB7A were treated with cisplatin as indicated. Cell apoptosis was analyzed by flow cytometry. The data represent the mean ± SD of three independent experiments. *P < .05 versus CTR. (M and N) The ZBTB7A zinc finger mutant was constructed (M). U2OS cells with or without overexpressed ZBTB7A or ZBTB7A R399L were treated with cisplatin as indicated, and the cell lysates were then subjected to Western blotting analysis using the indicated antibodies (N).

Figure 2

ZBTB7A repressed LINC00473 expression. (A) U2OS cells with or without knocked down ZBTB7A were treated with 10 μM cisplatin for the indicated times. The filtered lncRNA array data were subjected to unsupervised hierarchical clustering analysis. The metric was set as the Euclidean distance. (B and C) U2OS and U2OSR cells with or without knocked down ZBTB7A were treated with cisplatin as indicated and the mRNA levels of LINC00473 were analyzed by qRT-PCR. The protein levels of ZBTB7A were detected by Western blotting. The data represent the mean ± SD of three independent experiments. **P < .01, ***P < .001 versus CTR. (D) U2OS cells with or without overexpressed ZBTB7A or theR399L mutant were treated with cisplatin as indicated. The mRNA level of LINC00473 was analyzed by qRT-PCR. The data represent the mean ± SD of three independent experiments. **P < .01versus CTR. (E) Schematic illustration of pGL3-based reporter constructs used in luciferase assays to examine the transcriptional activity of LINC00473. (F) The promoter of LINC00473 (F1) was transfected into U2OSR cells with or without ZBTB7A knockdown. The cells were then treated with cisplatin. Luciferase activity was measured by the luciferase assay. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (G) The promoters of LINC00473 (F, F2, F3) were individually transfected into U2OSR cells with or without ZBTB7A knockdown. The cells were then treated with cisplatin. Luciferase activity was measured. The data represent the mean ± SD of three independent experiments. ***P < .001 versus CTR. (H) ChIP analysis showed the binding of ZBTB7A to the promoter of LINC00473. An isotype-matched IgG was used as a negative control. (I) Schematic illustration of pGL3-based reported constructs used in luciferase assays to examine the transcriptional activity of LINC00473. The dots indicated the ZBTB7A binding sites. (J) The promoters of LINC00473 WT and mutants (MUT1, 2, 3) were transfected into U2OSR cells with or without knockdown ZBTB7A, and then the cells were treated with cisplatin. Luciferase activity was measured. The data represent the mean ± SD of three independent experiments. ***P < .001 versus CTR.

Figure 3

ZBTB7A enhanced cisplatin resistance by repressing LINC00473. (A-C) U2OS cells with or without knockdown LINC00473 were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies (A). Cell viability was analyzed by the CKK8 assay (B). LINC00473 RNA level was detected by qRT-PCR. The data represent the mean ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001 versus CTR. (D-I) U2OS and U2OSR cells with or without LINC00473 overexpression were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies (D and G). Cell viability was analyzed by the CKK8 assay (E, H).The LINC00473 RNA level was detected by qRT-PCR (F and I). The data represent the mean ± SD of three independent experiments. *P < .05, ***P < .001 versus CTR. (J-L) LINC00473 was inhibited using shRNA in U2OSR cells with or without knocked down ZBTB7A, and then the cells were treated with cisplatin as indicated. Cell lysates were subjected to Western blotting analysis using the indicated antibodies (J). Cell viability was analyzed by the CKK8 assay (K). The LINC00473 RNA level was detected by qRT-PCR (L). The data represent the mean ± SD of three independent experiments. *P < .05, ***P < .001 versus CTR. (M-O) LINC00473 was overexpressed in U2OS cells with or without ZBTB7A overexpression, and then the cells were treated with cisplatin as indicated. Cell lysates were subjected to Western blotting analysis using the indicated antibodies (M). Cell viability was analyzed by the CKK8 assay (N).The LINC00473 RNA level was detected by qRT-PCR (O). The data represent the mean ± SD of three independent experiments. *P < .05, **P < .01, ***P < .001 versus CTR.

Figure 4

ZBTB7A inhibited IL24 expression relying on LINC00473. (A) An unbiased genome mRNA expression profiling heat map between CTR and LINC00473 overexpression under cisplatin treatment as indicated. (B) The numbers of changed genes involved in different biological processes were statistically analyzed. (C) Comparison of the array data (more than two-fold) with apoptosis-related genes. The related genes are listed. (D-G) U2OS and U2OSR cells with or without overexpressing LINC00473 were treated with cisplatin as indicated. The protein and mRNA levels of IL24 were detected by Western blotting and qRT-PCR. The data represent the mean ± SD of three independent experiments. *P < .05, **P < .01 versus CTR. (H and I) U2OS cells with or without LINC00473 knockdown were treated with cisplatin as indicated. The protein and mRNA levels of IL24 were detected by Western blotting and qRT-PCR. The data represent the mean ± SD of three independent experiments. ***P < .001 versus CTR. (J and K) LINC00473 was inhibited in U2OSR cells with or without knocked down ZBTB7A. The expression of IL24 was analyzed by Western blotting and qRT-PCR. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (L and M) LINC00473 was overexpressed in U2OS cells with or without ZBTB7A overexpression. The expression of IL24 was analyzed by Western blotting and qRT-PCR. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR.

Figure 5

LINC00473 upregulated IL24 expression via interacting with C/EBPβ. (A) Schematic illustration of pGL3-based reported constructs used in luciferase assays to examine the transcriptional activity of IL24. (B) The promoter of IL24 (P1) was transfected into U2OSR cells with or without LINC00473 overexpression. Luciferase activity was measured. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (C) The promoter of IL24 (P1) was transfected into U2OS cells with or without LINC00473 knockdown. Luciferase activity was measured. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (D) The promoter of IL24 (P1, P2, P3) was transfected into U2OSR cells with or without LINC00473 overexpression. Luciferase activity was measured. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR. (E and F) C/EBPα and C/EBPβ were individually knocked down in U2OS cells with or without LINC00473 overexpression. The protein and mRNA levels of IL24 were analyzed by Western blotting and qRT-PCR. The data represent the mean ± SD of three independent experiments. *P < .05 versus CTR. (G and H) ChIP analysis showed the binding of C/EBPβ to the promoter of IL24 when LINC00473 was overexpressed or knocked down. An isotype-matched IgG was used as a negative control. (I and J) RIP analysis showed the interaction of C/EBPβ with LINC00473. The data represent the mean ± SD of three independent experiments. **P < .01 versus CTR.

Figure 6

ZBTB7A repressed cisplatin-induced apoptosis via regulating the LINC00473-IL24 axis. (A and B) U2OS cells with or without IL24 knockdown were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies (A). Cell viability was analyzed by CKK8 assay (B). The data represent the mean ± SD of three independent experiments. ***P < .001 versus CTR. (C and D) IL24 was knocked down in U2OSR cells with or without LINC00473 overexpression. The cells were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies. The mRNA level of LINC00473 was analyzed by qRT-PCR. (E and F) IL24 was inhibited in U2OSR cells with or without ZBTB7A knockdown. The cells were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies (E). The mRNA levels of LINC00473 and IL24 were analyzed by qRT-PCR (F). (G) IL24 was knocked down in U2OS cells with or without ZBTB7A overexpression/LINC00473 knockdown. The cells were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies.

Supplementary Figure 1

Fractionation of U2OS cells with or without treatment with cisplatin followed by qRT-PCR (left panel) and fractionation controls by Western blot (right panel). U1 RNA served as a positive control for nuclear gene expression. N, nuclear fraction; C, cytoplasmic fraction. The data are representative of at least three independent experiments.

Supplementary Figure 2

(A-C) MG63 cells with or without knockdown ZBTB7A were treated with cisplatin as indicated. Cell lysates were then subjected to Western blotting analysis using the indicated antibodies (A). Cell viability was analyzed by CKK8 assay (B). The mRNA levels of LINC00473 and IL24 were analyzed by qRT-PCR (C). The data represent the mean ± SD of three independent experiments. **P < .01, ***P < .001 versus CTR. (D and E) LINC00473 or HA-IL24 was transfected into MG63 with or without overexpressing Flag-ZBTB7A. The cells were treated with cisplatin as indicated, and cell lysates were then subjected to Western blotting analysis using the indicated antibodies.